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Apparent induction, kinetic and physical properties of the multiple species of ornithine decarboxylase,.. Janzen, Andrea K. 1992-12-31

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APPARENT INDUCTION, KINETIC AND PHYSICAL PROPERTIESOF THE MULTIPLE SPECIES OF ORNITHINE DECARBOXYLASE,FORMS A AND B, IN THE KIDNEY AND LIVER OF HORMONE-TREATED RAT.byANDREA K. JANZENB. Sc. (Hon., Biochemistry) University of British Columbia, 1988A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMaster of ScienceinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF BIOCHEMISTRYWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAJuly 1992© Andrea K. Janzen, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(SignaturDepartment of ^F 1 ISTP(fThe University of British ColumbiaVancouver, CanadaDate DE-6 (2/88)ABSTRACTOrnithine decarboxylase occurs in the kidney and liver of control andhormone treated rats as two ionic forms, designated ODC A and B in order ofelution by DEAE-Sepharose chromatography. These studies were designedto better characterize the multiple forms of ODC and to investigate thefunctions and possible origins of ODC A and B in growth hormone, prolactinand/or dexamethasone stimulated tissue. The two species of ODC existed inseparate and distinct proportions in control rat kidney and liver. Within thekidney and liver of growth hormone, prolactin and dexamethasonestimulated rats, the proportions of ODC A and B were altered, reflecting theincreased half-life of ODC B. In growth hormone-stimulated animals, form Bhad an increased half-life of 25 minutes in the kidney and 60 minutes in theliver. Hormone stimulated rats were also given either LiC1, actinomycin D orputrescine, which decreased total ODC activity. The ODC A:B ratio wasaltered in some instances, but only within the kidney. In these cases, theenhanced proportion and most likely the stability of the B form of ODC wasinhibited or removed, as in the response of ODC to LiC1 in the stimulatedkidney. Within the liver, there was no alteration of the A:B ratio. Possibly,the liver uses different signalling or regulatory mechanisms. ODC B did nothave any kinetic advantage over A in either the kidney or liver of growthhormone-treated rats, as Km°rn and Vmax values did not differ significantlyiibetween forms A and B in either case. The charge separation of ODC A andB from the kidney and liver was shown to be dependent on the state ofphosphorylation based on the evidence that prior treatment of supernatantcontaining ODC with alkaline phosphatase resulted in the elution of activeODC A only by DEAE chromatography. Finally, ODC A and B did not differin molecular weight (-48kD) as seen on an SDS-PAGE gel. In growthhormone-treated rats, ODC showed complex responses in terms of activity orkinetics. Not only did the ratio of liver ODC A and B not respond to lithium,actinomycin or putrescine treatment, but there was evidence of multiplespecies of ODC within forms A and B having different kinetic and half-lifeproperties.iiiTABLE OF CONTENTSAbstract ^ iiTable of Contents ^  ivList of Tables  viList of Figures ^  viiAbbreviations  ixAcknowledgements ^ xiidedication ^INTRODUCTION ^ 1General Introduction1Pathway of Polyamine Biosynthesis ^4Cellular Regulation of ODC ^ 8Form of ODC in Mammalian Tissues 12Current Study ^ 14MATERIALS AND METHODS ^ 15Treatment of Animals 16Tissue Preparation ^ 17ODC Assay ^17Protein Determination 18Separation of the Ionic Forms of ODC ^  19Determination of ODC Half-Life In Vivo 20Alkaline Phosphatase Treatment of Tissue Extract ^ 21Enzyme Purification ^  21Step 1-DEAE Sepharose CL-6B chromatography  22Step 2-pyridoxamine-5'-phosphate Affigel chromatography . . . . , 22Determination of Km°rn and Vmax for Kidney ODC B, and Liver ODC Aand B ^ 24Immunoprecipitation of ODC ^ 24Electrophoresis of ODC  25Immunoblotting of the Gel26Staining of the Gel ^ 27ivRESULTS ^ 28Relative Proportions of ODC Activity in the Multiple Forms of ODCDuring Hormonal Induction ^ 28Relative Proportions of the Ionic Forms of ODC During a Decrease in theTotal Activity of ODC 34Half-Lives of Liver and Kidney ODC A and B In Vivo ^ 38Kinetic Properties of ODC A and B from the Liver of Growth HormoneStimulated Rat ^ 43Role of Phosphorylation in the Charge Separation of ODC A and BB. . ^ .52Physical Properties of ODC A and B from the Liver and Kidney of GrowthHormone Treated Rat57DISCUSSION ^  59SUMMARY 66REFERENCES ^ 78LIST OF TABLESTable 1Specific Activity of ODC and the Proportions of the Multiple Ionic Formsin the Kidney and Liver of Normal Rats and Rats Treated withGrowth Hormone, Prolactin or Dexamethasone. ^ 37Table 2Kinetic Properties of Purified Multiple Ionic Forms of ODC from Kidneyand Liver of Growth Hormone-Treated Rats. ^ 51viLIST OF FIGURESFigure 1The Polyamine Metabolic Pathway. ^ 6Figure 2DEAE Sepharose Chromatography of ODC Activity in Kidney fromNormal and Hormone-Treated Animals. ^  30Figure 3Deae Sepharose Chromatography of ODC Activity in Liver from Normaland Hormone-Treated Rats. ^ 32Figure 4Effect of Cycloheximide on the Two Major Ionic Species of ODC inKidneys of Growth Hormone-Treated Rats. ^  39Figure 5Effect of Cycloheximide on the Two Major Ionic Species of ODC in Liversof Growth Hormone-Treated Rats. ^  41Figure 6Eadie-Hofstee Graphical Analysis of Ornithine Kinetics for B Form ODCPurified from Kidneys of Growth Hormone-Treated Rats. ^ 44Figure 7Eadie-Hofstee Graphical Analysis of Ornithine Kinetics for A Form ODCPurified from Livers of Growth Hormone-Treated Rats. ^46Figure 8Eadie-Hofstee Graphical Analysis of Ornithine Kinetics for B Form ODCPurified from Livers of Growth Hormone-Treated Rats. ^48Figure 9Effect of Alkaline Phosphatase on the Two Major Ionic Species of ODCfrom Kidneys of Growth Hormone or Growth Hormone plus LiC1Treated Animals.  53Figure 10Effect of Alkaline Phosphatase on the Two Major Ionic Species of ODCfrom Livers of Growth Hormone or Growth Hormone plus LiCI-Treated Rats. ^ 55viiFigure 11Electrophoresis of the Multiple Species of ODC on SDS-PAGE Gels. . . .58viiiABBREVIATIONSADOMET^S-adenosyl-L-methionineADOMETDC^S-adenosyl-L-methionine decarboxylaseAlkPase^alkaline phosphataseAMP adenosine monophosphateATP^adenosine triphosphateBCIP 5-bromo-4-chloro-3-indoyl phosphateBSA^bovine serum albuminDEAE diethylaminoethylDEX^dexamethasoneDFMO alpha-difluormethylornithineDNA^deoxyribonucleic acidDTT dithiothreitolEDTA^ethylenediaminetetra-acetic acidGH growth hormoneHepes^[4-(2-hydroxyethy101-piperazine ethanesulfonic acidIP^intraperitoneallyKmorn^substrate binding constant for ornithineKCL^potassium chloridekD kilodaltonsKI-12PO4^potassium phosphate, monobasicix1^litersLiC1 lithium chloridemCi^millicuriemg milligramml^millilitermM millimolarmmol^millimolemRNA^messenger ribonucleic acidMTA 5'-methylthioadenosineNaCl^sodium chlorideNaN3 sodium azideNBT^nitro blue tetrazoliumNa2HPO4^sodium phosphate, dibasicnmol^nanomoleODC ornithine decarboxylaseORN^ornithinePAGE polyacrylamide gel electrophoresisPLP^pyridoxal-5'-phosphatePMSF phenylmethylsulfonylfluoridePO^polyamine oxidasePoly A^polyadenylatedpmol picomolePRL^prolactinPUT putrescinerRNA^ribosomal ribonucleic acidSAT spermine/spermidine-5'-acetyltransferaseSDS^sodium dodecyl sulphateSPD spermidineSPM^spermineTCA trichloroacetic acidTris-HC1^tris(hydroxymethyl)aminomethanehydrochlorideTPA^12-0-tetradecanoylphorbol 13-acetatetRNA transfer ribonucleic aciduCi^microcuriesug microgramul^microliteruM micromolarxiACKNOWLEDGEMENTSI would like to thank all of those in and out of the biochemistrydepartment that aided in the production of this thesis-there are too many toname so I won't even begin to start (you know who you are). Specialacknowledgement goes to Dr. J. F. Richards for his guidance, support andpatience through it all. Finally, a thank you to Dr. Cullis for allowing me theextensive use of his computers."That which does not kill you, makes you stronger"NietchzexiiTo Mom and Jen,for making this possible.INTRODUCTIONGENERAL INTRODUCTIONOrnithine Decarboxylase (L-ornithine carboxyl-lyase, E.C., ODC) is the initial and rate limiting enzyme in the polyaminebiosynthetic pathway (1-4). The products of the pathway, putrescine (PUT),spermidine (SPD), and spermine (SPM) are normal constituents ofprokaryotic and eukaryotic cells. At physiological pH, PUT, SPD and SPMare protonated and possess 2, 3, and 4 positive charges respectively and thusare known as the organic cations of the cell. Polyamines have a variety ofphysiological and biochemical effects and have been shown to play a role inmembrane function, proliferation and differentiation (5, 7). By virtue of theircharge, they can complex with nucleic acids to provide conformationalstability. Spermine has been shown to bind to the DNA helix, spanning themajor or minor groove and stabilizing it (8). There has also been evidence ofPUT, SPD and/or SPM binding to and stabilizing the stem-loop structures inrRNA and mRNA; further, tRNA conformation is stabilized through bindingof polyamines to specific sites (9). Additionally, they can influence theconformation of structural proteins as well as the activity and location ofenzymes. Spermine and spermidine were shown to induce the1polymerization of spectrin and dramatically decreased the lateral diffusionsof transmembrane glycoproteins (10). Polyamines can also affect the activityof certain enzymes involved in phosphorylation and dephosphorylation, suchas protein kinase C, casein kinase II and alkaline phosphatase. Finally, byassociation with phospholipids, polyamines can affect the cellular plasmamembrane and influence the action of enzymes in the endoplasmic reticulum(11). Polyamines affect the activity of membrane bound cholinesterase andare shown to decrease the hydrolytic activity of phospholipases A2 and C onmitochondrial membranes (10). Submillimolar concentrations of polyaminescan stabilize spheroplasts and mitochondria against osmotic shock andinduce the aggregation of subcellular organelles (11). It has been welldocumented that polyamines are essential for cell growth (12-14).Specifically, putrescine has been shown to be a growth factor for bothprokaryotes and eukaryotes and has been demonstrated to be conjugated todiscrete nuclear proteins intracellularly (15-18). Spermidine has been shownto increase the rate of chain elogation of DNA and RNA, increase proteinsynthesis and to be a specific translation factor for fidelity of proteinsynthesis (19-23). Finally, spermine increases the efficiency of acylation oftRNA, a process required in protein synthesis (24, 25).The physiological functions of cells vary widely in response to stimuli;however, in any stimulated cell, a large and rapid increase in the activity ofODC, which is then followed by an increase of the polyamines, is almostalways part of the initial response. This has been observed in tissuesresponding to stimuli as varied as growth factors, viral infections, mitogens2and foreign toxic substances (26-30). The increase in ODC activity resultsmainly from an increase in the amount of ODC protein and in the half life ofthe induced ODC. The amount of the ODC protein is increased primarily byincreased transcription of the gene, along with increased translation of themRNA (4, 7, 31). The relative contributions of the increases in translationand transcription seem to be stimulus specific. Additionally, in somesystems, induced ODC protein has been found to be more stable (32, 33).It is now well documented that ODC is present in mammaliantissue, in two ionic forms which are separable by ion exchangechromatography. These two forms have been observed in rat liver, kidney,heart, and thymus, mouse kidney and HTC cells under varied stimuli,including steroids and heavy metals (34-40). The two forms are named ODCA and ODC B in order of elution from DEAE Sepharose column. Evidencesuggests that these ionic forms are not artifactual (41). There has been muchspeculation as to the origin of these two species. In theory, since ODC hasbeen shown to be a multigene family (42-45), these two forms could beseparate gene products with the charge difference of the proteins attributableto differing amino acid content. Conversely, the gene transcripts may notdiffer and instead, differences between the two species could depend on apost-translational modification of the protein. While the regulation of ODChas been extensively investigated at the molecular level, the connectionbetween the regulation of ODC and the form in which it occurs inmammalian tissues has not yet been defined. The physiological significanceof ODC A and B and their origin are major interests in the field of3polyamines. If their occurrence is biologically important, it might beexpected that stimuli which affect total ODC activity would also affect theproportions and/or properties of ODC A and B. Properties of ODC directlyinvolved with its stability or kinetic function would most likely be associatedwith changes in cell activity. Additionally, the physical properties of theenzymes which cause the interconversion of the two species of ODC couldalso be related to a specific stimulus. Thus, changes in post-translationalmodifications, such as phosphorylation, or in size of the ODC monomer couldprovide the basis for the charge separation of ODC A and B.PATHWAY OF POLYAMINE BIOSYNTHESISODC, the initial enzyme in the polyamine biosynthetic pathway(see figure 1), has the shortest half life, in the range of 10-20 mins (46), of anyof the enzymes involved in the polyamine biosynthesis; indeed, it has theshortest half life recorded for any known mammalian enzyme. ODC is alsothe most highly inducible enzyme involved in polyamine biosynthesis and assuch the activity of this enzyme mainly determines the activity of thepathway. ODC is thus the rate limiting enzyme of this pathway (46).Decarboxylation of the amino acid ornithine by ODC results in the formationof putrescine, a diamine. Aminopropyl groups, donated by decarboxylated S-adenosyl-L-methionine (ADOMET), are subsequently added to the diamine,4by spermidine and spermine synthase, to give spermidine and spermine. Thedecarboxylation of ADOMET is catalyzed by ADOMET decarboxylase. Thisreaction is rate limiting in the synthesis of spermidine and spermine. Thechanges in ADOMET decarboxylase activity that occur after stimulus areusually similar to, but less than, those of ODC. Although the half life of S-adenosyl-L-methionine is longer than that of ODC, it is still much shorterthan that of the spermidine and spermine synthases. These synthasesexhibit considerably higher activities and longer half lives, so it is unlikelythat they are involved in the control of polyamine synthesis (47). Thereactions forming spermidine and spermine are effectively irreversible butspermidine and spermine can be converted back to putrescine by thepolyamine acetylation/oxidation pathway which involves the two enzymesspermicline/spermine-N1-acetyltransferase and polyamine oxidase. Theactivity of the polyamine oxidase appears not to change significantly inresponse to growth stimulus, although the conversion of spermine tospermidine and spermidine to putrescine appears to be enhanced in tissuesundergoing rapid proliferation (47).5Figure 1: The Polyamine Metabolic Pathway:ODC, ornithine decarboxylase; PUT, putrescine; SPD,spermidine; SPM, speini,ine; PO, polyamine oxidase; SAT,spermidine/spermine-N 1-acetyltransferase; ADOMET, S-adenosyl-L-methionine; ADOMETDC, S-adenosyl-L-methioninedecarboxylase; MTA, 5'-methylthioadenosine67CELLULAR REGULATION OF ODCThe complexity of the regulation of ODC would appear to berelated to the physiological importance of the polyamines. Besides beingpresent as only 0.00014% of soluble cellular protein in thioacetamidestimulated liver (48), ODC also has the shortest documented half life (10-20min) (46). As ODC is tied intimately with cell growth, its activity can changerapidly in response to a wide range of stimuli which affect cell growth, suchas tumor promoting agents, viral transformations, hormones and mitogens(26-30). ODC is rapidly induced, beginning within 2-3 hours of treatment inrat tissues, and increases in ODC activity of 5-500 fold have been reported(49-51). In stimulated systems, studies with a radiolabelled suicide substrateinhibitor of ODC, known as DFMO (difluoromethylornithine), have shownthat the increased ODC activity is mainly due to concomitant increases in theamount of ODC protein (52-54). Also seen in androgen stimulated mice andgrowth hormone stimulated rats, subsequent treatment with cycloheximide,an inhibitor of protein synthesis, prevented ODC induction (55, 56).Therefore, the accumulation of ODC protein seems to depend on an increasedrate of synthesis. The increased rate of ODC synthesis can result fromstimulation of the transcription of the gene and/or stimulation of translationof mRNA and this is dependent on the stimulus used. Increased gene8transcription has been observed in androgen stimulated BHK cells, withoutconcomitant increase in mRNA levels (57). On the other hand, in rathepatoma cells stimulated with TPA, increases in ODC activity could beblocked by actinomycin D, an inhibitor of mRNA synthesis (58). Also, thestability and the efficiency of translation of the ODC mRNA may beincreased, as can be seen in serum stimulated mouse S49 cells and in thekidneys of testosterone stimulated mice (94). In addition to the increase inprotein concentration, the increased ODC activity also depends on theincrease in the stability of the ODC protein, as shown by an increase in halflife. This prolonged stability has been shown in many systems, including ratkidney stimulated with dexamethasone and mouse kidney stimulated withandrogen (32, 36, 37, 59, 60).Because of its important role in cell growth, ODC also has anetwork of negative regulatory factors which affect it. Besides the inherentcontrol of its extremely short half life, there are other negative effectorspresent at almost every level of ODC synthesis. At the primary level,transcription of the gene itself can be suppressed. Additionally, it has beenshown that there are binding sites for regulatory factors on the ODC mRNA.The most important of the regulatory factors are most likely the polyaminesthemselves. Normal polyamine concentrations vary between types of cellsand tissues, but have been observed in the micromolar to millimolar range.At micromolar concentrations, the polyamines are able to depress ODCsynthesis by selectively inhibiting the translation of the ODC mRNA (61, 62).9Also, the addition of polyamines to HTC cells was shown to induce anoncompetitive protein inhibitor of ODC, which was termed antizyme. Thisprotein binds to ODC, directly inhibiting its activity (63). In the complex, theODC molecule seems more susceptible to degradation. Antizyme to ODC hasnow been induced in a variety of cells, including fibroblasts, nerve andhepatic cells, as well as in thyroid and plant cells (63). Another possible wayto regulate ODC is through phosphorylation of the enzyme. A polyaminedependent protein kinase, identified in the slime mold Physarumpolycephalum, was capable of the phosphorylation and subsequentinactivation of ODC (64). However, this polyamine dependent kinase has notbeen found in mammalian tissues.HORMONAL INDUCTION OF ODCODC induction exhibits a remarkably constant pattern ofexpression in response to stimulation with hormones even though theyinteract with cells by very different mechanisms. With most hormonestimulated rat tissues, the time course of ODC induction is quite similar;once stimulated, ODC activity is rapidly induced to a maximum valueapproximately 4-6 hours post stimulation, whereupon the activity diminishesto control values by 8 hours (65-67). Induction of ODC is related to theamount of hormone in a dose dependent manner. One of the first suchhormones to be studied extensively was growth hormone (GH). Of related10interest is the similar polypeptide, prolactin (PRL). The majority of studiesindicate that new rRNA synthesis is required for significant increases inprotein synthesis in response to trophic hormones, such as GH and PRL (68-70). Most of these hormones induce ODC at least in part by a cyclic AMPdependent mechanism (71). Trophic agents that elevate cyclic AMPconcentration also promote an elevation in de novo synthesis of ODC (71). Inmany of these systems, the addition of cyclic AMP analogs and/orphosphodiesterase inhibitors to tissues and cells elevates ODC activity (72-76). More recent evidence indicates that the phosphatidylinositol pathwayalso has a role in the hormonal induction of ODC. Increases in inositolphosphates have been shown to precede the increase in ODC activity in somesystems. Additionally, LiC1, primarily an inhibitor of the phophatidylinositolpathway through its uncompetitive inhibition of inositol phosphatephosphatase (108), has been shown to decrease ODC activity (92). In themajority of these animal and cell systems, ODC has been shown to betranscriptionally and translationally regulated as determined by the effectsof actinomycin D and cycloheximide on its activity (72-76).Another group of hormones having well documented effects on ODCare the steroids. As with the trophic hormones, early stimulation of ODC inthe tissue is related to the steroid in a dose dependent manner (71, 77).Steroids elicit response, not by cyclic AMP or phosphatidylinositol mediatedsignals but by interaction with high affinity cytosolic receptors. Thehormone-receptor complex translocates to the nucleus to directly affect genetranscription (78). However, the effects of the steroids can be expressed at11the transcriptional, translational or both, levels in the regulation of ODC (57,77, 79). This is in marked contrast to the effect of the trophic hormones,which regulate ODC by both transcriptional and translational stepsregardless of the system involved (80). Although the trophic hormones andthe steroids regulate ODC through different avenues, in both systems ODCexists in the two ionic forms, A and B.FORM OF ODC IN MAMMALIAN TISSUESRegulation of ODC has been well characterized at the molecularlevel in terms of ODC synthesis and stability. However, the form in whichODC exists in mammalian tissue has not yet been associated with aregulatory function. In mammalian tissues, ODC exists in two forms;isozymes, with differing pI's. These two forms are named ODC A and ODC Bin the order of their elution in DEAE Sepharose chromatography. Initiallyseen in the liver of dexamethasone-treated calf and in the liver ofthioacetimide-treated or partially hepatectomized rat, multiple species ofODC have now been observed in 3T3 cells, in the kidney of testosteronestimulated mouse, in the liver of chloroform, maleate or cobalt treated ratand in the heart of isoproterenol treated rat (34, 35, 37-39, 81-83). Theoccurrence of two forms is not confined to stimulated tissue; they have alsobeen observed in the heart, liver, thymus and kidney of untreated rats (34,1235, 37-39, 81-83) . Since these forms are present even with the additon of 8Murea and are eluted from Fast Protein Liquid Chromatography after littlepretreatment of cellular cytosol, it is unlikely that the ionic forms are a resultof nonspecific binding of ODC with other substances or nonspecific proteindegradation in the tissue preparations (41). Additionally, ODC A and Bisoforms from every source examined to date have had the same molecularweight and cannot be distinguished on a SDS denaturing gel (71). Animaltissues usually contain 2-3 species of ODC mRNA, but when isolated, thesemRNA have only differed at their poly A sites or the 5' untranslated regionand so are probably not responsible for the charge disparity of ODC A and B(84-86). Finally, the active ODC gene itself has been sequenced and appearsto contain only one start sequence for transcription (86, 109, 110).Further, there has been some evidence that the charge disparitybetween A and B may be due to phosphorylation. Native ODC has beenisolated from Friend murine erythroleukemia cells as a phosphoprotein andappears to be predominantly phosphorylated the single serine residue, ser303 (87, 88). Additionally, ODC A isolated from testosterone treated mousekidney can readily be labelled by 32P-ATP when incubated with caseinkinase II in vitro. However, ODC B, the more acidic form, cannot accept 32Plabel without prior alkaline phosphatase treatment (89).13CURRENT STUDYThis study was undertaken primarily to better characterize themultiple forms of ODC and to investigate the origins and possible functions ofODC A and B. The experiments reported here were designed with threemajor objectives;1) to establish patterns of expression of the two forms of ODC in theliver and kidney of control rats and of animals treated with dexamethasone,prolactin or growth hormone, to see if their proportions were changed onstimulation. The effects of agents which were known to alter the level ofODC activity in tissue of hormone treated rats, eg actinomycin D, putrescineand LiC1, were also used to define any possible changes of A and Bisoformsduring a decrease in ODC activity.2) to compare some kinetic and physical properties of ODC A and Bisolated from hormone stimulated tissue to see if change in proportion of Aand B is associated with a change in properties of ODC.3) and finally, to investigate the occurence of phosphorylation in theorigin of the charge disparity of ODC A and B in hormone stimulated tissue.14MATERIALS AND METHODS[1-14Q-L-ornithine hydrochloride was obtained from AmershamCorporation (Oakville, Ontario). DEAE Sepharose CL-6B was purchasedfrom Pharmacia Fine Chemicals (Dorval, Quebec). Affigel 10, Bradford dye,silver stain reagents, molecular weight standards and any otherelectrophoreisis reagents were bought from Biorad Laboratories (Richmond,California). L-ornithine hydrochloride, pyridoxa1-5'-phosphate,pyridoxamine-5'-phosphate, dithiothreitol, dexamethasone, actinomycin D,putrescine, lithium chloride and cycloheximide were obtained from Sigmachemical Company (St. Louis, Missouri). BCIP (5-bromo-4-chloro-3-indoylphosphate), NBT (nitro blue tetrazolium), prestained high molecular weightstandards, alkaline phosphatase conjugated goat anti rabbit immunoglobinsand any other immunological reagents were obtained from BethesdaResearch Laboratories.15TREATMENT OF ANIMALSImmature female Wistar rats, age 4-8 weeks and adult male CD-1 micewere obtained from Animal Care Unit (U.B.C. strain) and Canadian BreedingFarm (Charles River, Quebec). Animals were kept in a 12 hr dark/light cycleroom for 48 hrs prior to experiment and were given standard laboratory chowand water ad libitum. Dexamethasone, dissolved in ethanol at 2 mg/ml wasadministered by intraperitoneal injection as a suspension in 500u1 of 0.9%(w/v) NaCl. Each rat, assuming an average body weight of 80 g, received 200ug of the hormone. Both growth hormone (bovine or ovine) and prolactin(bovine) were dissolved in saline and adjusted to pH 8.2-8.3 with sodiumbicarbonate prior to IP injection. Each animal received 500 ug of GH or PRLin a volume of 500 ul. Rats were killed 5 hrs after treatment with eitherdexamethasone, growth hormone or prolactin. Other hormone treated ratswere additionally treated at hour 4 with either LiC1 (5 umol/g in 500 ulsaline), actinomycin D (2.5 mg/kg in 500 ul saline) or putrescine (125 mg/kgin 500 ul saline) and then killed at hour 5 post hormone. Testosteronepropionate in saline (100 mg/kg) was administered by subcutaneous injectionand the mice were killed 24 hrs later. Control animals were untreated. Ratsand mice were killed by cervical dislocation following brief exposure to CO2.16TISSUE PREPARATIONAfter killing, the kidney and or livers were removed immediately fromthe animals, quick frozen in liquid nitrogen and stored at -70°C. Prior toexperiment, tissues were partly thawed and homogenized in ice-cold buffer A(3.5 ml/g tissue for kidney or 4 ml/g tissue for liver). Buffer A contained 50mM Hepes, 3 mM dithiothreitol, 0.1 mM EDTA and 0.1 mM PMSF at a pH of7.3 at room temperature. Homogenization was performed using a Potter-Elvehjem homogenizer with the pestle being driven at approximately 600rpm during the 6-8 strokes. The homogenate was kept on ice throughout thisprocedure. The resulting kidney homogenate was centrifuged at 20000 xgavat 4°C for 20 minutes. The resulting liver homogenate was centrifuged at100000 xgau at 4°C for 35 min. After centrifugation, the supernatant wasdecanted and used in subsequent experiments.ODC ASSAYAliquots of the supernatant fractions were used for assay of ODC. TheODC activity was measured by a slight modification of the previous method(67). The reaction mixture, in a final volume of 400 ul, contained pyridoxalphosphate (0.2 mM) and L-ornithine (0.25 mM containing 0.150 uCi L-1- 14CORN (58 mCi/mmol)). All assay tubes were kept on ice until starting thereaction with the addition of enzyme. In the assay 14C-0O2 was collected in17200 ul hyamine hydroxide following acidification of the reaction mixture with2 M citric acid. All assays were carried out in duplicate or triplicate and anaverage value was used in data analysis. One unit of enzyme activity wasdefined as 1 pmol CO2 released per 30 min incubation at 37°C and specificactivity was expressed as units of activity per mg protein.PROTEIN DETERMINATIONProtein concentration was determined by the method of Bradford (90).A standard curve for the protein assay based on BSA was also done withevery set of samples.18SEPARATION OF THE IONIC FORMS OF ODCA settled volume of 40 mis of DEAE Sepharose CL-6B suspended inbuffer B (50 mM Hepes, 3 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 150 mMNaC1, pH 8.0 at room temperature) was degassed for 30 min. After decantingthe fines, the gel was packed in a Pharmacia column (1.5x28 cm) to a heightof 23 cm. The column was then moved to a 4°C cold room and washed with 4volumes of buffer. Supernatant prepared from either the kidneys or livers oftreated animals was loaded directly and the column washed for at least 20x5ml fractions with buffer B at a flow rate of approximately 35 ml/hr. Thecolumn was then eluted with a linear gradient composed of 200 mis of bufferB and 200 mis of buffer C (50 mM Hepes, 3 mM DTT, 0.1 mM EDTA, 0.1 mMPMSF, 250 mM NaC1, pH 8.0 at room temperature) with an additional170x2.5 ml fractions being collected. Aliquots of the eluent were taken fromevery second fraction for assays of ODC activity. If ODC A and B wereneeded for further experiments, enzyme activity of each ionic form wasseparately pooled and concentrated by ultrafiltration under a nitrogenpressure of 50 psi with a Diaflo YM10 membrane. Just prior toultrafiltration, Brij 35 was added to the pooled enzyme to a finalconcentration of 0.02% in order to stabilize the enzyme. All ultrafiltrationprocedures were carried out at 4°C.19DETERMINATION OF ODC HALF LIFE IN VIVOCycloheximide (25 mg/kg) was administered intraperitoneally in 500 ulof saline to rats given growth hormone 5 hrs previously. At intervals of 15,30 and 60 minutes post cycloheximide, the animals were killed and the liversand kidneys removed. Subsequently, DEAE elution profiles of the ODC inthe rat liver and kidney were determined. Groups of 3-6 rats were sampledand the tissues pooled for DEAE chromatography. After chromatography,ODC A and B were pooled separately and concentrated by ultrafiltration.ODC activity was then assayed in the ODC A and B samples. This activitywas expressed as a percentage value of the activity measured in similarlytreated samples of ODC A and B from rats not treated with cycloheximide.These activites were correlated with the duration of cycloheximide treatmentand graphed on semilog plot. Enzyme decay is exponential in nature andthus to interpret the data, a semilog graph had to be used. Any deviationsfrom first order rate of decay on semi-log plot will be observed as a curve.20ALKALINE PHOSPHATASE TREATMENT OF TISSUE EXTRACTThe supernatant from liver and kidney of growth hormone and growthhormone + LiC1 treated rats was prepared as described earlier. To theresulting supernatant, alkaline phosphatase (final concentration of 200Units) and magnesium chloride(final concentration 1.0 mM) were added. Themixture was then incubated in a slowly shaking water bath at 37°C for 30minutes. Subsequently, DEAE profiles of the ODC in the samples wasdetermined. Separation or inhibition of the alkaline phosphatase from thesupernatant prior to chromatography was not necessary as it eluted from thecolumn within the first five fractions collected i.e. void volume.ENZYME PURIFICATIONODC A and B from the livers and kidneys of rats treated with growthhormone for 5 hours were partially purified prior to kinetic studies. ODCwas purified by two main steps; DEAE Sepharose CL-6B chromatographyand pyridoxamine-5'-phosphate Affigel chromatography.21Step 1-DEAE Sepharose CL-6B chromatographyDEAE chromatography was performed as described earlier. Thetissues from 10 rats were pooled for ODC purification. Both ODC A and ODCB were pooled individually from the column and concentrated to a volume ofapproximately 10 ml for Affigel chromatography.Step 2-pyridoxamine-5'-phosphate Affigel chromatographyThe affinity gel was prepared by adding 110mg pyridoxamine-5'-phosphate hydrochloride to 25 ml of Affigel 10 in 0.10 M sodium phosphatebuffer, pH 7.0. The coupling reaction was allowed to proceed forapproximately 24 hr at 4°C with rotational mixing. The unreacted groups onthe agarose were then coupled with 1 M ethanolamine for an hour and the gelwashed extensively on a buchner funnel with 1 M NaC1 in 0.01 M sodiumphosphate buffer, pH 7.0. The gel was then degassed for 30 minutes andpacked into a column of 1.5 cm diameter to a height of 15 cm. The columnwas washed with 5 volumes of buffer D (50 mM tris-HC1, 3 mM DTT, 0.1 mMEDTA, 10 mM NaC1, 0.1 mM PMSF, 0.02% Brij 35, pH 7.3) at a flow rate of30 ml/hour. ODC A or ODC B from previous DEAE chromatography wasloaded directly onto the column and washed with buffer D for 30x5 mlfractions. The ODC activity was then eluted from the column with buffer E(50 mM Tris-HC1, 3 mM DTT, 0.1 mM EDTA, 10 mM NaCl, 0.1 mM PMSF,220.02% Brij 35, 50 uM PLP, pH 7.3) at the same flow rate and an additional80x5 ml fractions were collected. Aliquots were taken from every secondfraction for the determination of ODC activity. All Affigel chromatographywas done at 4°C. The ODC activity was pooled and further concentrated to avolume of approximately 3 mls by ultrafiltration as stated earlier, butwithout any further addition of Brij 35.The buffer of the resulting partially purified ODC was exchanged forbuffer K (50 mM Hepes, 3 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 0.02%Brij 35, 50 mM NaC1 pH 7.3) by elution through a Biorad ECONO-PAC 10DGdesalting column which had been washed with buffer K. The resultingvolume of the partially purified ODC was approximately 10 mis.23DETERMINATION OF Km°rn AND Vmax FOR KIDNEY ODC B, ANDLIVER ODC A AND BAliquots of 100u1 from the resulting partially purified ODC wereexamined for ODC activity in the presence of 5, 10, 25, 50, 75, 100, 250 and500 uM ornithine. These assays were done in duplicate and at two differentsalt concentrations (50 mM and 150 mM NaC1) in order to observe anykinetic differences due to possible changes in subunit structure due to affectsof NaC1 on binding of substrate or possible dimerization of ODC monomers.All other parameters for the ODC assay were as stated previously. Theexperimental values for Km°rn and Vmax were determined by Eadie-Hofsteeplot (111).IMMUNOPRECIPITATION OF ODCTo further concentrate ODC for gel electrophoresis, samples of kidneyODC A and B and liver ODC A and B from the desalting column eluent wereprecipitated using an anti-ODC antibody that had been previously prepared.This polyclonal antisera had been raised in a rabbit host using purified ODC24from the kidneys of testosterone stimulated mice as the antigen. Aliquots of200 ul of ODC (semipurified by DEAE Sepharose chromatography) werecombined with 100u1 of a 1/5 dilution of rabbit anti ODC serum in anEppendorf tube.. This was gently shaken for 30 minutes at 4°C. A 450 ulaliquot of 1:2 suspension of protein A Sepharose CL-4B in buffer A was addedand the mixture was shaken for another 30 minutes at 4°C. The suspensionwas washed with 1.0 ml of buffer A and spun briefly in an Eppendorfcentrifuge to collect the protein A Sepharose CL-4B immunocomplex. Thiswash was repeated 2-3 times. The protein A Sepharose Cl-4B was eluted bythe addition of 500u1 of buffer S (0.58% glacial acetic acid, 0.15 M NaC1) andthe suspension was spun once again to collect the supernatant. Thesupernatant was treated with 250 ul TCA(30%) and 100 ul 0.1% BSA andspun to collect the resulting precipitate. The precipitate was washed oncewith 500 ul of ethanol and dissolved in PAGE sample buffer.ELECTROPHORESIS OF ODCThe molecular weights of kidney ODC B, liver ODC A and B wereestimated by electrophoresis using a discontinuous polyacrylamide gel(PAGE). The method used was that of Laemmli (91) with some modifications.The sample buffer (63 mM Tris-HC1 pH 6.8, 10% glycerol and 0.01%bromophenol blue) was not made with any beta-mercaptoethanol, as anyreducing agent in the sample buffer seemed to interfere with later transfer of25the proteins to nitrocellulose. All gels were run using the BioRad MINI-PROTEAN II DUAL SLAB CELL system. All gels were done in duplicate sothat protein could be detected by both antibody and protein staining methods.IMMUNOBLOTTING OF THE GELAfter the gel had been developed, it was immunoblotted ontonitrocellulose using the BioRad MINI TRANS-BLOT ELECTROPHORETICTRANSFER CELL. The method was as stated in the BIORAD TRANS-BLOTinstructions. The transfer buffer was 25 mM Tris, 192 mM glycine and 20%(v/v) methanol. Transfers were done at 150 mA constant current for 1 hr.The nitrocellulose was then incubated with 5% bovine serum albumin in KBSbuffer (137 mM NaCl, 1.5 mM KH2PO4, 7.2 mM Na2HPO 4, 0.02% NaN3, 2.7mM KC1, 0.05% Tween 20) while gently shaking for 1 hour at roomtemperature or overnight at 4°C. The blot was then washed 3 times withKBS and further incubated with 25 mis of a 1/500 dilution of rabbit ODCantibody in KBS, with gentle shaking for 1 hour. The blot was again washed3-5 times with KBS and finally incubated with 1/3000 dilution of alkalinephosphatase conjugated to anti-rabbit antibody in KBS for an additional hourwith gentle shaking. The blot was washed a final 3-5 times with KBS and10mls of alkaline phosphatase substrate buffer (100 mM Tris pH 9.5, 100 mMNaC1, 5 mM MgC12) with NBT and BCIP substrate were added as per BioRad26IMMUNO-BLOT kit instructions. The blot was left in the substratecontaining buffer until color development occurred. The reaction wasterminated by extensive washing with distilled water.STAINING OF GELAfter electrophoresis, the gel was fixed and stained for protein usingthe BioRad SILVER STAIN kit. The stained gel was placed between twoacetate sheets which had previously been thoroughly wetted with distilledwater and then air-dried.27RESULTSRELATIVE PROPORTIONS OF ODC ACTIVITY IN THE MULTIPLEFORMS OF ODC DURING HORMONAL INDUCTIONPreviously, it has been demonstrated that multiple forms of ODC,which are separable by DEAE Sepharose chromatography, exist inmammalian tissue. In each case, A is designated as the form eluted earlier inthe gradient, and B is the more acidic. Thus, these experiments were firstdirected towards establishing the patterns of appearance of ODC A and B intissues of control and hormone treated rats. The data presented in figures 2and 3 demonstrate that ODC A and B occur in distinct, specific proportions inrat liver and kidney; and further, these proportions are changed when the ratis treated with different hormones. In each case, with the treatment ofhormone, the activity of ODC rapidly increases to a maximum of 8X controlfor rat kidney and 15X control for rat liver (see table 1). The specificactivities observed are in agreement with previous studies (65, 66, 67, 71).ODC A and B are both induced in response to hormone treatment, but notequally. The change in proportion of ODC A and B varied with hormonetreatment and tissue. In figure 2, the ratio of ODC A:ODC B in control rat28kidney is seen to be 50:50. However, in the kidney of rat treated with GH,PRL or DEX for 5 hrs, the pattern has been shifted to an ODC A:ODC B ofapproximately 40:60. The proportions in liver are somewhat different (fig 3).In control rat liver, the ODC A:ODC B ratio is 70:30, notably different fromcontrol kidney. Additionally, the response of the ODC A:ODC B ratio tohormonal stimulation differs in the liver. In rat treated for 5 hrs with DEXor PRL, the ODC A:ODC B ratio is shifted to 60:40. In the liver of ratsstimulated with GH, the ODC A:ODC B ratio is affected to a greater degreewith a shift to 50:50. Even though the ODC A:ODC B ratios differed betweenkidney and liver and to some extent, hormone treatment, in each case whereODC activity was induced, ODC B activity was increased to a greater degreethan ODC A activity. Each of the represented DEAE Sepharose profiles wasrepeated at least 3 times, with a minimum column efficiency of 75%. Theonly exception was that of the control kidney and liver which had the lowercolumn efficiencies of 65 and 60%. As ODC is so unstable in theunstimulated tissue, attempts to obtain higher column efficiencies wereunfeasible.29Figure 2: DEAE Sepharose Chromatography of ODC Activity inKidney from Normal and Hormone-Treated Animals: Kidneyextracts, prepared from 3-5 a) normal, b) growth hormone-treated, c) prolactin-treated and d) dexamethasone-treatedanimals were applied to DEAE Sepharose CL-6B columns andthe different forms of ODC separated as described in Materialsand Methods. The hormone treatment used was also asdescribed in Materials and Methods.30a)contni...1\11b)5\‘‘,hr GHa) 5 hr PRLcl) 5hr DV1iIa4§025002000■i 1500I 1000§500III'4§1II1Z§0000 50Figure 3: DEAE Sepharose Chromatography of ODC Activity inLiver from Normal and Hormone-Treated Rats: Liver extracts,prepared from 3-5 a) normal, b) growth hormone-treated, c)prolactin-treated and d) dexamethasone-treated animals wereapplied to DEAE Sepharose CL-6B columns and the differentforms of ODC separated as described in Materials and Methods.The hormone treatment used was also as described in Materialsand Methods.325003:i 4000 —■aI1 2000 -.2§ moo-IiI§I.i§I,iiI:-_-__--__-_-__300,, 20010003000250020001500100050001000750500250°antralaRELATIVE PROPORTIONS OF THE IONIC FORMS OF ODC DURING ADECREASE IN THE TOTAL ACTIVITY OF ODCSince the patterns (ratios) of ODC A:B isoforms were established in thecontrol and hormone stimulated tissues, other agents which are known toaffect ODC activity were also injected in vivo to observe their effects on thedistribution of isoforms A and B. Three agents which appear to act bydiffering mechanisms were chosen: actinomycin, an inhibitor of RNAsynthesis; LiC1, an inhibitor of the phosphatidylinositol and other signaltransduction pathways; and PUT, a natural negative feedback inhibitor ofODC which acts through stimulation of antizyme and repression oftranslation of ODC mRNA. Each of these agents were given 1 hour beforekilling the rats treated 4 hrs earlier with either GH, PRL or DEX. Previousexperiments have shown the effect of these agents on ODC activity underthese conditions (92, 71) and the results illustrating the effects of theseagents in the present study are seen in table 1.LiC1 had the same overall effect in opposing the effects of each of thethree hormones on kidney ODC. The specific activity of ODC was decreasedto approximately half of the value observed in hormone stimulated tissue andthe A:B ratio reverted to the ratio observed in control kidney (50:50).Actinomycin D also decreased the ODC specific activity, which had previouslybeen increased by each of the hormones, by at least 40%, but the effect ofactinomycin D on the ratio of ODC A:B appears to vary with the hormone34used. Actinomycin D, caused the A:B ratio in ODC from the kidneys of PRLtreated rat to revert to the 50:50 in control rats, but did not change the 40:60ratio in the kidneys of both GH and DEX treated animals. PUT affected thetotal ODC activity in the kidneys of hormone stimulated rat in the sameoverall manner as LiC1, with the concomitant 50% decrease in ODC activity.Treatment with PUT also resulted in an ODC A:B ratio of 50:50 in kidneysfrom rat treated with GH. The effect of PUT on the distribution of ODC fromthe kidneys of DEX treated rats however was not definitive.In its response to agents which inhibit ODC, the liver again differedfrom the kidney. As seen in table 1, none of the inhibiting agents seemed tochange the original stimulated profiles for ODC A and B from the livers ofDEX, GH or PRL treated rats. ODC from the livers of both DEX and PRLstimulated rats kept to the A:B ratio of 60:40 regardless of LiC1, ACT D orPUT addition. ODC from GH stimulated rat liver also kept to its unique A:Bratio of 50:50 regardless of agent. As with the kidney however, these agentsall decreased the ODC specific activity. Thus, the agents appeared to equallyaffect the activity of ODC A and B in liver.To further test the effects of hormones on A:B ratio, two hormones ofdiffering method of action, DEX and GH, were injected at the same time intothe rats. The resulting A:B profile for kidney was 40:60, similar to that ofsingle dose hormone stimulated kidney (see table 1). However, the profile ofODC from livers of GH+DEX treated rats was 50:50, similar to ODC from GH35stimulated rat liver, and not similar to ODC from DEX stimulated liver (seetable 1) , as with the ODC specific activity.36Table 1: Specific Activity of ODC and the Proportions of theMultiple Ionic Forms in the Kidney and Liver of Normal Ratsand Rats Treated with Growth Hormone, Prolactin orDexamethasone.KIDNEY LIVERTreatment Specific(unite) A:B Treatment Specific(units) A:BOf Animals Activity Ratio Of Animals Activity RatioCONTROL 1000 50:50 CONTROL 100 70:305hGH 8000 40:60 5hGH 1500 50:505hPRL 7000 40:60 5hPRL 900 60:405hDEX 8000 40:60 5hDEX 500 60:405hGH + 1hLiC1 3000 50:50 5hGH + 1hLiC1 800 50:505hPRL + 1hLiC1 4000 50:50 5hPRL + 1hLiC1 400 60:405hDEX + 1hLiC1 4000 50:50 5hDEX + 1hLiC1 300 60:405hGH + lhAct.D 6000 40:60 5hGH + lhAct.D 900 50:505hPRL + lhAct.D 4000 50:50 5hPRL + lhAct.D 550 60:405hDEX + lhAct.D 5500 40:60 5hDEX + lhAct.D 300 60:405hGH + 1hPUT 5000 50:50 5hGH + 1hPUT 600 50:505hDEX + 1hPUT 5000 45:55 5hDEX + 1hPUT 300 60:405hDEX + GH 8800 40:60 5hDEX + GH 1500 50:5037HALF LIVES OF LIVER AND KIDNEY ODC A AND B IN VIVOTo assess the possible significance of the two forms in vivo, the halflives for both liver and kidney ODC A and B were determined. The half lifeof ODC was determined by measuring the activity of enzyme following theadministration of cycloheximide. Rats were treated with GH for 5 hrs andthen with cycloheximide for 15, 30 or 60 mins. DEAE Sepharose profileswere then determined for each tissue at each time period. From figures 9and 10, it is shown that both kidney ODC A and ODC B activities decay in alinear fashion, indicating a species with a single half life. The half lives forkidney A and kidney B were found to be 7 mins and 25 mins respectively.However, the decay of both liver ODC A and ODC B is curvilinear andindicates the presence of two components with different half lives. The halflives for liver A appear to be 10 min and approximately 25 mins while liver Bis more disparate with half lives of 10 mins and approx 60 mins.38Figure 4: Effect of Cycloheximide on the Two Major IonicSpecies of ODC in Kidneys of Growth Hormone-Treated Rats.Rats were given 500 ug GH in 0.9% NaCl. After 5 hrs, wheninduced ODC activity had reached a peak, the animals wereinjected with 25 mg/kg cycloheximide. The two ionic species ofthe enzyme were separated by DEAE-Sepharose CL-6B columnchromatography as described under Materials and Methods.Samples were prepared at 15 min, 30 min and 60 min afterprotein synthesis was blocked. Results are shown for form A (•-•) and form B (o-o). Each point represents ODC assays on 5animals.390^15^30 45^60^75Cycloheximide Treatment(min)40Figure 5: Effect of Cycloheximide on the Two Major IonicSpecies of ODC in Livers of Growth Hormone-Treated Rats.Rats were given 500 ug GH in 0.9% NaCl. After 5 hrs, wheninduced ODC activity had reached a peak, the animals wereinjected with 25 mg/kg cycloheximide. The two ionic species ofthe enzyme were separated by DEAE-Sepharose CL-6B columnchromatography as described under Materials and Methods.Samples were prepared at 15 min, 30 min and 60 min afterprotein synthesis was blocked. Results are shown for form A (•-•) and form B (o-o). Each point represents ODC assays on 5animals.411000.11010^15^30^45^60^75Cycloheximide treatment(min)42KINETIC PROPERTIES OF KIDNEY ODC A AND B FROM THE LIVER OFGH STIMULATED RATTo see if the two forms differ in other properties, the similarities anddifferences between ODC A and B at the kinetic level were investigated.ODC A and B from the liver and kidney of GH stimulated rat were separatelypurified by the procedures outlined in materials and methods. The two mainchromatographic procedures included DEAE-Sepharose C1-6B ion exchangeand pyridoxamine phosphate affinity. ODC A and B from the liver andkidney of GH stimulated rat were examined because the responses of ODCactivity to hormone induction and inhibition differed between these tissues.Ornithine concentration was varied from 5-500 uM and kinetic studies werealso done in the presence of 50 or 150 mM NaCl. Table 2 shows the results ofthe determination of the enzyme kinetic parameters for kidney B, liver A andliver B. Figures 6-8 show the Eadie-Hofstee plots for kidney B, liver A andliver B.43Figure 6: Eadie-Hofstee Graphical Analysis of OrnithineKinetics for B Form ODC Purified from Kidneys of GrowthHormone-Treated Rats.ODC was purified by DEAE Sepharose and PyridoxamineAffinity Chromatography from rat kidneys 5 hr after injection ofhormone. Assays were in triplicate and contained 0.2 mMpyridoxal-5'-phosphate with varied ornithine concentrationsover the range of 5-500 uM, and in the presence of 50 mM (o-o)and 150 mM (•-e) NaCl. For full details, see Materials andMethods4445Figure 7: Eadie-Hofstee Graphical Analysis of OrnithineKinetics for A Form ODC Purified from Livers of GrowthHormone-Treated Rats.ODC was purified by DEAE Sepharose and PyridoxamineAffinity Chromatography from rat livers 5 hr after injection ofhormone. Assays were in triplicate and contained 0.2 mMpyridoxal-5'-phosphate with varied ornithine concentrationsover the range of 5-500 uM, and in the presence of 50 mM (o-o)and 150 mM (41-.) NaCl. For full details, see Materials andMethods4647Figure 8: Eadie-Hofstee Graphical Analysis of OrnithineKinetics for B Form ODC Purified from Livers of GrowthHormone-Treated Rats.ODC was purified by DEAE Sepharose and PyridoxamineAffinity Chromatography from rat kidneys 5 hr after injection ofhormone. Assays were in triplicate and contained 0.2 mMpyridoxal-5'-phosphate with varied ornithine concentrationsover the range of 5-500 uM, and in the presence of 50 mM (o-o)and 150 mM (•-.) NaCl. For full details, see Materials andMethods48From figures 7 and 8, it can be seen that ODC does not respond in afirst order manner to increases in substrate. The Eadie-Hofstee plot ofkidney ODC B is probably first order in nature (figure 6) as a least squaresanalysis of these points puts a straight line within the error bars of this data.The subsequent plots for liver ODC A and B are not. From figures 7 and 8, itappears that A and B from liver have two different V max and K morn values;signifying that there may be two species with different kinetic propertieswithin A and B. An increase in ionic concentration seems to accentuate thisproperty, for in each case the plots became more curved at the higher saltconcentrations. Previous studies have shown that higher salt concentrationswill increase the apparent value of the Km°rn (93).0) Ionic concentrationaffected both the Km and Vmax in each case. See table 2 for actual Kmornand Vmax values. From table 2, it seems as though kidney B possesses thehighest Vmax at either salt concentration. The lowest Vmax values werethose corresponding to liver B. Within liver A and liver B there were twodifferent kinetic components of ODC, consisting of one species with a low K mvalue and a low Vmax and another species with a high Km value and a highVmax.50Table 2: Kinetic Properties of Purified Multiple Ionic Forms ofODC from Kidney and Liver of Growth Hormone-Treated Rats.Tissue[Nea](mM)Korn(uM)Vmax(units/100u1)A B A B5h GH KIDNEY 50 — 37 — 4350150 — 87 — 42005h GH LIVER 50 9 6 940 485167 136 2500 2400150 14 9 500 150224 320 2250 750ROLE OF PHOSPHORYLATION IN CHARGE SEPARATION OF A AND BAs stated earlier, phosphorylation may play a role in the chargeheterogeneity of ODC A and B. To further test if phosphorylation is involvedin the charge separation of A and B, tissue supernatant from 24 hrtestosterone treated mouse kidney was treated with alkaline phosphatase for1 hr before loading onto a DEAE Sepharose column. The resulting profileshowed that all of the ODC activity eluted in the region corresponding toODC A (figure 4). Supernatants from the kidneys and livers from GHstimulated Wistar rats were treated with alkaline phosphatase for 30 minwith the same results (figure 4 and 5). Additionally, supernatants wereprepared from the livers and kidneys of rats treated with GH and LiC1 andalso subject to alkaline phosphatase treatment. However, phosphatasetreatment of liver and kidney supernatant again gave the identical results ofthe seeming conversion of all ODC to ODC A. A supernatant from the livertreated with GH+LiC1, was incubated without alkaline phosphatase, with aresulting profile of 77:27, thus indicating that any seeming conversion fromODC B to A is not solely due to unspecific breakdown.All the DEAE Sepharose column profiles of phosphatase treated tissuehad efficiencies of 75% or higher. ODC activity was decreased about 40% bythe incubation, regardless of phosphatase treatment. However, the decreasein ODC activity in form B is not wholly accounted for by this nonspecificdecrease.52Figure 9: Effect of Alkaline Phosphatase on the Two Major IonicSpecies of ODC from Kidneys of Growth Hormone- or GrowthHormone plus LiCL-Treated Animals.The rats were treated as described in Materials and Methods.Supernatants from the kidneys were incubated with alkalinephosphatase (200 Units) for 30 min at 37°C and were applied toDEAE-Sepharose C1-6B columns. Chromatography was run asdescribed in Materials and Methods.53o4.0.1.'Web•=1.1mall.1101MIMIOMMOB0b) 5 hr GHAlkPase.....300.•S7514a00.-.=00I275. a '1ooo. -.5ooI273.a%180Figure 10: Effect of Alkaline Phosphatase on the Two MajorIonic Species of ODC from Livers of Growth Hormone- orGrowth Hormone plus LiCI-Treated Rats.The rats were treated as described in Materials and Methods.Supernatants from the livers were incubated with alkalinephosphatase (200 Units) for 30 min at 37°C and were applied toDEAE-Sepharose Cl-6B columns. An additional GH + LiC1Supernatant was incubated without alkaline phosphatase priorto chromatography as a control. Chromatography was run asdescribed in Materials and Methods.553000250020000038c80•0008a--^ a) 5 hr GH..-----b) 5 hr GH+ AlkPase-.•14t1144111•a) 5 hr GH +1 hr ua +AlkPase•.IIIIc1) 5 hr OH +1 hr UCI —AlkPase000 503ction NumPHYSICAL PROPERTIES OF ODC A AND B FROM THE LIVER ANDKIDNEY OF GH TREATED RATSPartially purified ODC A and B from liver and kidney of growthhormone stimulated rats were run on a SDS denaturing PAGE and detectedvia western blotting. As seen in figure 11, there appears to be no differencein the apparent molecular weight of kidney A and B and liver A and B, whichis seen as approximately 48 kD. The corresponding protein gel is not shownas the BSA added to the ODC supernatants prior to immunoprecipitation forstabilization, overshadowed the ODC on the protein stained gel.571 2 3 4 5 668 -'43- 1.4Figure 11: Electrophoresis of the Multiple Species of ODC onSDS-PAGE Gels.The positions of the standards are shown by arrows. The resultsshown are forms A and B of enzyme purified from kidney andlivers of GH treated rats as described in Materials and Methods.The standard purified ODC was from the kidneys of 24 hrtestosterone treated mice. Aliquots were run on a discontinuousSDS gel and then electrophoretically transferred tonitrocellulose. Blots were reacted against the anti-ODCantibody as described in the materials and methods. Thecontents in the lanes are as follows: 1) prestained highmolecular weight standards 2) ODC A from GH kidney 3) ODCB from GH kidney 4) ODC A from GH liver 5) ODC B from GHliver 6) ODC standard.58DISCUSSIONThe physiological role of the multiple species of ODC observed inmammalian tissue is thus far undefined. Stimuli which affect thefunctioning of the cell also affect the activity of ODC. If the multiple speciesof ODC are biologically significant, alterations in total ODC activity mightinvolve changes in the proportions, or changes in the physical and/or kineticproperties of these forms.The results reported here suggest that multiple species of ODC inmammalian tissue are physiologically important. Hormonal stimulation ofthe ODC activity in rat kidney resulted in increases in activity in both A andB, and an increase in the proportion of form B. The ratio of ODC A and Bchanged with hormone stimulation to a value of 40:60 with each of thehormones studied. This increase in the proportion of form B reflects theincrease in half life of that species, as seen in the cycloheximide studies(figure 4). This finding, in hormone stimulated rats is similar to findings inother stimulated systems such as serum stimulated HTC cells, and kidneys oftestosterone stimulated mouse and liver of the cobalt stimulated rat (34).When rats were first treated with hormone for 4 hrs and then subsequentlytreated with agents which decreased the total specific activity of ODC, the59proportions of ODC A and B again changed. In the kidney of hormonestimulated animals, LiC1 was able to change the ratio of ODC A and B to thecontrol value of 50:50, indicating that the half life advantage of hormonestimulated ODC B is removed in the prescence of lithium. This has beencorroborated with half-life studies (Richards, J. F. R., unpublished data).Actinomycin D did not have the uniformity of effect in the kidney that LiC1showed. While ODC activity was decreased within each of the hormonallytreated groups, actinomycin D caused reversion of the A:B ratio to 50:50 inthe PRL stimulated rat only. In both the GH and DEX stimulated animaltreated with actinomycin D, there was no change in A:B ratio from theirprevious hormone stimulated values. As actinomycin D inhibits RNAgeneration, these results may indicate that PRL requires transcription toinduce ODC activity whereas GH and DEX can induce ODC activity throughboth transcription and translational pathways. Putrescine had limitedeffects on the proportions of forms A and B. In spite of the decrease in ODCactivity, the ratio of A:B in the kidney reverted back to the control value of50:50 only in GH stimulated rats.As actinomycin D and putrescine had differing effects on hormonestimulated tissue, this would corroborate the existence of stimulus specificregulatory processes regarding ODC, as seen in other stimulated systems(94-96), such as the kidneys of the testosterone stimulated mouse and theregenerating rat liver.60The kinetic properties of ODC from the kidney of growth hormonestimulated rat were investigated to see if form B might have an advantageover form A in function as well as stability. From the results, this is not thecase. The kinetic values observed for ODC B from the kidney of GHstimulated rat were well within values previously reported for thosecorresponding to ODC A (38, 39, 81, 97). Thus, ODC B does not have kineticadvantage over ODC A in the kidneys of GH stimulated animals, and kineticproperties alone cannot be the basis for the biological relevance of the twoionic forms. This is further substantiated by the kinetic studies of ODC Aand B from HTC cells and the kidneys of testosterone stimulated mice (96,36).In the livers of hormone stimulated animals, increases in the totalspecific activity of ODC also resulted in the preferential increase of form Brelative to form A. The proportions of A:B seen in the liver differed fromthose seen in the kidney, both for unstimulated and stimulated tissues. Also,GH was able to stimulate the increase in activity of form B to a greaterextent in the liver than the other hormones. As within the kidneys ofhormone stimulated animals, ODC B had a longer half-life than form A.However, the activity of form B was proportionally less in the liver than inthe kidney and experimentally it was found that total ODC activity from theliver was less stable than that from the kidney. Similar A:B ratios as well asincreases in half-life of form B have also been observed in the livers ofchloroform and cobalt stimulated rats (34, 37). Interestingly, there was61evidence of multiple half life species within both ODC A and B in the liver ofthe growth hormone-stimulated rat which has been undocumented as yet.With the use of the inhibiting agents, the total specific activity of ODCwas decreased in all three hormone stimulated systems. However, neitherLiC1, actinomycin D nor putrescine changed the A:B ratio from the hormonestimulated values. This is perhaps indicative of differing regulatorymechanisms involved with ODC between the liver and kidney, the pathwaysin the liver relying less on regulation of ODC by half-life.The kinetic properties of forms A and B from the liver of growthhormone-stimulated rats were also examined. The results show evidence ofmultiple kinetic species. These two kinetic species consist of a low Kmorn,low Vmax species and a high Kmorn, high Vmax species. These species, seenin both ODC A and B, are unusual but do not seem to give kinetic advantageto one ionic form over the other. These results could also imply dimerizationof A and B and subsequent cooperativity within the dimers. However, theelevated salt conditions and extreme enzyme dilution that is required in theconventional DEAE chromatography which was used to separate A and Bcontraindicate the formation of dimers (41, 113). There is some possibilitythat the dimers, A:A and B:B, could form within the assay conditions of thekinetic experiments; however,as the ODC assays for the kinetic experimentswere done under the same conditions for ODC purified from both kidney andliver and anomalous results were not observed for ODC from the kidney, it is62unlikely that the results observed for the ODC purified from the liver are dueto dimerization.The multiple half life species observed in both form A and B might beassociated with these kinetic species. There would seem to exist additionalmultiple functional forms of ODC within the liver of growth hormonestimulated animals. The liver from growth hormone stimulated rats appearsto have many distinguishing features when compared to those from thekidney. The effects of growth hormone in mammalian tissue are partiallyregulated by its intermediaries, the somatomedins. It has been documentedthat there are at least 3 types of somatomedins; A, B, and C. The kidney hasa preponderance of receptors for somatomedin C whereas the majority ofreceptors on the liver recognize form A (98). Possibly this could account forthe tissue specific responses of ODC to growth hormone.The origin of the charge separation of ODC A and B is still uncertainand under investigation. Of the possible mechanisms to explain this chargedifference, the post-translational modification, phosphorylation, has been themore fully investigated. First evidence of the phosphorylation of ODC wasthrough the work of Donato et al which used polyclonal and monoclonalantibodies to distinguish subpopulations of phosphorylated ODC frommammalian ODC immunoprecipitated from many species (112). Evidence ofthe possible phosphorylation of ODC was then investigated through the invitro phosphorylation of ODC by casein kinase II from RAW 264 cells (99).63Additionally, native ODC has been isolated from Friend murineerythroleukemia cells as a phosphoprotein being predominantlyphosphorylated at a serine residue (87, 88). In vivo evidence ofphosphorylation was observed in ODC from the kidneys of testosteronestimulated mice (89). ODC A isolated from this system was shown to readilyaccept 32P from 32P-ATP when incubated with casien kinase II; however,ODC B could not accept the 32P label without prior treatment with alkalinephosphatase. Further in vivo evidence of phosphorylation has been observedin ODC from HTC cells and mouse myeloma cells (87,100). To furtherconfirm the role of phosphorylation in the charge disparity of A:B, a series ofsupernatants from the kidneys and livers of hormone-treated rats wereincubated with alkaline phosphatase and then subject to DEAEchromatography. The resulting ODC had the characteristics of ODC A on aDEAE column. This indicates that the charge difference between A and B, asseen on the DEAE column, is attributable to phosphorylation. Similarresults were obtained in HTC cells, liver from mouse and rat and normal andtumorigenic colonic mucosa from mouse and human (100, 101). This effect ofalkaline phosphatase was also noted on crude extract from GH treatedkidney and liver also treated with LiC1, once again indicating thatphosphorylation is responsible for the charge difference between A and B andnot some other post-translational modification such as the attachment of asmall peptide tail (102). Additionally, an incubation without alkalinephosphatase was run on a column and although ODC B had beenpreferentially degraded or acted upon by a cellular phosphatase, the total64activity loss of ODC was greater than in the previous results and ODCeluting in the area corresponding to form B was still evident in thisuntreated supernatant. However, a recent report found that an alaninesubstitution for serine 303, the primary site of phosphorylation in ODC, didnot affect the half life of the ODC enzyme in vitro (103); indicating thatalthough A and B may be separated on DEAE chromatography on the basis oftheir phosphorylated states, the phosphorylation state is not importantbiologically. However, a previous study showed that ODC synthesized invitro from a cDNA differed from the native enzyme in certain properties,including stability of the enzyme (104).Finally, the physical properties of liver and kidney ODC A and B werefound to be similar on an SDS-PAGE Western. Their molecular weight forthe monomer was found to be 48 kD, which is low (-13%) according to theexpected weight of 55kD calculated from the mRNA sequence. However,there have been several reports of ODC with Mr of 50 kD (105-107), in whichcase the value reported here is low by only 4%. This could be due to minordegradation or the slightly different conditions of running the SDS-PAGEWestern which was used. These four samples were also run on an isoelectricfocusing gel and these preliminary results indicated at least two differentlycharged isoforms within ODC A and B from the liver of growth hormonestimulated animals. This experiment was not repeated and thus is not65presented. However, it provides suggestive evidence of the existence offurther forms of ODC.SUMMARYWithin the kidney and liver of both hormone stimulated rat andhormone stimulated rat given other agents, the proportions of ODC A and Bwere altered. This reflects the change in half life of ODC B as seen in thedata presented. When ODC activity is increased, so is the proportion andstability of ODC B. This is seen in both the liver and kidney of growthhormone treated animals. In colonic mucosa, (101) it has been shown thatwith tumorigenesis comes an increase in the proportion of ODC B, providingfurther evidence for the biological importance of the multiple forms of ODC.However, in some instances, only seen in the kidney, where ODC activity isdecreased, the enhanced stability of B is inhibited or removed, as in theresponse of ODC to LiC1 in the stimulated kidney (Richards, J. F.unpublished data). In the liver, there was no additional change in thehormone stimulated A:B ratio, although ODC activity was decreased. Thiscould represent different signalling mechanisms or perhaps differentregulatory mechanisms in the liver. Growth hormone stimulated rat liverseemed to be a complex system in that ODC did not show typical responses interms of activity or kinetics. 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