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Cyclic amp-dependent protein kinase : a potential target for actions of vanadium Jelveh, Kioumars Ahmadreza 2004

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CYCLIC AMP-DEPENDENT PROTEIN KINASE: A POTENTIAL TARGET FOR ACTIONS OF VANADIUM by KIOUMARS AHMADREZA JELVEH B.Sc. Hon., University of British Columbia, 1996 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 2004 © Kioumars Ahmadreza Jelveh, May 2004 ABSTRACT Vanadium salts and organic complexes diminish or reverse many of the consequences of insulin deficiency and insulin resistance in vivo. It is widely assumed that the inhibition of protein tyrosine phosphatases can explain biological effects of vanadium; however, there is considerable evidence in vitro that vanadium can act significantly downstream from the protein tyrosine phosphorylation "level" of signal transduction. The aim of these studies was to focus on the ability of vanadium to inhibit hormone-sensitive triglyceride (TG) hydrolysis because this action is observed at rather low vanadium concentrations (typically 10-100 uM) and the mechanisms involved in the control of TG hydrolysis are well defined. Based on the balance of prior studies, this thesis focused on the possibility that cAMP-dependent protein kinase (PKA) might be a viable target for inhibition by vanadium. These studies confirmed that PKA could be potently inhibited by vanadium. Due to the complexity of interactions between vanadate, vanadyl and reagents used in assay mixtures, it was essential to define the experimental conditions carefully to allow unambiguous characterization of the effects of vanadium. Following initial optimization of enzyme activity, PKA was found to be inhibited by high concentrations of vanadyl sulphate (VS) (IC50 > 400 uM). However, PKA inhibition was seen at dramatically lower VS concentration (IC50 < 25 uM) when sequestration of vanadyl ions was minimized. Under these conditions, the true concentration of vanadyl was lower than the threshold for detection by EPR spectroscopy (~ 15 uM). The derived kinetic constants (Xi values < 20 uM) must still be considered "apparent" values and the true affinity constant of vanadyl for PKA is probably even lower. The effective PKA inhibitor species is likely to be vanadyl because a range of divalent cation chelators abolished PKA inhibition by VS. Vanadyl was both a weak cofactor and a strong inhibitor of PKA, perhaps replicating the dual roles hypothesized for magnesium. From the results of EPR and kinetic studies, it was concluded that the vanadyl EPR signal is enhanced in the presence of glutathione at physiological pH. Significantly, the combination of reduced and oxidized glutathione (GSH and GSSG) was more effective than either form in maintaining the vanadyl EPR signal at pH 7-9. The most effective combination of GSH and GSSG observed in these studies is similar to that expected within mammalian cells. In conclusion, these studies provide evidence that PKA could be an important target for vanadyl action in vivo, the vanadyl being produced and stabilized through the actions of GSH and GSSG. ii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xiii ACKNOWLEDGEMENT xix CHAPTER ONE: INTRODUCTION 1 1.1 INSULIN AND DIABETES 1 1.1.1 Insulin Actions 1 1.1.2 The Complexity of Insulin Signaling Pathways 1 1.1.3 Control of Triglyceride Hydrolysis 4 1.1.4 Properties of PKA 7 1.1.5 Diabetes Mellitus 11 1.2 CHEMICAL PROPERTIES AND BIOLOGICAL ACTIONS OF VANADIUM 14 1.2.1 Discovery and Basic Properties of Vanadium 14 1.2.2 Vanadium Coordination and Physical Chemistry 14 1.2.3 Properties of Vanadium to Be Considered in the Context of In Vitro Enzyme Assays 19 1.2.4 Biological Effects of Vanadium 20 1.2.5 Vanadium Metabolism 21 1.2.6 Specific Proteins Affected by Vanadium Salts 22 1.2.7 "Insulin-Like" Properties of Vanadium In Vivo 23 1.2.8 "Insulin-Like" Properties of Vanadium In Vitro 25 iii 1.2.9 Mechanism(s) and Putative Site(s) of Vanadium Action 27 1.2.10 Examples of Other "Insulin-Like" Elements 29 1.3 BIOLOGICAL LIGANDS THAT MAY BE INVOLVED IN VANADIUM ACTIONS 31 1.3.1 Rationale for Binding of Vanadyl to Endogenous Ligands 31 1.3.2 Vitamin C (Ascorbic Acid) Interactions With Vanadium 32 1.3.3 Glutathione Interactions With Vanadium 33 1.3.4 Vanadium Binding to Serum Proteins 36 1.4 RATIONALE AND HYPOTHESES FOR PROPOSED STUDIES 37 CHAPTER TWO: MATERIALS AND METHODS 39 2.1 MATERIALS 39 2.1.1 Vanadium Salts 39 2.1.2 Enzymes .' : 39 2.1.3 Peptide or Protein Substrates and Inhibitors 39 2.1.4 Other Materials 39 2.2 ENZYME KINETICS : 40 2.2.1 Assay of PKA by the Filter Paper Method. 40 2.2.2 Phosphocellulose Paper (P-81) Washing Method 41 2.2.3 Methods of Analysis of the Enzyme Kinetic Results 41 2.2.4 Measurement of Assay Buffer pH 44 2.2.5 PKA Assays Involving N2(g) Purging 44 2.3 SPECTROMETRY 44 2.3.1 Electrospray Ionization Mass Spectrometry (ESI MS) 44 2.3.2 Electron Paramagnetic Resonance (EPR) Spectrometry 45 iv CHAPTER THREE: OPTIMIZATION OF PKA ASSAY AND EFFECTS OF VANADYL SULPHATE 49 3.1 RATIONALE 49 3.2 RESULTS AND DISCUSSION 49 3.2.1 Steady-State Kinetics 49 3.2.2 Effects of Buffer on PKA 51 3.2.3 Effects of pH on PKA 52 3.2.4 Effects of Salt Concentration on PKA 53 3.2.5 Effects of Reducing Agents on PKA 54 3.2.6 Effects of EDTA and BSA on PKA 55 3.2.7 Effects of Magnesium on PKA 57 3.2.8 Linearity of PKA Reaction Versus Time 59 3.2.9 Effects of Peptide or Protein Substrates on PKA 61 3.2.10 Effects of VS on PKA Using Conditions Optimized for PKA 64 3.3 SUMMARY 65 CHAPTER FOUR: FACTORS THAT LIMIT THE APPARENT INHIBITION OF PKA BY VANADYL SULPHATE 66 4.1 RATIONALE 66 4.2 RESULTS AND DISCUSSION 66 4.2.1 Effects of VS on PKA Using Minimal Concentrations of BSA, DTT, and EDTA 66 4.2.2 Effects of BSA, DTT, EDTA, and MES on the Inhibition of PKA by VS 68 4.2.3 Effects of Protein Substrate on PKA Inhibition by VS 72 4.2.4 Effects of Magnesium on PKA Inhibition by VS 73 4.2.5 Effects of VS on Different Preparations of PKA 74 v 4.2.6 Comparison of the Effects of Vanadium Salts and Compounds on PKA 76 4.2.7 What Is the True Concentration of Vanadyl Ions in PKA Assays? 79 4.2.8 Further Evidence that PKA Is Inhibited by Vanadyl Ions 82 4.3 SUMMARY 83 CHAPTER FIVE: POSSIBLE MECHANISMS OF ACTION OF VANADYL ON PKA 84 5.1 RATIONALE 84 5.2 RESULTS AND DISCUSSION 85 5.2.1 Does Vanadyl Substitute for Magnesium as a Cofactor for PKA? 85 5.2.2 Studies to Test the Binding of VS to Kemptide or Histone 86 5.2.3 Effects of ATP on PKA Inhibition by VS 90 5.2.4 Effects of VS on PKA Activity Following Nitrogen Purging 97 5.2.5 Studies of Possible Binding of Vanadyl to PKA 107 5.2.6 Effects of Glycyl and Glycine-Loop Peptides 112 5.3 SUMMARY 117 CHAPTER SIX: PRESERVATION OF VANADYL AT PHYSIOLOGICAL pH: IN SEARCH OF 'NATURAL" LIGANDS 118 6.1 RATIONALE 118 6.2 RESULTS AND DISCUSSION 119 6.2.1 Unprotected Vanadyl Is Unstable and Fails to Inhibit PKA 119 6.2.2 VS Has Limited Stability Even at pH 4 120 6.2.3 Effects of Glutathione on PKA Inhibition by VS 122 6.2.4 Analysis of Vanadyl in PKA Assay Buffers by EPR 128 6.3 SUMMARY 138 vi CHAPTER SEVEN: MAJOR CONCLUSIONS AND FUTURE DIRECTIONS 139 7.1 PKA IS POTENTLY INHIBITED BY VS 139 7.2 ASSAY CONDITIONS PROFOUNDLY INFLUENCE THE POTENCY OF INHIBITION OF PKA BY VS 139 7.3 VANADYL AND NOT VANADATE POTENTLY INHIBITS PKA 141 7.4 STUDIES OF THE MECHANISM OF INHIBITION OF PKA BY VANADYL 142 7.5 WHAT IS THE TRUE CONCENTRATION OF VANADYL IN PKA ASSAYS? 145 7.6 DOES GLUTATHIONE MEDIATE THE EFFECTS OF VANADYL ON PKA? 146 7.7 RELEVANCE OF THE CURRENT STUDIES TO VANADIUM ACTIONS IN VIVO 148 REFERENCES 152 vii LIST OF TABLES Table 1.1 Insulin Actions in Coordination of Metabolism 2 Table 1.2 Overview of cAMP/PKA-Mediated Physiological Functions 9 Table 1.3 Overview of the Treatments for Types 1 and 2 Diabetes 13 Table 1.4 Metabolic Effects of Vanadium on Adipocytes In Vitro 26 Table 1.5 Ligands and Antioxidants That May Protect Vanadyl In Vivo 32 Table 1.6 Interactions Between Vanadyl and GSSG or GSH 36 Table 2.1 Graphical Methods Used to Characterize the PKA Reaction and the Effects of VS 43 Table 2.2 Settings of the Bruker ESP300E EPR Spectrometer 48 Table 3.1 Binding of Vanadyl or Vanadate to Various Buffers 52 Table 4.1 Interactions of Vanadyl With Selected Anions 67 Table 4.2 Glycyl Peptides Specifically Bind Vanadyl Not Vanadate 88 Table 5.1 Effects of ATP on PKA Inhibition by VS - Summary of Kinetic Parameters.... 107 Table 5.2 Examples of the Glycine-Rich (Rossmann) Motif in Selected Protein Kinases..! 13 viii LIST OF FIGURES Figure 1.1 A Simplified Overview of Insulin Signaling Pathways 3 Figure 1.2 Key Proteins Involved in Control of Triglyceride Hydrolysis 6 Figure 1.3 The 3-Dimensional Structure of PKA Catalytic Subunit Complexed With Mn-ATP and PKI 10 Figure 1.4 Effects of pH on the Speciation of Vanadium (IV) and (V) in Aqueous Solution 15 Figure 1.5 Chemical Structures of Vanadium Salts and Compounds 18 Figure 1.6 Possible Interactions Between GSH, GSSG, Vanadyl, and Vanadate 34 Figure 1.7 Structure of Reduced Glutathione at Physiological pH 35 Figure 2.1 Configuration of the Continuous-Flow Mixing Apparatus and Electrospray Ionization Quadrupole Mass Spectrometer 47 Figure 3.1 Time-Dependent Phosphorylation of Histone by PKA 50 Figure 3.2 Time-Dependent Phosphorylation of Kemptide by PKA 50 Figure 3.3 Effect of PKA Concentration on Histone Phosphorylation 51 Figure 3.4 Effect of pH on PKA Activity 53 Figure 3.5 Effects of Salt Concentration on PKA Activity 54 Figure 3.6 Effects of Reducing Agents on PKA Activity 56 Figure 3.7 Effects of BSA and EDTA on PKA Activity 57 Figure 3.8 ATP-Magnesium Complexes 58 Figure 3.9 Effects of Magnesium on PKA Activity 59 Figure 3.10 Linearity of PKA Reaction Versus Time 60 Figure 3.11 Phosphorylation of Various Substrates by PKA 62 Figure 3.12 Effects of Kemptide Concentration on Phosphorylation by PKA 63 Figure 3.13 Effects of VS on PKA Activity 64 ix Figure 4 .1 Effects of VS on PKA Kemptide Kinase Activity in a Buffer Containing Minimal Concentrations of BSA, EDTA, and DTT 6 7 Figure 4 . 2 Effects of BSA, DTT, EDTA, and MES on PKA Inhibition by VS 6 9 Figure 4 .3 Effect of Increasing the Ratio of DTT to VS on PKA Inhibition 7 0 Figure 4 . 4 Effects of ATP or MES on X-Band EPR Spectrum of VS 7 1 Figure 4 . 5 Effects of VS on PKA Histone Kinase Activity in a Buffer Containing Minimal Concentrations of BSA, EDTA, and DTT 7 2 Figure 4 . 6 Effects of Magnesium Concentration on PKA Inhibition by VS 7 4 Figure 4 . 7 Effects of VS on Different Preparations of PKA 7 5 Figure 4 .8 Comparisons of the Effects of Vanadium Salts and Compounds on PKA 7 7 Figure 4 . 9 X-Band EPR Spectra of BPOV, BMOV, and VS 7 8 Figure 4 . 1 0 Absence of Vanadyl Signal by EPR in PKA Assay Mixtures 8 0 Figure 4 . 1 1 Threshold Sensitivity for Detection of Vanadyl by EPR 8 1 Figure 4 . 1 2 Effects of Glycyl Peptides on PKA Inhibition by VS 8 3 Figure 5.1 Comparisons of VS, OV, and Magnesium as Potential Cofactors for PKA 8 5 Figure 5 .2 ESI Mass Spectra of Kemptide in the Absence or Presence of VS 8 7 Figure 5.3 Effects of Kemptide Concentration on VS Inhibition of PKA 8 8 Figure 5 .4 ESI Mass Spectra of Histone in the Absence or Presence of VS 8 9 Figure 5 .5 Effects of ATP on PKA Inhibition by VS (I)-High ATP and VS Concentrations 9 1 Figure 5 .6 Effects of ATP on PKA Inhibition by VS (II) -Low ATP and VS Concentrations 9 1 Figure 5 .7 Effects of VS and ATP on PKA Activity With Chloride as the Major Anion 9 2 Figure 5 .8 Effects of VS and ATP on PKA Activity With Acetate as the Major Anion 9 5 Figure 5 .9 Effects of N2(g) Purging, DTT, GSH, and (3-EtSH on PKA Activity 9 8 x Figure 5.10 Retention of Significant PKA Activity With "Minimal" Buffer 99 Figure 5.11 Effects of VS and ATP on PKA (Batch I) Activity With the "Minimal" Buffer 100 Figure 5.12 Effects of VS and ATP on PKA (Batch II) Activity With the "Minimal" Buffer 103 Figure 5.13 ESI Mass Spectrum of PKA 109 Figure 5.14 ESI Mass Spectra of PKA With Various Concentrations of VS 110 Figure 5.15 ESI Mass Spectra of Cytochrome c: Effects of VS orNPLSO^ I l l Figure 5.16 The 3 -Dimensional Structure of the Glycine-Rich Loop of PKA 113 Figure 5.17 Effects of Di- and Tri-Glycyl and Glycine-Loop Peptides on PKA Inhibition by VS 115 Figure 5.18 X-Band EPR Spectra of VS in the Presence of the Glycine-Loop Peptide 116 Figure 6.1 VS Fails to Inhibit PKA Following Pre-incubation at pH 7 119 Figure 6.2 X-Band EPR Spectra of VS 120 Figure 6.3 Time-Dependent Decay of X-Band EPR Spectra of VS Solutions at pH 4.6 121 Figure 6.4 Loss of PKA Inhibition on VS Storage 122 Figure 6.5 Lack of Effect of GSH or GSSG on PKA Activity 123 Figure 6.6 Effects of GSH and VS on PKA 124 Figure 6.7 Effects of VS and OV on PKA when Combined with GSH or GSSG 125 Figure 6.8 Effects of EDTA on PKA Inhibition by VS 127 Figure 6.9 Effects of VS on PKA Are Enhanced by GSSG and Blocked by EDTA 128 Figure 6.10 X-Band EPR Spectra of Vanadyl in the Absence or Presence of GSSG 131 Figure 6.11 X-Band EPR Spectra of Vanadyl in PKA Assay Mixture 132 Figure 6.12 X-Band EPR Spectra of Vanadyl in the Absence or Presence of GSH 133 Figure 6.13 X-Band EPR Spectra of Vanadyl in the Absence or Presence of BSA 134 Figure 6.14 X-Band EPR Spectra of Vanadyl in the Presence of GSSG, GSH, or BSA 135 xi Figure 6.15 Time-Dependent Changes in X-Band EPR Spectra of VS in PKA Assay Mixture 136 Figure 6.16 X-Band EPR Spectra of Vanadyl at High pH, with GSH and GSSG 137 xii LIST OF ABBREVIATIONS AC acac ADA ADP AKAP AMPSO amu ANOVA Arg Asp ATP Asn P-AR p-EtSH BES Bicine BIS BMOV BPOV BSA BSO C CaM-KII cAMP cat Cbl CDK-1 cpm cps adenylyl cyclase acetylacetonato American Diabetes Association adenosine diphosphate A-kinase anchoring protein 3-([l J-dimethyl-2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid atomic mass unit analysis of variance arginine aspartate adenosine trisphosphate asparagine beta adrenergic receptor beta-mercaptoethanol N,N'-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid N,N' -bis(2-hydroxymethyl)glycine bis(2-hydroxyethyl)imino methane bis(maltolato)oxovanadium(IV) bis(picolinato)oxovanadium(IV) bovine serum albumin buthionine sulphoximine PKA catalytic subunit Ca /calmodulin-dependent protein kinase-II 3',5'-cyclic adenosine monophosphate catecholato the Cbl family of proteins (proto-oncogene products) are negative regulators of tyrosine kinase-coupled receptors cyclin-dependent protein kinase-1 counts per minute counts per second xui CREB cAMP-response element binding protein Crk adaptor protein identified as chicken tumor virus (CT10), regulator of kinase Csk COOH-terminal Src kinase Da Dalton DEA diethanolamine dH 20 deionized water DIPSO N,N' -bis(2-hydroxyethyl)-3 -amino-2-hydroxypropanesulfonic acid DM diabetes mellitus dpm disintegrations per minute DTT dithiothreitol EC enzyme classification number EDTA ethylenediamine tetraacetic acid EGTA ethylene glycol bis(P-aminoethyl ether) tetraacetic acid EGF-R epidermal growth factor receptor elF eukaryotic initiation factor EPR electron paramagnetic resonance ema ethylmaltolato EPPS N-(2-hydroxyethyl)piperazine-N' -3 -propanesulfonic acid EPR electron paramagnetic resonance ERK extracellular signal-regulated kinase ES enzyme-substrate complex ESIMS electrospray ionization mass spectrometry FFA free fatty acid G6Pase glucose 6-phosphatase G6PDH glucose 6-phosphate dehydrogenase GDP guanosine diphosphate GG glycylglycine GGG glycylglycylglycine GI gastrointestinal GLUT glucose transporter protein xiv Gly Grb2 GS GSH GSK-3 GSSG GTP GTPase H 2 0 2 HEPES HLA HPLC HSA HSL Hz ICP IEA IFG IGF-1 IRK IRS IUB IUPAC K kcat Kd Ki K\c Klu Km glycine growth factor receptor binding protein 2 glycogen synthase reduced glutathione glycogen synthase kinase-3 oxidized glutathione guanosine triphosphate guanosine triphosphatase hydrogen peroxide 2-(N-hy(koxyemyl)piperazme-2-(N'-ethanesulfonic acid) human leukocyte antigen high pressure liquid chromatography human serum albumin hormone sensitive lipase Hertz inductively coupled plasma iminoethanolamine impaired fasting glucose insulin-like growth factor-1 insulin receptor kinase insulin receptor substrate International Union of Biochemistry International Union of Pure and Applied Chemistry temperature unit, Kelvin, 1 K = - 272.15 °C; molecular weight cut-off unit for the membrane filtration technique, 1 KDa = 1000 Daltons (Da) catalytic constant or enzyme turnover number or enzyme molecular activity dissociation constant inhibition constant competitive inhibition constant uncompetitive inhibition constant Michaelis-Menten constant xv ma maltolato MALDI matrix-assisted laser desorption ionization MAPK mitogen activated protein kinase MDEA N-methyldiethanolamine MEK MAP kinase/ERK kinase MES 2-(N-morpholino)ethanesulfonic acid MLCK myosin light-chain kinase Mnk-1 MAPK signal integrating kinase 1 MOPS 3-morpholinopropanesulfonic acid mRNA messenger ribonucleic acid ms millisecond MS mass spectrometry mTOR mammalian target of rapamycin MV metavanadate MW molecular weight NADP nicotinamide adenine dinucleotide phosphate NADPH nicotinamide adenine dinucleotide phosphate (reduced form) NaV0 3 sodium metavanadate Na 3 V0 4 sodium orthovanadate Nek 47-kDa adaptor protein with 3 SH3 and 1 SH2 domain NF National Formulary is an independent group of scientists and experts that prepare and update standards for ingredients used in pharmaceutical products ov orthovanadate ( V O 4 3 ) p21 Ras "rat sarcoma" 21-kDa GTPase p70 S6K p70 S6 kinase p90 rsk p90 ribosomal protein S6 kinase P phosphate pa picolinato PAGE polyacrylamide gel electrophoresis PDB protein data bank PDE phosphodiesterase xvi PDK-1 phosphoinositide-dependent kinase 1 PEPCK phosphoenolpyruvate carboxykinase PF pre-filter rods PFK-2 phosphofructokinase-2 PHAS-1 phosphorylated heat- and acid-stable protein 1 PI phosphatidylinositol PI3K phosphatidylinositol-3'(OH) kinase PIP2 phosphatidylinositol 3,4-bisphosphate PIP3 phosphatidylinositol 3,4,5-trisphosphate PIPES piperazine-1,4-bis(2-ethanesulfonic acid) PKA cAMP-dependent protein kinase PKB protein kinase B PKC protein kinase C PKG protein kinase G PKI cAMP-dependent protein kinase inhibitor PKM catalytic fragment of PKC PS/TPase phospho serine/threonine phosphatase PTPase protein tyrosine phosphatase pV peroxovanadium compounds Q quadrupole rods R PKA regulatory subunit Raf-1 ser/thr-specific protein kinase related to viral oncogene v-raf RF radio frequency s.c. subcutaneous SDS sodium dodecyl sulphate SEM the standard error of the mean She src-homology/collagen-related protein Ser serine SOS son of sevenless Src mammalian homolog of viral protein tyrosine kinase derived from avian sarcoma xvii STZ streptozotocin Syp SH2-containing protein tyrosine phosphatase TAPS N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid TAPSO N-[tris(hydroxymethyl)memyl]-3-ammo-2-hyo^oxypropanesulfonic acid TEA triethanolamine TES N-[tris(hyolroxymemyl)memyl]-2-aminomethanesulfonic acid TG triglyceride Thr threonine TNF tumor necrosis factor TOF time-of-flight Tricine N-[tris(hydroxymethyl)methyl]glycine TRIS 2-amino-2-hydroxymethyl-l,3-propanediol, also known as TRIZMA® Tyr tyrosine U unit of enzyme activity UV ultraviolet V vanadium V2O4 vanadium tetroxide V2O5 vanadium pentoxide VLDL very low density lipoprotein V m a x maximum reaction rate VO(ma)2 bis(maltolato)oxovanadium(IV) VO(pa)2 bis(picolinato)oxovanadium(IV) VS vanadyl sulphate trihydrate xviii ACKNOWLEDGEMENT I would like to extend my sincerest gratitude to my friend Dr. Roger W. Brownsey for his invaluable, supportive and caring assistance, encouragement, instructions and patience throughout this project. (In addition, I would like to thank him for displaying an outstanding moral and personal character that, in my opinion, a great scientist and scholar must have.) I also immensely thank Dr. Jerzy Kulpa for his uninterrupted support and friendship throughout my time in this lab, as well as his help with experimental procedures. Equally, I would like to thank Rachel Zhande, Adrienne Wood, Susan Collins, Jason Elliott, Weissy Lee, Mary Pines, Kenneth Chan, Sasha Cuk, Andy Chan and Jason Louis for their tolerance, their patience and their extension of friendship to me. I am also very grateful to Dr. Don J. Douglas for teaching me a great deal about mass spectrometry and allowing me to share his MS equipment for well over two years. I also immensely thank Dr. Chris E. R. Orvig for allowing me to use his lab facility for EPR measurements as well as for the supply of BPOV and BMOV. I am also very grateful to Dr. A. Grant Mauk for his invaluable help with my comprehensive exam and for allowing me to use his personal library as well as the EPR and CD equipment. I am also very thankful to Dr. John H. McNeill for his invaluable and important suggestions and recommendations throughout my graduate studies. I would also like to thank all the members of my supervisory committee for their invaluable suggestions, recommendations, and help throughout the course of my project. I am also very grateful to John Sanker, who taught me much about UBC's administrative policies and interdepartmental "politics", and to Jack Louis who taught me many electronic "tricks", and to both who extended their friendship to me. I also sincerely appreciate Dr. Richard Barton's guidance throughout my undergraduate and graduate studies at UBC and his permission to use equipment in the graduate lab, including valuable books from his library. I am also indebted to my friend Ben Clifford at the Analytical Chemistry Laboratory for allowing me to share his computer, and for help with graphical, and statistical and analytical work. I am also indebted and thankful to Nham Thi Nguyen who carried out countless X-ray crystallography experiments and who taught me many invaluable lessons in this technique. Equally, I would like to thank Lillian Tamburic who taught me cell culture techniques. I am very grateful to both Lillian and Nham for extending their friendship to me. I would also like to thank Drs. Caroline Astell and Masayuki Numata for allowing me to use their cell culture facility. xix At last, I would like to thank all my family members for their moral and their personal support during my entire academic study period to date, especially my parents for all they taught me and for encouraging me to be the best that I can be. I will end this section with a famous Persian poem/proverb that translates into the following: From knowledge, an incompetent person becomes gifted and an old and dreadful spirit becomes young and blissful. J J J tijj J^J J J (j&b j J J J Ul J A S j * Ajj U I J J xx C H A P T E R O N E I N T R O D U C T I O N 1.1 INSULIN AND DIABETES Among the many biological actions attributed to vanadium, the induction of cellular responses similar to those of insulin has attracted particular attention. The general aim of the work presented in this thesis has been to define how vanadium salts bring about responses in vitro and in vivo that can compensate for diminished insulin action or availability. To provide the context for understanding the mechanism(s) by which vanadium influence metabolism a brief review of the literature related to insulin action and diabetes is given. 1.1.1 Insulin Actions Insulin exerts a wide range of acute and chronic effects on many mammalian cell types. Of most relevance to the aims of this thesis are the immediate metabolic effects of insulin, notably those in liver, skeletal muscle, heart and fat cells. These effects are summarized in Table 1.1. Overall, insulin promotes the biosynthesis of glycogen, complex lipids and proteins while suppressing the opposing catabolic pathways. Briefly, insulin stimulates glucose and amino acid uptake into adipose, muscle and heart cells and also stimulates several metabolic pathways through its effects on specific enzymes (for reviews see Denton et al, 1981; Fritz, 1988; Randle et al, 1966; and references cited in Table 1.1). Not surprisingly, defects in the actions or supply of insulin have profound effects on intermediary metabolism as manifest in the various forms of diabetes. 1.1.2 The Complexity of Insulin Signaling Pathways Insulin contributes to the control of many metabolic processes, the regulation of the transcription of specific genes, and the modulation of cellular growth and differentiation (Saltiel and Kahn, 2001; Taha and Klip, 1999). Since the discovery of insulin more than 80 years ago by Banting, Best, MacLeod and Collip, our understanding of the intricate web of proteins involved in the actions of the hormone has advanced considerably. However, insulin signaling pathways and their impact on metabolic pathways are still incompletely elucidated. A simplified overview of the insulin-signaling pathway is provided in Figure 1.1. 1 Table I.I Insulin Actions in Coordination of Metabolism Metabolic Effect Tissue Selected References Glucose Transport Adipose, Skeletal Muscle, Heart (Martin and Carter, 1970) (Czech, 1995) (Cushman and Wardzala, 1980) Glycogen Metabolism Adipose, Skeletal Muscle, Heart, Liver (Cohen, 2002) (Lamer, 1988) Glycolysis & Pyruvate Oxidation Adipose, Liver, Heart, Skeletal Muscle (Denton etal, 1975) (Pilkis etal, 1979) Lipogenesis & Lipolysis Adipose, Liver (Rodbell, 1966) (Denton etal, 1977) (Fain etal, 1979) Protein Metabolism Heart, Skeletal Muscle (Jefferson etal, 191 A) (Flaimef a/., 1983) Gene Expression All tissues (Granner and 0' Brien, 1992) (Sutherland etal, 1996) 2 Figure 1.1 A Simplified Overview of Insulin Signaling Pathways* Activating Inhibiting p21 Ras Raf-1 MEK MAPK (ERK) p90 rsk Transcription factors Protein Synthesis Glycogen Synthesis Gene Expression * Adapted from (Granner, 2003), (Saltiel andKahn, 2001), and (Taha and Klip, 1999) The cellular actions of insulin are initiated by the binding of insulin to the exofacial domain of the transmembrane OC2P2 receptor (IRK). Insulin binding leads to receptor autophosphorylation, in trans, by the intrinsic cytoplasmic tyrosine kinase domains of the |3-subunits. IRKs attract and promote tyrosine phosphorylation of a variety of intracellular substrate proteins, notably insulin receptor substrate (IRS) proteins and She isoforms (Figure 1.1 and reviews by Granner, 2003; Saltiel and Kahn, 2001; Taha and Klip, 1999). In turn, the receptor substrates interact with the SH2 domains of adaptor proteins such as Grb2, Crk, Nek, Syp, and the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K). She and IRS proteins can each interact with Grb2 and the associated Sos protein to induce activation of the p21 Ras-GTPase, MAPK (also known as extracellular signal-regulated kinase, ERK) and the subsequent downstream pathway. The ERK pathway is probably most important in the control of gene expression and is evidently not crucial for the acute metabolic effects of insulin (Denton and Tavare, 1995). For this reason this branch of insulin signaling is not considered in detail here. Another major branch of insulin signaling arises from the activation of PI3K via IRS proteins. Based on the effects of specific inhibitors (Wortmannin and LY29004) and mutational analysis, PI3K activation is essential for all the acute metabolic effects of insulin (Evans et al, 1995; Moule etal, 1995). Generation of PI-3,4, 5 triphosphate by PI3K leads to recruitment and activation of phosphoinositide-dependent kinase-1 (PDK-1, and perhaps PDK-2) and protein kinase B (PKB) at the cell membrane (Filippa et al, 2000; Scheid et al, 2002). PKB is an important enzyme because it propagates further signals by phosphorylating and inhibiting glycogen synthase kinase-3 (GSK-3) as well as by activating the mTOR/p70 S6K pathway, which is important in stimulation of protein synthesis (Brazil et al, 2002; Cho etal, 2001; Vanhaesebroeck and Alessi, 2000). PKB may also be required for regulation of glycolysis by phosphorylating and activating phosphofructokinase-2 (PFK-2) (Bertrand et al, 1999; Deprez et al, 1997) as well as regulating triglyceride (TG) hydrolysis (lipolysis) by acting on phosphodiesterase-3B (PDE-3B), as discussed later. The central action of insulin, stimulation of glucose uptake by recruitment of glucose transporter protein-4 (GLUT4) to the plasma membrane, is also dependent on PI3K and PKB. However, this signaling pathway alone is not sufficient for GLUT4 activation. Rather, insulin also activates at least one additional pathway involving Cbl and a separate guanosine triphosphatase (GTPase) (Baumann et al, 2000; Inoue et al, 2003; Jiang et al, 2002; Onuma et al, 1998). Because of the multitude of metabolic and other actions of insulin, it is clear that any study of "insulin-like" actions of vanadium must be selective. The model system chosen for the studies described in this thesis was the control of TG hydrolysis. 1.1.3 Control of Triglyceride Hydrolysis Among the metabolic pathways affected by insulin, the control of TG hydrolysis has been particularly well defined and is particularly sensitive to vanadium. Consequently, this pathway is a useful representative model for studies of vanadium action (see Figure 1.2 for details). The binding of lipolytic hormones to P-adrenergic receptors (p-ARs) and the consequent conformational change in the receptor induces activation of the heterotrimeric guanylyl nucleotide-binding protein, G s (a, P, y). In this process, G s a acquires GTP in exchange for GDP, 4 resulting in the dissociation of the a-subunit from the fJy-subunits. G T P - G s a then stimulates adenylyl cyclase (AC), leading to increased conversion of ATP to cAMP and subsequent allosteric activation of P K A . The most important substrates of P K A in the context of stimulation of TG hydrolysis are hormone-sensitive lipase (HSL) (Holm et al, 1997; Nilsson et al, 1980) and the perilipins (Londos et al, 1999; Su et al, 2003; Tansey et al, 2003). Perilipins are located at the surface of lipid droplets and, when non-phosphorylated, effectively inhibit access to HSL. Thus P K A has dual effects leading to dramatic stimulation of hydrolysis of triglycerides to glycerol and free fatty acids (FFAs) (Nilsson et al, 1980). In principle, TG hydrolysis might be inhibited by insulin in several ways. A particularly crucial role of insulin appears to involve activation of cyclic nucleotide phosphodiesterases (Castan et al, 1999; Eriksson et al, 1995; Fukui et al, 1998). This effect of insulin depends on PI3K and leads to activation of a specific isoform of cyclic nucleotide phosphodiesterase, known as the "particulate", low Km, cGMP-inhibitable isoform, abbreviated cGI-PDE or PDE-3B (Fukui et al, 1998). During insulin stimulation, PI3K activation leads to subsequent activation of PDKs and then P K B (Figure 1.2) (Castan et al, 1999). In turn, P K B or a related, perhaps downstream, kinase phosphorylates and activates PDE-3B. Activation of PDE-3B leads to inhibition of P K A , by removal of its activator, cAMP. Finally, loss of P K A activation leads to reduction of the HSL activity and to restoration of HSL inhibition by perilipins (Brady et al, 1997; Delibegovic et al, 2003). In addition to its ability to suppress P K A function, insulin may promote the activation of protein Ser/Thr phosphatases (PS/TPases) that act on HSL and perilipins (Cohen, 2002). Insulin might also exert effects at the level of the heterotrimeric G-protein. By analogy to the actions of prostaglandins and nicotinic acid that can lead to activation of an inhibitory G-protein (Gi), insulin actions also seem to be Gi-dependent (Moxham and Malbon, 1996; Zheng et al, 1998). Gi can suppress the activity of A C and thus prevent the production of cAMP and activation of P K A (Rodbell, 1997). Finally, insulin appears to be able to inhibit glucocorticoid-mediated activation of HSL that occurs by a cAMP-independent pathway (Mayes and Botham, 2003). From the perspective of this thesis it is important to note that vanadium may target one or more of the key proteins in Figure 1.2, namely G-protein, A C , PDE, P K A , HSL, perilipins, or PS/TPases. These possibilities are discussed more fully in Section 1.2.9. 5 Figure 1.2 Key Proteins Involved in Control of Triglyceride Hydrolysis 6 1.1.4 Properties of PKA From the studies reviewed below, PKA (EC 2.7.1.37), was identified as a potential target for vanadium action in the current studies. The properties of PKA, therefore, merit consideration. In the absence of cAMP, PKA exists as an inactive holoenzyme of 2 regulatory (R) and 2 catalytic (C) subunits (Francis and Corbin, 1994). The binding of two cAMP molecules to each R subunit (Taylor et al, 1993a) causes the complex to dissociate into an R 2 dimer and two free and active C monomers (Equation 1.1): 4cAMP + R 2 C 2 > R2(cAMP)4 + 2C (1.1) PKA was the second protein kinase to be identified following the recognition of phosphorylase kinase, and it was the first protein kinase for which the 3-dimensional structure was solved (Knighton etal, 1991a, 1991b; Taylor etal, 1990, 1993b, 1999). The enzyme is bilobal with a deep cleft between the lobes. The smaller lobe consists mostly of amino-terminal sequence, and its largely antiparallel P-sheet architecture contains the nucleotide-binding motif. The larger lobe is dominated by helical structure with a single P-sheet at the domain interface and is primarily involved in peptide binding. The cleft between the two lobes of the PKA structure provides the Mg-ATP binding site and critical catalytic residues (Figure 1.3). Both domains/lobes contribute to catalysis, and residues 40 through 280 constitute a conserved catalytic core that is shared by more than 100 protein kinases (Hanks and Hunter, 1995; Hanks et al, 1988). Most of the invariant amino acids in this catalytic core are clustered at the sites of nucleotide binding and catalysis. PKA has been the focus of considerable study, and the actions of cAMP (Equation 1.2) and the sites of cAMP binding as well as subunit contacts have been well defined (Taylor et al, 1993a, 1993b). The consensus sequence recognized for phosphorylation by the C subunit is Arg-Arg-X-[Ser or Thr]-Z, where X is any small residue, Z is a large hydrophobic group and the phosphorylated seryl or threonyl residue bracketed. However, this sequence is not sufficient to dictate substrate selectivity, and other structural determinants are essential for binding to the C subunit of PKA. The conserved catalytic core, as in all known protein kinases, is contained within this relatively simple monomeric C subunit, and it includes the ATP-binding and peptide or protein binding sites (Figure 1.3, Knighton et al, 1991b; Madhusudan et al, 2002; Zheng et al, 1993a). The ATP-binding site is located towards the N-terminus of the catalytic core domain and is characterized by the Rossmann motif, with the consensus sequence GXGXXG where X is any 7 amino acid (Kemp and Pearson, 1990; Rossmann et al, 1975). Although none of the glycines in this motif makes contact with the peptide substrate, mutation of any of them has been reported to lower the affinity for the substrate by about 10-fold (Grant et al, 1998). In addition to ATP, magnesium and peptide/protein substrates, the active site accommodates six water molecules that constitute a conserved structural element of the active site (Shaltiel et al, 1998). One of these water molecules is locked into place by interactions with ATP, the peptide or protein substrate, the small and large domains of the conserved core and a Tyr residue from the carboxy-terminal "tail" (Figure 1.3, Shaltiel et al, 1998). PKA is primarily a cytoplasmic enzyme. This localization is determined by tethering to a family of A-kinase anchoring proteins (AKAPs) (Rubin, 1994; Scott et al, 2000). The N-termini of R subunits dock to AKAPs and act as cytoplasmic anchors that prevent C subunits entering the nucleus (Adams et al, 1991). Thus, free C subunits not only phosphorylate cytoplasmic targets but also nuclear targets involved in the regulation of gene transcription. For example, the phosphorylation of cAMP response element binding protein, CREB, by PKA is limited by the entry of PKA C subunits into the nucleus (Hagiwara et al, 1993). For example, the activation of hepatic gluconeogenesis depends, in part, on the PKA-dependent phosphorylation of CREB and subsequent regulation of CREB-dependent gene expression. Other cAMP/PKA-mediated physiological functions, other than TG hydrolysis (discussed above), are listed in, but not limited to, Table 1.2 (for review see Tasken and Aandahl, 2004). For example, platelet aggregation is not part of the Table 1.2 but is a cAMP/PKA-mediated pathway. 8 Table 1.2 Overview of cAMP/PKA-Mediated Physiological Functions* Regulation and/or Function Known Targets/Substrates of PKA Metabolic pathways Examples include phsphorylase kinase, glycogen synthase, HSL, perilipins, acetyl-CoA carboxylase, liver pyruvate kinase Cardiac excitation-contraction coupling 111 L-type Ca channel, ryanodine receptor, troponin I, myosin binding protein C, phospholamban Steroid synthesis in adrenal glands and gonads Sterol ester hydrolase, AKAP also interacts with the peripheral-type benzodiazepine receptor which regulates steroid synthesis Initiation and maintenance of sperm motility Roles for several AKAPs have been identified HCI secretion from gastric parietal cells AKAP interacts with tubulovesicles of parietal cells Insulin secretion from pancreatic p-cells Several targets including synapsin 1 H 2 O reabsorption in renal principial cells H 2 O channel (aquaporin-2) Inhibition of T-cell activation COOH-terminal Src kinase (Csk) * Adaptedfrom (Tasken andAandahl, 2004) 9 Figure 1.3 The 3-Dimensional Structure of PKA Catalytic Subunit Complexed With Mn-ATP andPKI* The structure of PKA catalytic subunit consists of a-helices (light blue), /3-sheets (cyan), and loops, turns, etc. (dark blue). Oxygen, phosphorus and carbon atoms are depicted in red, orange and gray spheres, respectively. PKI (5-24), a 20-amino acid peptide inhibitor of PKA, is shown in green. Three phosphorylation sites (Ser 139, Ser 338, and Thr 197) on PKA are highlighted. The glycine-rich loop (residues 49-57), the catalytic loop (residues 166-171) and the Mn +-positioning loop (residues 184-187) are shown in brown, in magenta and with a red arrow, respectively. One of the conserved water molecules is indicated (black dot). Ser 338 Conserved H2O * Adapted from (Zheng et al, 1993a). The structure was prepared using Chime ™(MDL Information Systems, Inc.) and Protein Explorer ™. The structural information was obtained from the Protein Data Bank (PDB) at www.rcsb.org (PDB identification code: I ATP). 10 1.1.5 Diabetes Mellitus The term diabetes mellitus (DM) covers a group of metabolic disorders that result from an absolute or relative deficiency in insulin secretion, usually in combination with a resistance of target tissues to the action of insulin (Gutteridge, 1999; Kuzuya and Matsuda, 1997). The most common types of diabetes are referred to as type 1 and type 2. Type 1 diabetes (5-10 % of all cases) is immune-mediated usually with juvenile-onset (Gutteridge, 1999; Kuzuya and Matsuda, 1997). Type 2 diabetes (~ 90 % of all cases) is usually with adult-onset (Gutteridge, 1999). Additional types of diabetes, though less common, are also important. For example, gestational diabetes mellitus may occur in women with no previous history of diabetes and often persists after pregnancy (Gutteridge, 1999; Kuzuya and Matsuda, 1997). The incidence of both major types of diabetes is increasing internationally. In addition, many people probably have prediabetes or are still undiagnosed. In North America, type 1 diabetes is more common in Caucasians than people of African, Hispanic, Asian, or Indian origins. In contrast, several ethnic groups exhibit markedly greater risk of type 2 diabetes (e.g. native North Americans) (Harris et al. 1997, 1998, 1999). Cardinal features of diabetes include hyperglycemia and elevated blood levels of fatty acids and TG-rich lipoproteins. Clinically, a firm diagnosis of diabetes is made when two consecutive blood analyses show fasting glucose levels above 7 mM (normal ~ 5 mM) (Gutteridge, 1999). Intermediate values of blood glucose may indicate impaired glucose regulation, potentially a "precursor" to full diabetes (Borch-Johnsen, 2001). Untreated insulin deficiency leads to increased thirst, a frequent desire to urinate, blurred vision, and fatigue. In the longer term, people with diabetes suffer from markedly increased risk of cardiovascular disease (Wahi et al, 1997), renal failure (Friedman, 2001), retinopathy (Collins et al, 1995), and peripheral neuropathy (Collins et al, 1995). Type 1 diabetes is caused by autoimmune destruction of pancreatic P-cells, but the complexity of regulation of the immune system means that the factors leading to this autoimmune response are poorly defined. In general terms, the autoimmune process could result from the failure of the natural immune suppression known as "self recognition." On the other hand, the immune system might be inappropriately "tricked" into reacting to autoantigens. Some of the theories being actively evaluated include the following: 1. Generation of autoantibodies that recognize P-cell proteins such as insulin (Davison et al, 2003) or glutamic acid decarboxylase (Hoeldtke et al, 2000) might lead to type 1 diabetes. Some 11 human leukocyte antigens (HLAs) might also tag P-cells for destruction by T cells (Hathout et al, 2003); 2. Stresses that lead to the generation of reactive oxygen species are also implicated in the onset of type 1 diabetes because pancreatic islet cells have a limited capacity to detoxify free radicals (Gillerye/a/., 1989); 3. Several groups of viruses may be inducers of type 1 diabetes, including members of the Rubella (Menser et al, 1978) and Coxsackie families (Field et al, 1987); 4. Proteins in bovine milk might induce production of diabetogenic autoantibodies by "molecular mimicry". Wheat and soy proteins may also increase the risk of type 1 diabetes as studies in diabetes-prone rats showed that removal of these proteins from the diet helps delay or prevent diabetes (Kimpimaki et al, 2001). The risk of Type 2 diabetes is also strongly influenced by genetic predisposition as shown by susceptibility in certain ethnic groups and by the concordance between siblings. Obesity is one of the most important risk factors for type 2 diabetes (Hu, 2003). Although a sedentary lifestyle and consumption of high-calorie diets undoubtedly make a major contribution to human obesity, biochemical defects might also contribute significantly to the predisposition to insulin resistance and ultimately type 2 diabetes. Some of the candidate genes generating considerable interest include those encoding insulin signaling proteins such as the insulin receptor, receptor substrates, etc., (Ramachandran and Kennedy, 2003), as well as regulatory proteins such as tumor necrosis factor (Moriwaki et al, 2003), leptin (Wauters et al, 2003) and various centrally or peripherally acting neuropeptides (Niskanen et al, 2000). Some of the treatments commonly used for diabetes are listed in Table 1.3 (ADA, 2002; Bloomgarden, 2003; Bolli et al, 1999; Burden, 2003; Davidson and Peters, 1997; Peraldi et al, 1997; Pfendler and Kawaze, 2003). In conclusion, none of the available therapeutic approaches is perfect and, considering the long term morbidity and mortality associated with diabetes, new approaches to the treatment of this disease are required. 12 Table 1.3 Overview of the Treatments for Types 1 and 2 Diabetes* Treatment Mode(s) of action Limitations include but not limited to Type 1 Diabetes Insulin injection Direct restoration of plasma insulin Requirement of great discipline, risk of hypoglycemia, hypokalemia and long-term resistance Insulin analogues (e.g. lispro: LysB28, ProB29 ) Has a more rapid onset and shorter duration of action than normal insulin Same as above Pancreas or islet transplantation New source of insulin Stress of surgery, potential rejection, immuno-suppression requirements, limited number of donors Stem cell transplantation Stem cells differentiated to P-cells Requirements for further research and development Type 2 Diabetes Diet and exercise Reduce body weight and insulin resistance Problems with compliance a-Glucosidase inhibitors Slow the digestion of complex carbohydrates GI side effects (most common is flatulence), its requirement at start of each meal Sulfonylureas Stimulate pancreas to release more insulin Severe hypoglycemia, excessive water retention, and facial flushing Meglitinides Stimulate pancreas to release more insulin Hypoglycemia, GI and respiratory side effects (upper respiratory tract infection) Biguanides (e.g. Metformin™) Suppress hepatic glucose output GI side effects (most common is diarrhea), renal and hepatic function impairments, efficacy declines over time Thiazolidinediones Improve peripheral insulin sensitivity Liver and heart toxicity, weight gain, carcinogenesis in adipose tissue * Adapted from (ADA, 2002), (Bloomgarden, 2003), (Bollietal, 1999), (Burden, 2003), (Davidson and Peters, 1997), (Peraldi et al, 1997), and (Pfendler and Kawaze, 2003) 13 1.2 CHEMICAL PROPERTIES AND BIOLOGICAL ACTIONS OF VANADIUM 1.2.1 Discovery and Basic Properties of Vanadium Vanadium, V, has been discovered twice: first in 1801, when it was mistaken for a chromium (Cr) allotrope and then formally in 1830 (Butler, 2003). Named after the Nordic goddess Vanadis, vanadium is a grayish, ductile transition metal, a member of Group 5 of the Periodic Table with an atomic number of 23 and an atomic mass of 50.9415 (Lide, 2003). Vanadium has a melting point of 1910 °C, a boiling point of 3407 °C at atmospheric pressure and a specific gravity of 6.11 at 18.7 °C (Lide, 2003). Vanadium has two natural isotopes, 5 0 V and 5 1 V , and several artificially generated radioactive isotopes (Lide, 2003). 1.2.2 Vanadium Coordination and Physical Chemistry Vanadium chemistry is complex in several respects, notably because of the existence of a number of oxidation states from -III to V (except -II), and of anionic and cationic forms. The oxidation states III, IV, and V are the most common and are presently the only forms of vanadium found in biological systems (Boas and Pessoa, 1987a). At typical physiological concentrations (low micromolar), vanadyl (V0 2 +) and vanadate are probably the most relevant species in mammalian intracellular and extracellular fluids. Vanadate is a generic term that applies to all protonated and oligomeric forms of orthovanadate and metavanadate as discussed below. The speciation of V(V) and V(IV) in aqueous solution is a function of pH as shown in Figure 1.4. Four important qualifications must be recognized in interpreting this speciation diagram. First, our understanding of the hydrolysis of V(IV), especially in the neutral pH range, is incomplete. Second, vanadium speciation, in either oxidation state, is also concentration-dependent. The speciation depicted in Figure 1.4 would occur with vanadium concentrations in the millimolar range. Somewhat different speciation patterns would be observed at lower concentrations. In the submillimolar range, for example, [VO(OH)]3~ predominates at pH ~ 10 and not at pH ~ 13 as depicted. Third, V(V) oligomers form at pH 3-13, especially when the concentration is in the millimolar range or higher. V(IV) also tends to oligomerize, some oligomers being insoluble at pH ~ 4-8. Fourth, for simplicity, coordinated water molecules were omitted from Figure 1.4. For example, vanadyl and [VO(OH)3]" would be more accurately expressed as [VO(H20)5]2 + and [VO(OH)3(H20)2]", respectively (Boas and Pessoa, 1987b; 14 Chasteen, 1981,1983; Crans, 1994; Francavilla and Chasteen, 1975; Iannuzzi and Rieger, 1975; Komura et al, 1977; Labonnette, 1988; Rossotti and Rossotti, 1955; Willsky, 1990). Figure 1.4 Effects ofpH on the Speciation of Vanadium (IV) and (V) in Aqueous Solution* This figure illustrates the major species observed over the indicated pH range when vanadium salts (V, Panel A) and (IV in deoxygenated water, Panel B) are present at millimolar concentrations in the absence of any other reagent. In Panel B, the dimeric divalent cation^ and the water insoluble oligomer^ are EPR-silent. The oligomer is in equilibrium with other species in the pH range 3-12. Where more than one species is stated they are in equilibrium with each other. (?) Additional species may also be present. % figures represent approximate percentage of each species in a given column. V(V): V 0 2 + H3VO4 H2VO4 HVO4 V 0 4 B {VO(OH) 2}„ ( b ) {VO(OH)2}n(b> (95%) (50%) V(IV): V 0 2 + [VO(OH)]+ [(VO)4(OH)9]-(2.5%) (40%) [{VO(OH)}2]2+<a> (?) (2.5%) [(VO)4(OH),o]2-(45%) [(VO)2(OH)5r (45%) (?) PH 13 [VO(OH)3]-(95%) (?) PH 13 * Adapted from (Boas and Pessoa, 1987b), (Iannuzzi and Rieger, 1975), (Komura et al, 1977), (Labonnette, 1988), and (Willsky, 1990) From Figure 1.4 and additional studies it is clear that, depending on the ionic strength, the pH of the solution and the overall vanadium concentration, V(V) can exist as an equilibrium mixture of monomer, dimer, cyclic tetramer, cyclic pentamer and decamer (Crans, 1994,1995). For example, dissolving sodium metavanadate (NaVO )^ in water leads to a solution containing predominantly oligovanadates, commonly referred to as metavanadate (MV) (Rehder, 1995). 15 As well as showing distinct speciation patterns, vanadium (IV) and (V) can also be interconverted according to Equation 1.2. The standard reduction potential (E0) for the conversion of vanadate into vanadyl (Equation 1.2) is +1.31 Volts (Rehder, 1992). H 2V0 4" + 4H + + e > V 0 2 + + 3H 20 (1.2) In aqueous solutions, above pH ~ 2, especially in the presence of oxygen and in the absence of chelating agents, V 0 2 + undergoes a time-dependent auto-oxidation to orthovanadate (V043", OV) and its protonated forms such as H2V04". (Crans et al, 1995). V(V) exists predominantly as H 2V0 4" monomers at neutral pH and at concentrations below ~ 1 mM even if NaVOb or V2Os (vanadium pentoxide) salts are used to prepare the stock solutions (Crans et al, 1995). The complexity of vanadium chemistry presents significant problems in understanding the forms that may be adopted in biological systems. The main purpose of this work was to address the actions of vanadium on mammalian cell metabolism. In this regard, vanadium most likely exists in a variety of forms and complexes physiologically and these may differ in extracellular and intracellular environments. In extracellular fluids, including blood plasma and interstitial fluids, vanadium is exposed to an oxidizing environment, therefore, it is likely that V(V) predominates as anionic species that resemble phosphate (Gresser and Tracey, 1990; Plass, 1999; Tracey and Gresser, 1986), largely as OV or its protonated forms. In contrast, intracellular conditions are much more reducing and it is likely that V(I V) predominates, largely as vanadyl, although other minor cationic forms also exist. In addition to interconversions between oxidation states and ionic forms, vanadyl may also interact with a number of intracellular ligands. From a chemical perspective, "naked" non-oxo V 4 + exists in tris(catecholato)vanadate(IV) ([Vicafh]2") complexes (Bulls et al, 1990; Cooper et al, 1982) and V 4 + exists in halides, VX4 (where X is any halogen), amines, V(NR2)4 (where R is a hydrogen or an alkyl group), and alkoxide oligomers, [V(OR)4]n (Cotton et al, 1999). Corresponding compounds might form physiologically because of the existence of many natural compounds containing amines, catechols, and halides among other possible ligands. Further biologically important interactions might be possible because vanadyl is considered to resemble Mg 2 + in several respects (Banerjee et al, 1998). Thus, vanadyl can substitute for divalent ions in metal-activated enzymes and in metalloproteins. In fact, the electron paramagnetic resonance (EPR) spectra of vanadyl can provide a very useful spin probe in biological systems (Chasteen, 1981; Makinen and Mustafi, 1995); over the pH range 5-11, the 16 vanadyl EPR signal intensity is proportional to ligand-bound vanadyl. Significantly, the EPR signal of free vanadyl is rapidly lost at neutral pH (see Section 1.2.3). Vanadyl is one of the most stable ions in solution (Chasteen, 1981), and the electronic structure of vanadyl has been studied extensively (Ballhausen and Gray, 1962; Chasteen, 1981). Nearly all complexes of V(IV) are derived from the vanadyl ion (Cotton et al, 1999). The chemical structures of vanadium salts and complexes that were used in the present studies are illustrated in Figure 1.5. Among the vanadium salts studied, MV and OV both have V(V) with an [Ar] valence shell electronic configuration, and both are, therefore, diamagnetic and EPR-"silent". Vanadyl sulphate (VS), BMOV, and BPOV, have V(IV) with an [Ar] 3d1 valence shell electronic configuration and are, therefore, paramagnetic and EPR-"visible". Vanadyl, having a nuclear spin (I) of 7/2, exhibits an eight-line (2nl + 1, n = number of valence electrons) first-derivative EPR spectrum (Chasteen and Francavilla, 1976). The characteristic EPR spectrum of vanadyl has been invaluable in the studies reported here. Because vanadium forms complexes with many biological macromolecules and with common buffers and assay components, the apparent effects of vanadium in enzyme assays might be accounted for by one or more of the species formed under the experimental conditions used (Crans, 1995, 2000). 17 Figure 1.5 Chemical Structures of Vanadium Salts and Compounds C H 3 Bis(maltolato)oxovanadium(IV) (BMOV) or (VO(ma)2) Bis(picolinato)oxovanadium(IV) (BPOV) or (VO(pa)2) 18 1.2.3 Properties of Vanadium to Be Considered in the Context of In Vitro Enzyme Assays It is important to re-emphasize the fact that vanadyl is more susceptible to oxidation, oligomerization and precipitation near neutral pH, at which most enzyme assays are carried out. Consequently, the behavior of vanadyl at neutral pH is an important issue in designing and interpreting experiments that involve this species. At present the behavior of vanadyl in aqueous solution at pH 7 is incompletely understood owing to its complexity. Nevertheless several aspects of vanadyl speciation are known: 1. Precipitation, oligomerization and oxidation are all possible at neutral pH depending on the experimental conditions. The formation of vanadyl precipitates and EPR-silent vanadyl dimers has been demonstrated upon the gradual addition of base to millimolar, acidified, anaerobic vanadyl solutions in the absence of any other reagents (Chasteen, 1981, 1983; Francavilla and Chasteen, 1975; Labonnette, 1988; Rossotti and Rossotti, 1955). Vanadyl precipitation is possible at neutral pH (Francavilla and Chasteen, 1975). However, complex formation with oxygen-, nitrogen-, and sulfur-containing ligands can prevent precipitation (Chasteen, 1981). Significantly, even buffer components, e.g. 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid (HEPES), may afford sufficient chelating ability to prevent precipitation (Chasteen, 1981; Crans etal, 1989; Crans and Tracey, 1998); 2. The products of vanadyl hydrolysis formed at neutral pH have not all been fully identified; probably because the speciation of hydrolyzed vanadyl species is a complex function of pH and vanadium concentration (Chasteen, 1981,1983; Iannuzzi and Rieger, 1975; Komura etal, 1977; Labonnette, 1988); 3. Finally, aqueous vanadyl may be oxidized to vanadate by dissolved oxygen (Chasteen, 1983). Below pH 3, the rate of oxygenation of vanadyl, which exists predominantly as a penta-aqua species, is ~ lxlO"5 NT's"1. However, as the pH is increased and [VO(OH)]+ predominates (Figure 1.4), the rate of vanadyl oxygenation increases by a factor of ~ 105 (k = 1.07 NT's"1). The rate of vanadyl oxidation by oxygen is, therefore, greatly enhanced by hydrolysis (Wehrli and Stumm, 1989). Because not all hydrolyzed species of vanadyl are known, it is difficult to assess their individual rates of oxidation. Oxidation of vanadyl species in neutral aqueous solutions even in the absence of chelating agents is avoided by excluding atmospheric oxygen. 19 1.2.4 Biological Effects of Vanadium Vanadium is essential for normal development in chicks and rats. Dietary deficiency of vanadium in these species leads to reduced growth and impaired reproduction. Vanadium deficiency has also been shown to have adverse effects on lipid metabolism although results are inconsistent. For example, feeding chicks with vanadium-depleted diets reduced plasma concentrations of cholesterol and triglycerides at age of 28 days followed by an increase in plasma cholesterol at age of 49 days (Hopkins and Mohr, 1974). Other studies on vanadium deprivation revealed increases in plasma cholesterol in chicks (Nielsen, 1995). Biochemical changes observed in vanadium-deficient goats included decreased serum p-lipoproteins (Nielsen, 1995). Vanadium-deficient rats developed increased thyroid mass, with consequent effects on glucose and lipid metabolism (Nielsen, 1998). Despite the abundance of vanadium in the biosphere (Ramasarma and Crane, 1981), biological functions for vanadium are best identified in enzymes of lower organisms (Vilter, 1984). These enzymes include bacterial nitrogenases (Hales et al, 1986; Smith et al, 1988) and algal and lichen haloperoxidases (Plat et al, 1987; Vilter and Reader, 1987). In mammals, vanadium is an ultratrace element. It is not known if vanadium is essential for humans. For mammals vanadium deficiency is observed at subnanomolar plasma vanadium concentration (Hopkins and Mohr, 1974) and vanadium toxicity is observed at millimolar plasma vanadium concentration (Boyd and Kustin, 1984; Domingo, 2000, 2002). Studies with intact cells or tissues have revealed stimulatory effects of vanadium on NADPH oxidation reactions (Coulombe et al, 1987), lipoprotein lipase (LPL) activity (Sera et al, 1990), AC activity, amino acid transport (Hajjar et al, 1987), and mitogen-activated protein kinase (MAPK) activity (Daum et al, 1998). Further pharmacological actions of vanadium include but are not limited to inotropic effects on the myocardium (Ramasarma and Crane, 1981), stimulation of cell division (Carpenter, 1981), and anti-carcinogenic properties (Naylor et al, 1987). In vivo, vanadium can be toxic after acute parenteral administration but much less so after oral administration due to poor absorption by the gastrointestinal (GI) tract. Nevertheless, vanadium has been approved for clinical trials, and administration of ~ 100 mg/day of either VS or MV for a period of 2-4 weeks to both type 1 and 2 diabetic patients resulted in minor to severe GI symptoms (nausea, diarrhea, abdominal cramps, flatulence, and stool discoloration) (Domingo, 2000). The toxic effects of twenty-two vanadyl and vanadate compounds, including BMOV and OV, on transformed murine fibroblast lines were time-dependent and dose-dependent in the mM range (Rehder et al, 2002). 20 These studies showed that the toxicity of vanadium probably depends on the nature of ligands. Oxidation state may also be important because in one study V(V) complexes were less toxic than those of V(IV) (Rehder et al, 2002), but the opposite was found in other studies (McNeill et al, 1995; Thompson et al, 1999; Thompson and Orvig, 2000). Little is known about the extent or mechanism of toxic systemic effects of vanadium in humans (Baran, 1997), and the long-term effects of a marked elevation of plasma vanadium levels in humans remains unknown. Some effects in vivo might be related to the fact that vanadium can induce diuresis and natriuresis (Vivas and Chiaraviglio, 1986). In addition, the effect of vanadium on protein tyrosine phosphorylation, which is crucial in cell growth and differentiation, raises the potential risk of carcinogenicity. In longer-term studies in rats, however, no sign of carcinogenic action of vanadium was evident (Dai et al, 1994b). Because vanadyl salts and compounds seem to be somewhat more effective than vanadate in promoting insulin-like actions, when given acutely to rats in vivo, and because vanadyl could be the active form in target tissues, an argument can be made for the preferential use of vanadyl for therapeutic purposes. More studies are required to evaluate the validity of this preference. Furthermore, attempts are being made to develop vanadyl compounds with higher potency and lower toxicity than the inorganic salts. For example, BMOV was effective in decreasing the plasma glucose levels of diabetic rats and did not induce any signs of toxicity during six months of administration and was more than twice as potent as VS alone (Caravan et al, 1995; Thompson et al., 2003). 1.2.5 Vanadium Metabolism Vanadium is readily available in many foods (Byrne and Kosta, 1978,1979; French and Jones, 1993; Myron et al, 1977; Soremark, 1967), notably in seafood, mushrooms, dill, parsley, black pepper, and wine. Polluted air (Duce and Hoffman, 1976) and water (Korkisch and Krivanec, 1976) also contain significant trace amounts of vanadium. Vanadium can be absorbed via the lungs, small intestine, and skin although the vanadium species absorbed is uncertain (Nielsen, 1998). Overall, vanadium is absorbed through the respiratory and GI tracts with relatively low efficiency. For example, most studies indicate less than 5 % absorption of ingested vanadium with the remainder being excreted in the feces, most likely because of strong association of vanadyl with dietary fiber (Nielsen, 1987). Allowing for the indicated efficiency, net dietary intake of vanadium is probably in the range 10-60 ug per day for adult humans (Wiegmann et al, 1982). Vanadium uptake is generally balanced by excretion, mostly in urine 21 with lesser amounts in the bile (Wiegmann et al, 1982). Vanadyl may be the major form of vanadium taken up in the gut, based on the assumption that V(V) is substantially reduced to V(IV) in the low pH environment of the stomach (Chasteen et al, 1986b). The higher pH of intestine, however, means that vanadate is absorbed three to five times more effectively than vanadyl via the small intestine (Parker and Sharma, 1978). There is no clear evidence for the transport system that might mediate vanadium uptake. Vanadium also exists significantly as vanadyl in the intracellular environment, due to the presence of glutathione and other endogenous reducing agents (Chasteen et al, 1986a; Macara et al, 1980). This fact may be relevant during passage of vanadium across cells of the gut wall prior to entry into the bloodstream. During transport in the blood, vanadium is carried in erythrocytes (probably mainly in the form of vanadyl) and also exists in blood plasma in both oxidation states. The median concentration of vanadium in plasma was reported to be ~ 50 ng/L (Heinemann and Vogt, 1996). Although transferrin, albumin and ferritin can all bind vanadium, transferrin is the predominant vanadium carrier in plasma (Nielsen, 1995). With respect to vanadium transport across cell membranes, it seems likely that vanadate enters cells through the phosphate transport system owing to its resemblance to phosphate, although uptake via the transferrin receptor is also probable. In two studies with4 8 V, the abundance of vanadium in various rat tissues was as follows: bone > kidney > liver > spleen > intestine > stomach > muscle > testis > lung > brain (Parker et al, 1980; Ramanadham et al, 1991). In another recent study with 4 8 V , it was found that kidney contained more vanadium than bone (De Cremer et al, 2002). Most human tissues contain less than 10 ng of vanadium per gram wet weight, and the total body pool in adults has been estimated to be in the range 100 ug - 1 mg (French and Jones, 1993). Vanadium concentrations in specific human tissues (ng/g wet weight) have been estimated to be 0.55 (fat/muscle), 1.1 (heart), 3 (kidney), 7.5 (liver), 2.1 (lung), and 3.1 (thyroid glands) (Badmaev et al, 1999; Setyawati et al, 1998). In general, these values reflect intracellular concentrations of less than 1 uM and plasma concentrations of ~ 20 nM (Nechay, 1986). 1.2.6 Specific Proteins Affected by Vanadium Salts The first experimental evidence that vanadate influences a specific protein was reported in the late 1970s and involved studies showing that vanadate inhibits Na+/K+-ATPase in isolated membranes and in intact human erythrocytes (Beauge and Glynn, 1978; Cantley et al, 1977, 1978; Cantley and Aisen, 1979). Interestingly, in many studies with intact cells, there is little 22 evidence that the Na+/K+-ATPase is in fact inhibited (English et al, 1983; Erdmann et al, 1979a, 1979b). Subsequently, it has been demonstrated that vanadate is reduced to vanadyl by cytoplasmic reducing agents such as glutathione and that vanadyl is not an ATPase inhibitor (Erdmann et al, 1979b). This discovery probably explains the apparent resistance of the Na +/K +-ATPase to vanadium in intact cells. Indeed in further studies in vitro, the effect of vanadate on Na+/K+-ATPase is partially prevented by the reducing agents ascorbic acid and glutathione (Grantham and Glynn, 1979). Vanadium compounds also affect a variety of other enzymes, including ribonuclease (Sabbioni et al, 1983; Stankiewicz et al, 1995), respiratory enzymes (Mendz, 1999), several enzymes of glucose metabolism (Liu et al, 1992; Nour-Eldeen et al, 1985; Reul etal, 1999; Stankiewicz et al, 1987), and protein tyrosine phosphatases (PTPs) (Fantus et al, 1995; Mohammad et al, 2002; Shechter et al, 1995; Shisheva et al, 1994; Swarup et al, 1982; Tracey, 2000; Tracey and Gresser, 1986). Many other physiological and biochemical processes are also believed to be sensitive to vanadium (Nechay, 1984). 1.2.7 "Insulin-Like" Properties of Vanadium In Vivo Based on many studies, it is clear that vanadium salts exert insulin-like (or insulin-enhancing) actions, leading to the proposition that vanadium might be useful in the treatment of diabetes. Crucial early studies with Wistar rats that had been treated with streptozotocin (STZ) to destroy the insulin-producing p-cells of the pancreas demonstrated that oral administration of sodium orthovanadate in the (kinking water (0.6-0.8 mg/mL) for 4 weeks, normalized blood glucose levels (Heyliger et al, 1985). In diabetic rats treated with vanadate, basal circulating insulin levels were 8.8 ± 0.9 |tiU/mL, very similar to the levels in untreated diabetic animals and much lower than in non-diabetic animals (27.3 ± 4.2 uU/mL). The performance of isolated perfused hearts from vanadate-treated animals did not differ significantly from that of non-diabetic controls while hearts from untreated STZ-diabetic rats exhibited depressed contractility, indicating diabetic cardiomyopathy. It was concluded that vanadate controlled blood-glucose levels and prevented the decline in cardiac performance in STZ-diabetes (Heyliger et al, 1985). Since this early study, many others have documented similar effects of vanadate, vanadyl, or vanadium complexes with organic ligands in vivo (Badmaev et al, 1999; Brichard and Henquin, 1995; Brichard et al, 1991; Cros et al, 1992; McNeill et al, 1995; Poucheret et al, 1998; Sekar et al, 1996; Shechter, 1990; Thompson et al, 1993,1999; Yao et al, 1997; Yuen et al, 1993). Continuing efforts are being made to improve the pharmacological and other properties of 23 various forms of vanadium for potential use as human therapeutic agents (Thompson et al, 1999, 2003; Thompson and Orvig, 2003). For healthy individuals not treated with vanadium, basal circulating vanadium levels are ~ 1 nM as determined by electrothermal atomic absorption spectrometry (Heinemann and Vogt, 1996) and by neutron activation analysis (Sabbioni et al, 1996). For example, in human blood, urine, and hair, the mean vanadium concentration was ~ 0.5 ng/mL, 0.3 ng/mL, and 150 ng/g, respectively (Martin and Chasteen, 1988). Although the very low "natural" abundance of vanadium can be enhanced by dietary supplements, the concentration of vanadium species is unlikely to exceed 10-20 uM (Cam et al, 2000). This finding will be crucial in considering the biologically relevant mechanism of action. Not all authors have agreed that the anti-diabetic or insulin-mimetic actions of vanadium are beneficial, and some have suggested that vanadium may have toxic effects (Domingo, 2002; Leonard and Gerber, 1994; Llobet et al, 1993). For example, Donryu strain rats administered ammonium vanadate by gavage (20 mg/kg body weight) or injected subcutaneously (5 to 30 mg/kg body weight) exhibited dose-dependent, reversible increases in TG concentrations in the liver and blood serum, a decrease in the serum-cholesterol level, and increases in glutamate-oxalo-acetate transaminase and glutamate-pyruvate transaminase activity (Kaku et al, 1971). On the basis of these and other studies, concerns about vanadium toxicity have been expressed (Domingo, 2000, 2002). At least some of these concerns have been addressed in long-term studies in which the anti-diabetic effects of vanadium were not accompanied by serious toxic effects (Dai et al, 1994a, 1994b). Furthermore, vanadium salts have been used in several clinical trials with some success, albeit with significant side effects (Boden et al, 1996; Cohen et al, 1995; Goldfine et al, 1995, 2000). Overall, there is compelling evidence that vanadium treatment of diabetic humans as well as animals with chemical or genetic diabetes improves metabolic status and prevents or diminishes at least some longer-term consequences of diabetes (Boden et al, 1996; Cohen et al, 1995; Goldfine et al, 1995,2000). Vanadium salts have not yet emerged as an effective treatment for humans with diabetes but work continues with the aim of developing effective vanadium compounds. Whether vanadium is truly an "insulin-mimetic" agent is an interesting issue. Such a designation implies actions with similar or identical mechanisms, a point that emerges at least partially, from studies presented in this thesis. Certainly, vanadium treatment does not reproduce all actions of insulin (Liu et al, 1997), and the growth of STZ-rats that is restored by administration of insulin is not reproduced by vanadium treatment (Cam et al, 2000). 24 1.2.8 "Insulin-Like" Properties of Vanadium In Vitro Since the demonstration that vanadate inhibits the Na+/K+-ATPase, a multitude of studies have defined the ability of vanadium to influence a range of cellular processes in isolated tissues and cultured cell lines. Many of these studies have implicated processes that are also sensitive to insulin. Examples of the processes activated by vanadium include glucose transport, glycolysis, glycogen synthesis, lipogenesis, protein synthesis, and gene expression. Similarly, vanadium salts have been found to inhibit the corresponding catabolic processes, notably TG hydrolysis. As an example, responses seen in adipocytes in vitro are noted in Table 1.4. Dose-dependency is a crucial issue in assessing which of the effects of vanadium observed in vitro might also occur in vivo. Are the effects seen in vitro evident at concentrations in the low micromolar range that might exist in vivo? In this regard, it is clear that a broad range of concentrations (10 uM to 10 mM) have been used in vitro (Table 1.4). Several effects of vanadium have been reported in the range 10-100 uM, including effects on glucose oxidation, glycogen synthesis, TG hydrolysis and lipogenesis (Table 1.4). Among these effects, the ability of vanadium to inhibit hormone-stimulated TG hydrolysis has been most consistently found (in 6 out of 7 studies) to occur at low vanadium concentrations. Although not listed in Table 1.4, "mitogenic" effects of vanadium have also been observed at vanadium concentrations in the low micromolar range (Chen and Chan, 1993; Etcheverry et al, 1997; Wang and Scott, 1994). The physiological relevance of these mitogenic effects may be questioned because they have been found largely with immortalized cell lines that, by definition, already have a predisposition to proliferation and typically show limited metabolic responses to insulin. Furthermore, there is no evidence for proliferative effects of vanadium in vivo; indeed, several studies have shown vanadium to have anti-proliferative or anti-neoplastic effects (Bishayee and Chatterjee, 1995; Cruz et al, 1995; Jackson et al, 1997; Sakurai et al, 1995). These anti-neoplastic effects might be related to the apoptotic effects of vanadium that have been observed following environmental or occupational exposure to high levels of vanadium (Chen et al, 2001). In contrast to the effects of vanadium on lipolysis or mitogenesis, many studies of other metabolic parameters in vitro reveal dose-dependencies in the range 100 uM -1 mM and some even as high as 10 mM (Table 1.4). Effects of vanadium seen at concentrations in the millimolar range are most unlikely to be physiologically relevant and indeed include activation of AC and glucose uptake into hepatocytes, neither of which are insulin-sensitive; indeed insulin actions oppose those of AC. 25 Table 1.4 Metabolic Effects of Vanadium on Adipocytes In Vitro* Metabolic Activity Affected by Vanadium Effective Vanadium Concentration (uM) Selected References Glucose transport 100-10000 (Elbergera/., 1997) (Green, 1986) GLUT translocation 1000 (Kono etal., 1982) (Paquetefa/., 1992) Glucose oxidation 10-10000 (Tolmanef a/., 1979) (Shechter and Shisheva, 1993) Glycolysis 1000-10000 (Duckworth et ai, 1988) Glycogen synthesis 10-1000 (Sekare*tf/., 1999) (Ueki etal, 1992a) Lipogenesis 10-10000 (Fantusef a/., 1990) (Duckworth et al., 1988) TG Hydrolysis 10-100 (Li etal, 1997) (Sekarera/., 1998) *Adaptedfrom (Cam et al, 2000) - All metabolic activities are stimulated by vanadium except TG hydrolysis, which is inhibited. Another important consideration is the form of vanadium that may be effective within intact cells. Some studies have indicated that vanadate is the effective insulin-mimetic species because vanadyl oligomerizes, precipitates or oxidizes to vanadate at physiological pH. However, intracellular conditions are highly reducing due to the presence of glutathione (generally above 2 mM) and other antioxidants (see Section 1.3). Interestingly, administration of STZ to rats led to a decrease in reduced glutathione in liver and treatment with 1-1.25 mg/mL VS restored glutathione levels to normal (Thompson and McNeill, 1993). 26 Importantly, EPR studies have demonstrated the presence of vanadyl within intact erythrocytes and adipocytes, even when cells were incubated with vanadate. It seems likely that following uptake vanadate is reduced to vanadyl by glutathione (Degani et al, 1981). In addition, vanadyl-nitrogen interactions were detected in organs of vanadyl-treated rats (Fukui et al, 1995). As noted earlier, the fact that most cells do not show evidence of major disturbances of ion balance following vanadium treatment suggests that insufficient intracellular vanadate exists to inhibit the N+/K+-ATPase. An exception might occur in the kidney, a site of particular vanadium abundance in vivo that shows signs of toxicity at high dose levels (De Cremer et al, 2002; Parker and Sharma, 1978; Russanov et al, 1994). In addition to the vanadate and vanadyl species discussed so far, peroxovanadium species appear to exhibit potent insulin-like actions (Fantus et al, 1989; Kadota et al, 1987; Kanamori et al, 2001; Li et al, 1995; Posner et al, 1994; Shaver et al, 1995; Shisheva et al, 1994). Of particular note is the possibility that peroxovanadium species are generated transiently during oxidative stress (Huang et al, 2001; Krejsa et al, 1997). There is intriguing work showing that insulin itself might promote formation of hydrogen peroxide (H2O2) which, in turn, could enhance the formation of peroxovanadium species (Hadari et al, 1993; Heffetz and Zick, 1989; May and De Haen, 1979a, 1979b; Wilden and Broadway, 1995; Zick and Sagi-Eisenberg, 1990). Insulin-induced production of H202and subsequent production of peroxovanadium species might be especially significant because peroxovanadium is a potent inhibitor of PTPases as discussed below (Cuncic et al, 1999; Huyer et al, 1997; Zhang et al, 1997). In fact, it has been demonstrated that H2O2 generated in response to insulin may transiently inhibit PTPases, reducing IRK dephosphorylation (Mahadev et al, 2001) In view of the evidence reviewed here, the role of vanadyl as an insulin-like species certainly merits consideration. 1.2.9 Mechanism(s) and Putative Site(s) of Vanadium Action As noted above, insulin signal transduction pathways are complex and incompletely elucidated. Similarly, the "insulin-like" mechanism or mechanisms of action of vanadium are unclear. Vanadium might act at one or more sites of the insulin signaling cascade or independently of this cascade. Several hypotheses have been advanced to explain the biological effects of vanadium: The major hypothesis, based on early studies of isolated membranes (Swarup et al, 1982), is that vanadium compounds inhibit PTPase(s). This inhibitory activity should enhance 27 the phosphorylation and activation of the intrinsic tyrosine kinase of the |3-subunits of the insulin receptor (Tamura et al, 1984), thus increasing phosphorylation of the receptor and the IRS proteins and, in turn, potentiating the actions of insulin. In support of this mechanism, a number of studies have demonstrated the effects of vanadate and peroxovanadium compounds on cellular protein tyrosine phosphorylation levels (Fantus et al, 1995; Posner et al, 1994; Salice et al, 1999). As a consequence, for example, vanadium prolongs insulin-receptor activation and in some cases enhances insulin sensitivity (Eriksson et al, 1992; Fantus et al, 1994,1996; Lu et al, 2001). These actions of vanadate and peroxovanadium compounds are the result of their interaction with the active site of PTPases as demonstrated by X-ray crystallography (Huyer et al, 1997; Zhang et al, 1997), probably by mimicking the transition state of the phosphoryl transfer reaction (Tracey and Gresser, 1986). Certainly, modulating PTPase activity in vivo (notably PTP-1B) has profound effects on insulin action and sensitivity and the PTPase system is an important therapeutic target (Kennedy, 1999). Related to the action of vanadium on protein tyrosine phosphorylation, is the activation of a distinct non-receptor tyrosine kinase by vanadium (Elberg et al, 1994, 1997; Li et al, 1996; Shisheva and Shechter, 1993). It is not, however, clear that the actions of vanadium in vivo necessarily depend on PTPase inhibition. In fact, several observations suggest that vanadium can act at a site(s) distal to IRK. First, the insulin-like effects of vanadate in adipocytes were still evident when insulin receptors were depleted by proteolysis (Green, 1986). Second, the phosphorylation of insulin receptors in response to treatment with vanadate is lower than with insulin, even when vanadate exerts metabolic effects such as antilipolysis as strongly as insulin (Blondel et al, 1990; Mooney et al, 1989; Strout et al, 1989; Venkatesan et al, 1991). Third, vanadate stimulation of MAP kinases, which play a central role in some downstream insulin signal transduction, can be independent of insulin receptor phosphorylation (D* Onofrio et al, 1994). Fourth, VS stimulates glycogen synthesis through the action of PI3K but independently of insulin receptor tyrosine phosphorylation (Pandey et al, 1998). Fifth, if vanadium were to inhibit PTPase activity, all the subsequent (downstream) effects of insulin should be equally reproduced. In fact, vanadium effects on metabolism are selective with particularly potent effects on lipolysis (Brownsey and Dong, 1995; Duckworth et al, 1988; Fantus and Tsiani, 1998). Finally, vanadate has effects distinct from those of insulin in that they are independent of PI3K activation and the lipolytic cascade (Li et al, 1997). Taken together, these findings argue that vanadium salts and 28 compounds influence metabolism independently of early steps in the insulin transduction pathway that involve protein tyrosine phosphorylation. A second hypothesis for the insulin-like actions of vanadium is that vanadium compounds activate cyclic nucleotide phosphodiesterase and consequently suppress intracellular cAMP levels. Insulin itself induces activation of cyclic nucleotide phosphodiesterase, thereby attenuating cAMP signaling (Loten and Sneyd, 1970,1973). This activation involves phosphorylation of PDE-3B by an insulin-stimulated protein serine/threonine kinase (perhaps PKB) and contributes strongly to the antilipolytic effects of insulin (Degerman et al., 1990; Shibata et al., 1991; Vasta et al., 1992). Furthermore, vanadium can activate PDE-3B directly in vitro (Soilness et al, 1992; Ueki et al, 1992b). Moreover, PDE inhibition by 3-isobutyl-l-methylxanthine blocked some effects of vanadium (Sera et al, 1990). In other studies, however, the antilipolytic effects of vanadium were still apparent following PDE-3B inhibition by cilostamide (Brownsey and Dong, 1995), and both vanadate and peroxovanadate inhibited lipolysis independently of both PI3K and PDE-3B (Castan et al, 1999). Overall, therefore, although insulin itself does interfere with cAMP metabolism, it is not clear if the insulin-like antilipolytic effects of vanadium depend entirely on this mechanism. An alternative hypothesis implicates a direct action of vanadium compounds on a distal step in the anti-lipolytic cascade, perhaps at the level of PKA or an even more distal step. Specifically, equimolar VS/GSH mixtures induced a dose-dependent inhibition of Kemptide phosphorylation by PKA (Brownsey and Dong, 1995). Others have also observed PKA inhibition by vanadium (Pluskey etal, 1997). Interestingly, chronic treatment with vanadium abolished the persistent PKA activation and protein phosphorylation seen in extracts of adipose tissue from STZ-diabetic rats (Ramanadham et al, 1989). Furthermore, BMOV normalized hepatic glucose output by inhibiting the expression of mRNA encoding the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) (Marzban et al, 2002). Interestingly, the expression of PEPCK is strongly induced by cAMP in liver (Roesler, 2000). 1.2.10 Examples of Other "Insulin-Like" Elements Cr, zinc (Zn), tungsten (W), cobalt (Co), molybdenum (Mo) and selenium (Se), have, like vanadium, all been tested for possible "insulin-like" and antidiabetic properties. In principle, therefore, knowledge of these other "insulin-like" agents might help in understanding how vanadium exerts its effects. Some evidence suggests that the mechanism of actions of these 29 elements may overlap, but there are still marked differences among them and the literature concerning their effects is not consistent. Various forms of chromium have been found to inhibit PTPases. The mode of inhibition was suggested to involve effects at the thiol-dependent active site of PTP-1B, a PTPase that has been shown to be a major regulator of insulin signaling pathways (Goldstein et al, 2001). On the other hand, studies on the impact of vanadium, tungsten, and selenium on isolated rat muscle showed that vanadium stimulated glycogen synthesis whereas tungsten and selenium did not (Purnsinn et al, 1996). Furthermore, the stimulation of glucose uptake by tungsten and selenium was associated with glycogen depletion and increased rates of glycolysis, catabolic responses that were inferred to represent nonspecific responses to toxic or osmotic stress (Furnsinn et al, 1996). Studies on STZ-diabetic rats and mice showed that treatment of these animals with selenium not only lowered their blood glucose levels but also controlled the fate of glucose by stimulating glycolysis and fatty acid synthesis (McNeill et al, 1991). Although the mechanism of this action of selenium has not yet been established, the activation of key enzymes in insulin signaling such as cAMP-PDE could be involved (Stapleton, 2000). Treatment of STZ-diabetic rats with cobalt chloride also led to lower blood glucose levels. This effect may be mediated by reduction in hepatic gluconeogenesis through inhibition of PEPCK or PEPGK gene transcription (Saker et al, 1998). The antilipolytic activity of various transition metals vary in potency in the order VS > MV > Zn » W > Mo (Li et al., 1997). Zinc has a unique role in the physiology of insulin. Prior to release from pancreatic secretory granules, insulin exists as hexamers that contain a variable number of zinc atoms. Variation of the zinc-to-insulin ratio within these hexamers alters the antigenic properties of the hormone such that removal of zinc reduces insulin antigenicity. Furthermore, the addition of zinc to insulin induces conformational changes that enhance insulin binding to its receptor and influence its biological potency (Salgueiro et al, 2001). It seems unlikely that insulin would remain associated with zinc once it is diluted in the blood. Nevertheless, it is interesting to speculate that zinc, along with other contents of the secretary granules, is a "co-secreted" product that may have additional roles. The studies discussed above provide no conclusive insights into possible mechanism(s) of action of the various elements, so, it is difficult to draw inferences from them to illuminate the anti-diabetic actions of vanadium. 30 1.3 BIOLOGICAL LIGANDS THAT MAY BE INVOLVED IN VANADIUM ACTIONS 1.3.1 Rationale for Binding of Vanadyl to Endogenous Ligands Vanadium exists primarily in the reduced, vanadyl form once it enters the cytosol. Even though the cytosol is reducing, however, oxidants can be produced (especially during cellular stresses), and it is conceivable that transient formation of vanadate or even peroxovanadium species might be biologically relevant. If vanadyl is to exert significant effects within cells, then it must be sufficiently stabilized to interact with effector molecules. However, this requirement is physiologically problematic because vanadyl ion may oxidize to vanadate at neutral pH and it has a very low solubility product that leads to its precipitation as VO(OH)2. Nevertheless, EPR studies provide clear evidence for the sustained presence of vanadyl within intact cells (Degani et al, 1981). Presumably, endogenous organic ligands protect vanadyl from oxidation, oligomerization, or precipitation and may have complex effects on the actions of vanadyl. On one hand, if the binding constant of a ligand for vanadyl is greater than that for the interaction between vanadyl and hydroxyl ion, then the ligand can protect vanadyl sufficiently for it to interact with appropriate target(s). On the other hand, if vanadyl binds extremely tightly to a biological ligand (e.g. as it does to EDTA), vanadyl actions could be blocked. In other words, to be effective, vanadyl must be "protected" but not "excluded" from appropriate interactions. A number of endogenous ligands might be physiologically important in binding vanadyl. Potential candidates include but are not limited to the list shown in Table 1.5. For completeness, enzymes and coenzymes are included in this summary even though their ability to protect vanadyl from oxidation is probably indirect. At present, there have been no studies of interactions of vanadyl or vanadate directly with the redox enzymes listed in Table 1.4 (Maritim etal, 2003; Mason, 2001; Polidori etal, 2001). Instead, glutathione peroxidase (Equation 1.3), glutathione reductase (Equation 1.4), and selenium (a cofactor for the former) probably protect vanadyl indirectly through their catalytic activity: 2 GSH + R-O-OH > GSSG + H 2 0 + ROH (1.3) GSSG + NADPH + Pf > 2 GSH + NADP + (1.4) As the interactions between vanadium and some of the potential ligands have not been studied in detail, only those that have been studied are discussed here. 31 Table 1.5 Ligands and Antioxidants That May Protect Vanadyl In Vivo* CLASS EXAMPLES Tocopherols a-, y-tocopherols (Vitamin E) Retinoids & Carotenoids Retinol, a-, P-carotenes (Vitamin A) Redox Enzymes Superoxide dismutase, Catalase, Glutathione Peroxidase, Glutathione Reductase Biothiols a-Lipoic acid, Glutathione (GSH and GSSG), Cysteine, Cysteine Methyl ester, Penicillamine Coenzymes Coenzyme Qio, NAD(P)H, FADH 2 , Folic acid, Vitamin B complexes Minerals Copper, Zinc, Manganese, Selenium Bioflavonoids Flavones, Flavonols, Anthocyanins, Aurones, Chalcones Serum Proteins Transferrin, Ferritin, Albumin Others Lutein, Zeaxanthin, Ascorbic acid (Vitamin C), Lycopene, p-Cryptoxanthin, Catecholamines * Adapted from (Maritim etal, 2003), (Mason, 2001), and (Polidori et al, 2001) 1.3.2 Vitamin C (Ascorbic Acid) Interactions With Vanadium Evidence for the interactions between vanadium and vitamin C is mixed. In one study, strong binding of vitamin C to vanadate or vanadyl was inferred from the finding that vitamin C was an effective antidote for vanadyl and vanadate toxicity in mice (Jones and Basinger, 1983). Based on the survival of 90-100 % of animals treated, vitamin C provided better protection from toxicity than any of the 16 reagents tested, including glutathione, EDTA and related chelators (Jones and Basinger, 1983). Furthermore, vitamin C was able to reduce V(V) to V(IV) about 2000 times faster than glutathione (Song et al, 2002). Because the intracellular concentration of glutathione is much higher than that of vitamin C, one might argue that vitamin C is unlikely to be a major ligand for vanadyl in vivo. This conclusion is consistent with the report that the stabilities of vanadyl-vitamin C complexes are low (Ferrer and Baran, 2001). Nevertheless, it remains possible that, if the concentration equals or exceeds that of vanadium, vitamin C may bind significantly, particularly in the extracellular space where reduced glutathione is minimal. 32 1.3.3 Glutathione Interactions With Vanadium The tripeptide glutathione (L-y-glutamyl-L-cysteinylglycine) was first named "philothion" and is the most abundant thiol-containing reducing agent within mammalian cells, typically being present at ~ 2 mM (Hagenfeldt et al, 1978; Janaky et al, 1999). Glutathione seems to have emerged in parallel with oxidative metabolism and has been conserved evolutionarily in aerobes. Glutathione exists in the reduced form (GSH) and as the oxidized, disulfide-linked hexapeptide (GSSG); GSSG is the predominant form in extracellular fluids where the concentration can approach ~ 4 mM (Hagenfeldt et al, 1978). The term "glutathione" is used to indicate the sum total of GSH and GSSG. In the intracellular environment, greater than 98 % of total glutathione is in the reduced form, the reduction being sustained by NADPH-dependent glutathione reductase (Equation 1.3). In contrast to the eukaryotic cytosol, GSSG may represent up to 20 % of total glutathione in mitochondria (Lenton et al, 1999). Glutathione has many general and specific cellular functions. Glutathione contributes significantly to the cell redox state by protecting against the effects of oxidizing agents such as reactive oxygen species. In this regard, glutathione is a substrate for glutathione peroxidases that reduce H2O2, lipid peroxides, and other toxic compounds to produce GSSG and reduced toxins. In turn, GSH is regenerated by the NADPH-dependent glutathione reductase reaction (Equation 1.4, Forman etal, 1995). Other functions of glutathione include catalysis, metabolism, and interorgan transport (Meister, 1989), as well as acting both as a neuromodulator and as a neurotransmitter (Janaky et al, 1999). In the context of vanadium action, glutathione facilitates reduction of vanadate to vanadyl (Equation 1.5, Degani et al, 1981; Grantham and Glynn, 1979; Macara et al, 1980; Sabbionie/a/., 1993). GSH + vanadate > GSSG + vanadyl (1.5) This reaction is believed to play a major role in the reduction of vanadate to vanadyl following uptake into cells as demonstrated by EPR studies with erythrocytes and adipocytes (Degani et al, 1981). In the absence of glutathione, NADPH-dependent reduction of vanadate to vanadyl can be catalyzed by glutathione reductase in vitro. In these studies, H2O2 was generated if the reaction was conducted aerobically (Shi et al, 1997). Glutathione-independent reduction of vanadate to vanadyl can also be catalyzed by flavoproteins (Shi and Dalai, 1991, 1993). These authors have argued that the formation of H2O2 and other forms of reactive oxygen might 33 account for some actions of vanadium, but this view has been questioned (Liochev and Fridovich, 1996). In addition to its role in reducing vanadium, glutathione is also likely to be an important endogenous vanadium ligand. A recent potentiometric and spectrometric study (Kiss et al, 2003) provided evidence for complex interactions between vanadate, vanadyl, GSH, and GSSG (Figure 1.6). Figure 1.6 Possible Interactions Between GSH, GSSG, Vanadyl, and Vanadate* Vanadyl + GSH + Vanadate > Vanadyl + GSSG ti tl tl Vanadyl-GSH Vanadate-GSH Vanadyl-GSSG ^Adaptedfrom (Kiss et al, 2003) GSH is a polydentate ligand, with six potential e' donors: 2 carboxylate oxygens, 1 amino nitrogen, 1 sulfhydryl group, and 2 amide groups, various combinations of which might provide as many as eight potential binding sites for vanadyl. The pKa's of the ionizable groups of GSH are indicated in Figure 1.7 (Pessoa et al, 2002; Rabenstein, 1989). Recent *H NMR studies suggested that the two carboxylic acid groups ionize simultaneously over the pH range of 0.5-6 and the sulfhydryl and the amino groups ionize simultaneously over the pH range of 7-12 (Armas et al, 2001). The disulphide bridge constrains GSSG more than GSH because the sulfhydryl groups are no longer available, and the N and O atoms on the amide groups can provide four ligands for divalent metal ions. In fact, Cu(II), Ni(II), Co(II), and Zn(II) have been reported to form stable complexes with GSSG (Rabenstein, 1989). Because of the number, variety, and pH-dependence of coordination sites in both GSH and GSSG, studies of metal-glutathione complexes are complicated, and a combination of techniques is required to define the nature of the complexes. 34 Figure 1.7 Structure of Reduced Glutathione at Physiological pH* P K 3 ~ 8 HS P K a ~ 9 O o PK a ~3 + H 3 N NH NH O-o PKa~2 * Adaptedfrom (Pessoa et al, 2002) and (Rabenstein, 1989) The interactions of GSH and GSSG with vanadyl have been studied with a variety of spectrometric methods and found to depend on pH and on the ligand-to-metal ratio. Specifically, GSH coordination to vanadyl was detected between pH 3-7 and was stable up to a GSH:V0 2 + ratio of 1.5:1 at pH 7 (Ferrer et al, 1991). As the concentration of vanadyl approached that of GSH, a precipitate was observed that was presumed to be VO(OH)2 (Ferrer et al, 1991). Further subtleties exist in the vanadyl-GSH interactions, however, and it is possible that various complexes can form as the GSH:Vanadyl ratio is varied. For example, a blue-colored complex exists when the ratio of GSH to vanadyl is ~ 10:1. Increasing this ratio to 100:1 (GSH:V02+) resulted in the formation of a violet complex. GSH-vanadyl complexes are not necessarily reversible. This situation is exemplified by the fact that once the violet complex is formed, decreasing the GSH:vanadyl ratio by increasing V 0 2 + concentration does not necessarily lead to reformation of the blue complex (Ferrer et al, 1991). The interactions of vanadyl with GSSG differ in some respects from its interactions with GSH. At a GSSG:V0 2 + ratio of 10:1 a blue complex is formed, which is evidently more stable than its GSH counterpart, as no VO(OH)2 precipitation was observed when the ratio of GSSG:V0 2 + was reduced to 0.5:1 at pH 7 (Ferrer et al, 1993). In addition, the complexes formed with GSSG were more readily interconverted by changing the metal-to-ligand ratio (Ferrer et al, 1993). Table 1.6 summarizes the reported interactions of GSH and GSSG with vanadyl, including modes of coordination and ligands (Ferrer et al, 1991,1993). Using 35 concentrations similar to those found in vivo for GSH (3 mM), GSSG (0.2-0.5 mM) and vanadyl (10-100 uM), speciation-modeling calculations showed that the amount of vanadyl bound to GSH at pH 6-7 was about the same as that bound to GSSG. Thus, GSSG could effectively bind vanadyl even when present at low concentrations at physiological pH (Pessoa et al., 2002). Evidently, much remains to be learned about the interactions between vanadyl, GSH and GSSG. Table 1.6 Interactions Between Vanadyl and GSSG or GSH* G: Glutathione, V: Vanadyl, ?: Not determined. G:V (Ratio) pH Stoichiometry Ligands Properties of Complex GSH 10:1 7 VO(GSH)2 2COO", one from each GSH Blue, Changed to violet at high [GSH] GSH 100:1 7 VO(GSH)? 1SH?, 1NH2?, and 2 amide N? Violet, very stable, no reversion to blue at high [V] GSSG 2.5:1 4.5 (VO)2GSSG ? Pale blue, Very unstable at pH 7 GSSG 10:1 7 (VO)2GSSG Mainly O from COO" or amide? Blue, more stable than with GSH GSSG 100:1 7 9 Mainly N from NH2 or amide? ? * Adapted from (Ferrer et al, 1991, 1993) 1.3.4 Vanadium Binding to Serum Proteins Vanadyl is transported within blood cells and also in complexes with serum proteins, notably albumin and transferrin. It has been established that human serum transferrin has two non-equivalent vanadyl binding sites with different binding constants and that fransferrin binds vanadyl six times stronger than does albumin in vitro (Sun et ai, 1999). However, albumin is far more abundant than transferrin, making up ~ 60 % of total plasma protein, at a concentration of ~ 42 g/L or ~ 0.7 mM (Peters, 1996). Human and rat albumins retain bound vanadium when isolated by conventional techniques (see values below). The presence of other metals can also influence vanadium binding to plasma proteins. For example, transferrin is involved in transport of iron and other transition metals, the concentrations of which presumably exceed that of vanadium. Transferrin will, therefore, be less available to bind vanadium in the presence of other metals. Albumin also binds other metals including Cu(II) (Masuoka et al., 1993), Zn(II) (Masuoka et al, 1993), Ni(II) (Glennon and Sarkar, 1982), Cd(II) (Trisak et al, 1990), Ca 2 + 36 (Pedersen, 1972), and Mg (Guillaume et al, 1999); consequently, the knowledge of the relative affinities and concentrations is important. EPR studies on vanadyl-albumin interactions led to several important findings. First, albumin contains two types of vanadyl binding sites, one strong binding site with a Ka of 2.6 x 106 M"1 and five weaker binding sites with a Kd of 2.5 x 104 M"1. Thus, a maximum of six vanadyl ions can be bound to each molecule of albumin (Chasteen and Francavilla, 1976). In addition to vanadyl, VO(acac)2 forms an adduct with BSA with a stoichiometry of 1:1 (Makinen and Brady, 2002). Vanadyl and vanadate both exist in plasma; vanadyl being carried by both albumin and transferrin and vanadate carried only by transferrin (Chasteen et al, 1986a). The insulin-like effects of organic complexes of vanadyl may be enhanced by formation of complexes with BSA or other serum transport proteins (Makinen and Brady, 2002). Interestingly, a recent report on the interactions between VS and BMOV with human serum albumin (HSA) in vitro indicated that the presence of chelating ligands might enhance vanadium binding to HSA (Liboiron et al, in preparation). Stronger chelating agents such as EDTA, however, can strip vanadium from serum albumins. Thus, EDTA has been used to estimate the natural abundance of vanadium in commercial albumins from several animal sources. All tested albumins contained at least some vanadium, the lowest levels being ~ 0.1 Hg/g protein and the highest levels, in the case of BSA, -17 ng /g protein (Sakurai et al, 1987). Recently, a 21 kDa vanadium-binding protein was isolated from a vanadium-reducing bacterium that exhibits a protein:V(IV) molar ratio of ~ 1:20 at neutral pH. The authors concluded that this complex provided an important mechanism to prevent V(IV) oxidation in this organism (Antipov et al., 2000). 1.4 R A T I O N A L E A N D H Y P O T H E S E S F O R P R O P O S E D S T U D I E S PTPases are clearly targets of vanadium action in vitro, and it is widely assumed that the inhibition of PTPases can explain biological effects of vanadium through enhancement of the tyrosine phosphorylation of key proteins. In the context of insulin action, these proteins include the insulin receptor and the IRS proteins. Despite the evidence in vitro, it is not clear that the anti-diabetic effects of vanadium in vivo involve the same mechanisms. Importantly, many effects of vanadium in vitro are observed at concentrations far higher than those achieved in vivo. Furthermore, there is considerable evidence in vitro that vanadium can act significantly downstream from the protein tyrosine phosphorylation level of signal transduction. The current work concerns one aspect of vanadium action: the ability of vanadium to inhibit hormone-sensitive TG hydrolysis, an action observed at rather low vanadium 3 7 concentrations in vitro ( 1 0 - 1 0 0 uM). The mechanisms involved in the control of TG hydrolysis are well defined and provide a convenient context to assess the effects of vanadium. A number of key proteins in the lipolytic cascade might respond to vanadium, including heterotrimeric G-proteins, adenylyl cyclase, cyclic nucleotide phosphodiesterases, PKA, hormone-sensitive lipase, perilipins, and protein serine/threonine phosphatases. From this list and the available literature, the present study focused on the possibility that PKA might be a target for inhibition by vanadium. The complexity of interactions between vanadate, vanadyl and various reagents typically used in biological buffers and enzyme assay mixtures made it essential to carefully define the conditions necessary to evaluate the potential effects of vanadium in vitro. The basic hypotheses of this work are as follows: 1 . The reduced form of vanadium (vanadyl) inhibits PKA with an effective Kj low enough to account for known potency of vanadium in vivo; 2. Specific assay components are required to form complexes with vanadyl that are sufficiently stable that vanadyl does not oxidize, precipitate or oligomerize at neutral pH. 38 CHAPTER TWO MATERIALS AND METHODS 2.1 MATERIALS 2.1.1 Vanadium Salts BMOV and BPOV were kindly donated by Dr. Chris E. R. Orvig (Department of Chemistry, The University of British Columbia). VS.3H20, VS.nH20, OV, V 2 0 4 (vanadium tetroxide) and V2O5 were obtained from Aldrich Chemical Co. (Milwaukee, WI, USA). 2.1.2 Enzymes PKA catalytic subunit was obtained from two sources: BOehringer Mannheim (Laval, P Q , Canada) and Calbiochem-Novabiochem (La Jolla, CA, USA). Recombinant and activated MAPK and the catalytic fragment of protein kinase C (PKC), also known as PKM, were from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA, USA). 2.1.3 Peptide or Protein Substrates and Inhibitors The specific peptide inhibitor of PKA (PKI), casein, and lysine-rich histones HIS (corresponds to IUPAC-IUB HI class) and IIS were from Sigma Chemical Co. (St. Louis, MO, USA). Of the various histones tested, HI supplied by Upstate Biotechnology was found to be the best substrate and was used in these studies. Kemptide was from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA, USA) or from Sigma Chemical Co. (St. Louis, MO, USA). Kemptide is a synthetic phosphate acceptor substrate for PKA with the following amino acid sequence: LRRASLG (Kemp et al, 1976). Glycine-rich peptide (GTGSFG) was obtained from PeptidoGenic Research & Co. (Livermore, CA, USA). 2.1.4 Other Materials All laboratory chemicals and biochemicals were of reagent grade or better. Laboratory chemicals and solvents, including HPLC-grade reagents, were obtained from Fisher Scientific (Vancouver, BC, Canada), ICN Pharmaceuticals Inc. (Costa Mesa, CA, USA), and BDH Chemicals Canada Ltd. (Vancouver, BC, Canada). Biochemical reagents including glycine and glycyl peptides (di- and tripeptide) were from Sigma Chemical Co. (St. Louis, MO, USA). P-81 phosphocellulose paper was obtained from Whatman International (Maidstone, Kent, UK). 39 Syringe filters were from Pall Gelman Laboratories (Ann Arbor, MI, USA). Centrifugal filtration devices and water filtering units were from Millipore (Bedford, MA, USA). N2(g) was of medical NF grade and was from Praxair (Delta, BC, Canada). Fatty acid-free BSA was from ICN Pharmaceuticals Inc., and Redivue™ [y-32P] ATP was from Amersham International (Oakville, ON, Canada). 2.2 ENZYME KINETICS 2.2.1 Assay of PKA by the Filter Paper Method Unless otherwise specified, all experiments were done using commercial PKA with specific activity of 7-8 U/mg protein (Boehringer Mannheim), which was shipped in potassium phosphate buffer (10 mM, pH 6.5) containing KC1 (70 mM), DTT (0.5 mM), and EDTA (0.1 mM), and filled under N 2 (g). PKA purity was checked by SDS-PAGE, Mono-Q ion-exchange chromatography and electrospray ionization mass spectrometry (ESI MS). Assuming an isotopic averaged MW of 40,488.61 g/mol, PKA concentration averaged 13 uM. Based on the reaction shown in Equation 2.1, one Unit of PKA was defined as the activity required to transfer 1 umol phosphate in 1 min from ATP to Kemptide at 30 °C in a reaction mixture containing MES buffer (50 mM, pH 6.9), MgCl 2 (10 mM), EDTA (0.5 mM), Kemptide (160 uM), DTT (1 mM), and ATP (150 uM). Mg 2 + Kemptide + [y-32P]-ATP > Kemptide-[y-32P] + ADP (2.1) PKA was usually diluted in a "storage" buffer containing MES buffer (20-50 mM, pH 7), DTT (0.5-5 mM), EDTA (0.1-0.2 mM), BSA (5-20 uM), and KC1 or NaCI (70-100 mM) and kept in small portions at - 80 °C. Depending on the nature of each experiment, diluted PKA stock was further diluted with assay buffer. Assays were normally carried out in triplicate with a final volume of 30 to 50 ul/assay. Reaction temperature was 30 °C or room temperature as indicated. Unless otherwise specified, vanadyl solutions were prepared in dH 20 without any pH adjustments immediately prior to use and were quickly added to PKA in the absence of any other assay component to minimize time-dependent changes. Sodium orthovanadate solutions were prepared in dH 20 and pH adjusted with HCI to neutrality.Test compounds (e.g., vanadium salts, GSH, MgS04), PKA, and substrates were mixed (in the order indicated) on ice in microcentrifuge tubes. Following a brief centrifugation (12,000 xg, 2-3 sec) to ensure that the 40 small volumes were mixed at the bottom of the tube, assay mixtures were incubated at 30 °C for 5 min. The reaction was then initiated by adding [y-32P]-ATP at a final concentration of 100 uM and specific activity of 400-800 dpm/pmol. The reaction mixture was further incubated at 30 °C for the indicated time, and the reaction was stopped by adding the total assay volume to small squares of P-81 phosphocellulose paper (~ 4 cm2 in area). P-81 papers were then immediately immersed and washed four times, each time for a period of 10 min in 1.7 % (v/v) ortho-phosphoric acid to remove non-incorporated 3 2P from the papers. P-81 papers were then counted in deionized water (dtkO) using the settings for 3 H (Cerenkov method) in a liquid scintillation counter (Beckman Coulter, LS 6000IC), which had a maximum count rate of 2x106 dpm for 3 2 P isotope in standard 20 mL vials. PKA activity was then calculated and expressed as picomoles (pmol) of phosphate (P) incorporated from ATP into the peptide or protein substrate per minute (Casnellie, 1991). 2.2.2 Phosphocellulose Paper (P-81) Washing Method In previous studies using the P-81 phosphocellulose paper method (Casnellie, 1991; Pluskey et al, 1997) the washing solution used was either phosphoric acid, acetic acid or a combination of the two acids. The efficiency of the two acids in the washing of P-81 papers was, therefore, investigated. PKA assays were carried as described above except for variations in the washing procedure. Filter papers were subjected to one of four washing procedures: a) Four washes with phosphoric acid (0.85 %, v/v); b) Four washes with phosphoric acid (1.7 %, v/v); c) One wash with acetic acid (10 %, v/v) followed by three washes with phosphoric acid (0.85 %, v/v); d) As (c) but with phosphoric acid at 1.7 %, v/v. Assay results were linear with respect to PKA concentration over the range 0-1.0 mU/assay regardless of the washing procedure. Phosphoric acid alone was, therefore, chosen as the wash medium at 1.7 % (v/v) for all the kinetic experiments, and PKA activities were kept below 1.0 mU/assay. 2.2.3 Methods of Analysis of the Enzyme Kinetic Results Several graphical methods have been used to illustrate the features of the PKA reaction and the effects of vanadium salts (Table 2.1). In addition to graphical methods, Leonora® 41 software (Cornish-Bowden, 1995) was used to derive apparent Km and kcat values and to fit Equations 2.2,2.3, and 2.4, representing competitive, uncompetitive and mixed inhibition modes, respectively, to the inhibition kinetic data. Km is the Michaelis-Menten constant and other parameters are defined in Table 2.1. V = V m a x [ATP] / (Km(l + [VS]/#ic) + [ATP]) (2.2) V = V m a x [ATP] / (Km + [ATP](1 + [VS]/Km)) (2.3) V = V m a x [ATP] / (Km(l + [VSVKic) + [ATP](1 + [VS]/^)) (2.4) The Pearson correlation factor, R2, has been used as the measure of the co-linearity of the data points. For experiments with n = 2 the range of the measurements is included along with the mean as bars, for n > 3 the standard error of the mean (SEM) is reported. Where indicated, the results have been subjected to analysis of variance (ANOVA) using the Fisher test. 42 Table 2.I Graphical Methods Used to Characterize the PKA Reaction and the Effects of VS Ordinate (y) Abscissa (x) Shapes Names Notes Velocity, V Concentration of ATP, [ATP] Rectangular Hyperbola Michaelis-Menten* A 1/V 1/[ATP] Family of Straight Lines Lineweaver-Burke or Double-Reciprocal B Slopes of LB plot Concentration of VS, [VS] Straight Line C 1/V [VS] Family of Straight Lines Dixon D [ATP]/V [VS] Family of Straight Lines Cornish-Bowden D * Michaelis-Menten plot was originally presented as V versus log (fSJ), nevertheless, the plot of V versus [SJ is commonly referred to as the Michaelis-Menten plot by many authors. A. The primary plot was used to show that the reaction rate reaches a maximum at high [SJ. In addition, a family ofprimary plots obtained in the presence of different concentrations of inhibitor would expose competitive inhibition with a set of hyperbolic curves that approach the same maximum at high substrate concentrations; B. Because reciprocals are used, this plot gives large errors at low substrate concentrations. Nevertheless, this plot is useful to expose or eliminate uncompetitive inhibition as the family of lines generated would be parallel; C. Plotting the slopes of the Lineweaver-Burke plots against concentrations of the inhibitor can provide an estimate of the competitive inhibition constant (KiC) from the point at which the line intersects the abscissa; D. Dixon and Cornish-Bowden plots together can provide the basis to distinguish between competitive, mixed and uncompetitive modes of inhibition as follows: 1. Dixon (parallel lines) + Cornish-Bowden (intersecting lines) = uncompetitive. 2. Dixon (intersecting lines) + Cornish-Bowden (intersecting lines) = mixed. 3. Dixon (intersecting lines) + Cornish-Bowden (parallel lines) = competitive. The points of intersection of lines in Dixon and Cornish-Bowden plots are interpreted as competitive (KiC) and uncompetitive (KiJ inhibition constants, respectively. 43 2.2.4 Measurement of Assay Buffer pH To ensure that the effects of vanadium salts or other reagents on PKA activities were not due to pH changes during assays, pH measurements were performed after each kinetic experiment. Each assay was duplicated at double the normal volume (60-100 ul), and the pH was recorded using an Accumet™ pH meter (Fisher Scientific, model 620) equipped with a microelectrode. The only difference between the mixtures used for enzyme assay and pH determination was the omission of radioactive ATP from the latter to prevent contamination of the electrode; the non-radioactive ATP was still added. The omission of the radioisotope should not be significant because according to the supplier the pH of [y-32P]-ATP solution was 7.5. 2.2.5 PKA Assays Involving N2(g) Purging To reduce the chance of vanadyl oxidation to vanadate at neutral pH, a "minimal" assay buffer, containing MES (10 mM) and KCH 3COO (120 mM) and purged with N2(g) for 30 min, was used. This "minimal" buffer was prepared immediately prior to carrying PKA assays. This buffer was used not only to dilute all assay components from their representative stocks but also used to carry out PKA reactions. An exception was VS stock solutions which were diluted with dH 20 and not this "minimal" buffer. 2.3 SPECTROMETRY 2.3.1 Electrospray Ionization Mass Spectrometry All mass spectral data were collected and analyzed with the help of Dr. Don Douglas and Dr. Bruce Collings (Department of Chemistry, UBC) using an ESI quadrupole mass spectrometer (Figure 2.4) constructed by them (Collings and Douglas, 1996). All ESI MS experiments were carried out at room temperature. HPLC-grade methanol was added to aqueous solutions of Kemptide and HPLC-grade glacial acetic acid was added to those of PKA, BSA, and Histone. All solutions were filtered with Amicon™ centrifugal membrane filters (9500 xg) at room temperature. Solutions of Histone, PKA and BSA were filtered and/or desalted five times, each time for 5 min, with 10 K, 30 K, or 50 K Microcon™ membranes, respectively. The pH of each solution was measured with an Accumet™ pH meter (Fisher Scientific, model 15). Protein or peptide mass spectra were obtained with or without the indicated concentrations of VS. Sample solutions were injected by pumping at a flow rate of 1 p.l/min (syringe pump model 22, Harvard Apparatus), through a fused silica capillary (Polymicro Technologies, i.d. 75 + 3 um). 44 Gas phase ions were formed by pneumatically assisted electrospray ionization. The ions were passed through a dry nitrogen "curtain" gas, a 0.25 mm-diameter sampling orifice directly into a radio frequency (RF) only quadrupole (ion guide, Q0). The ions exited the ion guide through a differential pumping aperture, through a short RF only pre-filter (PF) and into the mass analyzing quadrupole (QI) operated at a pressure of 0.02 mTorr. Ions were detected using a channeltron detector. Typical electrospray conditions were 4500-5000 Volts on the sprayer and a potential difference of 2-100 Volts between the sampling orifice and Q0. In addition to routine ESI MS experiments, spectral titrations were also performed on BSA and PKA using a continuous-flow mixing apparatus (Figure 2.4). Syringes were automatically pumped together to generate a total flow rate of 2 uVmin and a reaction time of 54 ± 4 sec. The reaction time was controlled by the length of the reaction capillary between the mixing point and the electrospray source (Konermann et al, 1997). Uncertainties in time are related to the uncertainties in the capillary internal diameters. Each syringe was connected to a fused silica capillary (TSP075150, Polymicro Technologies, i.d. 75 ± 3 um, Phoenix, AZ) by a connector (P742, Upchurch Scientific, Oak Harbor, WA). These two capillaries were connected to a third "reaction" capillary of the same diameter by a custom-built tee junction (the mixing point), which had a dead volume of ~ 3 nl. This volume corresponds to a calculated mixing time (dead time) of 90 msec for the flow rate of 2 ul/min. One syringe was filled with a protein sample while the second was filled with VS solutions (1-100 uM). Between VS treatments, the syringe was washed first with N2(g)-treated CIH2O and then with the next concentration increment. For 0 uM VS, the syringe was filled with N2(g)-treated dH20. Spectral analyses, including the deconvolution of mass spectra, were performed with the Biomultiview™ software from Perkin-Elmer SCIEX (Calgary, AB, Canada). 2.3.2 Electron Paramagnetic Resonance (EPR) Spectrometry These experiments were carried out in the laboratories of Dr. Grant Mauk (Dept. of Biochemistry and Molecular Biology, UBC) and Dr. Chris Orvig (Dept. of Chemistry, UBC) with help from Antonio Villegas, Dr. Federico Rosell, Dr. Paul Wittig (Biochemistry), and Dr. Nicholas Aebischer (Chemistry). EPR spectra were obtained with an X-band Bruker ESP 300E spectrometer equipped with an Oxford Instruments ESR900 continuous flow cryostat, an ITC4 temperature controller, and a Hewlett Packard 5352B microwave frequency counter. Samples were injected into a KIMAX capillary tube (1.5-1.8 x 90 mm, KIMAX-51 glass, Kimble-Kontes, 45 Vineland, NJ, USA), and spectra were collected at room temperature. The machine parameters for experiments involving studies of glutathione, BSA and/or titrations against VS are listed in Table 2.2. Typically, solutions were prepared fresh, and the pH was adjusted with either KOH or HCI. For the interaction studies, the time of addition of vanadium salts to any solution was t = 0, and the mixtures (~ 70 ul in total volume) were immediately introduced to the capillary tube with a P200 Gilson Pipetman™ fitted with a rounded gel loading tip. 46 Figure 2.4 Configuration of the Continuous-Flow Mixing Apparatus and Electrospray Ionization Quadrupole Mass Spectrometer The two syringes were operated synchronously to generate a total flow rate of 2 pl/min and a reaction time of 54 sec. The concentration of VS could be changed during the experiment by flushing and re-filling (for details see the text.) PF: Pre-fdter rods; QO: RF only quadrupole rods (ion guide); QI: Mass analyzer quadrupole rods. Curtain Plate Ion Sampling Orifice Detector Syringe #2 Variable [VS] Syringe #1 Fixed [Protein] 47 Table 2.2 Settings of the Bruker ESP300E EPR Spectrometer Number of Scans 5 Receiver Gain 2x 104 Modulation Frequency 50 kHz Modulation Amplitude 20.305 Gauss Conversion Time 20.480 ms Time Constant 5.120 ms Centre Field 3450 Gauss Sweep Width (Field) 1600.00 Gauss Spectrum Resolution 4096 Points Microwave Frequency 9.6008 GHz Microwave Power 99-101mWatt Phase Angle 0 degree # Cycles/Loop 40950 Resolution of Field Axis 1024 48 CHAPTER THREE OPTIMIZATION OF PKA ASSAY AND EFFECTS OF VANADYL SULPHATE 3.1 RATIONALE It was necessary to optimize the activity and stability of PKA by careful selection of buffer and other assay components. At the same time it was important to define the tolerance of PKA to changes in reagent concentrations because these might later be changed to minimize possible interactions with vanadium ions. 3.2 RESULTS AND DISCUSSION 3.2.1 Steady-State Kinetics To study the characteristics of the PKA reaction the usual assumptions were made (for review see Dixon and Webb, 1979). Importantly, the concentration of substrate should be in large excess over the concentration of enzyme so that the concentration of enzyme-substrate complex, [ES], remains constant over time, i.e. d[ES]/dt« 0. This condition was met in all studies: [E] was typically < 0.01 uM whereas the concentration of the substrate, [S], exceeded 50 u.M (histone HI) or 100 uM (Kemptide). To avoid complications of substrate depletion or product accumulation, assays were carried out during the initial steady state. Because kinetic analyses in these studies could only measure product formation, a time-course study of PKA with both histone HI and Kemptide substrates was performed (Figures 3.1 and 3.2). In addition to the time-course study, histone phosphorylation was carried out with a range of PKA concentrations as shown in Figure 3.3. With increase in PKA concentration there was an increase in the rate of histone HI phosphorylation. 4 9 Figure 3.1 Time-Dependent Phosphorylation of Histone by PKA PKA assays were carried out (30 °C) in the following mixture: MES buffer (65 mM, pH 7), ATP (0.1 mM), histone HI (55 pM), DTT (25 pM), EDTA (10 pM), BSA (1 pM), MgS04 (1.5 mM), and KCl (70 mM). Results are presented as mean and range of two independent experiments, each carried out in duplicate. 70 i 60 50 40 30 20 10 12 16 20 24 28 Reaction Time, min 32 36 40 44 Figure 3.2 Time-Dependent Phosphorylation of Kemptide by PKA PKA assays were carried out (30 XI) in the following mixture: MES buffer (25 mM, pH 7), ATP (0.1 mM), Kemptide (0.5 mM), DTT(0.5 mM), EDTA (5 pM), BSA (5 pM), MgS04 (1.5 mM), and KCl (85 mM). Results are presented as mean and range of two independent experiments, each carried out in duplicate. i-. o B o (J a 3000 2500 H 2000 1500 ~r- 1000 I H 500 •I .A -5 -10 15 20 25 30 35 40 45 Reaction Time, min 50 50 Figure 3.3 Effect of PKA Concentration on Histone Phosphorylation PKA assays were carried out (30 min, 30 °C) in the following mixture: MES buffer (25 mM, pH 7), ATP (0.1 mM), histone HI (65 pM), DTT (0.5 mM), EDTA (0.25 mM), BSA (15 pM), MgS04 (1.5 mM), and KCl (70 mM). Results are represented as mean and range of two independent experiments, each carried out in duplicate. 9 i 8 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 [ P K A ] , m U 3.2.2 Effects of Buffer on P K A The choice of buffer was important because a survey of the literature (Crans et al, 1989) showed that many buffers commonly used for biological assays, such as TRIS and tricine, are able to form complexes with either vanadate or vanadyl or both (Table 3.1). For example, TRIS interacted strongly with vanadyl and tricine with vanadate. Based on established criteria shown in Table 3.1, HEPES, barbitol and perhaps 2-(N-morpholino)ethanesulphonic acid (MES) were considered the best candidates to use for PKA assays because of weak interactions with either vanadate or vanadyl. Barbitol is a restricted (and expensive) option and was therefore considered undesirable. Discussions with the PKA suppliers revealed that MES was a good option in terms of PKA stability and storage. Although MES had not previously been tested for vanadium binding, the related compound N-ethylmorpholine showed weak or no interactions with either vanadyl or vanadate. This is important because MES has a convenient pK a (6.1 at 25 °C) and is little affected by temperature changes. Consequently, MES was used as the buffer and the results of experiments using EPR (see Section 4.2.2) confirmed that this was an appropriate choice. Interestingly, strong interactions between buffer and vanadate or vanadyl could be exploited in 51 order to remove that particular form of vanadium from solution. This concept was in fact used later to remove vanadyl with glycyl peptides (see Section 4.2.8). Table 3.1 Binding of Vanadyl or Vanadate to Various Buffers* Vanadate Vanadyl Weak or No Moderate Strong Weak or No Moderate Strong Interaction Interaction Interaction Interaction Interaction Interaction HEPES TES DIPSO Barbitol Tricine TRIS N- PIPES, BES, IEA HEPES TAPS DIPSO ethylmorpholine TEA BIS-TRIS Gly TAPSO Gly-Gly EPPS Gly-Gly Tricine Gly-Gly-Gly Barbitol Pyridine TAPS DEA, Bicine MDEA AMPSO * Adapted from (Crans etal, 1989) 3.2.3 Effects of p H on P K A The pH profile of PKA was determined by diluting PKA in solutions with the following compositions: MES buffer (20 mM), KC1 (140 mM), and MgS0 4 (1 mM) and with final pH over the range 4.5-7.5. Figure 3.4 shows that PKA is a robust enzyme withstanding a wide range of pH change ~ 5.5-7.5. Below pH ~ 5.5 a sharp decrease in PKA activity is observed. When added at the highest experimental concentrations, acidic stock solutions of VS were found to perturb the PKA assay mixture at most by 0.2-0.3 pH units, too little to have a significant effect on PKA activity. Thus any change in PKA activity in the presence of the highest vanadium concentration would be unlikely to result from a change in pH. In any event, the pH of both control and vanadium-containing solutions was determined routinely to confirm that little or no pH change had occurred (see Materials and Methods). 52 Figure 3.4 Effect of pH on PKA Activity PKA assays were carried out (30 min, 30 X) in the following mixture: MES buffer (20 mM), ATP (0.1 mM), Kemptide (0.5 mM), DTT (0.4 mM), EDTA (5 pM), BSA (5 pM), MgS04 (1 mM), and KCl (140 mM). Results are presented as mean ±SEMfor three independent experiments, each carried out in triplicate. 35 -, 30 25 20 15 ^ 10 5 4 —a 5 6 7 pH of PKA Assay Buffer 8 3.2.4 Effects of Salt Concentration on PKA It was important to understand the impact of salt concentration on PKA activity because addition of multiple components to assay mixtures all contributes to specific as well as overall ionic strength. For example, ATP contributes 2 mol Na+/mol ATP, Kemptide contributes 1.5 to 2 mol AcetateVmol Kemptide, pH adjustment entails addition of KOH and/or HCI, etc. From the results shown in Figure 3.5 it is clear that PKA activity was very similar when assayed at salt concentrations of 50,100 or 150 mM. Above 150 mM, however, increasing salt concentration progressively and seriously impaired PKA activity. Very similar results were obtained when KCl was fixed at 30 mM and NaCI was used as the variable salt. PKA showed very low activity when the total cation concentration was below 30 mM. In subsequent studies the final concentration of any vanadium salt or compound was never more than 0.5 mM, that of Mg 2 + was never greater than 2.5 mM (except when effects of Mg 2 + were studied as in section 3.2.6), and that of Na + from ATP was never more than 0.2 mM (except when the effects of ATP 53 were studied as in sections 5.2.3 and 5.2.4). Based on these results, the final total salt concentration in all subsequent experiments was kept in the range of 70-150 mM. Figure 3.5 Effects of Salt Concentration on PKA Activity PKA assays were carried out (30 min, 30 X!) in the following mixture: MES buffer (10 mM, pH 7), ATP (0.1 mM), histone HI (55 pM), DTT (0.25 mM), EDTA (3 pM), BSA (3 pM), MgS04 (1 mM), and NaCI (30 mM). KCl was then added to give total monovalent cation concentrations over the indicated range. Results are presented as mean and range of two independent experiments, each carried out in triplicate. 18 i 16 14 12 10 8 6 4 2 0 O H & - • a •a--0 100 200 300 400 500 600 700 Final [K4] + [Na+], mM 800 900 1000 3.2.5 Effects of Reducing Agents on PKA PKA is stabilized by agents that ensure cysteinyl residues remain reduced and do not form disulfide bridges. It is, therefore, generally recommended that PKA should be diluted and stored with buffers containing DTT (as high as 100 mM). It is not so clear if high concentrations of reducing agents are also required for the relatively short duration of PKA assays. The reducing agents were all made up freshly on the day and adjusted to pH 7. Because of the unavoidable presence of DTT in the stored PKA preparations, all samples contained at least 20uM of DTT. Of the three reducing agents tested at the same final concentration, DTT was the most effective in preserving PKA activity (Figure 3.6A). Using either GSH or vitamin C, PKA activity was less than 50 % of that seen in the presence of DTT. Thus, although DTT does not exist in vivo, it outperformed these two endogenous antioxidants in the assay conditions. In fact, the DTT concentration could be reduced to 280 uM or 180 uM (Figure 3.6B) with 54 substantial retention of PKA activity (80 % or greater) in the presence of either GSH or vitamin C at concentrations that, alone, were less effective (Figure 3.6A). In fact, PKA activity was substantially retained even when the concentration of DTT was reduced as low as 50 uM. This is of interest because such low DTT concentrations are insufficient to protect PKA during longer-term storage. Thus, during storage over a period of days to weeks, PKA is irreversibly inactivated if the DTT concentrations drop below the millimolar range. 3.2.6 Effects of EDTA and BSA on PKA Two further reagents normally recommended in the assay of PKA are EDTA and BSA. EDTA is a chelating agent normally used in assays to bind divalent heavy metals, e.g. Pb 2 + and Hg 2 + , thereby preventing the inhibition of enzymes by these metals. BSA is included to stabilize PKA and to minimize losses due to the potential adherence of PKA to the surfaces of containers. It was not possible to eliminate EDTA as the PKA commercial batch already contained 0.2 mM EDTA. The minimal concentration of EDTA was therefore 5 uM. In the absence of BSA, EDTA was not able to stabilize PKA and activity was very low. In the presence of merely 5 uM BSA, PKA activity was well preserved and further addition of EDTA did not enhance PKA activity achieved by the presence of BSA alone (Figure 3.7). This suggests that the presence of BSA was more important than that of EDTA to reach optimal PKA activity. In the absence of BSA, increasing the concentration of EDTA appeared to slightly decrease PKA activity. It is not clear why this might be the case, because the Mg 2 + concentration (1.2 mM) was much higher than that of EDTA, so sequestering of Mg 2 + is unlikely and no other divalent metal ions are required for PKA activity (Figure 3.7). Because the effect of EDTA was not seen with BSA present, it is possible that these two reagents interact in some way. Studying the effects of BSA is potentially complicated by the fact that BSA can itself be phosphorylated by PKA. However, this interference is extremely small because the rate of Kemptide phosphorylation was found to be ~ 4200-fold faster than that of BSA (Chen and Kim, 1985). In addition, the optimal pH for BSA phosphorylation was found to be 9.0 and the rate was ~ 60-fold slower at pH 7.0 (Chen and Kim, 1985). Taken together with the fact that Kemptide concentration greatly exceeded that of BSA, these findings suggest that BSA phosphorylation should be insignificant under current assay conditions. 55 Figure 3.6 Effects of Reducing Agents on PKA Activity PKA assays were carried out (30 min, 30 X) in the following mixture: MES buffer (20 mM, pH 7), ATP (0.1 mM), histone HI (55 pM), EDTA (20 pM), BSA (15 pM), MgS04 (2 mM), andKCl (70 mM) with indicatedfixed (panel A) or varying (panel B) concentrations of GSH, DTT, and vitamin C. Results are presented as mean and range of two independent experiments, each carried out in triplicate. In these experiments, the maximum activity of PKA was 3.6pmol P/min. > o < B 3 B _> o 1 1 0 1 0 0 9 0 8 0 7 0 -6 0 5 0 -4 0 -3 0 -2 0 1 0 -0 -110 100 90 80 70 60 50 40 30 20 10 0 DTT GSH VIT.C X Final [Reducing Agent] = 680 uM X X X DTT GSH Vit.C 680 280 400 280 180 500 400 Final [Reducing Agent], uM 180 500 56 Figure 3.7 Effects of BSA and EDTA on PKA Activity PKA assays were carried out (30 min, 30 X) in the following mixture: MES buffer (20 mM, pH 7), ATP (0.1 mM), Kemptide (0.5 mM), DTT(0.5 mM), MgS04 (1.2 mM), andKCl (70 mM). Results are presented as mean and range of two independent experiments, each carried out in triplicate. In these experiments, the maximum activity of PKA was 3.9 pmol P/min. o < 1 1 0 1 0 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 EDTA, uM BSA, 5 uM 5 + 15 15 50 50 + 150 150 + 3.2.7 Effects of Magnesium on PKA Protein kinases and other nucleotide-dependent enzymes are inactive unless the nucleotide is present as a complex with a divalent metal ion (usually Mg2 +). The principle role of Mg 2 + as a cofactor is to aid the transfer of y-phosphate from ATP to the peptide or protein substrate. To achieve this transfer, ATP must be positioned in the correct orientation for nucleophilic attack to occur at the p-phosphate. Mg 2 + contributes to the reaction within the Mg-ATP binding site of PKA by forming a ring with oxygen atoms of both p- and y-phosphates, thereby forming two stereoisomers as shown in Figure 3.8 (Hengge, 1998). PKA activity increased over the range 0-1 mM Mg 2 + and optimum activity was observed 94-between 0.5-2.0 mM (Figure 3.9). With Mg concentrations above 2 mM, PKA activity declined and concentrations greater than 10 mM caused more than 50 % loss of PKA activity. It is not certain why higher Mg 2 + concentrations might inhibit PKA. One possibility is that Mg 2 + might bind to the substrate, in this case Histone, which is known to bind metal ions (Michele et al, 1997) and this could perhaps interfere with PKA action. Another possible explanation for PKA inhibition is that, at high concentrations, Mg 2 + may form a ring with a- and y-phosphates in 57 addition to the ring formed with the p4- and y-phosphates as depicted in Figure 3.8 (Armstrong et al, 1979; Granot et al, 1980). With this alternative binding, the glycine-rich loop, which serves to stabilize the (3-phosphate by hydrogen bonding (Taylor et al, 1993a), might not function properly and therefore the transfer of the y-phosphate could be impeded. Figure 3.8 ATP-Magnesium Complexes* The structure shows the coordination geometry of the A and A isomers during y-phosphate transfer to the peptide or protein substrate. Greek letters refer to position of the phosphate groups on ATP. O O Y Y P O P P o p AMP O A Stereoisomer A Stereoisomer * Adaptedfrom (Hengge, 1998) 58 Figure 3.9 Effects of Magnesium on PKA Activity PKA assays were carried out (36 min, 30 °C) in the following mixture: MES buffer (10 mM, pH 7), ATP (0.1 mM), histone HI (55 pM), DTT (0.25 mM), EDTA (5 pM), BSA (5 pM), and KCl (70 mM). Results are presented as mean ± SEM for three independent experiments, each carried out in triplicate. 2i 0 w i 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Final [Mg2+], mM 3.2.8 Linearity of PKA Reaction Versus Time A time course study was carried out to verify whether PKA is stable for the duration of typical assays in which individual components (EDTA, BSA, and DTT) were all minimized. The results show that the rate of reaction was linear for at least 30 min in the presence of minimized concentrations of EDTA, BSA and DTT (Figure 3.10). The initial rate of reaction was ~ 2-fold faster at the higher BSA concentration (75 uM). Between 30-60 min of assay, the reaction rate at the lower BSA concentration (0.04 uM) remained stable but the rate at 75 uM BSA actually declined somewhat. It is not clear why the decline was seen with 75 uM BSA but conceivably at this high rate the substrates might have become depleted or the accumulation of products might have exerted a feedback effect. 59 Figure 3.10 Linearity of PKA Reaction Versus Time PKA assays were carried out (30 X) in the following mixture: MES buffer (65 mM, pH 7), ATP (0.1 mM), histone HI (55 pM), DTT (25 pM), EDTA (1 pM), BSA (75 pM (filled squares) or 0.04 juM (open diamonds)), MgS04 (2 mM), andKCl (70 mM). Assays were quenched at eight-min intervals up to 64 min. Panel A: Time course of total phosphate incorporation into histone. Panel B: Rate of phosphate incorporation into histone calculatedfor each eight-min interval. The results are presented as mean and range of two independent experiments, each carried out in duplicate. A 0 i 1 i 1 : 1 1 1 i 8 16 24 32 40 48 56 64 Reaction Time, min o -I , 1 , , , 1 1 8 16 24 32 40 48 56 64 Reaction Time, min 60 3.2.9 Effects of Peptide or Protein Substrates on PKA Histone is an inexpensive and convenient substrate for PKA, but looking ahead to studies with vanadium salts, this substrate might be problematic because of its ability to bind metal ions (Michele et al, 1997). The phosphorylation of other substrates, Kemptide (LRRASLG) and casein was therefore investigated. Kemptide gave the greatest P incorporation, followed by histone HI and casein (Figure 3.8), although the concentration of Kemptide (1.2 mM) was higher than that of the protein substrates histone and casein (approximately 50 uM). This result confirms that Kemptide is an excellent substrate and has the important advantage of homogeneity (as demonstrated in results shown later in Figure 5.16). Even highly enriched preparations of histone HI contain four subtypes: 21263, 21374, 22761, and 22606 amu (Berger et al, 1995). Of some concern, the rate of phosphorylation differs for the different histone fractions (Walsh and Krebs, 1973). Casein is the least purified of the substrates tested and contains many isoforms, the a isoform being the most abundant. As well as being the least pure, casein was also the least effective substrate (Figure 3.11), in agreement with an earlier study in which histones and casein was compared (Walsh and Krebs, 1973). Because Kemptide is a pure and effective substrate and would be less likely to possess non-selective metal-binding properties, it was chosen for further work. To confirm the K m for Kemptide, PKA was assayed with the indicated range of Kemptide concentrations. Results (Figures 3.12 A, B) are shown as the primary plot of rate versus Kemptide concentration and as the derived Lineweaver-Burke plot (Equation 3.1). 1/ V = Km/Wmax • l/[Kemptide] + 1/Vm a x (3.1) From the Lineweaver-Burke plot (Figure 3.12 B and Equation 3.1), the Km for Kemptide was calculated to be 12.4 uM, close to the values reported by others of 4.7 uM (Kemp et al, 1976) and 7.7 uM (Wecker and Rogers, 2003). Although the term Km is less specific than the individual rate constants, it is still commonly employed and useful for descriptive purposes. Because rapid quench flow measurements were not feasible with this assay, the individual rate constants were not determined. 61 Figure 3.11 Phosphorylation of Various Substrates by PKA PKA assays were carried out (30 min, 30 X) in the following mixture: MES buffer (20 mM, pH 7), DTT (0.4 mM), EDTA (5 pM), BSA (5 pM), KCl (100 mM), MgS04 (1 mM), ATP (0.1 mM), and in the presence of the indicated substrates (1 mg/mL). Results are presented as mean and range of two independent experiments, each carried out in triplicate. 30 -, 25 20 15 10 Histone Kemptide Casein 62 Figure 3.12 Effects ofKemptide Concentration on Phosphorylation by PKA PKA assays were carried out (36 min, 30 °C) in the following mixture: MES buffer (20 mM, pH 7), DTT (0.4 mM), EDTA (3 pM), BSA (3 pM), KCl (100 mM), ATP (0.1 mM), andMgS04 (1 mM). Panel A: Primary plot of rate versus Kemptide concentration. Panel B: Lineweaver-Burke plot. Results are the average of two independent experiments, each carried out in triplicate. kcat was derived using Vmax /Et, when Et is the total PKA concentration (4.8 nM). R2 represents the Pearson correlation coefficient. A 3 5 -i .3 3 0 -PH "o 2 5 -a 5 2 0 -ivi < 1 5 -PH 1 0 -5 -O -O 1 0 2 0 3 0 -40 5 0 6 0 [Kemptide], uM 3.2.10 Effects of VS on PKA Using Conditions Optimized for PKA Based on the studies carried out to this point, effects of VS were assessed under conditions optimized for PKA activity. Under these conditions, VS gave a dose-dependent inhibition of PKA with an IC50 value > 400 uM (Figure 3.13). Assuming that vanadyl is stable as added, this concentration dependency suggests that vanadyl would likely be a very weak PKA inhibitor in intact cells or in vivo, where concentrations of vanadyl would be unlikely to exceed 20 uM. Figure 3.13 Effects of VS on PKA Activity PKA assays were carried out (30 min, 30 °C) in the following mixture: MES buffer (35 mM, pH 7), DTT (0.5 mM), EDTA (10 pM), BSA (10 pM), KCl (85 mM), ATP (0.24 mM), Kemptide (0.4 mM), and MgS04 (2.5 mM). A fresh solution ofVS was prepared and added at the indicated concentrations just before initiation of reaction with ATP. Results are presented as mean +SEM for three independent experiments, each carried out in triplicate. 100 -, 30.H 20 4 10 H 0 0 100 200 300 400 500 600 Final [VS], uM 64 3.3 SUMMARY PKA was optimally active over the pH range of 5.5-7.5 in the presence of: (a) low micromolar concentrations of both BSA and EDTA (3-10 pM), (b) a minimum of 0.1-0.2 mM DTT (preferred over either GSH or Vitamin C), (c) a minimum of 70 mM but not exceeding 200 mM total salt concentration, (d) a minimum of 0.5 mM but not exceeding 5 mM of Mg 2 + ions, and ATP significantly above the Km of ~ 20 uM (Edelman et al, 1987). When the optimal buffer was used, VS showed an IC50 > 400 uM, i.e. much larger than concentrations measured in vivo (< 20 uM). 65 C H A P T E R F O U R F A C T O R S T H A T L I M I T T H E A P P A R E N T I N H I B I T I O N O F P K A B Y V A N A D Y L S U L P H A T E 4.1 RATIONALE Effective assay of PKA is dependent on a number of assay components, some of which may interact (or are already known to interact) with vanadyl or vanadate. The next phase of the work involved studies designed to minimize the concentrations of assay components that might bind vanadyl or vanadate. The working hypothesis was that reagents that bind and therefore reduce its effective concentration had obscured the true potency of vanadium as a PKA inhibitor. By minimizing the concentration of reagents that interfere, it might be possible to reveal if vanadium species inhibit PKA at concentrations that may be achieved in vivo. 4.2 RESULTS AND DISCUSSION 4.2.1 Effects of VS on PKA Using Minimal Concentrations of BSA, DTT, and EDTA It has been previously shown that BSA, EDTA, and DTT all interact with vanadyl. EPR measurements of vanadyl-BSA complexes at pH 5 showed the existence of one "strong" binding site and five equivalent weak binding sites (see Section 1.3.4), accounting for the binding of up to six vanadyl ions per molecule of albumin (Chasteen and Francavilla, 1976). In another study, it was found that HSA bound up to 20 vanadyl ions versus 4-5 vanadate ions per molecule and also interacted more strongly with vanadyl than vanadate (Heinemann et al., 2002). DTT has also been shown to interact with both vanadate and peroxovanadate, especially when the ratio of DTT to vanadium was 2:1 or higher (Tracey and Paul, 1997). The overall charge of vanadate:DTT complexes was -2 at neutral pH. Finally, EDTA binds vanadyl very strongly but does not bind vanadate, as illustrated in Table 4.1 (Smith and Martell, 1976b). Taking into account the binding properties of these reagents, PKA was assayed in the presence of minimal concentrations of BSA, EDTA, and DTT, final concentrations being 0.02 uM, 0.1 uM, and 0.7 uM, respectively (Figure 4.1). In these assays the concentrations of MES and ATP were also lower than in the previous experiment. Under these conditions, the IC50 for inhibition of PKA by VS was dramatically reduced from > 400 uM (Figure 3.10) to < 10 uM (Figure 4.1). Although these modified conditions were 66 not entirely optimal for P K A , the maximal activity (at zero vanadyl) was still in the range ~ 70 % of the activity that was observed using conditions to fully optimize P K A activity (Figure 3.7B). Table 4.1 Interactions of Vanadyl With Selected Anions* (Vanadyl + Ligand -K -> Vanadyl-Ligand Complex) Ligand Log K (25 °C, 1 M) Acetate 1.86 Chloride 0.04 EDTA 15.5 Hydroxide 7.9 Adapted from (Smith and Martell, 1976a, 1976b, 1976c). Figure 4.1 Effects of VS on PKA Kemptide Kinase Activity in a Buffer Containing Minimal Concentrations of BSA, EDTA, and DTT PKA assays were carried out (33 min, 30 *C) in the following mixture: MES buffer (10 mM, pH 7), DTT(0.7 pM), EDTA (0.1 pM), BSA (0.02 pM), MgS04 (1 mM), Kemptide (1 mM), andKCl (140 mM). Vanadyl sulphate was addedfrom a fresh solution just before initiating reactions with ATP (0.1 mM). Results are presented as mean ± SEM for three independent experiments, each carried out in triplicate. In these experiments, the maximum activity of PKA was 26.7 pmol P/min. 110 j 100 « 1 90 -"a 80 -70 --60 -50 -< 40 -\% 30 -20 --10 --0 -IC 5 0 < 10 u M H 1 h -8 16 32 Final [VS], u M 64 125 250 500 67 4.2.2 Effects of BSA, DTT, EDTA, and MES on the Inhibition of PKA by VS Results shown in Figure 4.1 demonstrate potent inhibition of Kemptide kinase activity of PKA by VS, when the concentrations of a number of assay components were minimized, notably the concentrations of BSA, EDTA and DTT. To determine if one or more of the minimized assay components was specifically able to block or diminish PKA inhibition, the concentration of each was varied in turn (Figure 4.2). The "minimal" final concentrations of DTT, EDTA, and BSA were 0.5 uM, 4.7 u.M, 4.5 uM, respectively. Under these conditions, VS (0.5 mM) inhibited PKA Kemptide kinase activity by more than 90 % ("H20" bars, Figure 4.2). The PKA inhibition was not diminished by increasing the concentration of MES from 10 mM to 25 mM (bars labeled "MES") or of DTT from 0.5 uM to 0.5 mM (Figure 4.2). When present in 12-fold excess over vanadyl, DTT did reduce PKA inhibition somewhat (Figure 4.3) similar to results reported previously (Tracey and Paul, 1997). In contrast to the lack of effect of MES or DTT, when EDTA was increased from 4.7 uM to 0.5 mM or when BSA was increased from 4.5 uM to 0.5 mM, PKA inhibition by VS was substantially reduced. The actions of EDTA are most likely explained by binding of V 0 2 + . This observation is potentially very important because it suggests that vanadyl rather than vanadate (or other anionic forms of vanadium) is the most probable inhibitory species. This possibility is further reinforced by subsequent experiments using glycyl peptides (Section 4.2.8). The actions of BSA are likely more complex than those of EDTA, but it is possible that at least some of the ability to protect PKA from inhibition is explained by binding of vanadyl ions by BSA. EPR studies also provide support for the possible interactions between vanadyl and assay components. X-band EPR spectra of VS in the presence of MES or ATP were compared to the spectrum of vanadyl alone (Figure 4.4). These spectra were markedly changed (distorted) in the presence of ATP but not in the presence of MES, indicating that there were little or no interactions between MES and vanadyl detected by EPR (Figure 4.4). An important caveat to the interpretation is that this EPR study was carried out with solutions at pH 4. This pH was chosen because the vanadyl EPR signal was lost above pH 6 for the reasons outlined in the Introduction. Thus, at pH 4 and at room temperature, one can qualitatively compare the EPR spectrum of vanadyl with that of vanadyl-ATP complex as both have an eight-line spectrum. The effect of ATP on the VS spectrum confirms the interaction observed in previous studies, which were done at neutral pH and at low temperature in the range 20-77 K (Elvingson et al, 1997; Woltermann et al, 1974). 68 In contrast to ATP, Kemptide had no detectable effects on the vanadyl EPR spectrum (data not shown), suggesting a lack of interaction that was confirmed by mass spectral analyses (see Section 5.2.2). Figure 4.2 Effects of BSA, DTT, EDTA, and MES on PKA Inhibition by VS PKA assays were carried out (33 min, 30 °C) in the following mixture: MES buffer (10 mM, pH 7), ATP (0.1 mM), Kemptide (0.25 mM), DTT (0.5 pM), EDTA (4.7 pM), BSA (4.5 pM), MgS04 (2.5 mM), GSH (2.5 mM), andKCl (70 mM). Control bars are labeled "H20". Where indicated below the bars, reagent concentrations were increased for MES (to 25 mM), EDTA (to 0.5 mM), DTT (to 0.5 mM), and BSA (to 0.5 mM). Assays were carried out in the absence (open bars) and presence (filled bars) of vanadyl sulphate (0.5 mM) as indicated. Results are presented as mean and range of two independent experiments, each carried out in triplicate. 3 5 80 70 60 50 40 30 20 10 0 T— — i — — i r MES MES EDTA EDTA DTT X DTT BSA BSA H 2 0 H 2 0 VS + + + + 69 Figure 4.3 Effect of Increasing the Ratio of DTT to VS on PKA Inhibition PKA assays (33 min, 30 X)were carried out in the following mixture: MES buffer (10 mM, pH 7.0), ATP (0.1 mM), Kemptide (0.25 mM), DTT (0.5 pM), EDTA (4.7 pM), BSA (4.5 pM), MgSC*4 (2.5 mM), GSH (2.5 mM), and KCl (70 mM). Where indicated DTT concentrations were increased to 0.5 mMor 6 mM, and MES concentrations were increased to 25 mM. Assays were carried out in the absence (filled bars) and presence (open bars) of vanadyl sulphate (0.5 mM). Results are presented as mean and range of two independent experiments, each carried out in triplicate. £ J £ I 90 80 70 60 50 40 30 20 10 0 6.0 mM DTT 6.0 mM DTT 0.5 mM DTT 0.5 mM DTT 25 mM MES 25 mM MES VS + + 70 Figure 4.4 Effects of ATP or MES on X-Band EPR Spectrum ofVS Solutions (pH 4) were preparedfreshly with CIH2O to the final indicated concentrations. Spectra were recorded at room temperature. 1 I I 1 1 1 1 L 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 71 4.2.3 Effects of Protein Substrate on PKA Inhibition by VS To test the potential importance of vanadyl binding to the peptide substrate, PKA was assayed exactly as described in Figure 4.1 except that Kemptide was replaced with histone HI (Figure 4.5). The result was strikingly different using histone HI as the PKA substrate in that the IC50 exceeded 250 uM, despite the fact that BSA, EDTA, and DTT concentrations were still minimal. A probable explanation is that histone HI binds vanadyl under assay conditions, therefore reducing the effective concentration and increasing the apparent IC50 for the inhibition of PKA by VS. Interestingly vanadate and decavanadate induced precipitation of histone HI under similar conditions, providing further evidence for a strong interaction (Michele et al, 1997). Although evidence from mass spectroscopy with histone HI was inconclusive, it is likely that vanadyl binds substantially to the histone HI used in these assays (Berger et al, 1995). Figure 4.5 Effects of VS on PKA Histone Kinase Activity in a Buffer Containing Minimal Concentrations of BSA, EDTA, and DTT PKA assays were carried out (33 min, 30 °C) in the following mixture: MES buffer (10 mM, pH 7), DTT (0.7 pM), EDTA (0.1 pM), BSA (0.02 pM), MgS04 (1 mM), histone HI (55 pM), and KCl (140 mM). VS was addedfrom a fresh solution just before initiating reactions with ATP (0.1 mM). Results are presented as mean ± SEM for three independent experiments, each carried out in triplicate. In these experiments, the maximum activity of PKA was 9.1 pmol P/min. I NO > < 110 100 90 80 70 60 50 40 30 20 10 0 IC 5 0 > 250 uM -<2 100 200 300 400 500 600 700 Final [VS], uM 800 900 1000 72 4.2.4 Effects of Magnesium on PKA Inhibition by VS OA-Mg is a cofactor for the phosphorylation reaction catalyzed by PKA (Equation 2.1). Although the chemical properties of vanadyl differ from those of Mg 2 + , vanadyl is able to substitute for Mg 2 + in reactions catalyzed by certain nucleotide-dependent enzymes (Banerjee et al, 1998). It is possible that vanadyl might replace Mg 2 + as a cofactor for PKA and it was therefore of interest to see if increases in Mg 2 + concentrations could reverse the inhibitory effect of vanadyl (Figure 4.6). In the absence of VS, 0.2 mM Mg 2 + was sufficient for optimal PKA activity. This concentration is somewhat lower than that required in the earlier experiments with histone and with higher concentrations of EDTA, BSA, and DTT (0.5 mM Mg 2 + , Figure 3.6). It is likely that the lower EDTA concentration used in this latest experiment was the most important factor in altering the apparent Mg 2 + dependency. In any event, optimal PKA activity was seen over the range of 0.2-2 mM Mg 2 + , still quite similar to the previous experiments. This concentration range is in keeping with normal physiological cellular levels, reportedly 0.4-0.8 mM in total Mg 2 + and 90-140 uM free cytosolic Mg 2 + (Fatholahi et al, 2000). Vanadyl alone (at zero Mg 2 +) could not support PKA activity at the concentration used here (0.1 mM). Further, PKA inhibition was not overcome by increasing the Mg 2 + concentrations. This result contrasts with that found in the previous report (Pluskey et al, 1997), perhaps because of differences in composition of the assay buffers used. The previous reported studies used PKA in a mixture containing 100 mM MOPS buffer, 150 mM KC1, 0.38 uM DTT, and 0.125 mg/mL BSA (~ 1.5 uM) (Pluskey et al, 1997). The most notable differences between the previous studies and those reported here (Figure 4.6) are in the concentrations of EDTA, salt, and BSA but it is not clear if this is enough to explain the differences. 73 Figure 4.6 Effects of Magnesium Concentration on PKA Inhibition by VS PKA assays were carried (36 min, 30 *C) out in the following mixture: MES buffer (10 mM, pH 7), Kemptide (0.1 mM), DTT (0.7 pM), EDTA (0.1 pM), BSA (0.02 pM), and KCl (70 mM), ATP (0.1 mM). Assays were carried out in the absence (open diamonds) or presence (filled squares) of vanadyl sulphate (0.1 mM). Results are presented as mean and range of two independent experiments, each carried out in triplicate. 25 n 0 5 10 15 20 Final [Mg2+], mM 4.2.5 Effects of VS on Different Preparations of PKA PKA supplied by different companies is produced by different techniques and supplied in different buffers. Thus, it was interesting to test whether the results were independent of the form of PKA used. The Calbiochem PKA preparations contained MES buffer (20 mM, pH 6.5), NaCI (100 mM), P-EtSH (30 mM), EDTA (0.1 mM), and ethylene glycol (50 %). The Boehringer Mannheim PKA preparations contained K 3 P0 4 buffer (10 mM, pH 6.5), KCl (70 mM), DTT (0.5 mM), EDTA (0.1 mM), and were filled under N 2 (g). The Calbiochem PKA contained additional residual components following dilution: NaCI (3.4 mM), P-EtSH (1 mM), and Ethylene Glycol (1.7 %). Assays were carried out in the absence or presence of VS, added at the indicated final range of concentrations (Figure 4.7). The IC50 value seemed to be slightly higher with Calbiochem PKA than with Boehringer Mannheim PKA, although both values were below 25 uM. 74 Figure 4.7 Effects of VS on Different Preparations of PKA PKA from Bdehringer Mannheim (Panel A) and Calbiochem (Panel B) were assayed (30 min, 30 °C) in the following mixture: MES buffer (10 mM, pH 7), Kemptide (0.5 mM), DTT (0.7 pM), EDTA (0.1 pM), BSA (0.02 pM), KCl (70 mM), MgS04 (1.5 mM), and ATP (0.1 mM). PKA from Calbiochem also contained additional low concentrations of NaCI, /3-EtSH, and ethylene glycol. Results are presented as mean ± SEM for three independent experiments, each carried out in triplicate. IC50 < 10 uM Bdehringer Mannheim PKA 10 20 30 40 50 60 70 80 90 100 B Final [VS], uM * 3 10 IC50 < 25 uM Calbiochem PKA 0 50 100 150 200 250 300 350 400 450 500 Final [VS], uM 75 4.2.6 Comparison of the Effects of Vanadium Salts and Compounds on PKA A range of vanadium salts and compounds have been employed in studies aimed at elucidating insulin-like actions in vitro and in vivo. VS, OV and MV have been tested in studies in vitro as well as in vivo and in clinical trials (Brichard and Henquin, 1995). The compounds BMOV and BPOV have also been used successfully to treat STZ-diabetic animals (Caravan et ai, 1995; Melchior et al., 1999). In view of the demonstrated actions of these preparations of vanadium, their effects on PKA activity were compared in vitro using Kemptide or histone HI as substrate (Figure 4.8). Several observations emerge: 1. As demonstrated earlier, VS was an effective inhibitor of PKA activity using either Kemptide or histone HI as substrate. In contrast, sodium orthovanadate did not significantly inhibit PKA activity against either substrate; 2. The compounds BPOV and BMOV both inhibited PKA when Kemptide was the substrate (though inhibition was less robust than with vanadyl sulphate) but neither inhibited histone HI phosphorylation by PKA. The most likely explanation for the lack of inihibition of histone kinase is the sequestration of vanadium by binding to histone. The overall rank order of inhibition of kemptide kinase activity of PKA was therefore VS > BPOV > BMOV > OV; 3. The difference between BPOV and BMOV suggests that the nature of the ligand environment of vanadyl is an important determinant of PKA inhibition; 4. The relatively modest effects of BMOV and BPOV compared to VS could be due to the ability of maltolato and picolinato ligands to reduce the effective concentration of vanadyl. The ability of BMOV and BPOV to "protect" vanadyl is further illustrated by results shown in Figure 4.9. In these EPR spectra, vanadyl X-band EPR signals are observed using neutral solutions of BMOV or BPOV, but not with a neutral solution of VS. 76 Figure 4.8 Comparisons of the Effects of Vanadium Salts and Compounds on PKA PKA assays were carried out (30 min, 30 X) using either histone HI (filled bars, 55 pM) or Kemptide (open bars, 1 mM) as substrate in the following mixtures: MES buffer (10 mM, pH 7), DTT(0.7pM), EDTA (0.1 pM), BSA (0.02pM), (140mMKCl), ATP (0.1 mM), andMgS04 (1.5 mM). Assays were carried out in the absence or presence of 0.5 mMOV, VS, BMOV or BPOV. Results are presented as mean ± SEM for three independent experiments, each carried out in triplicate. Values were compared by ANOVA; significant differences were as follows: (a) VS, BPOV, BMOV versus control (p <0.025); (b) VS versus BPOV (p <0.01); (c) BPOV versus BMOV (p<0.001). 30 -i 25 15 a _T_ 10 a,b a,c a,b VS BPOV BMOV OV Control Final Concentration of 0.5 mM 77 Figure 4.9 X-Band EPR Spectra ofBPOV, BMOV, and VS X-band EPR spectra were recorded at room temperature using solutions containing the indicated concentrations ofBPOV, BMOV or VS. 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 78 4.2.7 What Is the True Concentration of Vanadyl Ions in PKA Assays? The apparent K\ for inhibition of PKA by VS was dramatically lower when the concentrations of assay components were adjusted to minimize binding of vanadyl (Figure 3.10 vs. Figure 4.1). Quantitatively, however, an important discrepancy may exist. Thus, EDTA, BSA, and DTT (the key potential ligands) were reduced from 10 uM, 10 uM, 0.5 mM, respectively, to values in the sub-micromolar range. In contrast, the IC50 for VS decreased from >400uMto<10uM. EPR analyses were therefore carried out to examine the stability of vanadyl under conditions used to assay PKA (Figure 4.10). The EPR spectrum revealed essentially no evidence for a characteristic vanadyl signal in assay mixtures to which vanadyl sulphate had been added (0.5 mM). This contrasted sharply with the control vanadyl signals seen at pH 2-3 in aqueous solution (Figure 4.10). Importantly, the EPR analysis had a low threshold (~ 15 uM) below which vanadyl could not be detected even at pH 3 in aqueous solution (Figure 4.11). The combination of V2O4 and HCI was used to reassure that none of the vanadyl is converted to OV. The EPR studies combined with assays of PKA activity raise several important possibilities: 1. The inhibition of PKA may not be explained by vanadyl, but rather by some other species of vanadium. The most likely fates of vanadyl include oligomerization, precipitation or oxidation to vanadate. It is difficult to visualize how oligomerized or precipitated vanadyl might inhibit PKA. Furthermore, orthovanadate itself clearly does not inhibit PKA. The fact that EDTA reverses PKA inhibition provides further support for the fact that the effective PKA inhibitor is vanadyl; 2. Vanadyl might be present in PKA assay mixtures at a low concentration that is sufficient to inhibit PKA, but below the threshold for detection by EPR; 3. The vanadyl signal might be sufficiently broadened by binding to other assay components that bound vanadyl remains poorly detected. 79 Figure 4.10 Absence of Vanadyl Signal by EPR in PKA Assay Mixtures PKA assay mixtures (pH 6.5) identical to those, which were used for the kinetic experiment described in Figure 4.8, were prepared freshly with the indicated substrate but with non-radioactive ATP. X-band EPR spectra were recorded at room temperature. 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 80 Figure 4.11 Threshold Sensitivity for Detection of Vanadyl by EPR X-band EPR spectra were recorded at room temperature and at pH 2-3, using fresh aqueous solutions ofV204 at indicated final concentrations. 1 mM V 2 0 4 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 81 4.2.8 Further Evidence that PKA Is Inhibited by Vanadyl Ions To further address the relative significance of vanadyl and vanadate in PKA inhibition, experiments were carried out to test the effects of glycyl peptides that are able to selectively bind vanadyl. Glycylglycine (GG) and glycylglycylglycine (GGG) bind more strongly to vanadyl than to vanadate (Table 4.2). Table 4.2 Glycyl Peptides Specifically Bind Vanadyl Not Vanadate* Vanadium Species (0.01 uMtolOOuM) Amino Acid or Peptide Formation Constant (x 103 M-1) Vanadate G 0.07 Vanadate GG 0.1 Vanadate GGG 0.2 Vanadyl G 5 Vanadyl GG 20 Vanadyl GGG 40 * Adapted from (Crans et al, 1989) To test the effects of the glycyl peptides PKA assays were carried out in the presence of glycine (G), GG, and GGG (Figure 4.12). Both GG and GGG reversed the inhibition of PKA by VS, whereas G did not. The final concentration of glycyl peptides were 2.86 mM whereas the final concentration of VS was 15 uM, a final ratio of ~ 190:1 (glycyl peptide:VS). Therefore, because the glycyl peptides are able to bind vanadyl more strongly than vanadate, it is likely that vanadyl and not vanadate is the effective inhibitory species. Interestingly, the glycine buffer gave higher PKA activity than MES buffer in the absence of VS. 82 Figure 4.12 Effects of Glycyl Peptides on PKA Inhibition by VS PKA assays were carried out (30 min, 30 X) in the following mixture: MES buffer (10 mM, pH 7), Kemptide (0.5 mM), ATP (0.1 mM), MgS04 (1.5 mM), DTT(0.7pM), EDTA (0.1 pM), BSA (0.02 pM), and KCl (140 mM) in the presence (filled boxes) or absence (open boxes) ofVS (15 pM). Assays were carried out in the presence of either glycine (G) or the corresponding di (GG) and tri (GGG) peptides (all at 2.8 mM). Results are presented as mean and range of two independent experiments, each carried out in duplicate. X MES GG GGG 4.3 SUMMARY When assay conditions were established to optimize PKA activity (Chapter 3), VS inhibited PKA with an IC50 of ~ 400 uM. The apparent potency of VS was dramatically increased (IC50 ~ 20 uM or less) when assay conditions were adjusted to minimize sequestration of vanadyl ions (this chapter). EPR spectroscopy revealed that, regardless of the assay conditions, the characteristic X-band spectrum of vanadyl could not be detected in PKA assay mixtures above the threshold level for detection of ~ 15 uM. This presents a dilemma because the presumed inhibitory species could not be directly detected. Evidence that vanadyl ions inhibit PKA therefore remains indirect. Significantly, the likely alternative form of vanadium (vanadate) is itself, unable to inhibit PKA. In addition, reagents known to sequester V 0 2 + (EDTA, BSA, histone and glycyl peptides) all moderate or abolish the inhibition of PKA by VS. Because of the discrepancy between added and EPR-detectable vanadyl, it is important to stress that the derived kinetic constants (IC50, K\, etc.) must be considered tentative estimates. 83 CHAPTER FIVE POSSIBLE MECHANISMS OF ACTION OF VANADYL ON PKA 5.1 RATIONALE In the previous chapter, it was demonstrated that by adjusting assay conditions PKA inhibition could be achieved at very low VS concentrations. From a comparison with other vanadium salts and considering effects of divalent metal chelators, a tentative conclusion was also reached that the active inhibitory form of vanadium is likely to be vanadyl and not vanadate. Further studies were therefore carried out to explore several possible mechanisms by which vanadyl might inhibit PKA: 1. V 0 2 + resembles Mg 2 + in that it can substitute for Mg 2 + in some enzymes by mimicking the interactions of Mg 2 + with nucleotides. Thus, it is possible that V02+might compete with Mg 2 + , preclude the formation of the normal A stereoisomer of ATP-Mg 2 + (Figure 3.5) and consequently inhibit the transfer of phosphate catalyzed by PKA; 2. V 0 2 + can interact with a variety of peptides and proteins and might do so with PKA substrates thereby perhaps changing their conformation or influencing their properties such that they become non-receptive to phosphate transfer. V 0 2 + might even bind at or near to the Ser/Thr moiety on the peptide or protein substrate and directly block phosphoryl transfer; 9+ 3. VO might bind directly to the PKA active site or to other sites on PKA, in the latter case inducing allosteric inhibition. Multiple vanadyl binding sites have been defined in BSA (up to 6 VO per BSA), so multiple vanadyl binding sites might exist on PKA. To address these possible mechanisms, several specific aims were identified: 1. To verify whether V 0 2 + competes with and/or substitutes for Mg 2 + ; 2. To test potential binding of V 0 2 + to Kemptide and histone and to verify whether VS effects could be overcome by high peptide concentrations; 3. To test V 0 2 + binding to ATP and to verify whether VS effects on PKA were sensitive to ATP concentration; 4. To test the possibility that V 0 2 + binds directly to PKA; 5. To test potential binding of V0 2 + to glycyl and/or "glycine-loop" peptides modelled on the glycine-rich Rossmann GXGXXG motif within the active site of protein kinases. 84 5.2 RESULTS AND DISCUSSION 5.2.1 Does Vanadyl Substitute for Magnesium as a Cofactor for PKA? It had been reported that V 0 2 + (10-100 uM) could act as a weak cofactor for PKA in the absence of Mg 2 + , the V m a x being 40-fold higher with Mg 2 + than with V 0 2 + (Pluskey et al, 1997). This action of V 0 2 + was consistent with other studies, in which V 0 2 + was considered to act at the Mg2+-binding sites of pyruvate kinase (Lord and Reed, 1990) and S-adenosylmethionine synthetase (Markham, 1984). Because of these earlier studies, V 0 2 + was considered to potentially inhibit PKA by replacing Mg 2 + at the active site. Figure 5.1 Comparisons ofVS, OV, and Magnesium as Potential Cofactors for PKA PKA assays were carried out (30 min, 30 °C) in the following mixture: MES buffer (12 mM, pH 7), DTT (0.8 pM), BSA (5 pM), EDTA (0.2 pM), KCl (70 mM), GSH (2.5 mM), Kemptide (0.3 mM), and ATP (0.1 mM). Results are presented as mean and range of two independent experiments, each carried out in triplicate. 901 0 20 40 60 80 Final Concentration, uM From the results shown (Figure 5.1) it is clear that Mg greatly outperformed both VS and OV in facilitating the PKA reaction, the rate being at least 20-fold higher with Mg 2 + than VS. In fact, no PKA reaction could be detected above background in the presence of OV. From the results of this experiment, together with results from Section 4.2.4, the following conclusions can be reached: 1. V 0 2 + alone is at best a very weak cofactor for PKA. The weak action of VS is probably due to vanadyl and not vanadate; 2. High concentrations of Mg 2 + can not overcome 85 PKA inhibition by VO even though Mg is a superior cofactor; 3. Because high concentrations of Mg 2 + inhibit PKA, probably by binding to different phosphate groups on ATP, then vanadyl might also have two actions: acting as a very weak cofactor but also as a potent inhibitor, perhaps mimicking the inhibitory effect of high Mg 2 + concentrations. The potential dual actions of V 0 2 + might be rationalized by comparison to the actions of Mg 2 + . Thus, Mg 2 + appears to have an inhibitory effect on PKA at concentrations above about 2-5 mM (as in Figure 4.6, for example). This is probably explained by binding of a second Mg 2 + in the PKA active site, bridging a- and y-phosphates of ATP with the Asn 171 side chain (Zheng et al, 1993a, 1993b). Considering this additional, inhibitory action of Mg 2 + , it seems reasonable to suggest that VO might also act in the a- / y- bridging mode (Armstrong et al, 1979; Granot et al, 1980). If V 0 2 + does mimic this secondary effect of high concentrations of Mg 2 + , then it obviously does so with greater potency. This is consistent with the fact that the binding of V 0 2 + to ATP is considerably stronger than the binding of Mg 2 + to ATP (Nechay et al, 1986). 5.2.2 Studies to Test the Binding of VS to Kemptide or Histone It is known that decavanadate binds to Kemptide and competitively inhibits PKA (Pluskey et al., 1997). The possibility that V 0 2 + might bind to peptide or protein substrates and thereby block PKA action by a substrate-directed mechanism was next investigated. To study possible V 0 2 + binding, Kemptide (771.9 amu) and histone (average MW 21.5 kDa) were subjected to ESI MS. Fresh stock solutions of Kemptide (5 uM in cffi^ O) were prepared with or without the addition of VS (50 uM) and subjected to ESI MS as described in the Methods (section 2.3.1). Under the MS conditions studied, there was no evidence that the mass of Kemptide (773 amu) was influenced by the addition of VS. Furthermore, there was no evidence of new peaks at ~ 840 amu or at ~ 870 amu that would be expected for complexes of Kemptide with vanadyl or sulphate, respectively (Figure 5.2). In addition to the parent Kemptide peak, there were two minor additional peaks. One located at ~ 790 amu (as the shoulder of the major peak) that might be explained by the addition of a water molecule to the parent peak (772 + 18 = 790). The other peak at ~ 802 amu could not be identified. In any case, no peaks corresponding to the addition of vanadium (51 amu) or vanadyl (83 amu) to the parent peak could be detected, with predicted masses of ~ 823 amu (772 + 51) or ~ 855 amu (772 +83). Two other species, with m/z under 500 amu, were also observed: one at 386 amu, doubly charged Kemptide (774/2), and another at 258 86 amu, triply charged Kemptide (775/3). There were no additional peaks representing the addition of either 83 amu or 51 amu to these doubly and triply charged species (data not shown). Because decavanadate and Kemptide (LRRASLG) have respectively net charges of -4 and +2 at neutral pH, an electrostatic interaction between Kemptide and decavanadate is possible. A repulsion between positively charged VO and Kemptide at pH ~ 5.5 may explain lack of binding of vanadyl to Kemptide. Figure 5.2 ESI Mass Spectra of Kemptide in the Absence or Presence of VS Kemptide was either diluted in dH20 or mixed with VS to give final concentrations of 10 pM and 100 pM, respectively. Final pHwas 5.5 for the peptide and 5.3 for the mixture, respectively. The mass spectrum is from a single experiment that was repeated 3 times with similar results. To further investigate potential binding of VO to Kemptide, PKA assays were carried out with higher concentrations of Kemptide. For these studies the assay buffer, optimized for PKA activity, led to a high IC 5 0 for VS. Results in Figure 5.3 show that increasing the Kemptide concentration had no effect on the potency of VS inhibition of PKA. If there were interactions between Kemptide and V 0 2 + , this high Kemptide concentration would have been expected to diminish PKA inhibition. These results demonstrate that the effects of vanadyl on the PKA reaction could not be explained by interactions of vanadyl with Kemptide. This conclusion is supported by the fact that vanadyl does not bind Kemptide under MS conditions (Figure 5.2). 87 Figure 5.3 Effects of Kemptide Concentration on VS Inhibition of PKA PKA assays were carried out (30 min, 30 °C) in the following mixture: MES buffer (35 mM, pH 7), DTT (450 pM), BSA (10 pM), EDTA (10 pM), MgS04 (2.5 mM), KCl (80 mM), and ATP (0.24 mM). Kemptide was added at 200 pM (filled bars) or 400 pM (open bars). Results are presented as mean ± SEM for three independent experiments, each carried out in triplicate. 100 - i 200 400 600 Final [VS], uM Similar studies were also carried out to test for the possibility of significant vanadyl interactions with histone HI. After many trials with different conditions, interpretable mass spectra were obtained with preparations of histone at low pH and in the presence of acetic acid. It is possible that acetic acid stabilized histone and prevented its aggregation as it has been shown to do for egg white lysozyme (Yang et al, 1996). Interestingly, 0.35 M (~ 2 %) acetic acid even facilitated the refolding of lysozyme into its native state after denaturation with 6 M guanidinium-HCl treatment for 2 hours (Yang et al, 1996). Importantly, acetic acid binds vanadyl rather weakly (log K~ 2, Smith and Martell, 1976c). A fresh stock of histone HI was diluted in acetic acid (2 %, v/v) and mixed with VS to give a final ratio of 1: 2 (histone HI :VS) and final concentrations of 50 uM and 100 uM, respectively in acetic acid (1 %, v/v). Under these conditions of mass spectrometry, a number of species were observed, most notably at 21264,21370, 22602, and 22681 amu (peaks 1 to 4, respectively in Figure 5.4). The detected species match closely to those predicted for each of the four homogeneous histone HI subtypes: 21263, 21374,22606, and 22761 amu (Berger et al, 1995). In addition, extra species were detected that may represent some other subtypes as discussed in Materials and Methods. Similar to Kemptide, there was no evidence of vanadyl binding to any variant of histone HI. Unlike 88 Kemptide, however, some additional species were detected that might be explained by binding of sulphate. For example, the histone peak at 21,792 amu (#1) could have given rise to the peak at 21,892 amu by addition of one sulphate. Similarly, the histone peak at 22060 amu (*1) could have given rise to peaks at 22163 amu (plus one sulphate) and 22225 amu (plus two sulphates). The results with histone using ESI MS are therefore inconsistent with earlier studies showing that the presence of histone caused a marked increase in IC50 for PKA inhibition relative to that seen using kemptide (Figures 4.1 versus 4.5). Based on those earlier assay results, histone probably did bind vanadyl ions. Although the results are not conclusive, it remains possible that the complexity of the pattern of species detected in the presence of VS has, in some way, obscured the binding of vanadyl to histone. Alternatively, the binding might be too weak to withstand the conditions employed for the MS analysis. In conclusion, the binding of vanadyl ion to histone can not be unambiguously confirmed. Figure 5.4 ESI Mass Spectra of Histone in the A bsence or Presence of VS Solutions of Histone HI (50 pM) and histone + VS (100 pM) in acetic acid (1 %, v/v, pH 3) were analyzed by ESI MS as described in the Materials and Methods. Species numbered 1 to 4 correspond to the major histone HI variants (21264, 21370, 22602, 22681 amu, respectively). Peak #1 (21792 amu) may give rise to peak #2 (21892 amu) by the addition of one sulphate. Peak *1 (22060 amu) may give rise to *2 (22163 amu) and *3 (22225 amu) by the addition of one or two sulphates, respectively. The mass spectrum is from a single experiment that was repeated 3 times with similar results. 0 , u 3 .1 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 • Histone (0.05mM) Histone:VS (12) 21348 1 #1 *1 -A 1 I ' #2 *2 22143 1 20000 20500 21000 21500 22000 Mass (amu) 22500 23000 89 5.2.3 Effects of ATP on PKA Inhibition by VS Although V 0 2 + is not able to replace Mg 2 + as a cofactor in the PKA reaction, it may be able to mimic the inhibitory actions of high Mg 2 + concentrations, by binding a- and y-phosphates of ATP. If V 0 2 + does indeed interact with ATP in some manner, then inhibitory effects might be influenced by changes in ATP concentrations. To address this possibility, effects of VS were determined over a range of ATP concentrations. In initial experiments, using assay conditions optimized for PKA and in which the IC50 for VS was quite high (Figure 3.10), doubling the ATP concentration from 120 uM to 240 uM did not have any detectable effect on PKA inhibition caused by 200 uM, 400 uM or 600 uM VS (Figure 5.5). However, interactions between VS and ATP did become evident using conditions to minimize V 0 2 + binding to assay components; notably by reducing DTT, EDTA, and BSA < 1 uM, (Figure 5.6). For example, under these conditions, PKA inhibition by 40 uM VS was more substantial (~ 70 %) at 40 uM ATP than at 100 uM ATP (-30 %). Correspondingly, the apparent IC50 for PKA inhibition by VS was increased as ATP concentration was increased. Based on these studies, a more formal kinetic analysis was carried out. For these more detailed studies, ATP concentration-dependency for PKA activity was determined at each of several VS concentrations (range 0-200 uM). In the first set of assays chloride was used as the major anion (Figure 5.7) and another set with acetate as the major anion (Figure 5.8). With chloride as the major anion, the mode of inhibition of PKA by VS is clearly complex. According to the primary and derived plots, a "mixed" mode of inhibition appears most likely for several reasons. First, the primary and Lineweaver-Burke plots appear to exclude competitive and non-competitive modes of inhibition as both V m a x and Km changed with varying VS concentrations. Second, the Cornish-Bowden plot did not result in parallel lines or in an infinite Km, again excluding the competitive mode of inhibition. Third, the Dixon plot did not result in parallel lines or in an infinite KK, excluding the uncompetitive mode of inhibition. Therefore, the "mixed" mode of inhibition best explains the observed results. Both Lineweaver-Burke and Dixon plots resulted in a competitive K\ for VS of ~ 11 uM. The Cornish-Bowden plot resulted in an uncompetitive K\ of ~ 4.5 uM (Figure 5.7, Table 5.1). 90 Figure 5.5 Effects ofA TP on PKA Inhibition by VS (I) - High A TP and VS Concentrations PKA assays were carried out (30 min, 30 °C) in the following mixture: MES buffer (35 mM, pH 7), DTT (450 pM), BSA (10 pM), EDTA (10 pM), KCl (80 mM), MgS04 (2.5 mM), and Kemptide (400 pM). Reactions were initiated by adding ATP at 120 pM (open bars) or at 240 pM (filled bars). Results are presented as mean ± SEM for three independent experiments, each carried out in triplicate. 100 i 90 -200 400 600 Final [VS], mM Figure 5.6 Effects of ATP on PKA Inhibition by VS (II) - Low ATP and VS Concentrations PKA assays were carried out (30 min, 30 °C) in the following mixture: MES buffer (10 mM, pH 7), DTT (0.7 pM), BSA (0.02 pM), EDTA (0.1 pM), KCl (70 mM), Mg2* (1.5 mM), and Kemptide (250 pM). Reactions were initiated by adding the indicated concentrations of ATP. Results are presented as the average of two independent experiments, each carried out in triplicate. [ATP], uM [VS], uM 91 Figure 5.7 Effects of VS and ATP on PKA Activity With Chloride as the Major Anion PKA assays were carried out (36 min, 30 X) in the following mixture: MES buffer (10 mM, pH 7), DTT(0.7pM), BSA (0.02 pM), EDTA (0.1 pM), KCl (140 mM), Kemptide (0.5 mM), and Mg2* (1.5 mM). VS and ATP concentrations were as indicated. Primary plot (Panel A), Lineweaver-Burke plot (Panel B), Kt estimate (Panel C), Dixon plot (Panel D), and Cornish-Bowden plot (Panel E). Results are the average of two independent experiments, each carried out in triplicate. R2 represents the Pearson correlation coefficient. :s s O H "o 6 a *-*-» < O H 16 -] 14 -12 -10 8 6 4 2 0 0 [VS], uM o 0 • 12.5 A 25 • 50 A 100 o 200 9'"' .•• m o A-20 40 60 [ATP], uM 80 -A 100 B -0.2 " 2 < a at 23 18 13 [VS], uM o0 • 12.5 A 25 • 50 A 100 o200 <5° A ...A.. A A f -0.1 -2tJ 0.1 0.2 1/[ATP], 1/p.M 0.3 0.4 92 < 4 _ CO ° i CO S <U > OH h o <u .S o 50 -j 45 -40 35 -\ 30 25 -\ 20 15 10 5 -20/-3 t) R 2 = 0.9877 ~ i 1 20 40 K i c = l l u M 60 80 100 120 140 160 180 200 [VSLuM at 9i S D 25 20 15 10 5 •1^ " [ATP], uM o2.5 • 5 A 12.5 • 25 0 .0 • A A - • A f ' . . y— p-* 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 *10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 -20^1 K i c = l l ± l uM [VS], uM 93 245 •£ * 195 o o < S at 145 ^ 95 45 -5 [ATP], uM o 2.5 • 5 A 12.5 • 25 6 -«•• A A i^5 15 K i u = 4.6 ± 0.4 uM i i i i i i 1 1 1 1 1 1 1—:—i 1 1 1 1— 25 35 45 55 65 75 85 95 105 115 125 135 145 155 165 175 185 195 [VS], uM When assays were repeated with acetate instead of chloride results differed slightly in quantitative terms, but the primary and derived plots lead to the same general conclusion about the mode of inhibition. Both Lineweaver-Burke and Dixon plots resulted in a competitive K, for VS of - 60 uM and the Cornish-Bowden plot resulted in an uncompetitive K\ of - 10 uM (Figures 5.9 and Table 5.1). Based on log lvalues for complex formation, acetate (log K= 1.86 at 25 °C and I = 1 M) is able to bind vanadyl 66-fold more strongly than chloride (log K- 0.04 at 25 °C and 1=1 M). Consequently, the concentration of free vanadyl will be lower in the presence of acetate than in the presence of chloride. Consistent with these relative strengths of binding, the competitive K\ for PKA inhibition was higher in acetate buffer than in chloride buffer. As in the presence of chloride, inhibition of PKA by VS in the presence of acetate was also complex. From results in both set of assays a "mixed" mode of inhibition was inferred by elimination of other modes from interpretations of the derived plots (Figure 5.8). As mentioned earlier, it is important to stress that the derived kinetic constants (IC50, K\, etc.) must be considered tentative estimates. 94 Figure 5.8 Effects ofVS and ATP on PKA Activity With Acetate as the Major Anion PKA assays were carried out (36 min, 30 °Cj in the following mixture: MES buffer (10 mM, pH 7), DTT(0.7pM), BSA (0.02 pM), EDTA (0.1 pM), KCH3COO (140 mM), Kemptide (0.5 mM) and Mg2* (1.5 mM). VS and ATP concentrations were as indicated. Primary plot (Panel A), Lineweaver-Burke plot (Panel B), K, estimate (Panel C), Dixon plot (Panel D), and Cornish-Bowden plot (Panel E). Results are the average of two independent experiments, each carried out in triplicate. R2 represents the Pearson correlation coefficient. -.9 o S *+-» o < 35 n 30 25 20 -\ 15 10 5 0 [VS], uM • 0 p 12.5 A 25 o 50 • 100 % it . . i a X3 20 40 60 80 100 [ATP], uM B 2.5 1.5 0.5 [VS], uM o 0 A 12.5 o 25 A 50 • 100 A O 0 -0.2 -0.-1 : ; 1/ [ATP], 1/jiM 0.3 0.4 -0.5 -1 95 *S CQ g 3 .a 8 7 6 5H 4 3 2 R = 0.9757 1 1 1 r~ -70 -60 ~ i 1 -40 -30 -20 -10 6 10 20 30 40 50 60 70 80 90 100 [VS], uM K i c = 61 uM D [ATP], uM • 2.5 • 5 o 12.5 A 25 • H . : : - ) ; ; - - — F " • > O H PH VS T r 2 1.5 1 : : : 3 r-e- 1 1 1 1 i 1 i i r -90 -80 -70 -60 \-50 -40 -30 -20 -10 § 10 20 30 40 50 60 70 80 90 100 -0.5 [VS], uM K i c = 59 ± 1 uM 96 > O H O < B S B 5 3 16 14 12 10 8 6 4 2 [ATP], uM o2.5 A 12.5 A 25 • 100 -A" . & 0 A.. A 0 -20 10 \ 0 10 20 30 40 50 [VS], uM 60 70 80 90 100 K i u = 10 ±0.3 uM 5.2.4 Effects of VS on PKA Activity Following Nitrogen Purging In the experiments carried out so far reducing agents have been included to stabilize PKA activity. These reducing agents unfortunately complicate the interpretation of the results because they interact with vanadyl. N2(g) purging was therefore considered as an alternative method to stabilize PKA without introducing any further vanadyl ligands. The effects of N2(g) purging were therefore compared to the effects of adding reducing agents. PKA was diluted in buffers containing 0.5 mM DTT or GSH or [3-EtSH or was diluted in a buffer which lacked reducing reagents but was purged with N2(g) for 30 min. All other buffer components were identical (Figure 5.9). N2(g) purging yielded significantly higher PKA activity than the PKA activity observed in the presence of any one of the reducing agents. The retention of PKA activity was therefore in the following rank order: N2(g) > DTT > GSH > (3-EtSH (Figure 5.9), confirming and extending the results shown in Section 3.2.4. 97 Figure 5.9 Effects ofN2(g) Purging, DTT, GSH, and fi-EtSH on PKA Activity PKA assays were carried out (30 min, 30 X) in the following mixture: MES buffer (15 mM, pH 7), KCH3COO (30 mM), ATP (0.1 mM), Kemptide (0.6 mM), MgS04 (1.5 mM), and the indicated reducing agents (0.5 mM). When buffer was purged with N2(g) other reducing agents were omitted. Results are presented as mean ± SEM"for three independent experiments, each carried out in duplicate. Values were compared by ANOVA; significant differences were as follows: (a) versus N2(g) (p <0.001); (b) versus DTT (p <0.01). In these experiments, the maximum activity of PKA was 4.5 pmol P/min. < 100 80 60 •> 40 20 a,b a.b p-EtSH DTT GSH 30 minN2(g) Purging Having demonstrated that N2(g) purging substitutes for the addition of DTT or GSH, the possibility of avoiding the use of BSA and EDTA and yet retaining PKA activity was explored. Remarkably, the addition of potassium acetate (KCH3COO) alone led to appreciable retention of PKA activity. This led to the development of the "minimal" buffer, containing MES (10 mM) and KCH3COO (120 mM) and purged with N2(g) for 30 min. Using this buffer, PKA activity was ~ 80 % of that found in the presence of an "optimal" buffer (Figure 5.10). 98 Figure 5.10 Retention of Significant PKA Activity With "Minimal" Buffer PKA assays were carried out (30 min, 30 °C) using Kemptide (0.6 mM) and ATP (0.1 mM) as substrates in either "optimal" buffer containing: MES (10 mM, pH 7), DTT (0.5 mM), MgS04 (1.5 mM), EDTA (10 pM), BSA (10 pM), and KCl (70 mM) or in "minimal" buffer that was purged with N2(g)for 30 min and contained: MES (10 mM, pH 7) andKCH3COO (120 mM). Results are presented as mean ± SEM for three independent experiments, each carried out in duplicate. 30 - i P/mi 25 -(pmol 20 -15 -< 10 O H 5 -0 -Optimal Buffer Minimal Buffer The effects of VS were then tested at each of several ATP concentrations, as in Figures 5.7 and 5.8, except for the use of the "minimal" Na(g)-purged buffer. These assays were carried out with the indicated ATP and VS concentrations but with different batches of PKA. The primary and the derived plots are shown in Figures 5.11 and 5.12 and the derived inhibition constants are shown in Table 5.1. From both sets of Figures, Lineweaver-Burke and Dixon plots resulted in a competitive K\ for VS of ~ 4 uM. The Cornish-Bowden plot resulted in an uncompetitive K\ for VS of ~ 16 uM. A "mixed" mode of inhibition was again most likely based on the graphical analysis. 99 Figure 5.11 Effects ofVSandATP on PKA (Batch I) Activity With the "Minimal" Buffer PKA from Boehringer Mannheim was assayed (32 min, 30 X) in the following N2(g)-purged mixture: MES buffer (10 mM, pH 7), KCH3COO (120 mM), Kemptide (0.5 mM) andMgS04 (1.5 mM). VS and ATP concentrations were as indicated. Primary plot (Panel A), Lineweaver-Burke plot (Panel B), Ki estimate (Panel C), Dixon plot (Panel D), and Cornish-Bowden plot (Panel E). Results are the average of two independent experiments, each carried out in triplicate. R2 represents the Pearson correlation coefficient. 6 4 2 0 4* [VS], uM • 0 • 10 A 20 o 30 x 40 x 50 + 60 -70 A 80 f|:l - X --X" 4-—-o X - A 0 20 40 60 80 100 [ATP], uM 100 B > OH t5 o < S 21 OH a 3.5 3 2.5 2 1.5 1 0.5 [VS], o 0 + 10 A 20 • 30 X40 O50 • 70 • 80 i o X o 8 o a •A -0.04: -0.02 -0.5 -1 .0 0.02 0.04 0.06 1/ [ATP], 1/uM 0.08 0.1 J S § £ «> .3 •10 25 20 15 10 5 ^ R = 0.9737 0 Kj c - 6 u M 10 20 30 40 [VS], uM 50 60 70 80 101 D > O H •i"H , S 6 1.5 0.5 0 [ATP], uM o 100 • 50 A 25 • 12.5 • 10 A 9 o T r A A • O o O —i 1 1 50 55 60 io ;s 6 t 5 10 15 20 K i c =1.7 ±0.3 uM 25 30 35 [VS], uM 40 45 ' > O H t> o < a at 10 40 35 30 25 20 15 10 5 — -5 -10 0 [ATP], uM o 100 • 50 A 25 • 12.5 o 20 O ; a K i u = 1 6 ± 2 u M 30 [VS],uM "O a. 40 D A 50 o A 60 102 Figure 5.12 Effects ofVS and ATP on PKA (Batch II) Activity With the "Minimal" Buffer PKA from Boehringer Mannheim was assayed (32 min, 30 °C) in the following N2(g)-purged mixture: MES buffer (10 mM, pH 7), KCH3COO (120 mM), Kemptide (0.5 mM) andMgS04 (1.5 mM). VS and ATP concentrations were as indicated. Primary plot (Panel A), Lineweaver-Burke plot (Panel B), K{ estimate (Panel C), Dixon plot (Panel D), and Cornish-Bowden plot (Panel E). Results are the average of two independent experiments, each carried out in triplicate. R2 represents the Pearson correlation coefficient. A [VS], uM 0 10 20 30 40 50 60 70 80 90 100 [ATP], uM 103 B [VS], uM oO • 20 A 40 • 60 • 80 A 9 -0,04- >0.02 0.02 0.04 0.06 0.08 0.1 1/Final [ATP], 1/uM <H-H o m C O t-l lop " E H C/3 Linew 45 35 25 15 R = 0.9798 -10 10 K i c = 3uM 20 30 40 50 [VS], uJVI 60 70 80 104 D > P H tS o < S S i S .6. 3 2.5 H 2 1.5 1 H 0.5 [ATP], uM olOO A 40 *20 • 10 * -•"x - A -EJ 1 O -A-a -20 -lfi in K i c = 3.2 ± 0.6 uM 20 30 40 [VS], uM 50 60 70 80 tJ o < e fit 5 ^ 55 45 35 25 15 5 1 [ATP], uM • 100 A 60 A 40 • 20 ..1-t A A A A 10 " 5 0 20 K i u = 14 ± 1 uM 30 40 50 [VS],uM 60 70 80 The results of all three major experiments dealing with the effects of VS and ATP on the activity of PKA are summarized in Table 5.1. The most notable conclusion is that the inhibition of PKA by VS can be observed under a variety of conditions in vitro using concentrations of vanadyl in the low micromollar range. In addition, some apparent differences in K\ values are evident depending upon the experimental conditions. For example, the competitive inhibition 105 constant (Kic) was highest in acetate buffer and lowest with N2(g) purging. In contrast, the uncompetitive inhibition constant (£;„) was highest with N2(g) and lowest in chloride buffer. The different K\c values in acetate and chloride buffers could be explained by the difference in the stability constants (Table 4.1); acetate having a 66-fold greater binding constant for vanadium and a corresponding ~ 6-fold higher KJC. The effects of N2(g) purging are less straightforward to interpret because BSA was also omitted. From Figure 5.10, it is clear that acetate can serve to promote nearly optimal PKA activity, even in the absence of BSA. In addition, it is reasonable to conclude that N2(g) purging lowers the apparent KIC by minimizing the conversion of vanadyl to vanadate. In contrast, the derived Kiu values do not conform to the same pattern and the value obtained using the N2(g)-purged buffer was actually higher than values observed with other buffers. This underlines the fact that, although these initial kinetic studies do give some important insights, considerably more experimental work will be required to fully define the mechanism by which vanadyl inhibits PKA activity. In addition to the graphical methods, Leonora® software (Cornish-Bowden, 1995) was used to derive Km and k c a t values and to fit Equations 2.2, 2.3, and 2.4 to the inhibition kinetic data (Table 5.1). V m a x was within the range of stated activity of the purified PKA preparation (100 - 200 pmol P/min). Km (chloride and DTT) was within the range of reported literature values (20 - 50 uM). kcat (acetate and DTT) was highest but at higher cost of the higher Km value. Km values were similar but K1C value with acetate and DTT was somewhat higher than the other two conditions. When all data points were included no convincing fit of the above equations representing competitive, uncompetitive and mixed inhibition modes, respectively, to the data could be determined. In fact, in some cases, derived kinetic parameters were large negative numbers. These observations suggested that some of the data points might be "outliers" and, accordingly, the lowest values of PKA activity (low concentrations of ATP and high concentrations of VS) were removed. Subsequently, the lack fit of the Equation 2.4, representing the mixed inhibition mode, to the kinetic data was no longer significant. In other words, the data is most consistent with a mixed mode of inhibition. 106 Table 5.I Effects of ATP on PKA Inhibition by VS - Summary of Kinetic Parameters Results are derivedfrom Leonora0 software andfrom graphical methods using data from the indicatedfigures. Kt values were calculated either using Leonora® software or graphically from the point of intersection of each possible pair of lines (n), presented as the mean ±SEM(KU from Cornish-Bowden plots and KiCfrom Lineweaver-Burke and Dixon plots). Vmax and Km were calculated using Leonora® software. kcat = Vmax / Eb where Et is the total PKA concentration. Figure # 5.7 5.8 5.11,5.12 Experimental Condition Chloride DTT Acetate DTT Acetate N2(g) Purging Kinetic Parameter (Leonora) V m a x (pmol P min"1) 27 + 2 112 ± 2 9 78 ± 11 kcat (sec ) 3 + 0.2 11 ± 3 1 ±0.1 *m(uM) 74 + 6 241 ± 73 300 ± 50 k c a t / ^ m (uM"1 sec"1) 0.04 ± 0.004 0.1 ±0.04 0.003 ± 0.001 ^>c(pM) (Leonora) 15 ± 5 48 ± 1 1 12 ± 3 (Graphs) 11 ± 1 (n = 7) 60 ± 1 (n = 7) 3 . 5 ± l ( n = 1 8 ) Aiu(uM) (Leonora) 2 ±0.5 3 ± 1 6 ±2.5 (Graphs) 5 ± 0.4 (n = 6) 10 ±0.3 (n = 6) 1 6 ± 3 ( n = 1 2 ) 5.2.5 Studies of Possible Binding of Vanadyl to PKA Having investigated the potential interactions between vanadyl and the substrates Kemptide, Histone, ATP, and Mg 2 + , the possibility that vanadyl binds directly to PKA was examined using ESI-MS. A number of different experimental conditions were tried in order to achieve good quality mass spectra of PKA preparations. In the course of these studies, a number of variables had to be evaluated, including the purity of PKA, pH, ionic composition of the preparation and sample temperature. Eventually, the conditions that gave the best quality spectra involved dilution of PKA in acetic acid (2.5 %, v/v, pH ~ 2) with ESI MS being carried out as described in the Materials and Methods. The two factors that appeared to be especially important in obtaining good quality spectra were: a) low sample pH and b) presence of acetate. Under these conditions, the mass of PKA was estimated to be 40907 amu, close to the predicted value of 40800 amu (Figure 5.13). Interestingly, when PKA was subjected to MS analysis in the presence of VS, the spectrum changed in a complex manner, with band broadening and band shifting (Figure 5.14). This provided evidence for some form of interaction between VS and PKA, albeit under very non-physiological conditions. The broad and extensive shift in peak mass of approximately 300 107 amu suggested that either multiple vanadyl ions bind per PKA molecule or that sulphate might also bind and obscure any effect of vanadyl binding. In a final set of studies, ESI MS was carried out using a higher voltage difference (AV) between sample orifice and QO (ion guide rod). The aim was to try to impose conditions that would be expected to cause the dissociation of any complexes that might have formed between PKA and buffer components. However, PKA was evidently unstable at higher orifice voltages and no interpretable spectra were generated. As an alternative model, Cytochrome c was analyzed because this relatively small protein (~ 12,380 amu) was stable at higher voltages. In these studies, although not directly relevant to understanding PKA, it became clear that multiple species generated by the addition of VS were likely explained by binding of sulphate rather than vanadyl to Cytochrome c. As the results in Figure 5.15 show, peaks at ~ 12460 amu and ~ 12560 amu probably represent Cytochrome c with 1 and 2 sulphate groups bound, respectively. This presumed sulphate addition was eliminated at a AV of 90 Volts. At higher values of AV additional peaks became visible, although none of these could be explained by binding of vanadyl to Cytochrome c. The important point was that PKA could not stand this AV and would not give an interpretable spectrum, but the potential compounding effect of sulphate binding raises an important caution in interpreting any further studies along these lines. 108 Figure 5.13 ESI Mass Spectrum of PKA PKA was diluted in acetic acid (2.5 %, v/v, pH 2) and subjected to ESI MS as described in Materials and Methods. Charge states are depicted in Panel A and the "deconvolved" spectrum, prepared with the Perkin-Elmer SCIEXInc. BioMultiview™program, is shown in Panel B. The mass spectra are from a single experiment that was repeated 4 times with similar results. A 1200000 ^ 1000000 & 800000 'co S 15 600000 la .§> 400000 1/3 200000 0 +30 +29 +28 +27 \J +26 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 m/z (amu) B CU o (O C C/3 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 .200000 0 40907 39545 42644 44268 1—; r 1 n — — i r 35000 36000 37000 38000 39000 40000 41000 42000 43000 44000 45000 Mass (amu) 109 Figure 5.14 Effects of VS on the ESI Mass Spectra of PKA PKA was diluted in acetic acid (1.25 %, v/v, pH 2.5), in the presence or absence ofVSas indicated, and mass spectra determined as described in Materials and Methods. The mass spectra are from a single experiment that was repeated 3 times with similar results. 70000 60000 50000 40000 •a 30000 20000 10000 1350 1400 1450 1500 1550 1600 1650 m/z (amu) 110 Figure 5.15 Effects of VS or (NH4)2SC>4 on ESI Mass Spectra of Cytochrome c Cytochrome c was diluted in dH20 to yield a final concentration of 5 pM (Panel A). Separate dilutions of Cytochrome c (5 pM) were mixed with either 50 pM (NH4)2 SO4 (Panel B) or 50 pM VS (Panel C), pH 6. ESI spectra were recorded as described in Materials and Methods, at the indicated voltage differences between ion guide rod (QO) and sample Orifice. The mass spectra are from a single experiment that was repeated 3 times with similar results. A 1600000 1400000 r 1200000 DC £ 1000000 •f 800000 e • | 600000 1 — H 400000 c 01 rr 200000 0 11800 50 Volts 90 Volts 12360 Cytochrome c (5 uM) 12000 12200 12400 12600 Mass (amu) 12800 13000 13200 1200000 %> 1000000 \% •53 800000 c 2 600000 a a 00 400000 200000 0 11800 50 Volts 90 Volts — p — 12000 12358 12457 Cytochrome c + (NH4)2S04 (50 uM) 12558 12200 12400 12600 Mass (amu) 12800 13000 13200 111 C 250000 g 200000 •g 150000 a rt c/3 100000 50000 0 11800 50 Volts 90 Volts 12356 Cytochrome c + VS (50 uM) 12455 12556 12000 12200 12400 12600 12800 13000 13200 Mass (amu) 5.2.6 Effects of Glycyl and Glycine-Loop Peptides As described earlier, the ATP-binding site of PKA is located near a conserved glycine-rich loop (Table 5.2, Figures 1.5 and 5.16) which is responsible for securing the nucleotide in the correct position for reaction (Rossmann et al., 1975; Zheng et al., 1993a). Although none of the glycine residues make direct contact with ATP, it was concluded from mutational analysis that: a) Gly 50 and Gly 52 were more important in catalysis than Gly 55, b) mutation in any glycine resulted in ~ 10-fold decrease in kemptide affinity, c) loss of Gly 52 selectively affected ATP binding and lowered the rate of reaction (Grant et al., 1998). This glycine-rich loop is therefore considered to play an important role in stabilizing PKA structure and in maintaining normal affinity for substrates (Knighton et ai, 1991a, 1991b). Because of the importance of the glycine-rich loop of PKA and the ability of vanadyl to selectively bind to di- and tri-glycyl peptides (Table 4.2), it was hypothesized that vanadyl might inhibit PKA by binding to the glycine-rich loop within the active site. To test this hypothesis, experiments were carried out to determine the effects of a model "glycine-loop" peptide on the inhibition of PKA by vanadyl. The glycine-loop peptide corresponded to the PKA motif GTGSFG (Table 5.3). The effects of the glycine-loop peptide, as well as the effects of di- and tri-glycyl peptides, were examined in PKA assays. In these experiments the peptides were added either at the same final concentration as VS (Figure 5.17A), or at a concentration exceeding that of VS (Figure 5.17B). 112 Table 5.2 Examples of the Glycine-Rich (Rossmann) Motif in Selected Protein Kinases Kinase N-terminal Continuation Sequence C-terminal Continuation Consensus GXGXXG "glycine-loop" Peptide GTGSFG PKA-a,p RKKTL GTGSFG RVMLV PKG IIDTL GVGGFG RVELV PKC-a,P,y FLMVL GKGSFG VLMAD Phosphorylase Kinase PKEIL GRGVSS VRRCI CaM-KII LFEEL GKGAFS VVRRC MLCK SKEAL GSGKFG AVCTC CDK-1 KVEKI GEGTYG VVYKA Src LEVKL GQGQFG EVWMG Insulin Receptor LLREL GQGSFG MVYEG Figure 5.16 The 3-Dimensional Structure of the Glycine-Rich Loop of PKA 113 Under these conditions, none of the peptides gave any appreciable protection of PKA from inhibition by VS. In contrast, EDTA and BSA were both able to sequester vanadyl and significantly protect PKA from inhibition. Interestingly, EGTA, unlike EDTA, did not significantly reverse the inhibition caused by VS. This provides further evidence for the importance of the vanadyl cation and its relationship to magnesium because EGTA binds Mg 2 + ions (and evidently also V0 2 + ) rather poorly. Log K values (25 °C, 0.1 M) for the interaction between EGTA and Mg 2 + or Ca 2 + are 10.86 and 5.28, respectively (Smith and Martell, 1976b). It is worth stressing that the protective effects of glycyl peptides seen earlier (Figure 4.12) were observed with much higher peptide concentrations and under conditions when vanadyl was incubated with the glycyl peptides before addition of other assay components. In the current series of experiments, increasing the final concentrations of glycyl peptides (to 5 mM) or glycine-loop peptide (to 1 mM), still did not lead to protection of PKA from inhibition by VS. Together with the earlier studies (Figure 4.12) it seems clear that the glycyl peptides compete very poorly for vanadyl in the PKA assay mixture. Furthermore, the glycine-loop peptide was unable to protect PKA from inhibition or to protect VS from oxidation as judged by EPR studies (Figure 5.18). In this study no vanadyl spectrum was detectable in the presence of any tested concentrations of Gly-Loop peptide (up to 22 mM). As noted elsewhere the vanadyl spectrum observed at pH 4 was rapidly lost at neutral pH in the absence or presence of Gly-Loop peptide. For these other controls see Figures 6.2 and 6.13. 114 Figure 5.17 Effects of Di- and Tri-Glycyl and Glycine-Loop Peptides on PKA Inhibition by VS PKA assays were carried out (30 min, 30 X) in the following mixture: MES buffer (25 mM, pH 7), MgS04 (2.5 mM), Kemptide (0.25 mM), ATP (0.1 mM), EDTA (5 pM), BSA (5 pM), DTT(0.5 pM), GSH (2.5 mM), and KCl (70 mM). In panel A, other reagents, as indicated, were added at a final concentration of 0.5 mM. In Panel B, the additional reagents were added at final concentrations of 5 mM except the glycine-loop peptide (1 mM). Assays were carried out in the absence (open bars) or presence (filled bars, 0.5 mM) of VS. Results are presented as mean and range of two independent experiments, each carried out in triplicate. A 90 80 70 £ ^ 6 0 :s 1 ^ 30 20 -10 -0 X X X B 9 0 80 70 60 H I I 5 0 <tZ 40 < | 30 20 10 0 X X X GG GG GGG GGG BSA BSA GLY GLY LOOP LOOP 115 Figure 5.18 X-Band EPR Spectra of VS in the Presence of the Glycine-Loop Peptide Solutions in MES buffer (60 mM, pH 6.8) were freshly prepared with or without VS (1 mM) and the indicated concentrations of glycine-loop peptide [GTGSFGJ. X-band EPR spectra were recorded at room temperature. 2.75 mM Gly-Loop I I 1 I I I I L 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 116 5.3 SUMMARY The studies described in this chapter were designed to define the inhibitory effects of vanadyl with respect to interactions with the natural substrates of PKA. The main conclusions drawn from the studies reported in this chapter are as follows: 1. Vanadyl can, at best, only very weakly replace Mg as a cofactor for PKA; 2. High concentrations of Mg 2 + do not overcome inhibitory effects of vanadyl on PKA. Indeed, high concentrations of Mg 2 + inhibit the PKA reaction and vanadyl might conceivably be a potent mimic of this additional effect of Mg 2 + ; 3. Vanadyl does not appear to bind appreciably to Kemptide but may bind directly to PKA itself although this remains a very tentative conclusion on the evidence available so far; 4. The binding of vanadyl to glycyl peptides led to the hypothesis that vanadyl might bind to the active site glycine-rich loop of PKA. Based on subsequent evidence, it now seems that this is very unlikely; 5. Based on evidence from kinetic analyses it is suggested that vanadyl interacts more appreciably with ATP. Thus, effects of vanadyl appear to be influenced by the concentration of ATP in PKA assay mixtures and detailed kinetic analysis suggests the nature of the interaction is complex and dependent on the assay conditions. With chloride as the major anion the apparent K1C was estimated to be ~ 11 uM. With acetate as the major anion, the apparent KK increased to ~ 60 uM. Interestingly, with acetate as the major anion but in the absence of BSA and with very low concentrations of DTT and EDTA, N2(g) purging of the MES buffer also led to a very low apparent Klc value of ~ 4 (Table 5.1). Based on the evidence obtained so far, the interaction of vanadyl with ATP and/or juxtaposed amino acids in the active site cleft appears the most likely explanation of inhibition of PKA by vanadyl. 117 CHAPTER SIX PRESERVATION OF VANADYL AT PHYSIOLOGICAL pH: IN SEARCH OF "NATURAL" LIGANDS 6.1 RATIONALE PKA, like most intracellular enzymes is most active and most stable close to the physiological pH ~ 7. In contrast, vanadyl is most stable at pH < 2, and above ~ pH 4 vanadyl has several fates depending on the conditions including oligomerization, precipitation and oxidation (see Introduction). Despite this intrinsic property, there is considerable evidence that vanadyl does exist within intact cells at neutral pH. Indeed, cells treated with extracellular vanadyl or vanadate contains predominantly EPR-detectable intracellular vanadyl. The predominance of vanadyl over vanadate within cells is further suggested by the fact that Na +/K +-ATPase remains active in intact cells treated with vanadate or vanadyl despite the fact that it is exquisitely sensitive to inhibition by vanadate (not vanadyl) in vitro. It is crucial to understand how vanadyl is protected from oxidation or other fates within cells and this knowledge should also help in the design of studies of vanadyl actions in vitro. Conceivably, vanadyl might bind predominantly to a single biological ligand or, alternatively, to multiple ligands. One strong candidate ligand is glutathione, which typically exists in the cytosol of mammalian cells at a concentration ~ 2 mM (Hagenfeldt et al, 1978; Janaky et al, 1999). As a major cellular "redox buffer", glutathione could have dual actions with the ability to bind vanadyl and also to reduce oxidized vanadium to vanadyl. While cells contain a number of other compounds with redox buffer or anti-oxidant properties (ascorbic acid, vitamin E, etc.), the interactions between glutathione and vanadyl were more of interest because of the cellular abundance of glutathione and existing literature that supports its important role. Another interesting question relates to the possible preservation of extracellular vanadyl in the reduced form. This is potentially much more challenging because the extracellular space is a far more oxidizing environment. From previous studies it is clear that vanadyl can be carried within blood cells and also binds to serum proteins, notably transferrin and serum albumin. Because of the utility of BSA in studies of PKA activity vanadyl-BSA interactions were also evaluated. 118 6.2 RESULTS AND DISCUSSION 6.2.1 Unprotected Vanadyl Is Unstable and Fails to Inhibit PKA Stock aqueous solutions of VS were typically made fresh and kept at pH 4 until addition to PKA assay mixtures immediately prior to initiating reaction with ATP. However, when the initial VS stock (pH 4 in water) was diluted into MES buffer (20 mM, pH 7), even for a brief time (< 1 min) prior to addition to assays, PKA inhibition was greatly diminished (Figure 6.1). Dilution of vanadyl solutions, per se, did not abolish PKA inhibition provided a low pH was maintained. The rapid loss of inhibitory activity confirmed, as expected from many previous studies, that vanadyl had probably oxidized to vanadate, oligomerized to an EPR-silent species or precipitated at neutral pH. This was confirmed by the rapid lost of typical X-band EPR signal of vanadyl at pH 7 (Figure 6.2). Figure 6.1 VS Fails to Inhibit PKA Following Pre-incubation at pH 7 VS (pH 4) was diluted into water (pH 4, filled squares) or MES buffer (20 mM, pH 7, open diamonds) for < 1 min prior to addition to PKA assay mixtures at the indicated final concentrations. Assays were carried out (36 min, 30 °C) with the following additional components: MES buffer (20 mM), ATP (0.1 mM), Kemptide (0.25 mM), DTT (0.7 pM), EDTA (0.1 pM), BSA (0.02 pM), MgS04 (1.2 mM), and KCl (140 mM). Results are presented as mean ± SEM of three independent experiments, each carried out in duplicate. 70 -, 60 A 0 0 10 20 30 40 50 60 70 80 90 100 Final [VS], uM 119 Figure 6.2 X-Band EPR Spectra of VS EPR spectra of freshly prepared VS (0.5 mM) were recorded at room temperature ~ 2 min after adjustment of pH to 4 or 7. i i i i i i i i 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 6.2.2 VS Has Limited Stability Even at pH 4 The previous experiments had confirmed that the vanadyl EPR signal is rapidly lost at pH 7 and that the ability to inhibit PKA is equally rapidly lost. For practical purposes, if vanadyl were completely stable at pH 4, stock solutions might be kept for reasonable lengths of time. Even at this low pH, however, vanadyl could be subject to oxidative conversion to vanadate or to oligomerization to an EPR-silent species or to precipitation. Thus the EPR spectrum of VS was significantly diminished over a period of 6 hours at pH 4.6 at room temperature. In fact, the intensity of the EPR signal diminished approximately 50 % within 6.5 hours despite the fact the sample was contained in an unstirred sample capillary with limited capacity for oxygen diffusion. The results in Figure 6.4 also show that storage of acidic VS solutions (pH 4) at 4 °C 120 for 2 days led to significant loss of the ability of VS to inhibit PKA. In these assays, PKA exhibited a high IC50 for inhibition by VS because of the presence of BSA, DTT, and EDTA. Figure 6.3 Time-Dependent Decay of X-Band EPR Spectra of VS Solutions at pH 4.6 X-band EPR spectrum of a 0.5 mM VS stock solution (pH 4.6) was recorded immediately after preparation of the solution (t = 0) and after 6.5 hrs incubation at room temperature in the unstirred capillary tube. t = 0hrs 1 I I I I I 1 L 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 121 Figure 6.4 Loss of PKA Inhibition on VS Storage PKA assays were carried out (30 min, 30 X) in the following mixture: MES buffer (35 mM, pH 7), DTT (0.5 mM), EDTA (10 pM), BSA (10 pM), KCl (85 mM), ATP (0.24 mM), Kemptide (0.4 mM), and MgS04 (2.5 mM). Solutions ofVS (pH 4) were prepared freshly on the day (filled bars) or were used after storage for 2 days at 4 X (open bars) and added to give the indicated concentrations. Results are presented as the mean and range of values from two independent experiments, each carried out in triplicate. 300 ! 250 200 $ I 150 100 50 h. ...... iiiiilii 111 | | | -vswm m 0.2 0.2 0.4 0.4 Final [VS], mM 0.6 0.6 6.2.3 Effects of Glutathione on PKA Inhibition by VS Several groups have studied the interactions between vanadyl and glutathione, as discussed in the Introduction. Glutathione probably plays an important role in preventing or reversing the oxidation of vanadyl in cells. In addition, glutathione can form a complex or complexes with vanadyl, judging from spectroscopic and potentiometric studies (Pessoa etal., 2002). Important questions remain; for example, it is not clear why complex formation, in vitro, evidently requires relatively high ratios of glutathione to vanadyl (50:1 or greater in some studies). Furthermore, me structure of the biologically relevant complexes formed between glutathione and vanadyl have not been defined. Experiments were therefore carried out to examine the interactions of glutathione and vanadyl under PKA assay conditions. For these studies it is important to bear in mind that two equilibria may be important: Vanadate + e" « * Vanadyl 2GSH » GSSG + 2Ff + 2e" 122 GSH and GSSG were first tested to be certain that neither alone inhibited PKA in the absence of vanadium. Neither oxidized nor reduced glutathione, up to 10 mM final concentration in the assay, had any significant effect on PKA activity. If anything, PKA activity was fractionally higher with GSH and slightly lower with GSSG but PKA activity changed < 10 % relative to control (Figure 6.5). Figure 6.5 Lack of Effect of GSH or GSSG on PKA Activity PKA assays were carried out (30 min, 30 °C) in the following mixture: MES buffer (20 mM, pH 7), DTT(0.5 mM), EDTA (10 pM), BSA (10 pM), KCl (70 mM), ATP (0.1 mM), Kemptide (0.5 mM) and MgS04 (1.5 mM). Assays were carried out with the indicated concentrations of GSH (filled bars) or GSSG (open bars). Results are represented as mean and range of two independent experiments, each carried out in triplicate. a l 15 -, 14 13 12 11 10 H 9 8 7 6 5 4 3 2 1 0 T , T X X X X 0 10 Final [GSH or GSSG], mM PKA activity was then assessed in the presence of different ratios of vanadium to glutathione. In these experiments, PKA assays were carried out under conditions designed to give relatively high IC50 for vanadyl (Figure 6.6) so that 100 uM VS inhibited PKA only ~ 10 %. PKA activity was not affected when the GSH:VS ratio was 1:1 or 10:1 but PKA was inhibited more than 50 % when the ratio of GSH:VS was higher (85:1). Based on the effects seen at the higher ratio of GSH:VS, the effects of VS and OV in combination with either GSH or GSSG were studied, again where conditions were such that the IC50 for VS was high (Figure 6.7). 123 Figure 6.6 Effects of GSH and VS on PKA PKA assays were carried out (30 min, 30 °C) in the following mixture: MES buffer (20 mM, pH 7), DTT (0.4 mM), EDTA (10 pM), BSA (10 pM), KCl (70 mM), ATP (0.1 mM), Kemptide (0.5 mM) and MgS04 (1.5 mM). Where indicated (+), VS was added at a final concentration of 0.1 mM, with the indicated concentrations of GSH. Results are presented as mean and range of two independent experiments, each carried out in triplicate. In these experiments, the maximum activity of PKA was 25.5 pmol P/min. 120 n VS - + . . + + + GSH - - 1 mM 10 mM 0.1 mM 1 mM 10 mM 124 Figure 6.7 Effects of VS and OV on PKA when Combined with GSH or GSSG PKA assays were carried out (30 min, 30 °C) in the following mixture: MES buffer (20 mM, pH 7), DTT (0.4 mM), EDTA (10 pM), BSA (10 pM), KCl (70 mM), ATP (0.1 mM), Kemptide (0.5 mM) and MgS04 (1.5 mM). Reactions were carried out in the absence (-) or presence (+) of 0.2 mM VS (Panel A) or OV(Panel B), together with the indicated concentrations (mM) of GSH or GSSG. Results are presented as mean + SEM for three independent experiments, each carried out in triplicate. Values were compared by ANOVA: (*) significantly different from control (p < 0.05). l l a l cu <y 30 25 -\ 20 15 H 10 5 0 VS GSH GSSG T 0.2 10 0.2 10 20 0.2 20 10 0.2 10 B 30 < 25 20 3 15 S io H o OV GSH GSSG 0.2 10 0.2 10 0.2 20 20 10 0.2 10 125 The main conclusions drawn from these experiments are as follows: 1. Under these conditions, 200 uM VS or OV alone induced no significant inhibition of PKA; 2. 200 uM VS induced significant inhibition of PKA (50 % or greater) when added in combination with 10 mM or 20 mM GSH, corresponding to GSH:VS ratios of 50:1 or 100:1. Surprisingly, OV also induced PKA inhibition when added in combination with 10 mM or 20 mM GSH. The results in Figures 6.7 and 6.8 differ slightly in this regard but overall the combinations of GSH with either VS or OV induced approximately the same degree of PKA inhibition. Because OV does not inhibit PKA, it seems likely that GSH may have induced some reduction of OV to vanadyl under these conditions; 3. Also notably, VS significantly and markedly inhibited PKA (~ 80 %) when combined with 10 mM GSSG. In contrast, 200 uM OV did not inhibit PKA in combination with 10 mM GSSG. A possible explanation for these latter observations is that the most effective complex of vanadyl is that with GSSG rather than with GSH. The presence of GSSG might even explain the inhibition of PKA seen when GSH is combined with VS because it is almost certain that some oxidation of GSH to GSSG occurs in the reaction mixture. The role of V 0 2 + as an effective PKA inhibitor when combined with GSH or GSSG was further tested by studying the effects of EDTA (Figures 6.8 and 6.9). In these experiments, the inhibition of PKA that was induced by adding VS in the presence of either GSH or GSSG was abolished by adding 130 uM EDTA (Figure 6.8). Importantly, EDTA also abolished the inhibition of PKA seen following the addition of GSH plus OV, thus providing strong support for the idea that some OV had been reduced to the cationic form of vanadyl, which had then been chelated by EDTA. The effects of EDTA were further confirmed in a different experimental design (Figure 6.9). Here, the dose-dependency for inhibition of PKA by VS revealed an IC50 of -150 uM in the absence of GSSG. The addition of GSSG altered the dose-dependency, reducing the IC5oto ~ 75 uM. Again, the PKA inhibition was completely abolished by EDTA (5 mM). Interestingly, the apparent IC50 obtained in Figure 6.9 (~ 150 uM) was considerably lower than that found in a related experiment in which the IC50 was > 400 pM (Figure 3.10). In comparing the conditions used in both settings; the most obvious difference was in the concentrations of GSH (2.5 mM in Figure 6.9). Other differences were the lower concentrations of DTT (0.5 uM versus 0.5 mM), EDTA (5 pM versus 10 uM), and BSA (5 uM versus 10 uM). The lower concentrations of EDTA and BSA, known chelators of vanadyl, in combination with the presence of GSH, some of which had likely been converted to GSSG, could explain the decrease 126 in IC50 from ~ 400 uM to ~ 150 uM. Overall these observations provide further support for the concept that vanadyl is the effective PKA inhibitor and that vanadyl can be effective at physiological pH if appropriate ligands are available: In this case, GSH and GSSG. Figure 6.8 Effects of EDTA on PKA Inhibition by VS PKA assays were carried out (30 min, 30 X) in the following mixture: MES buffer (20 mM, pH 7), KCl (70 mM), ATP (0.1 mM), Kemptide (0.5 mM), MgS04 (1.5 mM), BSA (3 pM), and DTT (0.3 mM). GSH (10 mM), GSSG (10 mM), VS (0.1 mM) or OV (0.1 mM) were added where indicated. Assays were carried out in the absence (-.filled bars) or presence (+, open bars) of EDTA (130 pM). Results are presented as mean and range of two independent experiments, each carried out in triplicate. £ 3 3 5 ^ o $ I CU <2 16 14 12 1<H 4 2 0 Control EDTA + -GSH GSH:VS GSH:OV GSSG GSSG:VS GSSG:0V + - + - + - + - + - + -127 Figure 6.9 Effects ofVS on PKA Are Enhanced by GSSG and Blocked by EDTA PKA assays were carried out (30 min, 30 °C) in the following mixture: MES buffer (15 mM, pH 7), KCl (70 mM), ATP (0.1 mM), Kemptide (0.25 mM), MgS04 (5.5 mM), GSH (2.5 mM), DTT (0.5 pM), EDTA (5 pM), and BSA (5 pM). Assay mixtures were further adjusted with no additions (open triangles), 5 mMEDTA (open squares) or 5 mMGSSG (filled squares). Results are represented as mean and range of two independent experiments, each carried out in triplicate. 0 50 100 150 200 250 3 0 0 3 50 4 00 Final [VS], uM In conclusion, these studies underline the importance of determining and controlling the concentrations of all relevant assay components in order to define the effects of vanadium salts on PKA activity in vitro. Understanding how vanadium works within intact cells, with many potential ligands is clearly a challenge. However, interactions with cellular GSH/GSSG could be extremely important. 6.2.4 Analysis of Vanadyl in PKA Assay Buffers by EPR In the experiments described so far, the true concentration of vanadyl under PKA assay conditions has not been accurately determined. EPR measurements have confirmed that the final vanadyl concentration is substantially lower than that expected, based on the added VS. Importantly, the calculated IC50 and Ki values remain estimates based on the added VS and not on the concentration of vanadyl that remains in solution. EPR analysis was used to obtain a more quantitative estimate of the concentration of vanadyl in experimental mixtures and to assess the effects of assay components including GSH, GSSG, and BSA. 128 GSSG alone, in the absence of GSH, was not sufficient to preserve a vanadyl EPR signal at ratios of GSSG:VS of 7:1 or 15:1. However, at a ratio of 30:1, a weak vanadyl signal was visible (Figure 6.10). A vanadyl signal was also preserved, albeit weak and distorted, when VS was analyzed in P K A assay buffer (Figure 6.11). The signal strength of vanadyl in P K A assay mixtures was equivalent to, or slightly stronger, than that in an aqueous solution with GSSG, despite the presence of a lower added concentration of VS (0.2 mM compared to 0.5 mM). This is perhaps explained by the additional components of the assay mixture that might interact with and "protect" vanadyl, notably 5 u M EDTA, 5 u M BSA, 5.5 m M GSSG, 100 uM ATP and 2.5 m M GSH. Interestingly, GSH alone, even up to a ratio of 50:1, was unable to preserve a vanadyl signal at pH 7 (Figure 6.12). In contrast, B S A (2.5 mM) was able to preserve a remarkably strong vanadyl signal at pH 7 (Figure 6.13). In fact, the vanadyl signal was still clearly detectable when the concentration of added VS was reduced to concentrations as low as 50 n M (Figure 6.13). B S A itself did not contribute to the EPR spectrum of mixtures because a solution of B S A alone gave a spectrum that was indistinguishable from that of an empty capillary tube (Figure 6.13). In serum, the concentration of albumin is in the range of 30-50 g/L (0.5-1 mM), theoretically enough to bind the highest concentration of vanadyl that might be present in vivo (up to 20 u M in treated animals, for example). In addition to the quantitative intensity of the vanadyl signal seen by EPR, interesting qualitative differences could also be detected. A particularly clear difference in spectrum was evident when comparing the EPR signals of VS in the presence of GSSG and B S A (Figure 6.14). The 8-line vanadyl EPR spectrum seen in the presence of B S A was very similar to the spectrum of pure vanadyl at pH 4. In contrast, the vanadyl EPR signal in the presence of GSSG revealed a pattern of only 5 prominent peaks that were broadened and adopted a sine wave form (see "diamond" shape in Figures 6.14 and 6.15). Clearly, the environment provided by binding to GSSG appears to have a greater impact on the vanadyl ion than the environment in the binding site of BSA. Inspection of the vanadyl spectrum in a P K A assay mixture revealed signs of interesting changes that take place over time. Thus at early times after addition of VS to P K A assay buffer, in the presence of GSSG and BSA, the vanadyl EPR spectrum resembled that of pure VS:GSSG mixtures (Figure 6.15). This pattern persisted for ~ 15 min at room temperature in the EPR capillary. Beyond this time period, however, the vanadyl EPR spectrum showed peak sharpening and a trend towards the full 8-line pattern characteristic of that seen in the presence of BSA. Although this change can only be interpreted with some caution, it suggests that the nature of the 129 vanadyl complex may change with time, from predominantly GSSG:VS to BSA:VS. The important general point is that the vanadyl signal could be seen when GSSG and GSH were present, among other components of a typical PKA assay mixture at physiological pH. Further experiments revealed that the spectrum typically seen with the GSSG:VS mixtures was even further stabilized in the presence of GSH (Figure 6.16). In this combination (GSH:GSSG:VS = 100:25:1), the broad, five-peak spectrum could still be detected even at pH 9. Interestingly, total cellular GSH is on the order of 2 mM and GSSG is approximately 50 uM. Assuming that vanadyl might be 5 uM in treated animals, this would give a GSH:GSSG:Vanadyl ratio of 400:10:1. 130 Figure 6.10 X-Band EPR Spectra of Vanadyl in the Absence or Presence of GSSG X-band EPR spectra of a freshly-prepared solution ofVS (0.5 mM) were recorded at room temperature in the absence (pH 4.5) or in the presence of indicated concentrations of GSSG (all at pH 7). VS V GSSG (3.75 mM) + VS pH7 i i i i i i i i 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 131 Figure 6.11 X-Band EPR Spectra of Vanadyl in PKA Assay Mixture X-band EPR spectrum of a freshly-prepared solution of VS (0.5 mM) was recorded at room temperature and pH 4.5. The EPR spectrum of same VS solution was also recorded after adjusting to pH 7 in the absence or presence of GSSG (15 mM, ratio 30:1). An EPR spectrum of a lower concentration ofVS (0.2 mM) was also recorded (bottom panel) in PKA assay mixture containing the following final concentrations: MES buffer (15 mM, pH 7), KCl (70 mM), ATP (0.1 mM), Kemptide (0.25 mM), MgS04 (2.5 mM), GSH (2.5 mM), DTT (0.5 pM), EDTA (5 pM), GSSG (5.5 mM), and BSA (5 pM). 0.5 mM VS pH 7 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 132 Figure 6.12 X-Band EPR Spectra of Vanadyl in the Absence or Presence of GSH X-band EPR spectra of a freshly-prepared solution ofV204 (0.8 mM) were recorded at room temperature in the presence of GSH (40 mM) at the indicated pH. I 1 I I 1 I I L 2 8 0 0 3 0 0 0 3 2 0 0 3 4 0 0 3 6 0 0 3 8 0 0 4 0 0 0 4 2 0 0 Field Strength, Gauss 133 Figure 6.13 X-Band EPR Spectra of Vanadyl in the Absence or Presence of BSA X-band EPR spectra of a freshly-prepared solution ofVS (0.5 mM) were recorded at room temperature in the absence of BSA (pH 4.5) or in the presence of BSA (2.5 mM, pH 7). The vanadyl signal was also recorded following dilution as indicated at constant BSA (2.5 mM, pH 7). For comparison, BSA (2.5 mM, pH 7) was also recorded in the absence ofVS (this signal is similar to that of the empty capillary tube). VS (0.5 mM) pH 4.5 VS (0.5 mM) + BSA VS (0.25 mM) + BSA BSA = Empty Capillary Tube I I I I I I 1 L 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 134 Figure 6.14 X-Band EPR Spectra of Vanadyl in the Presence of GSSG, GSH, or BSA X-band EPR spectra of a freshly-prepared solution ofVS (0.5 mM), except in bottom panel (0.05 mM), were recorded at room temperature in the presence of indicated concentrations of BSA, GSH and GSSG (all at pH 7). Spectra were derived by subtraction of spectra of control solutions that were identical except for omission of VS. GSSG (12.5 mM) + GSH (ImM) + VS GSSG (12.5 mM) + GSH (50 mM) + VS BSA (1.25 mM) +VS BSA (2.5 mM) + VS (0.05 mM) 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 135 Figure 6.15 Time-Dependent Changes in X-Band EPR Spectra of VS in PKA Assay Mixture X-band EPR spectra of a freshly-prepared solution ofVS (0.2 mM) were recorded in a PKA assay mixture containing the following final concentrations: MES (15 mM, pH 7), KCl (70 mM), ATP (0.1 mM), Kemptide (0.25 mM), MgS04 (2.5 mM), GSH (2.5 mM), DTT (0.5 pM), EDTA (5 pM), GSSG (5.5 mM), and BSA (5 pM). Spectra were recorded after incubation for the indicated times at room temperature and were derived by subtraction of the spectra of assay mixture devoid of VS. t = 14 min t = 22 min t = 30 min 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 136 Figure 6.16 X-Band EPR Spectra of Vanadyl at High pH, with GSH and GSSG X-band EPR spectra of a freshly-prepared solution of VS (0.5 mM) were recorded at room temperature in the presence of GSHand/or GSSG, added at the indicated concentrations and pH. GSSG (12.5 rrnVD + VS pH7 2800 3000 3200 3400 3600 3800 4000 4200 Field Strength, Gauss 137 6.3 SUMMARY From the results of EPR and kinetic Studies it is concluded that vanadyl can exist in the PKA assay buffer. Significantly, the combination of GSH with GSSG appears to be more effective than either form of glutathione alone in protecting vanadyl at neutral pH. The results obtained so far are consistent with the possibility that the two forms of glutathione might play different roles: GSH facilitating reduction and GSSG directly binding to vanadyl. However, further work is required to be certain of the exact roles of GSH and GSSG. The most effective combination of GSH and GSSG observed in these studies, in vitro, is rather similar to the combination that might be expected to exist within mammalian cells. BSA also protects vanadyl so effectively that, like EDTA, it is able to protect PKA from VS inhibition. 138 CHAPTER SEVEN MAJOR CONCLUSIONS AND FUTURE DIRECTIONS 7.1 PKA IS POTENTLY INHIBITED BY VS Vanadium salts are effective in reversing many effects of insulin deficiency or insulin resistance in vivo; under these conditions the circulating concentration of vanadium ions typically does not exceed 20 pM. The most generally accepted hypothesis to explain vanadium actions in vivo is that vanadium inhibits PTPases, thereby potentiating cellular protein tyrosine phosphorylation and reproducing or enhancing insulin signals. However, there is also considerable evidence that vanadium exerts selective effects on metabolism and may act downstream from PTPases and tyrosine kinases. The aim of this thesis was to focus on the control of TG hydrolysis, which is potently inhibited by vanadium and is well defined mechanistically. The initial working hypothesis was that vanadium inhibits TG hydrolysis by inhibiting PKA. Based on the results presented, the main conclusion is that PKA is indeed potently inhibited in vitro by vanadium salts, notably by vanadyl sulphate, with an IC50 in the low micromolar range (Figures 4.1 and 4.7). 7.2 ASSAY CONDITIONS PROFOUNDLY INFLUENCE THE POTENCY OF INHIBITION OF PKA BY VS During the course of this project, it was essential to define and control the concentrations of a number of reagents typically used in the assay of PKA, in order to minimize interference with the effects of vanadium. Experiments to define the possible interactions of vanadium with components of the PKA assay mixtures are now summarized. The role of substrate was investigated because some substrates might bind vanadium ions. VS inhibited PKA with an apparent IC50 that was much lower using Kemptide as a substrate than was found using histone HI (Figure 4.1 and 4.5). High concentrations of Kemptide did not diminish the inhibition of PKA by VS and ESI MS analyses provided ftirther evidence that Kemptide was unable to bind vanadium. In contrast, kinetic results are consistent with the possibility that vanadyl binds to histone, although this was not unambiguously confirmed by ESI MS (Figures 5.2 and 5.4). PKA is optimally active when the salt concentration of assay buffers is in the range 100 mM and the choice of buffer anion is also important. For example, chloride interacts very weakly if at all with any ionic forms of vanadium and is preferable to acetate, which can bind vanadyl 139 more appreciably. Consequently, assays carried out with chloride as the major anion revealed lower IC50 and K\ values for PKA inhibition by VS than were found using acetate as the major anion (Figures 5.7 and 5.8; Table 5.1). In many studies, PKA activity is enhanced in the presence of serum albumin. However, BSA has been found to bind both vanadate and vanadyl ions. Likewise, optimization of PKA activity depended on the presence of BSA, but BSA also tended to diminish the effectiveness of VS as an inhibitor (Figures 3.7 and 4.2). EDTA is another reagent often added to enhance PKA activity and/or stability, presumably by removing the risk of inhibition by heavy metal ions. Again, EDTA has the potential to bind vanadyl ions very strongly and results presented here confirmed this because PKA inhibition by vanadyl sulfate was completely abolished in the presence of EDTA (Figures 4.2, 5.17A, 6.8 and 6.9). Reducing agents, notably DTT, are essential for long-term storage of PKA; however, it was not clear from the literature if DTT was also essential in assay mixtures for optimal PKA activity. In this regard, PKA tolerated low concentrations of reducing agents for the short duration of enzyme assays, typically 30 min. Furthermore, not all reducing agents were equally effective; PKA activity being higher in the presence of DTT than in the presence of GSH or vitamin C (Figure 3.6). In terms of revealing PKA inhibition by VS, however, the presence of GSH was preferable. In contrast, high DTT concentrations could reverse PKA inhibition by VS (Figure 4.3). This suggests that DTT can bind vanadyl but that the DTT-vanadyl complex is unable to inhibit PKA. N2(g) purging of buffer revealed that this method was significantly better than the use of either DTT or GSH in maximizing PKA activity (Figure 5.9). The K\ for the inhibition of PKA by VS was also very low in N2(g)-purged buffer, suggesting that exclusion of oxygen and corresponding oxidative loss of vanadyl is otherwise significant. Overall, the importance of controlling the assay conditions was illustrated by comparing the effects of VS on PKA using two relative extremes of assay conditions. When concentrations of BSA, EDTA, and DTT were in the range 10 uM, 10 uM, and 500 uM, respectively, the IC50 for PKA inhibition by VS exceeded 400 uM (Figure 3.13). In contrast, when BSA, EDTA, and DTT concentrations were each reduced to less than 1 uM, the IC50 for VS inhibition of PKA was reduced from > 400 uM to < 25 uM (Figures 4.1 and 4.7). The two extreme conditions studied also differed in the concentrations of MES, ATP, and salt but the concentrations of BSA and EDTA appeared to be the most crucial. It was therefore concluded that BSA and EDTA are able to bind vanadyl more tightly than PKA and thereby diminish PKA inhibition by VS. 140 7.3 VANADYL AND NOT VANADATE POTENTLY INHIBITS PKA Because vanadium can exist in a variety of forms within intact cells, it is important to define the species that might inhibit PKA. The results of several experiments have provided strong support for the idea that a divalent cationic form, most likely vanadyl, is the inhibitory species; most directly VS inhibited PKA whereas OV did not (Figure 4.8). In a second experimental approach, agents known to bind vanadyl were found to diminish the inhibition of PKA. For example, high concentrations of di- or triglycyl peptides were mixed with VS prior to addition to PKA assay mixtures and both peptides were found to significantly diminish PKA inhibition by VS (Figure 4.11). These glycyl peptides bind vanadyl much more strongly than vanadate (Table 4.2) and if vanadate had indeed been the inhibitory species then PKA inhibition should not have been diminished in the presence of glycyl peptides. Similarly, EDTA, a strong chelator of divalent cations including vanadyl, also abolished PKA inhibition by VS (Figure 6.8), confirming that the inhibitory species is likely to be a divalent cation. Interestingly, the related chelator, EGTA binds vanadyl and magnesium much less strongly than EDTA and was unable to reverse the inhibitory effect of VS on PKA (Figure 6.8). Another interesting comparison comes from studies of other organic complexes of vanadyl namely, BPOV and BMOV (Figure 4.8). The lower potency of BPOV and BMOV in PKA inhibition, relative to VS, could be due to the fact that the picolinato and maltolato ligands bind quite strongly to vanadyl so that less free vanadyl is available to inhibit PKA. The stability constant for BMOV has been reported (Caravan et al, 1995) but that for BPOV is not known. Considering that BPOV was a more effective inhibitor of PKA than BMOV then it may be predicted that BMOV binds vanadyl more tightly. Vanadyl is stable at low pH, but unstable at neutral pH, being susceptible to precipitation, oligomerization and/or oxidation (see Introduction). Using this instability of vanadyl, equimolar solutions of VS were prepared at pH 4 or pH 7 and the inhibitory effects on PKA then compared. VS solutions that were prepared and kept briefly at pH 4 as usual inhibited PKA. In contrast, no inhibition of PKA was observed when VS was adjusted to pH 7, even for a few minutes, prior to addition to assays (Figure 6.1). Whatever the fate of vanadyl at neutral pH, it is clear that none of the products (vanadate, oligomers or precipitates) were effective PKA inhibitors; the lack of PKA inhibition by vanadate being directly confirmed. The kinetic analyses were supported by EPR studies designed to examine vanadyl stability. The vanadyl EPR signal was lost within 1 min at pH 7, and even when kept at pH 4 in a capillary tube at room temperature, a significant decrease (> 50 %) in the intensity of the vanadyl 141 EPR signal occurred over a period of 6.5 hours (Figure 6.3). Effects of VS on PKA activity were also determined by the instability of vanadyl during the storage. Thus, fresh VS solutions inhibited PKA whereas solutions that had been stored, failed to do so (Figure 6.4). 7.4 STUDIES OF THE MECHANISM OF INHIBITION OF PKA BY VANADYL Based on the preceding studies it was concluded that VS inhibits PKA through the actions of vanadyl; the potency of the inhibition being greatly influenced by the composition of the assay mixtures. In the course of subsequent studies several possible actions of vanadyl were addressed: 1. Binding to the peptide or protein substrate of PKA, 2. Interaction with the ATP-magnesium complex within the PKA active site, 3. Binding to the conserved glycine-rich loop within the PKA active site. 1. Binding of vanadyl to the peptide or protein substrate of PKA It is possible that vanadyl might bind at or near the phosphorylation site of a PKA substrate or, alternatively, at some distal site that could induce a conformational change affecting phosphorylation indirectly. In fact, ESI MS and kinetic results suggested that vanadyl does not bind to Kemptide. For example, the MS data did not show any peaks that might represent vanadyl complexes with Kemptide (Figure 5.2). Furthermore, if vanadyl inhibited PKA by binding to Kemptide, then a large excess of the peptide relative to vanadyl would be expected to reduce PKA inhibition. In fact the degree of PKA inhibition by VS remained relatively constant at all Kemptide concentrations (Figure 5.3). MS results were ambiguous with histone HI (Figure 5.4). Although the addition of VS did lead to the generation of additional species, these might have been explained by the binding of sulphate rather than, or as well as, vanadyl. More work would be required to confirm whether vanadyl itself does directly bind to histone HI. In contrast, the kinetic data strongly suggested that vanadyl interacted with histone HI because VS was a far less potent inhibitor of the phosphorylation of histone HI than of Kemptide (Figures 4.1 and 4.5). In other words, this observation suggests that histone HI might bind vanadyl sufficiently to reduce the effective concentration available to inhibit PKA. In order to further confirm or disprove the potential binding of vanadyl to Kemptide or histone HI, several techniques might be applied. For example, the relative strength of binding of vanadyl to Kemptide or histone HI could probably be determined using isothermal titration microcalorimetry. This technique can reveal the dissociation constant for the interaction between vanadyl and either Kemptide or histone HI. Similarly, equilibrium dialysis, using radioactive 142 vanadyl, might be used to assess direct binding. Finally, it might be possible to generate co-crystals of either substrate with VS or V2O4 in order to directly visualize the complexes formed. 2. Interaction of vanadyl with the ATP-magnesium complex within the PKA active site Because the inhibitory effects of vanadyl could not be readily accounted for by binding to peptide substrates, effects on other substrates were examined. High concentrations of magnesium could not relieve PKA inhibition by VS (Figure 4.6). In contrast, in the absence of magnesium, VS (up to 100 uM) did not lead to significant PKA activity. Vanadyl was therefore a very poor cofactor for the PKA reaction compared to magnesium, which was effective at concentrations in the range 60-100 uM (Figure 5.1). Interestingly, in the absence of VS, magnesium concentrations in excess of 5 mM caused significant PKA inhibition (> 50 %, Figure 4.6). This effect has been observed in studies by a number of other groups and may be explained by two possible modes of magnesium binding to ATP. Thus magnesium is able to bridge either the pVy-phosphates or the a-/y-phosphates of ATP. The former mode is required for effective phophotransferase reaction, but the latter mode of magnesium binding reduces PKA turnover (kcat) by ~ 5 fold. Under normal physiological conditions, magnesium acts primarily in the catalytic mode and only partially in the inhibitory mode (Cook et al, 1982). The dual mode of action of magnesium raises the intriguing possibility that vanadyl might act in a complex manner, being able to mimic the inhibitory effect of magnesium, whilst being a very poor mimic of the catalytic action of magnesium. Vanadyl certainly binds more strongly than magnesium to ATP (Nechay et al, 1986), although specific binding of vanadyl to pVy-phosphates or to cc-/y-phosphates of ATP has not been determined. Therefore, vanadyl might cause PKA inhibition in two ways: a) by binding to pVy-phosphates and blocking the catalytic actions of magnesium, b) by binding to a-/y-phosphates and causing a much stronger inhibition than magnesium. To resolve this issue, it would be necessary to specifically assess vanadyl binding to the different combinations of phosphate groups of ATP. For example, isothermal microcalorimetry or detailed kinetic analyses with various combinations of vanadyl, magnesium, and ATP might provide a method to discriminate specific vanadyl binding. Furthermore, rapid quench flow measurements might permit studies of PKA activity before significant losses of vanadyl have occurred in the reaction mixture (Grant and Adams, 1996; Shaffer and Adams, 1999). 3. Binding of vanadyl specifically to the conserved Glycine-rich loop within the PKA active site 143 The possibility that vanadyl might bind directly to PKA itself was studied using ESI MS. Although good quality mass spectra were obtained under acidic conditions, results were ambiguous with respect to vanadyl binding to PKA (Figure 5.14). The addition of VS did result in a clear change in PKA mass spectra, but it was not possible to determine if the changes were caused by binding of vanadyl or sulphate, or perhaps caused by some other indirect effect. Because of the recognized binding of vanadyl to glycyl peptides (Table 4.2), the hypothesis that vanadyl might bind to the glycine-rich loop within the active site of PKA was tested using two approaches. First, the stability of the vanadyl EPR signal was examined in the presence of the glycine-loop peptide. In this study, no protection of the vanadyl signal was evident, suggesting no effective interaction (Figure 5.18). Second, the ability of glycyl peptides to reverse PKA inhibition by VS was assessed. When added directly to assay mixtures, none of peptides were able to reverse PKA inhibition by VS (Figure 5.17). Taken together, the studies so far suggest vanadyl does not bind effectively to the glycine-rich loop of PKA. Of course, it is possible that the synthesized glycine-loop peptide may be too short. Thus further studies with different synthetic glycine-loop peptides might be worthwhile. For example, the use of longer peptides or the acetylation of the N-terminus, to avoid repulsion between the positively charged vanadyl and the N-terminal amine, could shed light on the potential binding of vanadyl to the glycine-rich loop of PKA. One or more of the six conserved water molecules visible within the crystal structure of PKA might provide another possible mode of binding of vanadyl within the PKA active site. One of these water molecules is locked into position by several interactions, including those with ATP and peptide substrate (Shaltiel etal, 1998). The stability of hydrated forms of vanadyl may give an interesting perspective on this issue. Specifically, a stock solution (10 mM, pH 4) of VS.3H2O showed considerably greater stability and retained its characteristic color at 4 °C for several days. In contrast, the corresponding solution prepared using VS.nFbO (n = 1 to 6), which is predominantly VS.5H2O (Chasteen, 1981), turned from sky blue to green either at 4 °C or at room temperature within 24 hours, indicating extensive oxidation to vanadate. X-ray structures of these two species were studied by several groups. VS.5H2O has a monomeric structure whereas VS.3H2O has a dimeric structure, molecular units consisting of two SO4 tetrahedra and two VO6 octahedra sharing corners, linked by hydrogen bonds (Tachez and Theobald, 1980; Theobald and Galy, 1973). Based on this difference in stability, it is tempting to speculate that the structured water molecules within the PKA active site might bind to and stabilize vanadyl. 144 In terms of further analytical approaches to test for vanadyl binding to PKA, isothermal titration microcalometry, high-resolution mass spectrometry, or X-ray crystallography may all give insights into this problem. For example, microcalorimetry might yield the inhibitor binding constant and the enthalpy of binding of vanadyl to PKA, as in the case of fluoride ion binding to urease (Saboury and Moosavi-Movahedi, 1997). Ultimately, of course, X-ray analysis of PKA in the presence of vanadyl or vanadate might be possible as with PTPase complexed with vanadate (Zhang et al, 1997) and with BMOV (Peters et al, 2003). The difficulty of preparing sufficient PKA for crystallization is a major limiting factor, of course. Further work with MS analysis might also be productive, perhaps the use of tandem MS or matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry, which can withstand large salt concentrations in the sample, may be beneficial to define the nature of the vanadium species bound to PKA. 7.5 WHAT IS THE TRUE CONCENTRATION OF VANADYL IN PKA ASSAYS? Because vanadyl is particularly sensitive to oxidation at the pH of PKA assays and because several assay components can act as vanadyl chelators, the quoted final vanadyl concentration in each experiment and the derived kinetic parameters must be considered to be apparent values. Independent analyses using EPR spectrometry confirmed the instability of vanadyl in PKA assay mixtures and provide some basis to estimate the possible vanadyl concentrations. Results shown in Figure 6.2, for example, confirmed that at room temperature, the EPR vanadyl signal diminished within minutes at neutral pH. Similarly, EPR analysis revealed that the true vanadyl concentration in PKA assay mixtures in the absence of BSA, EDTA or glutathione was below the detectable threshold of the EPR instrument (~ 15 uM, Figures 4.9 and 4.10). Despite the intrinsic instability of vanadyl at neutral pH, an EPR signal could be detected in PKA assay buffers provided certain reagents were also present. Thus a clear EPR vanadyl signal was visible in PKA assay mixtures containing BSA and a combination of GSH and GSSG (Figures 6.11 and 6.15). The combination of GSH and GSSG was also most effective in promoting PKA inhibition by vanadyl (Figure 6.7). To determine vanadyl concentrations more precisely, a freshly-prepared vanadyl solution might be concurrently subjected to EPR, 5 1 V NMR and inductively coupled plasma (ICP) mass analyses. By using these techniques, it could be possible to quantitatively define the ratio of vanadyl (measured by EPR) and vanadate (measured by 5 1 V NMR), as well as the total vanadium content (measured by ICP MS). 145 7.6 DOES GLUTATHIONE MEDIATE THE EFFECTS OF VANADYL ON PKA? EPR studies described in the Introduction have provided compelling evidence for the existence of vanadyl within intact cells. It has been suggested by several groups that glutathione plays an important role in reducing vanadate to vanadyl. Certainly, glutathione is an abundant reducing agent inside cells and can be readily regenerated. Glutathione may also act as a ligand for vanadyl, GSH and GSSG both forming complexes with vanadyl at certain ligand-to-metal ratios at physiological pH. Interestingly, vanadyl formed a more stable complex with GSSG than with GSH at pH 7, at a GSSG:vanadyl ratio of 100:1 (Figures 6.6 and 6.7). In an in vitro assay setting, glutathione may act in competition with vanadyl chelators, such as BSA, EDTA and DTT, providing a possible mechanism to "deliver" vanadyl to targets such as PKA. Accordingly experiments were carried out to test the effects of GSH and/or GSSG on vanadyl EPR signals and on PKA inhibition. Kinetic studies revealed that PKA was inhibited by vanadyl in the presence of either GSH or GSSG, the GSSG complex, surprisingly, being more effective (Figure 6.7). Interestingly, the combination of GSH plus vanadate also inhibited PKA, but the combination of GSSG plus vanadate did not, consistent with the possibility that some vanadate was reduced to vanadyl by GSH in the assay: GSH + vanadate • GSSG + vanadyl A most important consideration to emerge from the combination of activity and EPR analyses is that there are likely to be at least two pools of vanadyl present in PKA assay mixtures. One pool is protected by binding to strong chelators such as EDTA and BSA, while a second pool is bound less strongly (for example to GSH and GSSG) but remains available to inhibit PKA. Simply recording the total EPR-detectable vanadyl therefore does not necessarily predict the probability of inhibition of PKA. For example, mixtures containing significant concentrations of BSA and or EDTA but no GSH or GSSG retain detectable vanadyl EPR signals with little or no PKA inhibition. Indeed, the addition of excess BSA or EDTA can reverse or block PKA inhibition by VS completely. Under the conditions used to study the effects of VS on PKA activity, a vanadyl EPR signal could be detected whether or not GSH or GSSG were present, provided the assay mixtures contained sufficient BSA, EDTA and DTT to ensure a pool of tightly bound vanadyl. The addition of GSH and/or GSSG clearly enhanced the pool of vanadyl available to inhibit PKA. Importantly, GSH and GSSG influenced the vanadyl EPR signal in at least two ways: First, the intensity of the vanadyl EPR signal is increased, indicating a possible increase in the total vanadyl pool size. Second, the EPR spectrum is 146 qualitatively different in the presence of GSH and GSSG (Figures 6.14 and 6.15, and discussion in Chapter 6). In fact, the qualitative effects of GSH/GSSG, suggested by the prominent 5-line spectrum (see the "diamond" shape in Figures 6.14 and 6.15), appear to be lost over time in PKA assay mixtures, with the emergence of an 8-line EPR spectrum that most resembles binding to BSA (Figure 6.15). Of course, as demonstrated in earlier experiments (Figure 4.8), the addition of GSH or GSSG is not essential in order to observe PKA inhibition by VS. In these earlier studies, VS potently inhibited PKA in the presence of very low concentrations of BSA, EDTA and DTT (Figure 4.9). Further EPR studies using individual components of the PKA assay mixtures revealed that the preservation of vanadyl at pH 7 was most convincing when GSH and GSSG were both concomitantly present. In fact, with a GSH:GSSG:vanadyl ratio of 100:25:1 the vanadyl EPR signal was stable up to pH 9 (Figure 6.16). Interestingly, this ratio is quite similar to that expected in vivo, assuming concentrations of 2 mM GSH, ~ 50 uM GSSG, and ~ 5 uM vanadyl (predicted ratio 400:10:1). It is important to emphasize that the final GSH and GSSG ratios mentioned are also apparent, because some of the GSH almost certainly becomes oxidized to GSSG in experimental incubations. For this reason, results seen following the addition of GSH probably represent effects of GSH plus GSSG generated during incubation. It is therefore difficult to assess the effects of GSH per se. In contrast, the effects of added GSSG are probably reliable because GSSG is very unlikely to be spontaneously reduced to GSH. If glutathione were an essential natural ligand for vanadyl, one would predict that the loss of cellular glutathione should compromise the actions of vanadium. It is possible to deplete cellular glutathione by treating cells with buthionine sulphoximine (BSO), an inhibitor of y-glutamylcysteine synthetase, the rate-limiting step of glutathione synthesis (Drew and Miners, 1984). Other approaches might also be used to explore the role of glutathione in mediating vanadium actions in intact cells. For example, if vanadyl binding to oxidized glutathione is crucial, it might be possible to minimize the production of GSSG, perhaps under anaerobic conditions in white skeletal muscle, or in adipose tissue. In this setting, loss of GSSG would be predicted to block the metabolic effects of vanadyl. Although less direct, it might be informative to firmly establish the relative binding strength of vanadyl to potential physiological vanadyl ligands, for example by isothermal titration microcalorimetry techniques. By comparing the strength of binding of vanadyl to GSH, GSSG, ATP, cysteine, catecholamines, ascorbic acid and other potential cellular ligands, it should be possible to predict the relative binding under 147 physiological conditions. In this way, it might be possible to obtain an estimate of the relative importance of the most abundant cellular factors in preserving and mediating the actions of vanadyl. Considering a number of published studies that have indicated interactions between glutathione and vanadyl in vivo and in vitro (see the Introduction), together with the studies reported here, it is reasonable to conclude that glutathione, especially GSSG, is currently the most likely major intracellular ligand for vanadyl in vivo. In fact, because it is also abundant in serum, largely as GSSG, glutathione might also play a role in extracellular transport of vanadium. 7.7 RELEVANCE OF THE CURRENT STUDIES TO VANADIUM ACTIONS IN VIVO The studies described in this thesis demonstrate the effects of vanadyl on PKA in vitro. Although these in vitro effects do occur over a concentration range that could be achieved in vivo, many questions have to be resolved before we can be certain that PKA inhibition represents an important mechanism by which vanadium exerts metabolic effects in vivo. In other words, how significant is this in vitro inhibition of PKA in the context of actions of vanadium in vivo? In support of the significance of PKA inhibition, PKA inhibition and TG hydrolysis are seen in isolated adipocytes following the addition of 10-100 uM vanadyl sulphate to the culture medium (Table 1.4). In fact, because of the instability of the inhibitory vanadyl species, the true available concentration of vanadyl is likely to be substantially less, as confirmed by EPR analysis. By comparison, vanadium concentrations in treated animals are in the range of ~ 10 uM in vivo. On this basis, it is certainly feasible for PKA inhibition to occur in vivo. In fact, the enhanced rates of TG hydrolysis seen in adipose tissue of STZ-diabetic rats are indeed inhibited following vanadium treatment in vivo (Ramanadham et ai, 1989). Nevertheless, the work done so far can not exclude the possibility that vanadium also acts downstream from PKA, perhaps inhibiting TG hydrolysis through effects on other key regulatory proteins, namely HSL, perilipins or protein Ser/Thr phosphatases (Figure 1.2). Thus, vanadyl or vanadate, may inhibit HSL, may lead to dephosphorylation of perilipins or may lead to activation of protein Ser/Thr phosphatases. With regard to the last possibility, it has already been reported that vanadyl binds to protein phosphatase-2B, calcineurin, and leads to higher enzyme activity than calcium (Parra-Diazera/., 1995). Further support for the potential significance of PKA inhibition by vanadium in vivo is indirect. Thus, according to the glucose-fatty acid cycle proposed originally by Randle and co-148 workers, reduction in the release of free fatty acids (FFA) from adipose tissue could have a number of important consequences on other aspects of metabolism (Randle et al., 1966). For example, the reduced supply of FFA from adipose tissue would lead to enhanced glucose consumption by muscle cells, would diminish the substrate supply for very low density lipoprotein (VLDL) synthesis and release from the liver and would contribute to the reduction of gluconeogenesis and glucose export from the liver. All these metabolic changes are indeed seen in vanadium-treated STZ-diabetic animals. Overall then, the inhibition of PKA and TG hydrolysis in adipose tissue could contribute significantly to the overall metabolic effects of vanadium treatment in vivo. Of course, one anticipates that vanadium may have important effects on other tissues; whether these are also PKA-dependent is a major unresolved issue. Interestingly, vanadium treatment does lead to changes in expression of genes encoding key metabolic enzymes in the liver, including genes that are controlled at least in part by the cAMP/PKA pathway, including PEPCK and G6Pase (Marzban et al., 2002). It would be interesting to examine the possibility that vanadium might have more rapid and direct effects on the activities of these and other gluconeogenic enzymes. For example, the activation of hepatic gluconeogenesis depends, in part, on the PKA-dependent phosphorylation and inactivation of pyruvate kinase and phosphofructokinase-2. Acute vanadium treatment of hepatocytes may lead to the dephosphorylation (and activation) of these two enzymes and of the CREB transcription factor. In a broader sense, it would be very interesting to explore the effects of vanadium on expression of a wide range of genes, including those encoding key enzymes of other metabolic pathways that are insulin-sensitive as well as genes encoding proteins important in insulin signaling pathways. For this purpose, the best approach would be one of the DNA microarray techniques currently available. At the same time, and perhaps contingent on initial DNA array analysis, a screening of proteins expressed in vanadium-treated tissues could be most informative in pointing to changes that might not be otherwise predicted and that might provide evidence for or against the general significance of cAMP/PKA-mediated control. At the same time, it would be interesting to perform a parallel study to compare the effect(s) of other insulin-mimetic elements, discussed earlier (see Introduction), on PKA and/or TG hydrolysis. Another crucial consideration of the actions of vanadium in vivo is whether vanadium might also affect other protein kinases, especially considering the substantial conservation of structures of this large family of proteins. In this regard, some initial studies were carried out with two other protein Ser/Thr kinases, PKC and MAPK. The initial results obtained were 149 complex and it was decided that full optimization for each of these protein kinases would be required, as done for PKA, and that this was beyond the scope of the current studies. The effects of vanadyl on PKA may represent only part of the mechanism by which vanadium exerts its effects in vivo. For example, although the intracellular environment is highly reducing, it is very probable that other oxidation states of vanadium exist, including vanadate and peroxovanadium species. These, in turn, have the potential to exert profound effects on cells, including responses mediated by inhibition of PTPases. In this regard, it is becoming increasingly evident that cellular redox state can change dynamically in response to many cellular and environmental influences. Consequently, this could have a dramatic effect on the equilibrium between the various oxidation states of vanadium. For example, it has been suggested that insulin stimulates intracellular H2O2 production and that extracellular H2O2 can mimic insulin action. On this basis, it has been proposed that H2O2 may act as a "second messenger" for the observed effects of insulin (Hadari et al, 1993; Heffetz and Zick, 1989; May and De Haen, 1979a, 1979b; Wilden and Broadway, 1995; Zick and Sagi-Eisenberg, 1990). Insulin-induced production of hydrogen peroxide and subsequent production of peroxovanadium species might be especially significant because peroxovanadium is a potent inhibitor of PTPases (Cuncic et al, 1999; Huyer et al, 1997; Zhang et al, 1997). Interestingly, peroxovanadium species can be produced in vitro by the addition of either vanadate or vanadyl to H2O2 (Bevan et al, 1995; Fantus et al, 1989; Heffetz et al, 1990; Shankar and Ramasarma, 1993). Consequently, if insulin promotes rapid formation of H2O2 then this might lead to subsequent formation of peroxovanadium from endogenous vanadyl. In considering vanadium to be an "insulin-like", "insulin-mimetic", or "insulin-enhancing" agent, important qualifications must be kept in mind. First, a survey of literature (Table 1.2) suggests that not all vanadium actions occur with the same dose-dependency and low concentrations of vanadium might induce only a subset of cellular responses that would be induced by insulin itself. This is perhaps most obviously illustrated by the failure of vanadium treatment to restore overall rates of growth in STZ-diabetic rats (Cam et al, 2000). Is vanadium essential for normal insulin action? It is possible that vanadium plays important constitutive roles in normal physiology that are relevant to insulin action. This is obviously a hypothetical possibility but nevertheless it would be interesting to culture isolated mammalian cells under conditions that would lead to vanadium depletion. Assuming vanadium is not essential for cell viability then it would be possible to examine hormone responses in the absence of vanadium. 150 In conclusion, it is important to emphasize that many studies have provided evidence for an alternative mode of action of vanadium involving effects distal to the insulin receptor and PTPases. Current studies strengthen the concept that vanadium acts independently of the insulin receptor through downstream actions on PKA. 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