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Investigation of an ion transport peptide in desert locust ventral ganglia Bilgen, Tolga 1994

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INVESTIGATION OF AN ION TRANSPORT PEPTIDE IN DESERT LOCUST VENTRAL GANGLIA by TOLGA BILGEN BSc. Concordia University, Montreal, 1990 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1994 © Tolga Bilgen, 1994 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of " 2 L Q C $ U The University of British Columbia Vancouver, Canada Date fcjyosv <D \ ^ ^ DE-6 (2/88) ABSTRACT Previous observations have localised an antidiuretic peptide factor to the ventral ganglia (VG) of the desert locust, Schistocerca gregaria. Homogenates of VG increase active ion transport and fluid reabsorption across locust ilea in vitro. There is evidence to suggest that the VG peptide employs cAMP as a second messenger. As it is unstable at high temperatures and at low pH, the VG peptide is probably not Scg-ITP, an ileal transport peptide previously isolated from the corpora cardiaca (CC). Using a short-circuit current (Isc) bioassay, a direct measurement of active CI" transport across locust ilea, further characterization of the VG peptide was conducted. The peptide is unstable below pH 6.0 and loses all activity at pH 4.75. Extreme loss of activity under reducing conditions suggests the VG peptide requires intact disulfide bridges for its biological activity on assay. Reverse-phase cartridges and a high-performance chromatography column did not prove to be useful in purifying the VG peptide, owing to near complete losses of activity. Recovery of activity improved somewhat on anion-exchange cartridges, but results did not suggest useful separation of the peptide. Partial purification (36-fold increase in specific activity) of the VG peptide was accomplished using a combination of gravity-driven and high-performance size exclusion chromatography. These methods also provided an estimation of the peptide's molecular weight as 38 kDa. ii TABLE OF CONTENTS Page Abstract ii Table of Contents iii List of Tables v List of Figures vi List of Abbreviations viii Acknowledgements xi Chapter 1: Introduction 1 The excretory system of the desert locust (S. gregaria) 2 Ion and fluid transport in the hindgut 5 Insect endocrine system 7 Insect neurosecretory system 7 Neurosecretory cells 8 Neurosecretion and waterbalance 10 Localisation and isolation of anti/diuretic peptides: Bioassays 12 Peptide purification 12 Diuretic peptides 13 Antidiuretic peptides 16 Factors in locust ventral ganglia: objectives of this thesis 18 Chapter 2: Materials and Methods 20 Animals 20 Saline 20 Tissue Extracts 20 Flat sheet ileal assay 21 Assay procedures 23 Stability of VG factor 23 Ultrafiltration 24 Electrophoresis: SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 24 Isoelectric focusing (IEF) 25 Protein concentration 26 Liquid chromatography: Reverse-phase cartridges 27 Cation exchange 28 Anion exchange 28 Size exclusion 29 Reverse-phase HPLC 30 Statistical treatment 31 in Chapter 3: Results 32 Dose-response relationship of VG homogenate on ileum 32 VG homogenate activity at various pH values 36 VG homogenate activity under reducing conditions 36 Ultrafiltration 39 Reverse-phase cartridges 39 Isoelectric focusing gels 45 Cation exchange cartridges 48 Anion exchange cartridges 48 Size exclusion 55 RP-HPLC 66 Chapter 4: Discussion 70 References 78 iv LIST OF TABLES Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Effects of ultrafiltration fractions on ileal L,,. 40 Effects of reverse-phase cartridge fractions (C4 and Clg packings, acetonitrile/TFA eluant) on ileal Isc 42 Effects of reverse-phase cartridge fractions (C4, Cg and Clg packings, acetonitrile/citrate/sodium phosphate eluant) on ileal L 43 Effects of reverse-phase cartridge fractions (C4 packing, acetonitrile/sodium phosphate eluant) on ileal L^ . 44 Effects of non-denaturing isoelectric focusing gel slices exposed to VG homogenate on ileal I^ 46 Effects of cation-exchange cartridge fractions (sodium phosphate, increasing NaCl elution) on ileal L.c 49 Effects of anion-exchange cartridge fractions (sodium phosphate, increasing NaCl elution) on ileal L_c 50 Effects of anion-exchange cartridge fractions (Tris-HCl, decreasing pH elution) on ileal L^  52 Effects of anion-exchange cartridge fractions (Tris-HCl, saline elution) on ileal L_c 53 Effects of anion-exchange cartridge fractions (saline elution) on ileal L^  54 Effects of anion-exchange batch-separation fractions (Tris-HCl, increasing NaCl elution) on ileal 1^ . 56 VG equivalent per p.g protein, increase in specific activity and molecular weight estimation of active VG peptide at successive steps in size exclusion chromatography 64 Summary of current knowledge of VG factor 71 Insect diuretic factors of known molecular weight 74 Insect antidiuretic factors of known molecular weight 75 v LIST OF FIGURES Locust alimentary canal and central nervous system 3 Model of transport mechanisms across locust ileum 6 Ussing chamber assembly used to measure L,c, V, and Rj 22 Dose-response curve for maximum stimulation of ileal L.c by VG homogenate 33 Dose-response curve for maximum stimulation of ileal Vt by VG homogenate 34 Maximum change in ileal Rt during stimulation with VG homogenate 35 Stimulation of ileal L_c by VG homogenate at various pH values 37 Effects of reducing conditions on the ^(.-stimulating activity of VG homogenate 38 Non-denaturing isoelectric focusing gel of pi markers and VG homogenate 47 Elution profile (protein and activity) of VG homogenate from classical size exclusion column 58 Calibration curve for classical size exclusion column 59 Elution profile (protein and activity) of VG homogenate from high-performance size exclusion column 61 Figure 13 Calibration curve for high-performance size exclusion column 62 Figure 14 Standard curve for protein content microassay 63 Figure 15 SDS-PAGE of classical and high-performance size exclusion fractions of VG extract 65 Figure 16 Elution profile of high-performance size exclusion fractions from reverse-phase HPLC separation 68 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 vi Figure 17 Elution profile of classical size exclusion fractions from reverse-phase HPLC separation 69 vii LIST OF ABBREVIATIONS - ohms centimeter squared - microgram - microliter - microequivalents per square centimeter per hour - micrometer - Angstrom - antidiuretic - adipokinetic hormone - bovine serum albumin - degrees Celcius - corpus allatum - adenosine 3':5'-cyclic monophosphoric acid - corpus cardiacum - centimeter - central nervous system - corticotropin releasing factor - chloride transport stimulating hormone - diuretic hormone - diuretic peptide - glandular lobe of corpus cardiacum - hour viii - high-performance liquid chromatography HPSEC - high-performance size exclusion liquid chromatography IEF - isoelectric focusing IsC - short-circuit current JH - juvenile hormone kDa - kilodalton Kav - distribution coefficient M - molar (moles/liter) MAS-DP - Manduca sexta diuretic peptide min - minute ml - milliliter mM - millimolar (millimoles/liter) M-NSC - median neurosecretory cells mV - millivolts MW - molecular weight n - number NA - not applicable ng - nanogram NCC - nervous (storage) gland of corpus cardiacum NCC-I, II - nervi corpori cardiaci nm - nanometer Nps - neuroparsins NSC - neurosecretory cells pi - isoelectric point ix psi PMS PNS RER RIA RP R, Scg-ITP SDS-PAGE SE SEC SOG TFA UV V mV VG V, - pounds per square inch - premenstrual syndrome - peripheral nervous system - rough endoplasmic reticulum - radio immune assay - reverse-phase - transepithelial resistance - Schistocerca gregaria ion transport peptide - sodium dodecyl sulphide polyacrylamide gel - standard error - size exclusion chromatography - suboesophageal ganglion - trifluoroacetic acid - ultra violet - volt - millivolt - ventral ganglion - transepithelial voltage X ACKNOWLEDGEMENTS My thanks go to John Phillips for his enthusiasm and generous support, not to mention all the free lunches. Thanks to Chris Mcintosh for all of his time and help. Joan Martin provided invaluable assistance, (im)patience, conversation and various baked goods to help chase the blues and starvation. Megumi, Lloyd-baby, Nay-ohm and Ranj were the perfect cast for Gilligan's Island, here's to you all. A special tip of the hat goes to the everlovin' Baloney Club, the staff and patrons of the Colorado Harvest Room, and to the spectacular Bilgens, all of whom help keep life complex, colorful and contradictory. xi CHAPTER 1 INTRODUCTION Hormones and neuropeptides contribute to the control of virtually every aspect of physiological, biochemical and developmental processes in insects (Goldsworthy and Wheeler, 1985). The isolation and characterization of insect neuropeptides is highly relevant to the study of evolutionary relationships, insect physiology and control of insect populations (reviewed by Keeley et al., 1990; Masler et al, 1993). Osmotic homeostasis is a crucial factor contributing to the success of insects (reviewed by Phillips, et al, 1986), and the humoral factors which regulate it have been under investigation for over thirty years, since MaddreU's (1962, 1963) work with Rhodnius prolixus. Insect habitats frequently offer conditions of extreme salinity, aridity and temperature. This, coupled with high surface area to volume ratios, can place the animals under severe osmotic stress. Nevertheless, insects are able to maintain haemolymph composition within a narrow range. Schistocerca gregaria has been shown to vary its haemolymph osmolality by only 30% when fed hyperosmotic saline or tap water (Phillips, 1964). Terrestrial insects lose water through the cuticle and through the spiracles (e.g. respiration), but the major source of water loss is via excretion, and it is the excretory system which ultimately determines haemolymph solute composition and concentration (reviewed by Chapman, 1981; Phillips, 1981, 1983). Though dehydrated terrestrial insects are known to drink water (Bernays, 1977), water is gained mostly through feeding. In the case of S. gregaria, haemolymph volume can drop by up to 90% upon prolonged dehydration and starvation, but this can be corrected within hours of feeding on succulent plant materials (Phillips et al., 1986). The locust's food is often rich in K+ and low in NaCl, and the animals can consume their own weight every day. Upon digestion, these ions and other solutes enter the haemolymph by absorption in the midgut wall. This would cause an extreme perturbation in the normally NaCl-rich haemolymph if it were not corrected for by the excretory system (Phillips, 1981; Phillips et al., 1986). The excretory system of terrestrial phytophagous insects is typified by that of S. gregaria (see Fig. 1). The Malpighian tubules actively secrete a KCl-rich, Na+-poor isosmotic filtrate which contains most haemolymph solutes, including any toxic molecules (reviewed by Phillips, 1981, 1983). This primary urine then flows into the gut, where some may move anteriorly to the midgut for reabsorption (Dow, 1981). Most, however, flows posteriorly to the hindgut where the ileum and rectum selectively reabsorb ions, metabolites and water. The ileum absorbs a NaCl-rich, isosmotic fluid (absorbate), while that of the rectum is KCl-rich and can be hypoosmotic (Irvine et al., 1988). The rectum is the main site of water conservation and ion concentration regulation, and can produce a hypoosmotic or strongly hyperosmotic urine, or powder-dry faeces (reviewed by Phillips, 1983; Phillips et al., 1986). The excretory system of desert locusts (5. gregaria) The locust excretory system comprises approximately 250 Malpighian tubules lying free in the haemoceol, joined to the alimentary canal at the junction between the mid and hindgut segments. The hindgut consists of the ileum, colon and rectum. It is lined with a 2 Figure 1. Cross-section of locust, showing alimentary canal and central nervous system. CC= corpora cardiaca. CA=corpora allata. SOG= suboesophageal ganglion. Caeoa Midgut liaipighiaii tubules Ileum Oesophagus Brain Colon Rectum Yentral ganglia 3 porous cuticle 2-lO^im in thickness, which serves to protect the epithelial tissue from abrasive gut contents. The water-filled pores of this cuticle, with a diameter of 6.5A and a pK~4 (Phillips and Dockrill, 1968; Maddrell and Gardiner, 1980) allow the passage of hydrophilic ions and metabolites while excluding larger, often toxic molecules. These remain in the gut and are finally eliminated along with the excreta (reviewed by Phillips et al., 1986). Ultrastructural studies of the ileum were conducted by Irvine et al. (1988). This organ, approximately 6mm in length and with a macroscopic surface area of 0.4cm2, consists of a simple epithelium of one cell type (40x20|im). These are closely covered apically by the aforementioned cuticle and basally by a basal lamina. There is considerable infolding on both the apical and basolateral surfaces, with numerous associated mitochondria, typical of ion-transporting epithelia. The colon is made up of unspecialized epithelial cells and its cuticular lining displays low permeability. Rather than participating in fluid reabsorption, the colon serves to break luminal contents into suitable pieces for processing by the rectum (Maddrell and Gardiner, 1980;reviewed by Chapman, 1985). The rectum (ultrastructure reviewed by Chapman, 1985;Irvine et al., 1988) has a macroscopic surface area of 0.65cm2 and is covered loosely on the apical surface with cuticle, creating a subcuticular space. The rectum is made up of six radially arranged, thickened pads connected by reduced epithelium. The pads consist mostly of large (17xl00^im) columnar cells, which display abundant apical and lateral membrane infolding with associated mitochondria. The latter, known as the scalariform complex, supports the ion-recycling model of water reabsorption (reviewed by Bradley, 1985). The second type of cell present in the rectal pads, 'B' cells, contact the lumen only and their function is uncertain. 4 Ion and fluid transport in the hindgut Ileal and rectal epithelia of locusts possess similar mechanisms of CI", K+ and Na+ transport which drive fluid reabsorption (see Fig. 2)(reviewed by Phillips et al, 1986,1988). Apically, a unique electrogenic pump transports CI" from the lumen against large electrochemical gradients and, unlike vertebrate systems, does so independantly of co- or counter-transport with other major ions (Hanrahan and Phillips, 1983a;Irvine et al, 1988). This pump is stimulated by adenosine 3':5'-cyclic monophosphoric acid (cAMP), neuropeptide and luminal K+ (Spring and Phillips, 1980 a,b;Hanrahan and Phillips, 1983b, 1985). K+ traverses the apical membrane passively through channels opened by cAMP (Hanrahan and Phillips, 1983b, 1984,1985). In the ileum, apical Na+ entrance is believed to occur through co-transport with amino acids and glucose (Peach, 1991) and by cAMP-stimulated channels (Richardson, 1993). Na+ enters rectal cells via amino-acid co-transport as well, but some counter-transport with NH4+ (H+) also occurs (Black et al, 1987). NH4+ secretion by ileal cells is achieved through unknown mechanisms. Both rectal and ileal epithelia have apical proton pumps, which are probably vacuolar-type H+-ATPases (Phillips et al, 1994). Basolaterally, both ilea and recta possess CI" and K+ conductances as well as Na+-K+ ATPase (Audsley, 1990; Lechleitner and Phillips, 1988; Phillips et al, 1986). The rectum possesses a powerful apical proline pump and possibly a C1'-HC03" counter-transporter basolaterally (Phillips et al, 1994). These ion-transport processes were elucidated with an in vitro bioassay which voltage-clamps a hindgut epithelium using a short-circuit current (1^; for more detail, see Materials and Methods). Using radiotracer methods and saline substitution, I,.c has been shown to be a 5 Figure 2. Model of mechanisms and control of ion transport across locust ileum. Filled circles represent active transport, open circles are carriers, gaps indicate ion channels (After Richardson, 1992). LUMEN CELL HAZMOCOBL H CI' K + Na + Scg-ITP ariino D a c l d B I 3 p • <* -^AM?T Na •> K + Scg-ITP K H / • -© cAMPT, 6 direct measurement of active CI" transport (reviewed by Phillips et al., 1982). Insect endocrine system The insect central nervous system (CNS) was first proposed to have an endocrine function by Kopec" over 70 years ago (reviewed by Karlson, 1983; Goldsworthy and Wheeler, 1985). Since then the CNS has been the major focus of endocrine research in insects. The insect endocrine system (reviewed by Chapman, 1982;Cymberowski, 1992) comprises the brain, the corpora cardiaca (CC), the corpora allata (CA), the prothoracic gland and the ventral ganglia (VG, see Fig. 1). The CA and the prothoracic gland produce Juvenile Hormone (JH, a sesquiterpene) and ecdysone (a steroid) respectively, through their intrinsic secretory cells (Chapman, 1982). Neuropeptides, the third chemical type of traditional insect hormones, are produced by neurosecretory cells (NSC) throughout the CNS, but are also present in the peripheral nervous system (PNS)(Chapman, 1982). Biogenic amines, such as octopamine and serotonin (Orchard and Loughton, 1985; Raabe, 1989) and prostaglandins (Berridge, 1983; Raabe, 1989) may also have endocrine functions, but these have yet to be formally established. Insect neurosecretory system There are two paired clusters of NSC in the brain, the median (M-NSC) and lateral groups. Axons from these groups extend posteriorly, forming the nervi corporis cardiaci I and II (NCC-I, NCC-n) respectively (Cymborowski, 1992). These nerves extend to the CC. In S. gregaria, some axons in these nerves pass through the CC to terminate in the CA (Highnam, 1961). The corpora cardiaca are paired glands closely associated with the aorta. They contain 7 intrinsic secretory cells and the axon terminals of the brain NSC (Cymborowski, 1992). In S. gregaria, these are arranged into discrete glandular (GCC) and storage (NCC) lobes, though the degree of segregation varies in other insect species (Raabe, 1989). Thus the CC produces its own neurosecretions (the GCC) and is the major storage and release site of the brain's neurosecretory products (the NCC)(Orchard and Loughton, 1985). The corpora allata, paired organs associated with the oesophagus, are innervated by the nervi corpori allati I and II (NCA-I, NCA-II) and contain intrinsic secretory cells. The NCA-I comprises brain NSC axons from the CC, and the NCA-II consists of axons from the subesophageal ganglion (SOG)(Pipa, 1983). The ventral ganglia (VG), which include the SOG, are segmentally arranged and interconnected by the ventral nerve cord affixed to the body wall along the midline. The VG all contain neurosecretory cells (Delphin, 1965), many of which terminate in perisympathetic organs located along the paired segmental peripheral nerves (Raabe, 1989). The perisympathetic organs also contain intrinsic NSC (Cymborowski, 1992). Remy and Girardie (1980) showed that two NSC in locust SOG send axon branches throughout the CNS, suggesting that some NSC could have widespread axon terminals and physiological influence. Neurosecretory cells Neurosecretory cells differ from conventional neurons in that they are specialized for peptide secretion (Golding and Pow, 1988), though this distinction can be ambiguous (Orchard and Loughton, 1985). Indeed, the dual vesicle hypothesis put forth by Golding and May suggests that all neurons are capable of secreting both classical neurotransmitters as well as peptides to varying degrees (Golding and Pow, 1988). In addition, there is evidence for 8 colocalization, in which some NSC secrete both peptides and biogenic amines (Raabe, 1989). Generally, however, NSC have an abundance of rough endoplasmic reticulum (RER) and secretory granules (Golding and Pow, 1988). In the neurosecretory cell body, peptides destined for secretion are synthesized on the RER and packaged by the Golgi apparatus into secretory granules. The granules are 80-300nm in diameter and contain precursors of both carrier proteins and neurohormones (Golding and Pow, 1988;Loughton, 1983), which undergo processing as the granule is transported through the cell (Orchard and Loughton, 1985). The neuropeptides are fully processed by the time the granule reaches the neurohaemal area. The neurohaemal area, comprising the axon terminals, is the site of granule storage and release. It is also the only anatomical specialization of NSC, the axon displaying considerable branching and the endings, swollen and typically filled with secretory granules, lying outside the perineurium (the 'blood-brain barrier'; Maddrell, 1966). Peptide release is thus enhanced by increased surface area and closer contact with the haemolymph. Some NSC axons directly innervate organs (i.e., Malpighian tubules, hindgut, epidermis), while the axon terminals of others aggregate to form a neurohaemal organ (i.e., CC, perisympathetic organs)(Orchard and Loughton, 1985). Neurosecretion involves the fusing of granule and plasma membranes, with the characteristic formation of omega profiles (Orchard, 1983). Compound exocytosis, in which one or more granules fuse with another already engaged in exocytosis, has been demonstrated in annelids and S. gregaria (Golding and Pow, 1988). Regulation of neurosecretion (reviewed by Orchard and Loughton, 1985;Orchard, 1983, Raabe, 1989) is achieved by action potentials depolarizing the axon terminals. Studies in vitro and in vivo have demonstrated that electrical stimulation of the brain NSC groups 9 initiates release of assayable factors from the CC of several insect species, as well as increased appearance of omega profiles. Incubation in high K+ + Ca+ salines has been shown to cause depletion of stainable neurosecretory material (i.e. secretory granules) from NSC (Gosbee et al., 1968) and to cause secretion of assayable factors in vitro (reviewed by Phillips, 1982). Neurosecretion and waterbalance The link between NSC activity and waterbalance in insects was first indicated by histological staining techniques. Working with Iphita limbata, Nayar (1957) demonstrated a depletion of secretory granules in the M-NSC of waterloaded animals, while salt-loaded or dehydrated specimens displayed an accumulation. Highnam et al. (1965) obtained similar results for the M-NSC of 5. gregaria, and observed the apparent mobilization of secretory granules in the CC to the gland's periphery in waterloaded insects. Neurohaemal areas of recently fed R. prolixus were also shown to be emptied of secretory granules (Maddrell, 1966). These observations all suggest the accumulation and release of a diuretic factor from insect NSC. The participation of the VG in insect waterbalance was first proposed by Wall and Ralph (1962), and later in S. gregaria by Delphin (1965). The function of NSC cannot be determined from histochemical studies alone, however, as an abundance of secretory granules may indicate an increase in production or a decrease in release, and vice versa for an absence of granules (Orchard and Loughton, 1985). Incubation of glands in high [K+] solution with Ca+ provided further evidence of the NSC involvement in insect osmoregulation. The metathoracic ganglion of R. prolixus (Aston and White, 1974) and neurohaemal areas of NSC from that ganglion (Maddrell and Gardiner, 10 1976) both release diuretic factor in K+-rich saline. The tsetse fly Glossina austeni similarly releases a diuretic factor from isolated neurohaemal areas (Gee, 1975), as do heads of the mosquito Aedes taeniorhynchus (Maddrell and Phillips, 1978) and isolated CC of the desert beetle, Onymachris plana (Nicolson and Hanrahan, 1986). Osmoregulatory factors have also been detected in the haemolymph, suggesting the hormonal role of neurosecretions. Maddrell (1976) found assayable diuretic activity in the haemolymph of R. prolixus 15 seconds after feeding, and this study is considered the only 'convincing' proof of a factor's release in vivo (Coast and Wheeler, 1990). Whole and methanol extracted haemolymph of recently fed S. gregaria was shown to increase ion transport across recta in vitro, suggesting the release of an antidiuretic factor (Spring and Phillips, 1980). By radioimmunoassay (RIA), Picquot and Proux (1987) showed that fluxes in haemolymph titres of a diuretic peptide roughly correspond with excretion in L. migratoria. Despite these observations however, a satisfying demonstration of a peptide's release into the haemolymph and effect on a distant organ has yet to be conducted (reviewed by Coast and Wheeler, 1990). Thus the hormonal role of these peptide factors remains in question. This is also illustrated by the first insect peptide 'hormone' to be isolated, proctolin, which is currently categorized as a neurotransmitter (Stone and Mordue, 1980). Localisation and isolation of anti/diuretic peptides Generally, isolation of insect neuropeptides has been hampered by the minute quantities of active factor in each animal and the instability of these compounds (Stone and Mordue, 1980). Mitigating factors include the sensitivity and reliability of the bioassays and the purification methods employed (Schooley, et al. , 1990). 11 Bioassays Identification and purification of the osmoregulatory peptides has hinged primarily on the development of appropriate bioassays (reviewed by Phillips, 1983; Coast and Wheeler, 1990; Spring, 1990; Nicolson, 1993). Assays must give consistent results and be sensitive enough to ensure sample conservation at various steps of purification. Whole animal studies, monitoring haemolymph volume and dye clearance, are at best indirect indications of excretion/reabsorption, and may reflect the workings of processes other than excretion (Phillips, 1983). In vitro techniques involve measurement of fluid and ion movement as well as transepithelial potentials and levels of second messengers (e.g. cAMP) in Malpighian tubules and hindgut. These have the advantages of monitoring specific processes under the influence of factors acting directly on the organs in question, but leave doubt as to the in vivo (i.e. hormonal) role of the factors studied (Coast and Wheeler, 1990). Complete investigations of insect peptides comprise a combination of approaches. Peptide purification Liquid chromatography is by far the most popular method of protein purification. Proteins in a mixture (often including a buffer, the mobile phase or eluant) are separated by their differential rates of passage through a column of some stationary phase (packing). Migration through a column depends on the physical properties the packing was designed to exploit (reviewed by Schooley et al., 1990; Mordue and Siegert, 1988; Harris and Angal, 1989). Size exclusion chromatography, also called gel filtration, separates proteins on the basis of their molecular diameter, which strongly correlates with molecular weight. Ion exchange chromatography exploits the net charge and isoelectric points (pi) of proteins. A protein binds to a column by ionic interaction, then the pH or salt concentration of the eluant 12 is altered until the protein reaches its pi, at which point it is released. Reverse-phase chromatography separates proteins on the basis of their hydrophobicity. Proteins bind to a hydrophobic column packing in polar eluant. The eluant's polarity is decreased by the addition of some organic modifier (e.g., acetonitrile, methanol) until the protein is released. These chromatographic methods can be used in 'classical' or high-performance modes. 'Classical' applications of these techniques involve eluting columns by gravity or low-pressure pumps (Harris and Angal, 1989). High performance liquid chromatography (HPLC), operates under pressures of 1000-8000 psi and increases the resolution and reliability of protein separation while decreasing the amount of starting material required (Mordue and Siegert, 1988). HPLC versions of the three modes of chromatographies already discussed are usually key components of any peptide purification scheme (Schooley et al., 1990). Diuretic peptides Maddrell's (1963, 1964a,b) investigations on R. prolixus are often regarded as the seminal studies of an insect diuretic factor. Using an in vitro Malpighian tubule bioassay, he detected diuretic activity in the haemolyph of freshly fed animals, then went on to localise it in the posterior NSC groups of the metathoracic ganglion. The diuretic factor was subsequently shown to be peptidergic, soluble in aqueous solvents and unstable at high temperatures and in haemolymph (Aston and White, 1974; Hughes, 1979). Through neural ablation and histochemistry, the brain M-NSC were determined to be the source of diuretic activity in 5. gregaria, transported to and stored in the CC (Highnam et al., 1965). This was confirmed when only the NCC was shown to contain a methanol-soluble factor which stimulated dye secretion by Malpighian tubules in vivo (Mordue, 1969;Mordue 13 and Goldswoithy, 1969). Lehmberg et al. (1993) found Locusta-DP (LOM-DH, see below) immunopositive material in S. gregaria brain and CC, but this was not determined to be the same factor previously detected in this insect. Two diuretic peptides (Manduca-DP I and II), purified and sequenced from the trimmed heads (brain/CC complex) of tobacco hornworm, Manduca sexta, were found to belong to the vertebrate corticotropin releasing factor (CRF) family; peptides of approximately 45 amino acid residues in length with >40% homology to vertebrate CRF (Katakoa et al, 1989;Blackburn et al., 1991). Native and synthetic peptides were shown to increase fluid secretion of in vitro Malpighian tubules from M. sexta and Acheta domesticus (Audsley et al., 1993; Coast et al, 1992). In Locusta migratoria, a methanol-soluble diuretic factor was located in NCC homogenates (Mordue and Goldswoithy, 1969), and subsequently two thermolabile factors, DH I and DH II, were partially purified (Morgan and Mordue, 1983). A partial sequence was later obtained for another NCC factor, DP-1 (Morgan et al., 1987), which differs from the DH-factors by molecular weight (6-7 and l-2kDa respectively). An arginine vasopressin-like diurectic hormone (AVP-DH), localised to the SOG and VG 1-3 (Schooley et al, 1987), was the first insect diuretic peptide to be fully sequenced (Proux et al, 1987). Locusta-DP, the second locust diuretic factor to be fully sequenced, was purified from whole heads (Kay et al, 1991b). It was found to bear some similarity to DP-1, but sequence homology to Manduca-DP I and II places it in the CRF-like peptide family. Locusta-DP was localised to NCC, with some activity in the brain (as LOM-DH, Lehmberg et al, 1991, 1993). Diuretic activity in the cricket A. domesticus was first detected in the brain and CC (Spring and Hazelton, 1987; Coast, 1988), though peptide factors with diuretic activity were 14 also partially purified from all parts of the insect's CNS (Coast and Wheeler, 1990). The diuretic peptide AP-I was isolated from CC and fully sequenced, though synthetic peptide failed to elicit fluid secretion on in vitro Malpighian tubules, suggesting co-purification of a contaminating peptide (Coast et al., 1990b). Acheta-DP, purified from whole heads, was the second cricket diuretic factor to be sequenced and is another member of the CRF-like peptide family (Kay et al., 1991a). The achetakinins, a family of five myotropic octapeptides related to the leucokinins (see below) and purified from whole cricket heads, were also shown to increase in vitro Malpighian tubule secretion, though an in vivo role in water balance was not established (Coast et al., 1990a). A diuretic factor was detected in the terminal VG of the cockroach P. americana but not purified (Golbard et al., 1970). Periplaneta-DP, purified and sequenced from whole cockroach heads, belongs to the CRF-like peptide family (Kay et al, 1992). The leucokinins, a family of eight myotropic octapeptides isolated from the coackroach Leucophaea maderae, were found to stimulate fluid secretion by isolated Malpighian tubules of the mosquito Aedes aegypti (Hayes et al., 1989). Curiously, the leucokinins' possible role in controlling diuresis in L. maderae was left unexplored. Other diuretic factors have been detected but have not received much attention and remain unpurified. These include a CC factor in the honeybee, Apis mellifera (Altmann, 1956), a thermostable factor from G. austeni (Gee, 1975), a factor or factors detected throughout the CNS of O. plana (Nicolson and Hanrahan, 1986) and a factor detected in the brain and VG of the corn earworm moth, Heliothis zea (Bushman and Nelson, 1990). 15 Antidiuretic peptides A thermostable factor in homogenates of A. mellifera CC was found to decrease urine production when injected into bee haemocoel (Altmann, 1956). This uncharacterized assay is typical of those employed in early research on osmoregulatory factors. In P. americana, in vitro rectal fluid uptake was increased by all parts of the CNS (Wall, 1967; DeBesse and Cazale, 1968). The factor in the terminal VG was later confirmed and determined to be peptidergic (Goldbard et al., 1970). Antidiuretic (AD) activity was detected in the GCC of two other cockroaches, B. craniifer and L.maderae (Fournier, 1990). These factors were found to be thermolabile, unstable in acid and soluble in 70% methanol, making them similar to the GCC factor of L. migratoria (see below). Rectal sac preparations were used to detect antidiuretic activity in homogenates of L. migratoria brain, CC, SOG, perisympathetic organs and VG (Cazal and Girardie, 1968;Debesse and Cazale, 1968;Mordue, 1969). AD activity was later localised to both the NCC and GCC, but the SOG activity was not corroborated (Proux et al., 1984). The NCC factor was found to be soluble in acid and thermostable (Herault et al., 1985; Fournier et al., 1987). The neuroparsins A and B (NpA, NpB), subsequently isolated from the NCC (Girardie et al., 1985, 1987,1989) were the first sequenced insect peptides to have an effect on rectal fluid reabsorption. The GCC factor, found to be peptidergic, acid-labile, thermolabile, and methanol-soluble (Herault et al., 1988), remains unpurified. The existence of AD factors which affect secretion by Malpighian tubules is still contested. A factor which decreased in vivo dye uptake and in vitro fluid secretion by Malpighian tubules was localised to the CC, CA, SOG and perisympathetic organs of P. americana (Wall and Ralph, 1964;De Besse and Cazal 1968), and a similar factor was 16 detected throughout the CNS of L. migratoria (Cazal and Girardie, 1968), but no further investigations of these factors have been reported. In A. domes ticus, a factor which decreased tubule secretion was found in the haemolymph of dehydrated animals and was partially purified from CC (Spring and Clarke, 1990; Spring et al, 1988). However, control of primary urine secretion is thought to be achieved by the amount of diuretic peptide released into the haemolymph and by its rapid degradation (by the Malpighian tubules and haemolymph), making a direct Malpighian tubule deactivating peptide redundant (Mordue, 1970; Davey and Mordue, 1972). Considering the degree of redundancy in insect peptide function known to date however (i.e., the poly tropic neuroparsins and the acheta/leucokinins), doubt over the existence of such a Malpighian tubule-inactivation peptide should rather arise from the lack of subsequent reports of it in the literature. S. gregaria NCC and GCC were both shown to contain a thermostable factor that increased ion transport and fluid reabsorption in both the rectum and ileum (Spring and Phillips, 1980; Proux et al, 1984; Lechleitner and Phillips, 1989a,b; Lechleitner et al, 1989). Chloride transport stimulating hormone (CTSH), found primarily in the NCC and which acts on the rectum, was partially purified by Phillips et a/.(1980). This 8kDa peptide was found to be methanol soluble and unstable in acid. The effects of CTSH on ion transport, elucidated using voltage clamped in vitro recta, include an increase in CI" and K+ absorption, acting via cAMP (Chamberlin and Phillips, 1988). Ileal transport peptide (Scg-ITP) was isolated from CC and partially sequenced (Audsley et al, 1992a,b,c, 1994). This peptide, the first purified factor shown to stimulate the insect ileum, is found in the NCC, is not soluble in methanol, is acid-stable, thermostable and has a molecular weight of 8.6kDa (Audsley et al, 1994). Its effects on ileal ion transport 17 include increased CI", K+ and Na+ absorption, probably using cAMP as a second messenger. Scg-ITP also inhibits active ileal proton secretion, though through some second messenger other than cAMP (Audsley et al., 1994). The known sequence of Scg-ITP places it in a crustacean peptide family, which includes hyperglycaemic and moult-inhibiting factors (Audsley et al, 1992a). The presence of the two factors, CTSH and Scg-ITP, suggests that the locust produces different peptides to enhance antidiuresis in different hindgut segments. Factors in locust ventral ganglia: objectives of this thesis In dehydrated S. gregaria, the NSC of the VG 3-8 were found to be depleted of secretory granules, suggesting a role for VG in antidiuresis (Delphin, 1965). Early work with in vitro assays revealed a thermolabile factor in VG that ellicited negligible effects on rectal ion transport (Spring and Phillips, 1980; Proux et al., 1985). However, VG homogenates were later shown to contain a proteinacious factor that caused qualitatively similar changes in ion and water absorption in both recta and ilea (Lechneitner and Phillips, 1989; Lechleitner et al., 1989a,b; Audsley and Phillips, 1990). Its effects on ilea are mimicked with exogenous cAMP, suggesting it acts via this second messenger (Audsley and Phillips, 1990). Further characterization of the VG factor showed it to be acid labile and insoluble in methanol. The VG peptide was also shown to cause a faster time-course of ileal ion-transport stimulation than Scg-ITP ; this and its acid and thermolability strongly suggested that it was indeed a separate factor (Audsley, 1990). The goal of this thesis was to further characterize and purify the most potent peptide factor in VG that stimulates ileal ion transport. Methods employed, including the bioassay and protein purification techniques, are reported in Chapter 18 2. Chapter 3 covers the results from these attempts and interprets them in light of past observations. The discussion of Chapter 4 places this work in the context of present knowledge and outlines future directions. 19 CHAPTER 2 MATERIALS AND METHODS Animals Schistocerca gregaria were maintained under a 12:12h lightrdark cycle at 28°C and 55% relative humidity. They were given a diet of fresh lettuce and a mixture of alfalfa, bran and milk powder. Female adult locusts, 2-3 weeks past final moult, were used in all experiments. Saline The complex saline used in all experiments was based on the composition of locust haemolymph (Hanrahan and Phillips, 1983) and contained (mM) : 100 NaCl, 5 K2S04, 10 MgS04, 10 NaHC03, 5 CaCl2, 10 glucose, 100 sucrose, 2.9 alanine, 1.0 arginine, 1.3 asparagine, 5.0 glutamine, 11.4 glycine, 1.4 histidine, 1.4 lysine, 13.1 proline, 1.5 serine and 1.8 valine. Tissue extracts Ventral ganglia (VG) 4 through 7 were excised from adult male and female locusts, 2-6 weeks past their final moult. The ganglia were immediately frozen on dry ice and subsequently stored at -70°C. Crude extracts were prepared by homogenizing ganglia in complex saline using a 'Tissue Tearer' homogenizer (Bartlesville, OK). The homogenate was then put through 10 freeze-thaw cycles using dry ice, as this is thought to rupture secretory granules which may contain biologically active materials (Phillips et al., 1980). After 20 centrifuging for 20 minutes at 12 000 g at 4°C, the supernatant was removed and stored at -70°C until use. Standard stock solution was 0.5 VG/fil. The pellet was separated and stored at -70°C. Flat sheet ileal assay Active ion transport across locust ilea was measured by voltage-clamping to OmV an epithelial sheet mounted between two modified Ussing chambers (see Fig. 3)(previously described for locust recta by Hanrahan et al., 1984). Excised ilea were cut longitudinally, producing a flat sheet which was mounted over a 0.196 cm2 opening by means of tungsten pins. After securing the tissue with an overlaying neoprene O-ring, the two chambers were clamped together using elastic bands. Each chamber was filled with 2 ml complex saline which was vigorously stirred and oxygenated by bubbling with a 95% 0 2 : 5% C02 gas mixture. Transepithelial potential (V,) was measured via 3M KCl agar bridges (PE tubing size 90) situated close to the tissue through ports in the chamber walls. These agar bridges were connected by leads to a high input impedance differential amplifier (4253, Teledyne Philbrick, Dedham, MA), which continuously monitored Vt. Vt was clamped at OmV using a second amplifier (725, National Semiconductor Corp., Santa Clara, CA) which passed short-circuit current (Lc) between two Ag-AgCl electrodes on either side of the tissue. Locust ileal I,c has been demonstrated to be Cl"-dependent and is a direct measurement of electrogenic CI" transport (Irvine et al., 1988) ; it was continuously measured by a third amplifier (308, Fairchild, Mountainview CA ). Vt was measured during an experiment by stopping the Isc for 20-30 seconds to monitor voltage difference. Tissue resistance (R^ was calculated from L^  and Vt using Ohm's law. All assays were carried out at 22-25°C. A computer logging program 21 Figure 3. Ussing chamber assembly used to measure ileal 1^ and Vt. A flat-sheet ileal preparation (1) is mounted on a plexiglass collar (2) by means of tungsten pins (3) and secured with a neoprene O-ring (4). Neoprene chamber seals (5,6) prevent saline leakage. Ports (7) serve as inlets for gas, which aerates and mixes saline. V, is measured through agar bridges fitted through ports (8) and current sending electrodes (9) provide measured l^c (from Hanrahan et al., 1984). 22 (supplied by D.M Jones, Dept. of Oceanography, UBC) collected and simultaneously displayed the amplifier outputs at various selected intervals (5, 60 or 120 seconds). An IBM® compatible personal computer with CGA video monitor and 640K RAM was used to operate the program. The chambers were calibrated prior to mounting the tissue by correcting for series resistance of the saline and asymmetries between the voltage-sensing electrodes (Hanrahan et al., 1984). Assay procedures Assays were carried out 60-90 minutes after dissection, as by then both J^ and Vt in the ileum had declined to a steady-state level (Irvine et ah, 1988). Various volumes (1-200(0.1) of VG homogenate and semi-purified VG extracts were added to the haemocoel side of the ileum. Any resulting increase in Isc was monitored until it reached its maximum. Vt values were measured immediately prior to sample application (i.e. during the steady state) and at the maximum stimulated \c. Each preparation was used for only one assay, as any subsequent stimulations caused substantial variations in responses, often potentiation (Audsley, 1990). Bovine serum albumin (BSA, 50(0.1 of 0.1% w/v), which does not affect ileal 1^ ,., was applied to both chambers prior to adding VG extracts to reduce non-specific binding of active peptide to the sides of the chambers. Stability of VG factor I^-stimulating activity of VG homogenate at various pH values was investigated. The pH of VG homogenate was varied over a range of 4.75-8.0 using a series of citric acid/sodium phosphate buffers (100/200 mM respectively). Samples (80(0.1) of 9:1 v/v VG 23 homogenate:buffer were vortexed and allowed to stand for 15 minutes at room temperature. The final pH of the various samples was determined to within 0.25 units using pH indicator strips. Aliquots of these were assayed on flat sheet ileal preparations. As a control, the buffers alone were assayed and were found to have no effect on ileal \c. Biological activity of VG homogenate was tested on ileal l^ under reducing conditions, using a 9:1 v/v VG crude:B-mercaptoethanol mixture which was allowed to stand for 20 minutes at room temperature. These samples were not heated, as the VG factor has been shown to be unstable at high temperatures, losing I^-stimulating activity after 1 minute of boiling (Audsley and Phillips, 1990). It was assayed on flat sheet ileal preparations. Ultrafiltration Microcon Microconcentrators (Amicon, UK) were employed in an initial investigation of the VG factor's molecular weight. The Microcon-10 was used, which has a molecular weight cutoff of 10 kDa. One hundred and fifty gland equivalents of VG homogenate were appplied to the microconcentrator and centrifuged for 70 minutes at 12 OOOg at 4°C. The filtrate was assayed directly, while the retentate was resuspended in complex saline and vortexed before assay. Electrophoresis SDS-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was carried out using the Mini-PROTEAN® II Dual Slab Gel apparatus (Bio-Rad, Richmond,CA). Discontinuous (Laemmli) gels were cast, with stacking (4% total acrylamide (T), 1% cross-linking (C)) and separating (12% T, 1%C) zones, the latter zone optimal for resolving 10-100 kDa polypeptides. The gels were approximately 7 cm x 8 cm, 24 with 1 mm thickness. The buffer system consisted of 25mM Tris, 250mM glycine and 0.1% SDS at pH 8.3. Gels were run at 200V for approximately 45 minutes. Samples of VG homogenate or partially purified VG extracts were diluted 1:4 (v/v) in sample buffer consisting of 63mM Tris-HCl (pH 6.8), 5% SDS (w/v), 10% glycerol (v/v) and 5% B-mercaptoethanol (v/v) and heated to 95°C for 10 minutes before application to the gel. For non-reducing conditions, the same sample buffer minus 6-mercaptoethanol was employed. Two standard molecular weight marker systems were used. GIBCO BRL Prestained Protein Molecular Weight Standards, 2.9-42.2 kDa (Life Technologies, Inc., Gaithersburg, MD), were used initially. In separate gels, a customized mix of protein markers from a Gel Filtration Calibration Kit (Pharmacia, Sweden) was employed. The markers (with MW in kDa) were; bovine serum albumin (BSA; 67), ovalbumin (45), chymotrypsinogen a (25) and ribonuclease a (13.7). Proteins were detected using Coomassie Blue (capable of resolving 0.5|ig protein per band) and silver staining (capable of resolving lng protein per band). Staining procedures were those of Sambrook et al. (1989) and Harlow and Lane (1988). Isoelectric focusing (IEF) The PhastSystem (Pharmacia, Sweden) was used to perform isoelectric focusing. Phastgel IEF gels (homogeneous gels, pH 3.0-9.0, 5% T and 3% C) were selected for their non-denaturing properties, the pH gradient being established by carrier ampholites. Running conditions were 200V, 2.5mA, 3.5W at 15°C for approximately 30 minutes. VG homogenate (3.5 gland equivalents) was applied to the center of the gel, where pH 25 is neutral. The gel was run, then sliced into four equal strips from the anodal to the cathodal end. Each strip corresponded to different regions in the pH gradient (pH 3.0-4.5, 4.5-6.0, 6.0-7.5, 7.5-9.0), and was separately assayed on ilea for ^-stimulating activity. The gel strip that caused an increase in 1^ would indicate the pH range where the active peptide in VG homogenate stopped migrating. In this way the pi of the active VG peptide could be estimated. This procedure was repeated with the homogenate applied to acidic and basic regions of the gel. As a control, gels were run without sample, sliced as described and assayed. These gel slices proved to have no effect on ileal l,c. In subsequent attempts, partially purified VG extracts were applied to the gels. A Broad Isoelectric Focusing Calibration Kit, pi 3.5-9.3 (Pharmacia, Sweden) was used in conjunction with Fast Coomassie Staining (PhastSystem Technical File No.200) to determine banding of VG samples on the PhastGels. Protein Concentration The Bio-rad Protein Assay (Bio-rad, Richmond, CA) was used to measure protein content of VG homogenate and partially purified VG extracts. This dye-binding assay exploits Coomassie Brilliant Blue G-250's differential colour change at 595nm in solutions of various protein concentrations. BSA (stock solution 1.36mg/ml in complex saline) was employed as the protein standard, mixed in dilute coomassie dye over a range of 1.36 to 25.84|ig protein. The blank consisted of the dye in complex saline. An SP6-500 UV spectrophotometer (Pye Unicam, England) was used to measure optical density. 26 Liquid chromatography All chromatographic procedures were carried out at 22-25°C unless otherwise stated. Reverse-phase cartridges C4, Q and C18 Ultra-Sep (Phenomenex, Torrance, CA) 2.8ml polypropylene cartridge columns were used in the fractionation of VG crude, each containing 500mg of reverse-phase packing material. Three elution schemes were employed: 1). Acetonitrile and triflouroacetic acid (TFA). C4 and C18 columns were primed sequentially with 10ml each of the following; 60% CH3CN/0.1% TFA, 0.1% TFA, 0.1% w/v BSA, 60% CH3CN/0.1% TFA and 0.1% TFA. After sample application, columns were eluted stepwise with 10ml fractions, each of increasing acetonitrile content (0-60%, see Table 2). 2). Acetonitrile and citrate/sodium phosphate (100mM/200mM, pH 6.5). C4, Q and Q, columns were primed with 10ml each of 100% CH3CN, HzO and citrate/sodium phosphate. Elution was performed stepwise with 10ml each of increasing acetonitrile in buffer (0-60%, see Table 3). 3). Acetonitrile and sodium phosphate (200mM, pH 8.0). A C4 column was primed with 10ml each of 100% CH3CN, HzO, NajHPQ,. Sample elution was carried out with 10ml each of increasing acetonitrile in buffer (0-60%, see Table 4). All fractions were collected manually in 20ml polyethylene scintillation vials (Kimble, Toledo, OH) and stored at -70°C. Samples of fractions were divided into aliquots in 7ml polyethylene vials (Fisher, Ottawa, ON) containing 50|il of 0.1% w/v BSA and concentrated by centrifugal evaporation (Speed-Vac, Emerston Instruments Inc., ON). BSA was present to reduce non-specific binding of active peptide to the walls of the tube during concentration. 27 Concentrated samples were diluted in complex saline before bioassay. Cation exchange CM-Sephadex C-25 cation exchanger (Pharmacia, Sweden) was swollen in 0.5M NajHPO^ pH 7.0, at room temperature for 24 hours. A cartridge was made using 500mg of exchanger in a 3ml polypropylene filtration column (Supelco Inc., Bellefonte, PA). The column was first washed with 1ml 50mM NajHPCv Elution was stepwise, using 600jxl fractions of increasing NaCl concentration (0-1M) in 50mM NajHPCv Fractions were collected in polypropylene microcentrifuge tubes (Sarstedt, Germany) and tested using the ileal Ij,. assay. Anion exchange DEAE-Sephadex A-50 was employed in all anion exchange experiments, which involved preparatory cartridge columns as described for cation exchange. Several swelling and eluting conditions were used. In all schemes the exchanger was allowed to swell in initial solutions for up to 24 hours at room temperature and elution was stepwise. The exchanger was swollen in 0.5M Tris-HCl, pH 6.5. Elution was carried out using 600 .^1 fractions of increasing NaCl concentration in lOmM Tris-HCl (0-1M NaCl, see Table 7). This scheme was repeated using Tris-HCl at pH 7.0 and 8.0. A pH gradient was used in another experiment. The exchanger was swollen as before in 0.5M Tris-HCl at pH 8.0. Elution was carried out with 600|il fractions of lOmM Tris-HCl of decreasing pH (8.4- 7.0). Isocratic elution was tried next. The exchanger was swollen as before in 0.5M Tris-28 HC1 at pH 7.0, but washed for up to an hour in lOmM Tris-HCl at pH 7.0 prior to pouring the column. Elution was performed with either 1) an initial lOmM Tris-HCl (pH 7.0) fraction followed by several fractions of complex saline or 2) several fractions of complex saline alone. Batch operation anion-exchange was also carried out. Exchanger was swollen for 24 hours in 0.5M Tris-HCl at pH 7.0. After being washed thoroughly in 50mM Tris-HCl, pH 7, 250mg of the exchanger was placed into an Eppendorf microcentrifuge tube and centrifuged for 5 minutes at 12000 g. Excess buffer was removed and a sample of VG homogenate (40 gland equivalents) applied to the exchanger. After thorough mixing followed by 10 minutes of settling time, elution was carried out stepwise using 500|il of 50mM Tris-HCl solutions of sequentially increasing NaCl concentration (0-1M). Solutions were applied to the exchanger and after vortexing, allowed to settle for 10 minutes. After centrifugation, this fraction was assayed for I^-stimulating activity. Care was taken to prevent any exchanger from being applied to the bioassay. Size exclusion Sephadex G-100 size-exclusion packing (fractionation range 4-150 kDa, Pharmacia, Sweden) was swollen in complex saline for 5 hours at 90°C. This was poured into a 1x50 cm open-ended glass chromatography column fitted with a flow adaptor (Sigma, St.Louis, MO). The column was eluted at 4°C with complex saline by gravity-feed at an average of 8ml/hour. These classical columns were calibrated using the same Gel Filtration Calibration Kit (MW range 13.7-67 kDa) decribed for SDS-PAGE. VG homogenate was applied and 1ml fractions were collected with an LKB 2070 29 Ultrarac II (LKB Instruments Inc., Rockville, MD) into Eppendorf microcentrifuge vials. An elution profile at 280nm was measured using an SP-500 UV spectrophotometer (Pye Unicam, England). Fractions from several runs were pooled such that samples could be bioassayed for High performance size exclusion chromatography (HPSEC) was carried out using a Superdex 75 HR 10/30 column ( fractionation range 3-70 kDa, Pharmacia, Sweden) in conjunction with a Waters U6K loop injector, a Beckman 114M pump, a Beckman 421A system controller and a Waters 490 variable wavelength detector, set at 280nm. Complex saline was used as the mobile phase in all experiments, and the column was calibrated using the same protein standards as described above. BSA was eluted through the column before injecting VG extract to prevent any non-specific binding of active peptides. Flow rate was 0.5ml/minute. The most active fractions from the classical size-exclusion runs were pooled and applied to the HPSEC column. 500(j.l fractions were collected manually into Eppendorf microcentrifuge tubes and assayed for ^-stimulating activity. Reverse-phase HPLC The same HPLC system as described for HPSEC was used with a 250 x 4.6mm Nucleosil 10 C8 column (Phenomenex, Torrence, CA) fitted with a guard column (30 x 4.6mm) of similar packing material, except that an additional Beckman 114M pump was employed and the detector was set at 225nm. NajHPQi (20mM) at pH 7.0 and acetonitrile were used to elute the column with a gradient of 0 - 70% CH3CN for 10 minutes, 3 minutes of 70% CH3CN, 70 - 100% CH3CN over 3 minutes, 100% CH3CN for 3 minutes then 100 -30 0% CH3CN over 3 minutes. Flow rate was lml/minute. This column was used to fractionate pooled, active samples of partially purified VG extract from the classical size-exclusion column as well as those from HPSEC, resulting in two and three step purification schemes respectively. Fractions (500|il) were collected manually, concentrated by centrifugal evaporation and assayed for ^-stimulating activity. Statistical treatment Differences between treatments were considered significant when ANOVA indicated a P value less than 0.05. 31 CHAPTER 3 RESULTS Dose-response relationship of VG homogenate on ileum A dose-response relationship (see Fig. 4) was determined to permit quantitative estimates of VG activity during purification. The change in ileal ^ increases sharply with dosage of ventral ganglia over the range of 0-0.5 VG/ml. A maximum response could not be determined, as the \c readings continued to rise gradually beyond the upper range of the amplifier (150|iA) at doses greater than 4 ganglia/ml. This contrasts with an earlier finding that ilea could be maximally stimulated to 13+1.1 nequiv.cm^.h"1 with 1.5 VG5/ml (Audsley and Phillips, 1990). Though this value approximates the ALC of 13.8±1.07 at 1.5 VG/ml in this study, it should be noted that Audsley (1990) applied homogenates of the fifth ventral ganglion alone while the homogenate of VG 4-7 was used here. The changes in ileal V, as VG dosage increases is shown in Figure 5. A maximum change (AmV 29) is evoked by 2 VG/ml. Audsley (1990) found that lVG5/ml caused a 16.7mV increase in ileal V,. Using measured Lc and Vt, the maximum change in transepithelial resistance (RJ for various dosages of VG was calculated using Ohm's Law. Rt decreased by an average of 65Qcm2 over the range of dosages tested (see Fig. 6). This agrees with Audsley's (1990) observation that homogenates of VG5 (dosage 1 ganglion/ml) ellicted a decrease in ileal R, of 47.8Qcm2. 32 Figure 4. Dose-response curve for maximum stimulation of ileal \c (iiequiv.cm"2.!!"1) by VG homogenate (Mean±SE, n=5-7). ^ prior to stimulation was -1.47+0.31 (lequiv.cm"2.!!"1. CM I £ o > UJ 3. o < 25 20 -15 -10 -5 -0 6 0.000 1.000 2.000 3.000 Dose ( VG m l - 1 ) 4.000 33 Figure 5. Dose-response curve for maximum stimulation of ileal Vt (mV) by VG homogenate (Mean±SE, n=7-12). V, prior to stimulation was -3.4±0.66 mV. 40 UG • • ' ' — 0.000 1.000 2.000 3.000 4.000 Dose ( V G . m r 1 ) Figure 6. Maximum change in ileal Rt (Qcm2) during stimulation with VG homogenate (Mean±SE, n=5-7). Rj prior to stimulation was 127.5±12.2Qcm2. 0© CM E o a < -20 - 4 0 •60 -- 8 0 -100 0.000 1.000 2.000 3.000 . - 1 4.000 Dose ( VG m l - ' ) 35 VG homogenate activity at various pH values Knowledge of pH stability was crucial in selecting purification methods and conditions that would optimize recovery of VG activity. As shown in Figure 7, VG homogenate begins losing I,.c-stimulating activity irreversibly below pH 6.0. This loss becomes pronounced as acidity increases and activity is destroyed almost completely (95%) at pH 4.75. Audsley and Phillips (1990) found that crude homogenates of VG5 lost all activity when extracted in 0.2M acetic acid, and CTSH was also found to be unstable below pH 5 (Phillips et al., 1980). Further, Fournier (1990) reported that the antidiuretic factors in the CC of B. craniifer and L. maderae precipitated in 0.1M acetic acid. The acid lability of the VG peptide restricted the range of separation methods that could be employed in this project. VG homogenate activity under reducing conditions The known sequence of Scg-ITP places it in a crustacean peptide family that contains three disulfide bridges (Audsley et al., 1992). On the chance that the VG peptide may have some homology to members of this family, investigation of its biological activity under reducing conditions was undertaken. Also, this information could prove useful in evaluating protein separation procedures that require reducing conditions, such as SDS-PAGE. VG homogenate loses approximately 76% of its biological activity after being mixed with B-mercaptoethanol (see Fig. 8), suggesting that intact disulfide bridges are necessary for the 1^-stimulating activity of the peptide. However, it is possible that other peptides in the homogenate, denatured by the reducing agent, interfere with the active VG peptide. Other insect osmoregulatory peptides, including the AVP-like DH (Proux et al, 1987) and the neuroparsins (Hietter et al., 1991) from L. migratoria, also require intact disulfide bridges for their activity. 36 Figure 7. Stimulation of ileal \c by VG homogenate (4VG/ml) at various pH values (Mean±SE, n=4-5). IN I E o #> 'zs cr o (0 4,500 5.500 6.500 7.500 8.500 PH 37 Figure 8. The effects of reducing conditions on the I^-stimulating activity of VG homogenate (2.25VG/ml). Untreated homogenate and ilea served as the control. (A) Untreated VG homogenate on ilea previously exposed to 6-mercaptoethanol. (B) VG homogenate previously mixed with 6-mercaptoethanol. Concentration of 6-mercaptoethanol in bathing saline was 3. ImM. * indicates significant difference from control at P<0.05 (Mean±SE, n=6-8). si C M ' I E o O 20 15 -10 -5 -0 Control B 38 Ultrafiltration Ultrafiltration was employed primarily to obtain a rough estimate of molecular weight of the VG factor. It was also evaluated as a possible purification step. Foumier (1990), Foumier and Girardie (1987) and Foumier et al. (1987) used ultrafiltration in this manner while studying antidiuretic factors in L. migratoria, B. craniifer and L. madereae. The microconcentrators used in this study consisted of a microcentrifuge tube divided into two chambers by a semi-permeable cellulose membrane with a known exclusion limit of lOkDa. A sample of VG homogenate was placed in the upper chamber. Proteins small enough to pass through the membrane upon centrifugation were filtered into the lower chamber, becoming the filtrate, while those that exceeded the MW cutoff remained in the upper chamber as the retentate. Only the retentates of VG homogenate were found to stimulate change in ileal Is,.. Recovery of biological activity was poor ( 3.9%, see Table 1), probably due to non-specific binding to the membrane, although this was not tested. Foumier (1990) and Foumier et al. (1987) reported that 19% and 26% of ADH activity from CC homogenates of L. madereae and L. migratoria respectively remained bound to the membranes of microconcentrators. In this study, ultrafiltration did not appear to be a useful purification step. Nevertheless, results suggest that the active factor in VG is greater than 10 kDa in weight, making it greater than CTSH (8 kDa; Phillips et al, 1980) or Scg-ITP (8.6kDa; Audsley et al, 1994). Reverse-phase cartridges Reverse-phase cartridges allow bulk separation of crude tissue homogenates, de-fatting and removing various impurities which may bind irreversibly to any RP-HPLC column used subsequently (Schooley et al, 1987). Studies using the cartridges also serve to indicate roughly 39 Table 1. Effects of ultrafiltration fractions on ileal I^ (^lequiv.cm^.h1, Mean±SE, n=3). Retentate and filtrate represent materials >10kDa and <10kDa respectively. Recovery calculated as the percent of the applied sample (8 VG) detected on assay. Fraction AI^ . (p.equiv.cm"2.h"i) Retentate 2.8710.82 Filtrate 0 Recovery (%) 3.93 40 what elution conditions isolate the peptide of interest, and this information can be applied to RP-HPLC purification steps. Katakoa et al. (1987) and Audsley (1990) employed C4 cartridges as a preliminary step in the purification of MAS-DP I and Scg-ITP respectively, while Kay et al. (1991a,b, 1992) used C18 cartridges in the purification of various diuretic factors in the CRF-like insect peptide family. Using the acetonitrile/TFA stepwise elution, biological activity of VG homogenates could only be recovered from C4 columns (see Table 2). Activity was present in the 30% and 60% acetonitrile fractions, though it amounted to less than 1% of the total activity applied to the column. This loss of activity could be explained by the acidic conditions (pH<3) of the CH3CN/TFA eluant, and these results prompted the pH stability experiments described earlier (Fig. 7). To avoid acidic conditions, an acetonitrile/citrate/sodium phosphate elution scheme (pH=6.5) was used. It was thought that in the absence of TFA, the buffer itself (100/200mM) was adequate to prevent any unwanted ionic interactions on the column. As shown in Table 3, biological activity was detected only in the 30% acetonitrile fraction of the C4 column, and recovery improved to 7.23%. Similarly, when an acetonitrile/sodium phosphate elution scheme (pH=8) was used on Q packing, biological activity was detected in the 30% and 50% acetonitrile fractions (see Table 4). Recovery however was low at 2.1% of the ganglia in the applied sample. Biological activity was only recovered from the C4 cartridges. This may indicate irreversible denaturation of the VG peptide by the stronger hydrophobic interactions of the Cg and Cj8 packings. The peptide's size (>10kDa) as determined by ultrafiltration corroborates this idea, as smaller peptides are more suited to reverse-phase separation (Harris and Angal, 1989). When isolating hydrophobic peptides, shorter-chain packings have been shown to be more effective, 41 Table 2. Effects of reverse-phase cartridge fractions on ileal l^ (liequiv.cm^.h1, Mean±SE, n=4-6). Columns (C4 and C18 packings) eluted with acetonitrile/0.1% TFA in water. Recovery calculated as the percent of the initial sample ( 30 VG ) detected in the fractions. AI^ . (|iequiv.cm .h ) Fraction C4 '18 H20 0 0 15% CH3CN/TFA 0 0 30% CH3CN/TFA 1.7311.09 0 60% CH3CN/TFA 1.4310.74 0 Recovery (%) 0.7 0 42 Table 3. Effects of reverse-phase cartridge fractions on ileal \c (nequiv.cm"2.h"\ Mean±SE, n=4-9). Columns (C4, Q and C,8 packings) eluted with acetonitrile in citrate/sodium phosphate (100mM/200mM, pH 6.5). Recovery calculated as the percent of the initial sample (25 VG) detected in the fractions. AI^ (nequiv.cm .h ) Fraction C 4 "-8 *-18 Citrate/Sodium phosphate (100/200mM) 10% CH3CN 30% CH3CN 60% CH3CN Recovery (%) 0 0 11.37±1.93 0 7.23 0 0 0 0 0 0 0 0 0 0 43 Table 4. Effects of reverse-phase cartridge fractions on ileal 1^ (|iequiv.cm"2.h"J, Mean±SE, n=4-5). Column (C4 packing) eluted with acetonitrile in sodium phosphate (200mM, pH 8). Recovery calculated as the percent of the initial sample (20 VG) detected in the fractions. A ^ (|j.equiv.cm .h~) Fraction C4 Sodium phosphate (200mM) 0 10% CH3CN 0 30% CH3CN 3.6±0.78 50% CH3CN 0.25±0.19 60% CH3CN 0 Recovery (%) 2.1 44 while a packing like Clg would be optimal for separating hydrophilic peptides (Schooley et al., 1990). Isoelectric focusing gels Reverse-phase cartridges proved of limited use, so purification using ion-exchange chromatography was considered. Determining the isoelectric point (pi) of the VG peptide could be useful in developing suitable conditions and gradients for an ion-exchange protocol. Phillips et al. (1980) employed cellulose acetate strips in an attempt to determine the pi of CTSH. Finding that CTSH has a slight negative charge above pH 5 led them to consider anion-exchange chromatography as a possible separation technique. When submerged in the complex saline of the bioassay, slices of IEF gel dissolved within 30 seconds. As shown in Table 5, 100% of biological activity was recovered when VG homogenate (3.5 VG) was applied to neutral or basic regions of the gel, thus the active peptide is not denatured by the gel and is liberated into the bathing saline of the assay. When applied to the acidic region of the gel, however, all activity was lost. This supported earlier results, which indicated the instability of the VG peptide under acid conditions. Coomassie staining (sensitive to 0.5 g protein) showed that protein in VG homogenate could be separated into approximately 20 bands within the pi range of 3.5-9.3 when applied to neutral (see Fig. 9) and basic regions of the gel (data not shown). Biological activity, however, was only detected at the point of sample application, suggesting the peptide precipitates in the gel or that it has no apparent net charge. The VG factor's pi then, could not be determined using this technique. As other proteins in VG homogenate were shown to migrate freely while the active peptide remained stationary, isolectric focusing was later considered as a purification step. 45 Table 5. Effects of non-denaturing IEF gel slices exposed to VG homogenate on ileal 1^. ((iequiv.cm^.h1). Recovery calculated as the percent of the initial sample (3.5 VG) detected in the slices. Single observations. pH range of gel slice AI,,. (liequiv.cm"2.!!"1) Recovery (%) 4.5-6 0 0 6-7.5 18.18 100 7.5-9 20.63 100 46 Figure 9. Non-denaturing IEF gel of pi markers (1) and VG homogenate ((2), 0.5 VG). pi values indicated. Gel stained with coomasie blue. 9.30 8.65 8.15 — 7.35 — 6.85 — 655 — L • .• » V 5.85 5.20 455 330 - K - * (1) (2) 47 Partially-purified VG extract (0.5 VG equivalents) from a classical size exclusion column was applied to the gel at the neutral zone, but no biological activity could be recovered from any portion of the gel. Cation exchange cartridge As the pi of the VG peptide could not be determined, it was necessary to investigate the potentials of both cation and anion exchange chromatography. The cartridge method was used since it offered a relatively quick assessment of the separation, in terms of effectiveness (i.e. recovery of biological activity) and the optimal conditions to isolate the VG peptide. Using a stepwise gradient of 0-1M NaCl in sodium phosphate (pH=7), biological activity recovered from the cation exchange cartridge was only detected in the first fraction, that being sodium phosphate alone (see Table 6). Recovery was 71.16% of the gland equivalents applied to the column. These results imply the active peptide did not bind to the exchanger. This could mean the peptide has no apparent net charge or is negatively charged at pH 7, or that it has a weak positive charge and can be displaced by 50mM sodium phosphate. Anion exchange cartridges The cation exchange results prompted consideration of anion exchange. Phillips et al. (1980) turned to anion exchange in their attempt to isolate CTSH. The HPLC version of this technique was also used in the isolation of the neuroparsins (Girardie et al., 1990; Hietter et a/.,1991). In all these cases, an NaCl gradient was used. Table 7 summarizes the results of experiments using an increasing NaCl concentration in Tris-HCl. At pH 6.5 and 7.0, biological activity was detected in all fractions, indicating no 48 Table 6. Effects of cation-exchange cartridge fractions on ileal I,c (nequiv.cm'2.h"\ Mean±SE, n=4). Exchanger was swollen in sodium phosphate (500mM, pH 7) and eluted with increasing NaCl concentration in sodium phosphate (50mM). Recovery calculated as the percent of the initial sample (40 VG) detected in the fractions. Fraction AI^ (nequiv.cnV2.h'1) Sodium phosphate (50mM) 14.55±2.44 0.125M NaCl 0 0.375M NaCl 0 1M NaCl 0 Recovery (%) 71.16 49 Table 7. Effects of anion-exchange cartridge fractions on ileal I,,. (nequiv.cm"2.h"', Mean±SE, n=3-4). Different columns swollen with Tris-HCl (500mM) at pH values shown (6.5, 7 and 8), and eluted with increasing NaCl concentration in Tris-HCl (lOmM). Recovery calculated as the percent of the initial sample (40 VG) detected in the fractions. Alg,. (jiequiv.cm .h ) Fraction 6.5 7 8 0 0 0 0 Recovery 1.23 29.83 0 Tris-HCl (lOmM) 0.125MNaCl 0.375M NaCl 1M NaCl 0.4410.27 0.89±0.16 0.25±0.47 0.6710.34 1.9910.9 3.0311.25 4.4612.2 3.7110.78 50 particular separation of active peptide. Recovery was poor, amounting to 1.2 and 29.8% of the gland equivalents applied to the respective columns. At pH 8.0, no biological activity was recovered, suggesting that the active peptide was still bound to the column. Phillips et al. (1980) found that at pH 9.3, CTSH eluted from an anion exchange column between 15-150mM NaCl with 100% recovery of activity. Elution using a stepwise pH gradient was tried next. As previous results at pH 8 suggested the active peptide was retained on the exchanger, it was thought that unwanted proteins in the homogenate could be separated under this condition while the active peptide could be eluted at some lower pH. Biological activity was detected in all fractions and amounted to 3.4% of the original VG applied (see Table 8). The decreasing amount of activity in each successive fraction suggests that the active factor was simply being washed off the column by fluid flow rather than by actual ion-exchange. The exchanger from these columns was assayed by submerging portions of it in the bathing saline of the assay, and it caused increases in ileal 1^. This indicated that most of the biological activity was retained on the exchanger (up to 80%, results not shown). As a control, exchanger that had not been exposed to VG homogenate was also assayed and it caused no AI,C. Fournier and Girardie (1988), in their work on the Nps, reported that '(anion exchange) HPLC efficiency is approximately 50%', presumably in reference to recovery of biological activity. They did not report an assay of the exchanger itself, as was conducted in this study. Most of the biological activity was retained on the exchanger under salt and pH gradient elution, yet was released into the bathing saline of the bioassay, possibly by some undetermined interaction with some component(s) of the saline. This led to isocratic elution of cartridges with complex saline, to determine if the active peptide could be displaced as effectively from a cartridge column. Tables 9 and 10 summarize the results of these experiments. A combination of Tris-HCl with saline or saline alone were used as eluants. Biological activity was recovered 51 Table 8. Effects of anion-exchange fractions on ileal 1^ (ixequiv.cm^.h1, MeaittSE, n=3). Exchanger was swollen in Tris-HCl (500mM, pH 7), and sample was eluted with Tris-HCl (lOmM) at decreasing pH values. Recovery calculated as the percent of the initial sample (40 VG) detected in the fractions. Fraction (pH) Alg,. ((lequiv.cm^.h1) 8.5 2.1111.41 7.7 1.48±0.85 7.5 0.77±0.19 7 0.65±0.06 Recovery (%) 3.41 52 Table 9. Effects of anion-exchange cartridge fractions on ileal I,,. (^lequiv.cm^.h1, Mean±SE, n=4-7). Exchanger was swollen in sodium phosphate (500mM, pH 7) and eluted isocratically with sodium phosphate (lOmM, pH 7) then complex saline (pH 7, two fractions). Recovery calculated as the percent of the initial sample (30 VG) detected in the fractions. Fraction AI^ ((xequiv.cm'2.!!'1) Tris-HCl (lOmM) 3.02±0.94 Saline! 2.73±1.68 Saline2 2.4610.66 Recovery (%) 23.80 53 Table 10. Effects of anion-exchange cartridge fractions on ileal J^ (nequiv.cm^.h'1, Mean±SE, n=4-6). Exchanger was swollen in sodium phosphate (500mM, pH 7) and eluted isocratically with complex saline (pH 7, three fractions). Recovery calculated as the percent of the initial sample (30 VG) detected in the fractions. Saline fraction AI^ (nequiv.cm^.h1) 1 1.23±0.69 2 2.41i0.68 3 0 Recovery (%) 10.97 54 in both experiments and this decreased in each successive fraction. Total recovery of VG equivalents improved in these experiments to 23.8 and 10.9% respectively. However, the results suggest that most of the VG peptide remained bound to the exchanger. By determining the protein content in a sample of known biological activity, the specific activity of partially purified VG extracts could be estimated and compared to that of the initial homogenate. A protein assay was conducted on the first fraction of the saline-only anion-exchange separation, and suggested a 3.27 fold increase in specific activity over VG homogenate. This compares poorly with the 17-fold purification of CTSH activity using anion exchange (Phillips et al, 1980). In the anion-exchange cartridge experiments, the active factor from VG homogenate may have remained bound to the exchanger due to inadequate exposure (spacial and temporal) to various eluants. To investigate this, batch separation was carried out where thorough mixing and settling of eluants and exchanger was allowed. Recovery of biological activity in these experiments was high (see Table 11), but each successive fraction had decreasing activity. This suggested that the active factor in VG homogenate was simply being flushed off the exchanger by fluid flow rather by ion exchange, much like the results from the cartridge experiments implied. Size exclusion Size exclusion chromatography was used as a purification step and to determine the native weight of the VG peptide. Separation strategies which included size exclusion were used to purify CTSH from S.gregaria CC (Phillips et al., 1980) and stonustoxin peptide from Synanceja horrida venom (Poh et al., 1991), the latter to homogeneity. The ultrafiltration experiments suggested the VG peptide's molecular weight was greater than lOkDa, so size exclusion packing with a 55 Table 11. Effects of anion-exchange batch fractions on ileal Is,, (liequiv.cm^.h1, Mean±SE, n=3-4). Exchanger was swollen in Tris-HCl (500mM, pH 6.5 or 8) and eluted with increasing NaCl concentration in Tris-HCl (lOmM, corresponding pH). Recovery calculated as the percent of the applied sample (10 VG) detected in the fractions. AI^ (|iequiv.cm .h ) Fraction 6.5 8 Tris-HCl (lOmM) 23.56±5.31 16.23±3.04 0.125M NaCl 3.56+0.03 10.05±4.37 0.375M NaCl 2.42±0.85 8.32±2.44 1M NaCl 0 0 Recovery (%) 86.6 73.9 56 fractionation range of 4-150kDa (Sephadex G-100) was selected for the classical column. The protein elution (absorbance at 280nm) and biological activity (AI^) profile of VG homogenate from the classical size exclusion column is shown in Figure 10. Two protein peaks were observed ; the first at the void-volume (15ml), and another broad peak at 38ml, corresponding to molecular weights of >150kDa and 0.48kDa respectively. Biological activity eluted between >150 and 6.3 kDa. The peak of activity eluted at 20.5ml. From this a Ktv value of 0.30 was calculated and from the calibration curve generated for this column, the molecular weight of the active peptide in VG homogenate was estimated at 38kDa (see Fig. 11). This is much higher than the molecular weights determined for other putative antidiuretic factors. An antidiuretic factor from P. americana was determined to have a molecular weight of 8-10kDa (Goldbard et al., 1970), Neuroparsins B was found to be 14kDa (Girardie et al., 1989) and Scg-ITP was found to be 8.6kDa (Audsley et al., 1994). CTSH activity, however, was found in the 8kDa and >30kDa range (Phillips et al., 1980) and both high and low molecular weight forms of the diuretic factor of/?, prolixus were found (>60kDa and <2KDa, respectively, Aston and White, 1974). These authors suggest that these high molecular weight forms may be precursors to the lower MW forms. The same may be true for the VG peptide, though no low molecular weight form was apparent. The recovery from the size exclusion column was calculated as 77.5% of the VG applied. Subsequent experiments using classical columns showed variable recoveries, ranging from 10.75 to 81.25%. This corresponds to the finding of Phillips et al. (1980), who report that CTSH recovery from similar columns varied greatly (35-85%). In characterization of a diuretic factor from G. austeni, Gee (1975) could not recover any biological activity from a gel permeation column. This could reflect the instability of partially purified peptides (Phillips et al., 1980) or 57 Figure 10. Elution profile of VG homogenate (250 VG) from classical size exclusion chromatography column (Sephadex G-100, Vt=33.4ml, V0=15ml). Vertical bars indicate the I^-stimulating activity in each fraction (Mean±SE, n=3-4). (•) = absorbance of each fraction at 280nm. E c o CO CM © o c D .O i_ O v> < 2.500 2.000 -1.500 -1.000 0.500 -aooo 20 30 40 Elution Volume ( ml ) 58 Figure 11. Calibration curve for the classical size exclusion column. Distribution coefficient K,v for marker proteins (o) used are plotted against the log of their respective molecular weights, in order to determine K,v of the VG factor (•). The regression line is expressed as y= 2.62 - 0.5 lx. 0.600 0.400 > D 0,200 0.000 Ribonucleaaa A CtTymotripwrwgen A o VG Ovalbumin BSA 4.000 4.200 4.400 4.600 4.800 5.000 Log Molecular Weight 59 that ionic interactions on the column are preventing the elution of the peptide of interest (Aston, 1979). The most active fractions from the classical column were pooled and applied to high performance size exclusion and the resulting elution/activity profile is given in Figure 12. Protein in this sample eluted as one broad peak at 12.2ml, corresponding to 39.4kDa. Biological activity eluted from 11.5 to 14ml (54.7-19.7kDa). Activity was highest in the 12.25ml fraction and the resulting Kav of 0.208 yielded a molecular weight estimate of 37kDa from the calibration curve generated for this column (see Fig. 13). This compares favorably with the MW estimate obtained with the classical column. Recovery of VG equivalents from this column varied between 41.3 and 73.5%. The specific activity of VG extracts at various stages of this purification scheme were estimated through protein assay (see Fig. 14). Classical size exclusion alone increased specific activity of VG extract 5.8 times over the homogenate. Combining classical with HPSEC resulted in a 36.4-fold increase in specific activity (see Table 12). To further assess the degree of purification, active fractions from the classical and high-performance columns were subjected to gel electrophoresis (SDS-PAGE, see Fig. 15). With silver staining, approximately 50 bands are distinguishable in the classical column fraction, evenly extending over the lane. The HPLC fraction shows fewer bands (approx. 30), mostly with molecular weights less than 42.4kDa. With relatively few apparent bands, it was thought that these size exclusion fractions could be used in conjunction with IEF gels to further purify the VG extract. As the active peptide always remained at the point of sample application on the IEF gel, it was possible that it could be recovered after a number of unwanted proteins migrated freely to their pi. No biological activity could be recovered in these experiments, however, as previously described. 60 Figure 12. Elution profile of VG homogenate (11 VG equivalents) from high-performance size exclusion chromatography column (Superdex 75HR 10/30, Vt=25ml, V0-8.9ml). Vertical bars indicate the Isc-stimulating activity in each fraction (Mean±SE, n=3-4). Solid line indicates absorbance at 280nm. 0.600 0.400 -o 00 0.200 -0.000 5 10 15 Eluifon Volume (ml) 61 Figure 13. Calibration curve for the high-performance size exclusion column. Distribution coefficient Kav for marker proteins (O) are plotted against the log of their respective molecular weights, in order to determine Kav of the VG factor (•). The regression line is expressed as y= 1.82 - 0.35x. 0.400 0.300 P 0.200 0.100 0.000 Rfbonucteaae A o Chymotripsinogen A VG Ovalbumin BSA 4.000 4.200 4.400 4.600 4.800 5.000 Log Molecular Weight 62 Figure 14. Standard curve for protein microassay, the absorbance at 595nm plotted (o) against the amount of protein in the sample. VG extracts at various stages of purification (•) are indicated on the curve (0.5 VG equivalents each). The regression line is expressed as y= 0.15 + 0.04x. ID < 0.800 0.600 0.400 0,200 n non t -o/1 HPSEC i O s<^ Classical SEC oy^ • o / I _S o Homogenate 0 10 15 Protein (jug) 20 63 Table 12. VG equivalent per \ig protein, increase in specific activity and molecular weight estimation of the active VG peptide at each successive step in size exclusion purification. NA= not applicable. SEC = size exclusion chromatography. HPSEC = high performance size exclusion chromatography. Stage of VG/|ig protein Increase in MW estimation purification specific activity (kDa) (Fold) NA NA 5.8 38.07 36.4 37.08 Homogenate Classical SEC HPSEC 0.03 0.17 1.04 64 Figure 15. SDS-PAGE of classical (1) and high-performance (2) size-exclusion fractions of VG extract (0.21 and 0.05 VG equivalents in the respective lanes). Molecular weight markers not shown but their positions are indicated (i= ovalbumin, 42.4 kDa, ii= carbonic anhydrase, 27.5 kDa, iii= 6-lactoglobulin, 18.9 kDa). Gel stained with silver. *-™%i&t?x<i&*>?&&8&> 1 — 11 — 111 — '"' (1) (2) 65 RP-HPLC RP-HPLC is a powerful technique for isolating peptide hormones as it offers high resolution as well as adjustable separation selectivity (Harris and Angal, 1989; Schooley et al, 1990). RP-HPLC has been a component of all protocols in which insect osmoregulatory peptides were purified to homogeneity (Katakoa et al, 1989; Kay et al, 1991a,b, 1992; Blackburn et al., 1991; Morgan et al, 1987; Proux et al, 1987; Coast et al, 1990b; Girardie et al, 1990; Audsley et al, 1992a-c). Eluting VG homogenate through reverse-phase cartridges yielded poor recoveries of biological activity. It was thought that the great losses incurred might be overcome on a RP-HPLC column if enough VG equivalents were applied. The RP-HPLC column was employed as part of a three and two step purification scheme in conjunction with size exclusion chromatography. If activity could be recovered, but the peptide could not be isolated to homogeneity, the elution conditions would nevertheless give an estimate of its hydrophobicity. If no activity could be recovered, it was still possible that the relatively few proteins in the sample could be isolated to purity and sequenced, with the hopes of finding homology with other known insect antidiurectic peptides. Active fractions from HPSEC were pooled, concentrated and subjected to RP-HPLC (7 VG equivalents). The elution profile is shown in Figure 16. The initial peak, eluting before the introduction of acetonitrile, consists of proteins that were not retained on the column. The final two peaks observed are the result of solvent effects, appearing whether or not sample was injected into the column. Fractions (0.5ml) were collected throughout the elution, none of which showed any biological activity when assayed. A recovery of only 0.35 VG equivalents would have been detected on assay, thus the loss of activity was greater than 95%. Active fractions from classical SEC were pooled, concentrated and applied to RP-HPLC 66 in an attempt to increase the amount of biological activity injected into the column (27 VG equivalents). Several samples of these SEC fractions were applied to the reverse-phase column while it was being eluted isocratically with sodium phosphate. In this way proteins binding to the column would accumulate while those that didn't interact (i.e. the initial peak of Fig. 16) eluted from the column and were collected before the acetonitrile gradient was introduced. The resulting elution profile (see Fig. 17) shows a greater number of peaks and protein content compared to the three step scheme, but no I^-stimulating activity could be detected in the collected fractions. A recovery of even 2% (0.5 VG equivalents) could have been easily detected on assay. These results were strong indications that RP-HPLC was not a feasable purification step, and the mixture of proteins in the samples was too complex to isolate and sequence all peptides present. 67 Figure 16. Elution profile of high-performance size exclusion fractions (7 VG equivalents) from reverse-phase HPLC separation. Solid line indicates absorbance at 225nm. dashed line indicates acetonitrile concentration. 3ml fractions were collected throughout the elution. E c lO CM CM 1.000 0.500 r — / v / i I > t n i i ^ — • • — * I I / • 1 / II ' I I ' • / i / 1 s 1 t I / 1 ' 1 \ -'' \ _J v _ ^ J L _JUJ i • • 1 1 . 1 \ t * I > •\ ILL *. 90 70 50 30 10 10 20 Retention time (min.) 30 68 Figure 17. Elution profile of classical size exclusion fractions (27 VG equivalents) subjected to reverse-phase HPLC separation. Solid line indicates absorbance at 225nm. dashed line indicates acetonitrile concentration. 3ml fractions were collected throughout the elution. ' 1.000 E c •o CM < c.saa [J |y" f i r ' s / / / J t T 1 1 V ^ ^ ^ ^ / ^ V^A^U , » i 90 70 50 30 £ rt> r-t-o 1 r+* ~* tt tf • 10 10 20 Retention time (rnfn.) 30 69 CHAPTER 4 DISCUSSION The goal of this thesis was to purify to homogeneity the most potent ion-transport peptide from the locust ventral ganglia. Though this aim was not realized, the attempt has uncovered new information that serves to place the VG peptide in context with other known insect osmoregulatory factors. A brief summary of what is now known about the VG peptide is presented in Table 13. The isolation of insect peptides is frequently complicated by the minute quantities of peptide per animal, bioassay insensitivity, peptide instability and loss of active peptide on container surfaces (Stone and Mordue, 1980). In this study, the first two obstacles were compensated for by the use of large amounts of starting material (thousands of excised ganglia) and by the well-characterised and very sensitive Lc bioassay respectively. Loss on surfaces was reduced by adding BSA to partially purified VG extracts. The instability of the VG peptide, however, proved to be a tremendous impediment to its isolation. Peptide instability, whether inherent in the molecule or due to proteolytic activity, seems common among diuretic factors in insects, and in fact the diuretic peptide from R. prolixus, one of the first to be studied, remains unpurified (reviewed by Mordue and Morgan, 1985). The acid lability of the VG peptide places it among the ranks of many other unpurified insect osmoregulatory factors, including CTSH (Phillips, et al., 1980), antidiuretic factors from B. craniifer, L. maderae (Fournier, 1990) and L. migratoria (Herault et al., 1985), and the diuretic peptide from R. prolixus (Aston, 1979). 70 Table 13. Summary of current knowledge of VG factor. * indicates information from Audsley, 1990. Solubility* 70% Methanol — poor Distilled HzO — poor Temperature Stability* 4°C, 24hrs — no loss of activity Room Temp., 24hrs — 32% loss of activity 100°C, lmin -- 100% loss of activity pH Stability Unstable below pH 6 Reducing Conditions Stability 76% loss of activity Effects on Ileal Ion Transport* Increases CI" transport Increases K+ permeability Inhibits H+ secretion Molecular Weight 37 kDa 71 The main drawback of acid lability is that reverse-phase chromatography, arguably the most powerful mode of protein purification, cannot be employed at peak efficiency. Trifluoroacetic acid (TFA) is often used as an ion-pairing agent, preventing unwanted ionic interactions between a reverse-phase column and sample proteins. However, TFA makes the eluant intolerably acidic for the VG peptide. Reverse-phase HPLC was conducted in this study without TFA, in the hopes that the eluant buffer, sodium phosphate, was itself enough to prevent ionic binding. This may be one way to account for the loss of VG homogenate's biological activity on reverse-phase columns. Other peptides have been successfully isolated without RP-HPLC. Stonustoxin, the acid-labile proteinacious lethal factor from stonefish venom, was isolated using HPLC applications of ion-exchange and size exclusion (Poh et al, 1991). It should be noted however, that the venom used as starting material contained far fewer contaminating proteins than the tissue homogenates commonly used in insect peptide purification. Proctolin and adipokinetic hormone (AKH) were both purified prior to the development of reverse-phase chromatography and without the advantage of HPLC (reviewed by Schooley et al, 1990), however these peptides are extremely short and stable and can be successfully subjected to various batch-separation techniques involving precipitation in acidic conditions. Intact disulfide bridges appear to be crucial to the \c-stimulating activity of the VG peptide. This makes it similar to AVP-like DH and the neuroparsins, both isolated from L. migratoria (Proux et al, 1987; Hietter et al, 1991). The former is a dimer held together by disulphide bridges, while the dimeric character of the latter is controversial (Hietter et al., 1991). It is possible then that the VG peptide is a dimer as well. If this is the case, it is also possible that the subunits of the VG peptide are separated or some other irreversible 72 denaturation occurs on reverse-phase columns, explaining the loss of activity. If the VG peptide is monomelic, disruption of disulfide linkages would still account for the loss of activity. This problem did not arise in the RP-HPLC isolation of the AVP-like DH or neuroparsins, but these factors are smaller (14 and 16kDa, repsectively) than the VG peptide (37kDa) and may not be as easily denatured by reverse-phase packings. The molecular weight of the VG peptide is unusually high when compared to other known insect peptides that influence waterbalance (see Tables 14 and 15). Most peptides range from 2 to lOkDa while the VG peptide has been shown in this study to be 37kDa. The other peptide factors of considerable molecular weight include CTSH and diuretic peptides from R. prolixus and P. americana. In S. gregaria CC, 98% of CTSH activity was found in an 8kDa form, while the remainder was accounted for by a >30kDa fraction (Phillips, et al., 1980). It was suggested that this large form, if indeed the same factor, was in fact CTSH weakly bonded to some other larger proteins in CC homogenate. This seems unlikely in the case of the VG peptide, as no low MW form was observed. The two forms of DH in R. prolixus were both detected in homogenates of metathoracic ganglia, but only the low MW form (<2kDa) is released by ganglia exposed to high K+ in vitro (Aston and White, 1974). Furthermore, the high MW form (>60kDa) was demonstrated to give rise to the low MW form spontaneously in vitro (Hughes, 1979). These observations suggest the high MW form is in fact a precursor molecule to its low MW counterpart. It seems doubtful that the VG peptide is a precursor molecule, however, as no low MW Isc-stimulating factor was ever detected in this study. Also, prohormones are typically inactive on the process that the mature hormone controls (Harris, 1989). If the VG 73 Table 14. Insect diuretic factors of known molecular weight. Species Peptide Molecular weight (kDa) Reference Acheta domesticus Acheta-DP Kay et al, 1991a Glossina austeni 1.2 Gee, 1975 migratoria DH I, DH II DP-I AVP-DH Locusta-DP 1-2 6-7 2 5.3 Morgan and Mordue, 1983 Morgan et al, 1987 Proux et al, 1987 Kay et al, 1991b Lehmberg et al, 1993 Manduca sexta Periplaneta americana Rhodnius prolixus Schistocerca gregaria Manduca-DP I Manduca-DP II _ Periplaneta-DP -. 5 4 >30 5 <2, >60 2 Katakoa et al, 1989 Blackburn et al, 1989 Goldbard et al, 1970 Kay et al, 1992 Aston and White, 1974 Mordue and Goldswortl 74 Table 15. Insect antidiuretic factors of known molecular weight. Species Peptide Molecular Reference weight (kDa) Blaberus craniifer >6 Fournier, 1990 Leucophaea maderae >6 Fournier, 1990 Locusta migratoria Neuroparsins 14 Girardie et al, 1985 >14 Hdrault et al, 1985 Periplaneta americana Schistocerca gregaria CTSH Scg-ITP VG 8-10 8, >30 8.6 37 Goldbard et al, 1971 Phillips et al, 1980 Audsley et al, 1994 This study 75 peptide is indeed a precursor, it would have to be transformed to its active form during bioassay, as has been suggested for the high MW factor of R. prolixus (Hughes, 1979). It is possible that the VG peptide is a precursor for a factor that affects some other physiological purpose in vivo, but in its precursor form has an effect on ileal 1^. Goldbard et a/.(1970) reported a >30kDa peptide in the terminal abdominal ganglion of P. americana that decreased rectal reabsorption in vitro. It should be noted that diuretic peptides acting on the hindgut have not been substantiated. Nevertheless, no low MW form of this factor was found, and this may suggest it is a possible precursor peptide that has an incidental effect on a physiological process it would never be exposed to in vivo. It has already been mentioned that the 'hormonal' nature of many peptides can be questioned (see Introduction). In the same manner, the primary function of these peptides in vivo can also be challenged. Examples of polytropic peptides include the neuroparsins, which have been shown to increase rectal fluid reabsorption as well as mobilization of trehalose and lipid in locusts (Moreau et al, 1988). The achetakinins and leucokinins were isolated from cricket and cockroach respectively according to their myotropic qualities, but were later shown to have diuretic effects (Coast et al., 1990; Hayes et al., 1989). This may mean the full range of a peptide's influence can only be elucidated by the use of a broad range of bioassays. Furthermore, early stages of purification often show multiple factors which affect the employed assay, but only one peptide, often the most potent, is purified to homogeneity. This was the case in the isolation of Scg-ITP (Audsley, 1990) and AP-1 (Coast et al, 1990). The VG peptide may in fact have no in vivo role in ileal fluid transport, and its isolation may prove more practical using some other assay, especially if the VG factor is a precursor of another, more stable peptide which exerts an effect on a physiological process other than 76 hindgut ion-transport. Should purification of the VG peptide be attempted again, a possible protocol should involve size-exclusion chromatography coupled with ion-exchange (batch-separation or HPLC applications), should recovery of biological activity from the latter be improved. If these combined steps fail to isolate the VG peptide to homogeneity, a large-scale non-denaturing isoelectric focusing gel, or a polyacrylamide gel, may serve as a final step. The remaining peptides in the extract can be separated and assayed from the gel. Should biological activity be unrecoverable from the gels, it would still be possible to predict (e.g. by MW) which among several bands would most likely be the VG peptide. Bands could then be sequenced and observed for homologies to other known antidiuretic sequences. Another promising technique would be to investigate VG, through radioimmunoassay, for Scg-ITP-like peptides that stimulate ileal Lc. It is not unreasonable to assume some sequence homology between these two peptides, as they affect the same organ. Affinity chromatography could then be employed as an additional step in the purification. 77 REFERENCES Altmann, G. (1956). Die regulation des wasserhaushaltes der honingbiene. Insectes Soc. 3:33-40. Aston, RJ. (1979). 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