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Guanine nucleotide binding properties and attempted immunopurification of ras protein from dictyostelium.. Bramble, Sharyl Elizabeth 1987-07-07

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GUANINE NUCLEOTIDE BINDING PROPERTIES AND ATTEMPTED IMMUNOPURIFICATION OF RAS PROTEIN FROM DICTYOSTELIUM DISCOIDEUM By BHARYL ELIZABETH BRAMBLE B.Sc, The University of Saskatchewan, 1984 A THESIS SUBMITTED!IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Microbiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1987 © Sharyl Elizabeth Bramble, 1987 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 The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) ii ABSTRACT One purpose of this study was to determine whether the ras protein from Dictyostelium discoideum (p23) binds guanine nucle otides like the ras proteins from mammals (p21) and yeast. The other purpose of this investigation was to purify or enrich for p23ras from D. discoideum by immunoaffinity chromatography. A number of different approaches were used to determine guanine nucleotide binding by p23 . A simple filter binding assay, binding to Western blots, and photoaffinity labeling all failed to demonstrate specific binding with lysates of D. dis-ITcl S coideum cells. In contrast p21 from transformed NIH-3T3 cell lysate was successfully photoaffinity labeled in the pre sence of ^2^-cx-guanosine 5'-triphosphate (GTP) suggesting that the technique had been performed correctly. It was concluded that either p23 has a very low affinity for guanine nucleo tides such that GTP binding was not detectable in these experi ments or that the ras protein from D. discoideum simply does not bind guanine nucleotides. The purification of p23 from D. discoideum cells was attempted in order to provide a purified protein preparation for guanine nucleotide binding and for reconstitution studies. An anti^ras monoclonal antibody (Y13-259) was used as the ligand for the immunoaffinity chromatography. This approach was not successful in that the ras protein could not be en riched relative to other proteins because the immunoaffinity columns did not bind p23 iii TABLE.OF CONTENTS ABSTRACT ii LIST OF TABLES V LIST OF FIGURES vACKNOWLEDGEMENT vii INTRODUCTION 1 MATERIALS AND METHODS I. MATERIALS 7 II. METHODS 8 A. Organisms and Culture Conditions 8 B. Radiolabeling of D. discoideum cellsC. Preparation of Cell-Free Extracts 8 D. Immunoprecipitation 10 E. Polyacrylamide Gel Electrophoresis 10 F. Protein Determination 11 G. Filter Assay for Guanine Nucleotide Binding .. 11 1. Guanosine 51-Diphosphate (GDP) Binding .... 11 2. Guanosine 51-Triphosphate (GTP) Binding ... 11 H. Western Blot Assay for Nucleotide Binding .... 12 I. Western Immunoblot Assay for p23 13 J. Photoaffinity Labeling 14 K. Enzyme-Linked Immunoadsorbent Assay (ELISA) .. 16 L. Preparation of Antibodies 17 M. Removal of Triton X-100 8 N. Immunoaffinity Chromatography 10. Dot Blot ..: 20 iv RESULTS SECTION I. GUANINE NUCLEOTIDE BINDING A. Filter Assay 21 B. Western Blot Probed with GTP 23 C. Photoaff inity Labeling with GTPSECTION II. ATTEMPTED PURIFICATION OF p2 3ras A. Solubilization Conditions 30 B. Detection by EL ISA 34 C. Detection by Immunoprecipitation 39 DISCUSSION 43 REFERENCES 50 V LIST OF TABLES 1. Guanine Nucleotide Binding Filter Assay 22 2. ELISA Using Vegetative Membranes as Antigen 35 3. Two-Step ELISA with Goat Anti-Rat Immunoglobulin G (Garat) Conjugated to Alkaline Phosphatase 36 4. ELISA with Vegetative Membranes and Acetone-Preci pitated 0.3% Triton X-100 Membrane Extract 38 5. Immunoaffinity Chromatography with Y13-259 Column Equilibrated in Tris-Salts Plus 1% Triton X-100 and 0.5% Sodium Dodecyl Sulphate (SDS) 41 6. Immunoaffinity Chromatography with Y13-259 Column Equilibrated in Tris-Salts 4 2 vi LIST OF FIGURES 1. GTP Binding to D. discoideum Proteins 24 2. Direct Photoaffinity Labeling with GTP 26 3. Photoactivation and Immunoprecipitation in the Presence and Absence of 1 mM GTP 2 7 4. GTP Binding to p21 from Harvey Murine Sarcoma Virus (HaMSV) Transformed NIH-3T3 Cells 29 5. Solubilization of p23 with Triton X-100 31 6. Effect of Low Concentrations of Triton X-100 on the Solubilization of p23 32 7. Effect of High Salt, Ethylenediamine Tetraacetic Acid (EDTA), or High pH on Solubilization of p23 .. 33 vii ACKNOWLEDGEMENT I would like to thank three people for their assistance in the completion of this thesis: Dr. G. Weeks for his help ful suggestions and for the use of his office, Steve Robbins for the use of his computer, and Adam B. Lossing for his effi cient photographic work. INTRODUCTION Dictyostelium discoideum is a cellular slime mold that provides a simple model system for the study of development (Loomis, 1982). It is unique among eukaryotes because the processes of proliferation and differentiation are separated. The vegetative amoebae proliferate in the presence of a nu trient source and upon starvation, growth stops and differ entiation then occurs over a 24 hour period in the laboratory to produce two cell types. Cell-free extracts of D. discoideum contain a protein of molecular weight (Mr) 23,000 (p23 ) "(Pawson et al. , 1985) that is specifically immunoprecipitated by an antibody originally raised against the transforming protein (p21 ) encoded by the viral ras gene of Harvey murine sarcoma virus (Furth, 1982). The ras protein is development-ally regulated in D. discoideum, providing an opportunity to study the normal function of the ras protein during cell growth and differentiation in an organism considerably less complex than mammals. The human ras oncogene family consists of three members: Harvey, Kirsten, and N-ras. The first two were identified as homologues of the transforming ras genes of Harvey and Kirsten murine sarcoma viruses (Chang et al., 1982) whereas the N-ras gene was originally isolated from a neuroblastoma cell line (Hall et al., 1983). Since then, cellular homologues of the ras gene have been identified in organisms as diverse as Dic tyostelium discoideum, Aplysia, Drosophilia melanogaster, and Saccharomyces cerevisiae (Levinson, 1986) . 2 The presence of the ras gene in normal cells and its evolutionary conservation suggest that it has an important role in cellular physiology. Transformation is likely a result of the loss of control of this normal function (Bishop, 1983). The mammalian ras proto-oncogene can be activated to a trans forming oncogene by amplification of the gene's expression in the cell (Pulciani et al., 1985) or by acquiring a point muta tion that results in a single amino acid change (Bos et al., 1987; Forrester et al., 1987; Quaife et al., 1987) at one of five specific residues in the encoded protein (Fasano et al., 1984; Marshall, 1986). All three mammalian ras genes encode proteins of 21,000 Mr that are closely homologous in the N-terminal region and have a variable C-terminal domain (Gibbs et al., 1985). Al though little is known as to the physiological function of the mammalian ras proteins, they bind guanine nucleotides speci fically (Scolnick et al., 1979 ; Shih et al., 1980 ; Manne et al., 1984; Trahey et al., 1987) and possess a GTPase activity (Mar-shall, 1986). P21 is located at the cytoplasmic face of the plasma membrane (Papageorge et al., 1982), anchored by a fatty acid covalently linked to the C-terminus of the protein (Sefton et al., 1982). The ability to bind GTP, the GTPase activity, and the membrane localization are all characteristics that are very similar to those of a class of regulatory G proteins that function in signal transduction across the plasma membrane (Newbold, 1984). When GTP is bound, the protein is in an ac tive state and has an effect on a target enzyme. The hydrolysis 3 of GTP to GDP is thought to eliminate this signal. Transform ing ras genes have a reduced GTPase activity compared to proto-oncogenes (McGrath et al., 1984; Gibbs et al., 1984) which re sult in an incorrectly regulated signal. Also, membrane local-ization of p21 is required for transformation (Willumsen & Christensen, 1984) making this G protein analogy an attractive hypothesis. Yeast RASl and RAS2 genes encode proteins that also pos sess the biochemical characteristics of G proteins (Tatchell, 1986). The RAS proteins are fatty acylated and membrane asso ciated (Fujiyama & Tamanoi, 1986) but these properties are not essential for RAS function (Deschenes & Broach, 1987) in Sac-char omyces cerevisiae. Activated RASl (Temeles et al., 1985) and RAS2 (Tamanoi et al., 1985) genes, with mutations analog ous to those of oncogenic mammalian ras genes, encode proteins with reduced GTPase activity as compared to wild-type gene products. The conserved biochemical properties between ras proteins from yeast and mammals suggest that these character istics may be essential to the ras gene product's cellular function. In yeast, membrane reconstitution experiments have shown that the RAS protein is involved in the activation of adenylate cyclase (Broek et al., 1985). However, purified £* cl S p21 does not stimulate or inhibit the adenylate cyclase of mammalian membranes (Beckner et al., 1985). JL7 cl S Recently, mammalian p21 has been implicated in the phosphoinositide pathway of intracellular signal transduction. Inositol lipids are involved in signal transduction from 4 certain growth factor receptors on mammalian cell membranes to intracellular second messengers, initiating responses leading to cell growth. In response to growth facter occupation of its specific receptor, there is an activation of a phospholipase C that is specific for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to 1,2-diacylglycerol and inositol 1, 4 , 5-triphosphate (1,4,5-IP.j). These products stimulate pro tein kinase C and mobilize intracellular calcium, respectively (Michell & Kirk, 1986; Majerus et al., 1985). The PIP2 is generated by sequential phosphorylation of phosphatidylinosi tol, by two different kinases (Berridge, 1986). There is evi dence that a GTP-binding protein is involved in the regulation of phospholipase C (Cockcroft & Gomperis, 1985; Lapetina & Reep, 1987) but tehe protein has not as yet been characterized. I" 3. S Mammalian cells transformed with p21 possess elevated levels of 1,2-diacylglycerol and increased protein kinase C activity compared to untransformed cells (Wolfman & Macara, 1987) and the anti-ras antibody, Y13-259, inhibits the insulin-dependent induction of Xenopus laevis oocyte maturation (Deshpande & Kung, 1987) suggesting that p21ras is involved in the inositol signalling pathway in response to insulin. The GDP-bound form of p21 has been shown to attenuate the autophosphorylation of the insulin receptor (O'Brien et al., 1987) but its physio logical significance remains unclear. Although it is tempting ITcl S to postulate that p21 encodes the G protein that regulates phospholipase C and interacts with the insulin receptor to 5 transduce a signal to this enzyme, there is no direct evidence in support of this hypothesis. D. discoideum also has an intracellular signalling path way involving phosphoinositide lipids although, in comparison to mammalian cells, less is known about this pathway. The sig nalling system of D. discoideum does not involve hormones but rather cells communicate with cyclic adenosine 51-monophosphate (cAMP) and folate (Devreotes, 1983). Cells are attracted to folate during vegetative growth and as cells start to differen tiate, they lose their sensitivity to folate and gain sensitiv ity to cAMP, the chemoattractant responsible for aggregation. The binding of folate or cAMP to their respective cell surface receptors induces a transient accumulation of cyclic guanosine 51-monophosphate (cGMP), that peaks at 8 to 10 seconds after binding of the attractant (Mato et al., 1977; Wurster et al., 1977), presumably due to a transient activation of guanylate cyclase. Calcium (Small et al., 1986) and 1,4,5-IP^ (Europe-Finner & Newell, 1985), when added to permeabilized amoebae, mimic the action of the chemoattractants and induce cGMP for mation. More direct evidence for phosphoinositide involvement in the chemostatic signalling pathway was obtained when 1,4,5,-IP^ was shown to accumulate rapidly in response to stimulation by cAMP (Europe-Finner & Newell, 1987). A model for chemosensory transduction has been proposed in which the activated cAMP and folate receptors interact with a membrane phospholipase that hydrolyses PIP2 to 1/2-diacylglycerol and 1,4,5-IPo (similar to the response in mammalian cells to growth 6 hormones). The intracellular messenger 1,4,5-IPg then mobi lizes intracellular calcium stores and this in turn stimulates guanylate cyclase activity to produce cGMP. Recently, it has been shown that a Dictyostelium ras gene, mutated in an ana logous position to oncogenic mammalian ras gene, caused a re duction in the level of cGMP in D. discoideum cells (Van Haastert et al., 1987) after c7\MP or folate stimulation. This 3i S suggests a role for p23 in the chemotactic signalling path way of D. discoideum. One purpose of this study was to investigate the suppo-IT cl S sition that p23 binds guanine nucleotides. The other pur pose of this investigation was to immunopurify the ras protein from D. discoideum because a purified preparatin of p2 3 could be used in membrane reconstitution experiments to directly study its function. 7 MATERIALS AND METHODS I. MATERIALS Bacteriological peptone and yeast extract were from Oxoid and Bacto-Agar was from Difco. Dulbecco's modified. Eagle me dium and fetal bovine serum were from Gibco. Triton X-100 was from Amersham. N-tetradecyl-N,N-dimethyl-3-ammonio-l-propane-sulfonate (Z-14) was from Calbiochem. 5.0 ym pore size filters were from Nucleopore. Nitrocellulose (0.45 ym pore size) was from Schleicher and Schuell. All radioisotopes were from New England Nuclear. Formalin fixed Staphylococcus aureus (Staph) was from The Enzyme Centre. Rabbit anti-rat immunoglobulin G (RARIG) was from Cooper Biomedical. Goat anti-rabbit immuno globulin G (GARIG) and GARAT alkaline phosphatase conjugates were from Kirkegaard and Perry Laboratories, respectively. The rat myeloma cell line Y3-AG1.2.2 (Y3) was from the Amer ican Type Culture Collection. Y13-259 purified by high pres sure liquid chromatography (HPLC) was from Oncogene Science. Diethylaminoethyl (DEAE)-Sephace1 was purchased as pre-swollen beads from Pharmacia. SDS (specially pure) was from BDH. Bio-Beads SM-2, Affi-Gel 10, acrylamide, and tetramethylethy1-enediamine were from Bio-Rad. Bis-acrylamide and XAR-5 film were from KODAK. All other chemicals were of reagent grade from Fisher Scientific or Sigma Chemical Co. 8 II. METHODS A) Organisms and Culture Conditions Dictyostelium discoideum wild-type strain V12-M2 was used, throughout this study. Strain V12-M2 was grown in association with Enterobacter aerogenes on nutrient agar plates until clearing of the bacterial lawn was discernible (Sussman, 1966). The cells were harvested from growth plates using sterile phosphate buffer (20 mM KH2P04, K2HP04, pH 6.0) and then wash ed free of bacteria with four differential centrifugations (700 x g for 2 min). Harvey murine sarcoma virus (HaMSV) transformed, nonpro-ducer NIH-3T3 mouse fibroblasts were grown in Dulbecco modified Eagle medium containing 10% fetal bovine serum at 37°C. B) Radiolabeling of D. discoideum cells For the preparation of radiolabeled cell extracts, cells 35 were labeled with S-methionine at the outset of differentia tion as described previously (Weeks et al., 1987). C) Preparation of Cell-Free Extracts Crude cytosol and membrane fractions were prepared by g resuspending the washed D. discoideum cells to 1 x 10 cells per ml of Tris-salts (lOmM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2), containing 40 yg/ml leupeptin and 40 ug/ml antipain. The cells were broken by passing them through a 5.0 'um pore size polycarbonate filter 2 to 3 times, as described by Das and Henderson (1983), until over 99% of the cells had been broken as assessed by microscopy. The lysate was centrifuged at 9 100,000 x g for 1 h, at 4°C to separate a crude cytosol (super-nate) and a crude membrane (pellet) fraction. The 0.3% Triton X-100 membrane extracts were routinely prepared by resuspending the crude membrane fraction in anr-equal volume of Tris-salts plus 40 yg/ml: leupeptin, 40 yg/ml antipain, and 0.3% (v/v) Triton X-100. This suspension was incubated for 15 min on ice, and then centridluged at 100,000 x g for 1 h, at 4°C. Membrane extracts containing 1% Triton and 0.5% SDS were prepared for immunoaffinity chromatography using the same procedure except that the crude membrane fraction was resuspended in Tris-salts containing 40 yg/ml leupeptin, 40 yg/ml antipain, 1% (v/v) Tri ton X-100, and 0.5% (w/v) SDS. Whole cell lysates were prepared for immunoprecipitation as described previously (Pawson et al., 1985) except the lysis buffer contained Tris-salts, 40 yg/ml leupeptin, 40 yg/ml anti pain, and 1% (v/v) Triton X-100. The extracts were passed three times through an 18 gauge needle and particulate matter was removed by centrifugation at 100,000 x g for lh, at 4°C. These lysates will be referred to as Triton-solubilized whole cell lysate throughout the remainder of this thesis. HaMSV-transformed NIH-3T3 cells were harvested and lysed as described by Scolnick et al., (1979) except the cells were placed on ice after removal of the medium and washed with Tris-buffered saline. Cells were lysed by scraping them into 1 ml of Tris-salts, 40 yg/ml leupeptin, 40 yg/ml antipain, and 1% Triton X-100. The extracts were passed three times through a 23 gauge needle and clarified at 100,000 x g for 1 h, at 4°C. 10 D) Immunoprecipitation All steps in this procedure were performed at 4°C. Cell lysates were adjusted to 1% Triton X-100 and 0.5% SDS. For the solubilization experiments (Figures 5, 6, and 7) particulate matter was removed by centrifugation at 15,000 x g for 1 h. Aliquots (0.5 ml) were precleared by an initial incubation for 15 min with 50 ul of a 1:10 suspension of Staph, followed by a second incubation with 50 ul of a 1:10 suspension of RARIG-coated Staph (RARIG-Staph) for 15 min. The Staph was pelleted by centrifugation at 13,000 x g for 2 min and the supernate was transferred to a new tube. Samples were incubated with 50 to 100 ul of ammonium sulphate-precipitated Y13-259 for 1 h. Control immunoprecipitations contained an equal volume of nor mal rat serum or ammonium sulphate-precipitated supernate (Jonak, 1980) from the Y13-259 parental myeloma, Y3. All sam ples were incubated with 50 yfl' of a 1:10 suspension of RARIG-Staph for 1 h. The immune complexes were pelleted by centrif li gation at 13,000 x g for 1 min and the supernates were removed by aspiration. The immune complexes were resuspended by pipet ting in 0.5 ml of Tris-salts containing 1% Triton X-100 and 0.5% SDS and washed by repeated centrifugation (3 to 5 times). Where indicated, SDS addition or preclearing were omitted. E) Polyacrylamide Gel Electrophoresis All immunoprecipitates and lysates were heated at 100°C in SDS sample buffer for 3 min unless otherwise indicated. All samples were electrophoresed through 11.25% or 12.5% SDS-poly-acrylamide gels (Pawson et al., 1985). 11 F) Protein Determination Protein was determined by the Folin method, as modified by Sandermann and Strominger (1972), or with the amido black protein assay (Schaffner & Weissman, 1973) if the protein con centration was less than 1 mg/ml. G) Filter Assay for Guanine Nucleotide Binding 1) Guanosine 5'-Diphosphate (GDP) Binding. A 500 yl ali quot of Triton-solubilized whole cell lysate (2.36 mg) was in--5 3 cubated with 10 M H-GDP (10 Ci/mmol) for 1 h at 4°C. The samples were immunoprecipitated directly without the addition of SDS or preclearing, using either normal rat serum of Y13-2 59 followed by incubation with RARIG-coated agarose beads (100 yl of a 1:10 suspension) for 1 h at 4°C. After washing, the immune complexes were collected on 0.45 ym pore size filters and wash ed three more times with 5 ml of ice-cold Tris-salts containing 3 1% Triton X-100. The amount of H-GDP retained by the filters was determined by liquid scintillation counting. The purity 3 of the H-GDP was assessed by thin layer chromatography on poly-(ethyleneimine)-cellulose plates using 1.6 M LiCl in deionized H2O as solvent (Randerath & Randerath, 1964). Radioactive spots were detected by exposing the chromatographed thin layer to XAR-5 film and quantitated by<:cutting the spots out and dissolv ing them in scintillation fluid for counting. 2) Guanosine 5'-Triphosphate (GTP) Binding. Aliquots (200) yl of Triton-solubilized whole cell lysate (0.79 mg) were precleared and then 10 yl of 40 yM S-Y-GTP and 2 yCi (2.7' yl) 35 S—Y-GTP were added to each sample. The samples were incubated 12 for lh at 4°C and then directly immunoprecipitated with either normal rat serum or Y13-259 without addition of SDS. The im mune complexes were washed and collected as described above. Alternatively 600 ul aliquots of Triton-solubilized whole cell lysate (0.96 mg) were precleared and immunoprecipitated (in the absence of 0.5% SDS) with either normal rat serum or Y13-259. The immune complexes were washed and then resuspended in 200 "uiL of Tris-salts plus 1% Triton X-100. Two uM S-Y'-GTP (10 ul of 40 yM stock) and 2.7 ul 35-S- y-GTP (2 yCi) were added to each aliquot and incubated for lh at 4°C. The immune com plexes were then collected on filters, washed, and counted as described above. H) Western Blot Assay for Nucleotide Binding Proteins were electroblotted from SDS-polyacrylamide gels to nitrocellulose as described by Towbin et al. (1979) . Triton-solubilized whole cell lysate was immunoprecipitated in the ab sence of 0.5% SDS. The Triton-solubilized whole cell lysate samples and the immunoprecipitated samples were not heated be fore electrophoresis. The electrophoresed protein was electro-blotted and probed as described previously for p21 ras (McGrath 35 et al., 1984) except 50 yCi S-y-GTP was used in place of 32 P-a-GTP in 4 ml of binding buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 0.1% (v/v) NP40, 1 mg/ml bovine serum albumin). After incubation for lh at 37°C in binding buffer, the electro-blotted protein was washed for 10 min three times in 4 ml of binding buffer lacking bovine serum albumin (BSA) and GTP. 13 3 The dried filter was sprayed with En hance and was then subject ed to fluorography at -70°C. I) Western Immunoblot Assay for p2 3 Aliquots of 1% Triton X-100 membrane extracts (1.67 mg) were either directly electrophoresed, or immunoprecipitated with either Y13-259 or normal rat serum and then electrophoresed, through 12.5% SDS-polyacrylamide. The protein was electroblot-ted overnight at 30V in precooled transfer buffer (20 mM NaPO^, pH 8.0). The Western blots were incubated in 15 ml of blocking buffer (3.0% (w/v) BSA, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl) for 1 h at 37°C, on a gyratory incubator. The blot was then incubated in 15 ml of Y13-259 diluted 1/200 in blocking buffer, at 4°C, overnight. The filter was washed three times for 15 min, at room temperature with washing buffer (0.6% BSA, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl). RARIG (preadsorbed with metha-nol-fixed vegetative cells) was diluted 1/400 in 15 ml of block ing buffer and this was incubated with the filter for lh at 37°C/: shaking. The blot was then washed as described above and then incubated for lh at 37°C on a gyratory shaker with GARIG conju gated to alkaline phosphatase diluted 1/3000 in 15 ml of block ing buffer. The blot was washed twice as before and once for 15 min in washing buffer without BSA. The blot was then incu bated for 30 min at 37°C with 15 ml of developing solution (0.01 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 0.1 mg/ml nitroblue tetrazolium (NBT) in 1 M Tris-HCl, pH 8.8 containing 4 mM MgCl2), rinsed twice with deionized H20 and air-dried. 14 For the Western immunoblots and the Western blots used for guanine nucleotide binding, lanes containing size markers of known molecular weight were cut from the filter after electro-blotting for staining with India Ink (Hancock & Tsang, 1983). J) Photoaffinity Labeling Photoaffinity labeling with crude cytosol and membrane fractions was conducted as described previously (Schleicher et al., 1986), with a few modifications. The membrane pellet was resuspended in Tris-salts containing 40 yg/ml leupeptin and 40 yg/ml antipain. The particulate matter was removed by centrifu-gation at 100,000 x g for lh at 4°C before protein determination. Cell lysates were adjusted to 2 mg/ml protein and 50 yl aliquots 32 were preincubated on ice with 5 yCi P-a-GTP (750-800 Cifaimol) in microtiter wells for 30 min. Irradiation with short-wave ultraviolet (UV) light (maximal emission, 254 nm) was for 60 min, at 4°C. After irradiation, samples were either mixed with SDS sample buffer and electrophoresed directly or immunopreci-pitated with either Y3 or Y13-259 before electrophoresis through a 12.5% SDS-polyacrylamide gel. Size markers of known molecular weight were coelectrophoresed and their mobilities were determined by Coomassie blue staining. Gels were dried and autoradiographed with a screen at -70°C or -20°C. Til other experiments, Triton-solubilized whole cell lysate was immunoprecipitated before photoaffinity labeling with GTP. Triton-solubilized whole cell lysate was prepared in Tris-salts containing 40 yg/ml leupeptin, 40 yg/ml antipain, 1% Triton X-100, and 0.5% (w/v) SDS and 500 yl aliquots (3.34 mg protein) 15 were immunoprecipitated with either Y3 or Y13-259. The immune complexes were washed and then resuspended in Tris-salts. The immunoprecipitated samples were placed in microtiter wells for 32 photoaffinity labeling with P-a-GTP as described above. After irradiation, samples were mixed with SDS sample buffer and elec trophoresed through a 12.5% polyacrylamide gel as described above. To study the possible deleterious effect of photoaffinity 35 labeling on immunoprecipitation, S-methionine radiolabeled Triton-solubilized whole cell lysate was adjusted to 4 mg/ml protein and 0.5% Triton X-100. Aliquots (100 yg) were incu bated with or without 1 mM GTP on ice for 30 min and irradiated, immunoprecipitated and electrophoresed as described above. Nucleotide photoaffinity-labeling of HaMSV-transformed NIH-3T3 cell extract was performed as described by Stein etal. (1984) except for a few modifications. Aliquots (1 ml) were concentrated 10-fold with a Minicon B15 filter system at 4°C (about 2 h). After concentration, 40 yg leupeptin and 40 yg antipain were added and the sample was stored at -70°G. Each 32 sample was preincubated with 1.2 yM P-a-GTP (750-800 Ci/mmol) in microtiter wells for lh in the dark, on ice. Irradiation with short-wave UV light was for 30 min at 4°C. After irradia tion, samples were immunoprecipitated with either Y3 or HPLC-purified Y13-259 and electrophoresed as described above. A 0.3% Triton X-100 membrane extract was prepared from D. dis coideum vegetative cells and treated in the same manner as the transformed mammalian cell extract. 16 K) Enzyme-Linked Immunoadsorbent Assay (ELISA) The standard ELISA was performed using a crude membrane fraction from vegetative cells as antigen. The protein was diluted to 0.1 mg/ml in carbonate buffer (14 mM Na2CC"3, 35 mM NaHC03, 0.02% (w/v) NaN^, 5 mM MgCl2, pH 9.6) and 100 yl was added to each well of a 96-well microtiter plate (except the substrate blank wells in the first row) for adsorption at 4°C, overnight. The wells were then washed three times with 0.3% BSA in phosphate-buffered saline (137 mM NaCl, 1.2 mM KH2P04, 8 mM Na2HP04, 2.7 mM KCl, 0.02% (w/v) NaN^, pH 7.2) plus 0.05% (v/v) Tween 20 (PBS-Tween). Y13-259 ammonium-sulphate precipitated and control antibody (normal rat serum or Y3) were diluted 1/200 in PBS-Tween plus 0.3% BSA and 100 yl was added to the appropriate wells for incubation at 37°C for 1 h. No-antibody control wells contained PBS-Tween plus 0.3% BSA. Substrate blank controls that contained only substrate solution (p-nitro-phenyl phosphate) were also included in each assay. After the incubation, the wells were washed three times with PBS-Tween. RARIG (preadsorbed with vegetative cells) was diluted 1/2000 in PBS-Tween and 100 yl of this solution was added to each well (except substrate blank). The plates were incubated at 37°C for 1 h. The wells were washed three times with PBS-Tweenoand 100 yl of GARIG-alkaline phosphatase conju gate diluted 1/2000 in PBS-Tween was added to each well except the substrate blank. The ELISA plates were incubated for 1 h at 37°C and then washed three times with PBS-Tween. Finally, 17 100 yl of a 1 mg/ml solution of p_-nitropheny 1 phosphate dis solved in diethanolamine buffer (1 M diethanolamine, 0.0 2% NaN-j/ 0.5 mM MgCl^) was added to each well. After incubating with substrate at 37°C for 30 to 90 min, the O.D.^Q^ of each well was taken using a Titertek. The substrate blank wells gave an average O.D.^QJ- of 0.002 or less. The no-antibody control wells had an average O.D.^Q^ that was 0.0 5 to 1.0 unit lower than the average O.D.^Q^ obtained from the wells incu bated with control antibody (Y3 or normal rat serum). Any changes from the standard ELISA are noted in the text or the footnotes to the tables. Vegetative cells for the preadsorption of RARIG were pre pared by resuspending washed and pelleted V12-M2 cells in an-7 hydrous absolute methanol at 4°C to 10 cells/ml. Cells were fixed by end-to-end rotation for 30 min at 4°C. The methanol was washed from the cells by repeated centrifugation (three times) at 700 x g for 2 min in PBS. The pelleted cells were then resuspended in RARIG (reconstituted according to manufac-g turer's instructions) to 10 cells/ml and the sera was adsorbed on the end-to-end rotator overnight at 4°C. The cells were pelleted at 700 x g for 5 min and the RARIG was removed to an other tube for storage at -20°C. L) Preparation of Antibodies Ammonium sulphate-precipitated Y13-259 or Y3 was purified by ion exchange chromatography on a DEAE-Sephacel column as described previously (Fahey & Terry, 1973). Immunoglobulin G 18 (IgG) was detected in fractions eluted from the column using RARIG in an Ouchterlony test. M) Removal of Triton X-100 Triton X-100 was removed from samples prior to the adsorption of the protein to the ELISA plate using Bio-Beads SM-2 using the batch procedure described by Holloway (1973). Removal of Triton X-100 by acetone precipitation was done by adding an equal volume of ice-cold acetone to the membrane ex tract and incubating on ice for 30 min. The samples were cen-trifuged at 13,000 x g for 5 min and the supernates were poured off. The precipitates were placed under vacuum in a centrifuge (Speed-Vac) for 30 min to remove all the acetone. Pellets were resuspended in Tris-salts containing 40 yg/ml leupeptin and 40 yg/ml antipain. N) Immunoaffinity Chromatography Affi-Gel 10 was coupled to DEAE-purified Y13-259 or Y3 in 0.1 M MOPS, pH 7.5, for 4 h at 4°C. Between 5 and 12 mg antibody was coupled per ml gel. For the experiments utilizing detection of p23 by ELISA, 5 ml immunoaffinity columns were used. Columns were equili brated with Tris-salts. The membrane extract was applied to the column and the flow-through was recycled 3 to 5 times. The column was washed and eluted as described in the Results. One ml fractions were eluted into tubes containing 40 yg leupeptin and 40 yg antipain. The fractions were dialysed against 1 1 of Tris-salts plus 100 yg/ml p_-aminobenzamidine overnight at 4°C 19 and the protein content was determined. Either 10 yg of protein or 100 yl of each fraction was used as antigen for the ELISA and was adsorbed in buffer adjusted to pH of approximately 10 units. For experiments utilizing the detection of p23 by immuno-precipitatin of radiolabeled membrane extracts, columns of 1 ml bed volume were used. The columns were equilibrated with the buffers described in the Results. Membrane extracts were 8 3 5 prepared from 5 x 10 S-methionine labeled cells and applied to the columns in a volume of 1 ml. The flow-through was re cycled 3 to 5 times. Total protein content was estimated by trichloroacetic acid (TCA) precipitation of 5 yl aliquots that had been dried on glass fiber filters. The filters were washed with 20% TCA, 5% TCA and methanol, respectively, and then the radioactivity was determined by liquid scintillation in a Tri ton X-100 based scintillation fluid. The columns were washed with different detergent-containing Tris-salts buffers until all the TCA-precipitable radioactivity had been eluted. Each wash buffer was concentrated to 1 ml. The elution conditions that were used depended on the experiment and are described in the footnotes to tables. Eluted fractions were dialysed:against 2 1 of Tris-salts plus 100 yg/ml p_-aminobenzamidine overnight at 4°C unless they contained Tris-salts already. The samples were split into two 0.5 ml aliquots and were immunoprecipitated with either Y13-259 or Y3. The immunoprecipitated proteins were electrophoresed through a 12.5% SDS-polyacrylamide gel. 20 3 Following treatment with En hance, the dried gel was exposed against XAR-5 film for one to six weeks. The p2 3 bands were quantitated by densitometry and these values were used to evaluate recovery of p23 from the columns. O) Dot Blot The amount of p2 3 in vegetative membrane preparations was also determined by the dot blot method described by Jahn et al. (1984) except the blots were incubated ini .either Y13-259 or normal rat serum diluted 1/100 or 1/200 for 1 h at 37°C or overnight at 4°C. After washing and reblocking the nitrocellu lose sheets, they were incubated with RARIG (preadsorbed with vegetative cells) diluted 1/400 for 1 h. After washing for 5 min five times, the blots were incubated with GARIG-alkaline phosphatase conjugate that had been diluted 1/3000 in blocking solution containing 0.1% (v/v) Triton X-100 for 1 h. The blots were washed four times for 5 min in Tris/NaCl/0.1% Tri ton X-100 and then four times for 25 min in Tris/NaCl/0.1% Tri ton X-100. The blots were incubated for 30 min at 37°C in 10 ml of D'.M Tris-HCl, pH 8.8 containing 4 mg/ml MgCl2, 0.01 mg/ml BCIP and 0.1 mg/ml NBT. The blots were rinsed with deionized H^O and air-dried. DEAE-purified normal rat serum and different dilutions of RARIG were used unsuccessfully to try to lower the background. 21 RESULTS SECTION I. GUANINE NUCLEOTIDE BINDING 1" cl S The purified ras protein from mammalian cells (p21 ) -9 -9 has a binding constant of 8 x 10 M for GTP and 7 x 10 M for GDP (Shih et al., 1980). Using at least ten times higher concentrations of guanine nucleotide, a number of different approaches were used to try to detect guanine nucleotide binding by p23raS. A) Filter assay A previous study had shown that 100 ug of a cell-free lysate of Kirsten murine sarcoma virus (KiMSV) transformed NIH-3 3T3 cells will bind 1.10 pmoles of H-GDP when immunoprecipi-3 tated with anti-p21 antibody whereas only 0.05 pmoles of H-GDP are bound using control serum (Scolnick et al., 1979). In a filter assay using similar conditions, p23 did not bind GDP (Table 1). The level of bound GDP in cell lysates immunopreci pitated with the anti-ras antibody was identical to that in cell lysates immunoprecipitated by normal rat serum. Using RARIG-Staph instead of RARIG-coated agarose for immunoprecipi-tation did not change the result and adding SDS to the immuno-precipitation increased the background levels of GDP bound with both specific antibody and normal rat serum (data not shown). 3 The H-GDP used in these experiments was determined to be 93 to 96 per cent pure by thin layer chromatography therefore the low amount of GDP binding was not due to degraded or contaminated GDP. 22 Table 1. Guanine Nucleotide Binding Filter Assay 35S-7~GTP bound (pmol/mg)b H-GDP bound Incubate Immunoprecipitate Antibody (fmol/mg) First First Normal rat 21. .8 ± 9.2 1. .93 ± 0. . 32 12. .45 ± 4. . 47 serum Y-13-259 16. ,8 ± 13.2 1. .92 ± 0, .29 13. .21 ± 6, .69 a. The means and standard deviations of two determinations are given. b. The numbers are the means of four determinations ± the standard deviation. "Incubate First" indicates GTP bind ing followed by immunoprecipitation and "Immunoprecipi tate First" indicates immunoprecipitation followed by GTP binding. 23 Filter assays using the nonhydrolyzable GTP-analogue, S-y-GTP, revealed higher levels of binding than those obtained with GDP but there was no indication of any specific binding to p23 ras (Table 1) . When extracts were enriched for p23 by immu-noprecipitating first and then incubating with GTP, the overall amount of GTP bound was increased but there was no indication of specific binding to the protein immunoprecipitated with Y13-259 (Table 1). B) Western Blot Probed with GTP When..'proteins were separated by SDS electrophoresis and then electroblotted onto nitrocellulose, several of the blotted 35 proteins bound S-y-GTP. However, there was no labeling in IT cl S the region that corresponded to p2 3 . The number of GTP-binding proteins was decreased if the lysates were first immu noprecipitated prior to electrophoresis, but the same GTP-bind-ing proteins were immunoprecipitated by anti-ras antibody and by normal rat serum (Fig. 1). Again, there was no indication of GTP binding to a protein of the molecular weight of p23 C) Photoaffinity Labelling with GTP Nucleotides can be covalently cross-linked to nuclectide-binding proteins using photochemical methods. Under the direct action of ultraviolet light, the binding of the ligand requires active enzyme and only occurs at the ligand-binding site on the protein (Yue & Schimmel, 1977). This technique has been used to detect several GTP-binding proteins in D. discoideum, in cluding a strongly binding protein of M of 24,000 (Schleicher 24 66K-l i .« 3.6K-29K-t 20 K-14 K-A B Figure 1. GTP Binding to D. discoideum Proteins. Samples were electrophoresed through an 11.25% polyacrylamide gel and the protein was transferred to nitrocellulose. The blot was probed with 50 yCi S-y-GTP as described in Methods. Two samples of Triton-solubilized whole cell lysate (1.2 mg protein) were treated with normal rat serum (Lane A) or Y13-259 (Lane B) and the immunoprecipitated material was subjected to electrophore sis. Lane C: 120 yg total protein. Molecular weight markers were visualized by the India Ink staining method. 25 et al., 1986). In order to determine whether this GTP-binding 10 3, S protein was actually the p23 , extracts were photoaffinity labeled with GTP and then either subjected to SDS electrophore sis directly or immunoprecipitated and then subjected to SDS electrophoresis. The resulting gels were dried and autoradio-graphed. A number of proteins in crude cytosol and membrane fractions bound GTP, including a protein of Mr about 24,000 (Fig. 2) but none of these proteins were immunoprecipitated with either the specific Y13-259) or the control antibody (Y3). 10 Si s These results indicate that the p23 protein does not bind GTP under these conditions and that the previously identified 24,000 Mr GTP-binding protein is not the ras protein. To en-10 ci S rich for p23 in this assay, samples of Triton-solubilized whole cell lysate (3.34 mg protein) were immunoprecipitated with either control or Y13-259 first and then the immunopreci pitated proteins were photoaffinity labeled and electrophoresed (see Methods). The results obtained were the same as those TJ* Si S when p23 was photoaffinity labeled and then immunoprecipi tated . 10 Si s Although Y13-259 neutralizes p21 activity, it does not interfere with the binding of GDP (Hattori et al., 1987) or GTP (Lacal & Aaronson, 1986). However, it is possible that GTP 10 cl S binding prevented the immunoprecipitation of p23 in these 35 experiments. To address this question, S-methionine labeled Triton-solubilized whole cell lysate was photoaffinity labeled in the presence and absence of 1 mM GTP and then immunoprecipi tated with control or anti-ras antibody (Fig. 3) . In both 26 Figure 2. Direct Photoaffinity Labeling with GTP. Three ali quots each of 100 ug protein from crude cytosol and membrane., Inactions were incubated in the dark, on ice, with 1.4 x 10 M P-a-GTP for 30 min. The samples were then irradiated with short wave UV light for 60 min. One cytosol (Lane A) and one membrane (Lane B) sample was mixed with SDS sample buffer and stored at -20°C while two samples each of cytosol (Lanes C and D) and membrane (Lanes E and F) proteins were immunoprecipi tated with either control antibody (Lanes C and E) or Y13-259 (Lanes D and F). All samples were then subjected to electro phoresis through a 12.5% polyacrylamide gel. The gel was cut to separate Lanes A and B from Lanes C through F so that the dried gels could be' exposed to film (with a screen and at -70°C) for different periods of time. Lanes A and B: exposed for 23 h. Lanes C through F: exposed for one week. 27 20.1K 14.2K 12 3 4 Figure 3. Photoactivation and Immunoprecipitation in the Pre sence and Absence of 1 mM GTP. Equal amounts (100 yg protein) of S-methionine labeled Triton-solubilized whole cell lysate were incubated in the presence (1) and absence (2) of 1 mM GTP in the dark, on ice, for 30 min. The samples were then irra diated with short wave UV light for 60 min. Samples were then immunoprecipitated with either control (Lanes A and C) or anti-ras (Lanes B and D) antibody and electrophoresed through a 12.5% polyacrylamide gel as described in Methods. The dried gel was exposed to XAR-5 film for 10 days. 28 cases the p23 protein was iitununoprecipitated by the Y13-259 antibody suggesting that GTP binding does not interfere with immunoprecipitation. A positive control for GTP binding was provided by photo-affinity labeling a lysate from HaMSV-transformed rat cells and then immunoprecipitating with either anti-ras (Y13-259) or control antibody (Y3). The immunoprecipitates were then elec-2Tcl S trophoresed. The p21 protein did bind GTP under these con ditions (Fig. 4). A 0.3% Triton X-100 D. discoideum membrane extract was photoaffinity labeled under the same conditions and then immunoprecipitated with either Y13-259 or Y3 antibody. The immunoprecipitated proteins were then electrophoresed and autoradiographed. There is no indication of GTP binding by 3tr SL S p23 under these conditions. 29 66K -36K •29K •(24K) A B C D Figure 4. GTP Binding to p21 from Harvey Murine Sarcoma Virus (HaMSV) Transformed NIH-3T3 Cells. Equal amounts of protein (2.5 mg in about 1 ml volume) from either a 0.3% Tri ton X-100 D. discoideum membrane extract (Lanes A and B) or a lysate of HaMSV-transformed NIH-3T3 cells (Lanes C and D) were concentrated to 100 yl at 4°C. Two 50 yl aliquots of ^|ch sample were incubated in microtiter wells with 1.2o.pM P-crGTP in the dark for 60 min, on ice. The samples were then irra diated with short-wave UV light for 30 min. The photoaffinity-labeled samples were then immunoprecipitated with either 5 yl of Y3 (Lanes A and C) or 5 yl of HPLC-purified Y13-259 (Lanes B and D). The immunoprecipitated proteins were electrophoresed through a 12.5% SDS-polyacrylamide gel. The dried gel was exposed to film at -20°C with a screen for 4 days. 30 SECTION II. ATTEMPTED PURIFICATION OF p23raS Attempts were made to purify p23 since a partially purified preparation should provide more definitive results in a GTP-binding assay in that other GTP-binding proteins should be removed and there should be relatively more p23 protein. 3T cl S Ultimately purified p23 could be used in membrane reconsti-tution experiments to study the function of the protein. Immu noaff inity chromatography was chosen because it potentially provides a one-step purification procedure, the immunopreci pitation with Y13-259 works well, and there is an unlimited supply of the monoclonal antibody. A) Solubilization Conditions 2T cl S The p2 3 is exclusively found in the crude membrane fraction rather than the cytosolic fraction after high-speed centrifugation in the presence of the protease inhibitors leu-peptin and antipain (Weeks & Pawson, 1987). The p23 protein was removed from the membrane by Triton X-100 (Fig. 5), a con centration of 0.3% (w/v) being required for total solubiliza tion (Fig. 6). Triton X-100 solubilization resulted in an enrichment of p2 3 of approximately four-fold over Triton-solubilized whole cell lysates. The p23raS was not solubilized from the membrane by high pH, high salt, or the presence of EDTA (Fig. 7), suggesting that p2 3 is tightly bound to the membrane bilayer. In fact, p23 is acylated (Weeks et al., 1987) like the mammalian ras protein, and the acyl group may anchor the protein to the membrane. 31 1 2 3 4 5 6 7 8 9 10 11 12 — 66K — 45K 36K 29K p23 — 20.1 K 14.2K Figure 5. Solubilization of p23 with Triton X-100. Equal amounts of S-methionine-labeled crude membranes were resus-pended in 1 ml of Tris-salts containing 40 yg leupeptin, 40 yg antipain, and 0% (Lanes 1 to 4) , 0.3% (Lanes 5 to 8), or 1.0% (Lanes 9 to 12) Triton X-100 and mixed well by vortexing. After a 15 min incubation at 4°C, the samples were recentri-fuged to separate unsolubilized pellet (Lanes 1, 2, 5, 6, 9, 10) and solubilized supernatant (Lanes 3, 4, 7, 8, 11, 12). The pellets were then resuspended in 1 ml of buffer and each sample was split into two 0.5 ml aliquots for immunoprecipi-tation with 75 yl of either normal rat serum (Lanes 1, 3, 5, 1, 9, 11) or Y13-259 Lanes 2, 4, 6, 8, 10, 12). The immuno precipitated protein was electrophoresed as described in Methods and fluorographed for 9 days. 32 — 66K —45K —36K 29K p23 —20.1 K — 14.2K 1 2 3 4 5 6 7 8 9 10 11 12 Figure 6. Effect of Low Concentrations of Triton X-100 on the Solubilization of p23. Equal amounts of radiolabeled crude membrane fractions were resuspended in 1 ml of Tris-salts containing 40 yg leupeptin, 40 yg antipain, and 0.01% (Lanes 1 to 4), 0.03% (Lanes 5 to 8), or 0.1% (Lanes 9 to 12) Triton X-100. After a 15 min incubation, the samples were recentri-fuged to separate unsolubilized pellet (Lanes 1, 2, 5, 6, 9, 10) and solubilized supernatant (Lanes 3, 4, 7, 8, 11, 12). The pellets were resuspended in 1 ml of buffer and each sample was split into two 0.5 ml aliquots for immunoprecipitation with either normal rat serum (Lanes 1, 3, 5, 7, 9, 11) or Y13-259 (Lanes 2, 4, 6, 8, 10, 12). The immunoprecipitated protein was electrophoresed and fluorographed for 4 days. 33 — 66K — 45K — 36K —-29K <-p23 — 2WK — 14.2K 3 4 6 7 8 9 10 11 12 Figure 7. Effect of High Salt, Ethylenediamine Tetraacetic Acid (EDTA), or High pH on Solubilization of p23. Equal amounts of radiolabeled crude membranes were resuspended in 1 ml of Tris-salts containing 40 yg leupeptin, 40 yg anti-pain, and 0.5 M NaCl (Lanes 1 to 4), 10 mM EDTA (Lanes 5 to 8), or 100 mM Na^COg (Lanes 9 to 12). After a 15 min incu bation, the samples were recentrifuged to separate unsolu-bilized pellet (Lanes 1, 2, 5, 6, 9, 10) and solubilized v>. <•:•• supernatant (Lanes 3, 4, 7, 8, 11, 12). The supernatants were dialysed against Tris-salts overnight at 4°C and the pellets were resuspended in 1 ml of buffer and stored over night at -70°C. Each sample was split into two 0.5 ml ali quots and immunoprecipitated with either normal rat serum (Lanes 1, 3, 5, 7, 9, 11) or Y13-259 (Lanes 2, 4, 6, 8, 10, 12). The immunoprecipitated protein was electrophoresed and fluorographed for 4 days. 34 B) Detection by ELISA The ELISA Is a rapid and simple method that can be used to monitor protein purification. An assay was developed using vegetative membranes as the antigen, as described under Methods. In an attempt to lower backgrounds, a variety of modifications were tried: BSA, sheep IgG (Table 2), or 1% SDS (data not shown) as blocking agents, ther-use of DEAE-purified Y13-259, and the use of enzyme-antibody conjugate that had been first adsorbed with vegetative cells (data not shown). The best results were obtained with 0.3% BSA as blocking agent with Y3 as control antibody (Table 2). In addition, varying the incu bation times did not improve the activity over background. A two-step ELISA, using goat anti-rat IgG (GARAT) conju gated to alkaline phosphatase as the second antibody, gave high activity to control ratios but only at very high concentrations of GARAT (Table 3). For example, using Y3 as the control anti body, a 1/100 dilution of GARAT gave an activity to control ratio of 3.3 whereas the standard assay using the alkaline phosphatase conjugate at a dilution of 1/2000 gave an activity to control ratio of 1.7 (Table 2). However:, the two-step ELISA proved to be cost-prohibitive due to the high concentrations cf GARAT required for a high activity to control ratio. In the standard ELISA, concentrations of Triton X-100 in excess of 0.00 3% reduce the Y13-259 activity to the same level as the control activity (data not shown). Other detergents (polyoxyethylene (20) sorbitan monolaurate (Tween 20), 3-(3-cholamidopropyl)dimethyl-ammonio (CHAPS), Z-14, polyoxethylene Table 2. ELISA Using Vegetative Membranes as Antigen °'D-405 Condition Normal Rat Serum Y3 Y13 -259 Activity - Control A/C Ratio2 0.3% BSA 0.495 N/Db 0 . 710 0.215 1.4 ± 0.028 + 0 .024 ± 0.037 0.3% BSA N/D 0.426 0 . 710 0 .284 1.7 ± 0.037 + 0 .024 ± 0.044 0.001% 0 .374 N/D 0 .535 0 .161 1.4 Sheep IgG ± 0.021 + 0 .023 ± 0.031 0.003% 0.417 N/D 0 .510 0.093 1.2 Sheep IgG 1 0.033 + 0 .020 ± 0.0.39 a. Activity to control ratio. b. Not determined. The means and standard deviations of eight determinations are given. These results are from a single experiment but are representative of several experiments. Sheep IgG was added in place of BSA. The incubation with substrate was for 60 min. 36 Table 3. Two-Step ELISA with Goat Anti-Rat Immuno globulin G (GARAT) Conjugated to Alkaline Phosphatase O.D.4Q5 Dilution Normal Activity A/C of GARAT Rat Serum Y3 Y13-259 - Control Ratio 1/20' 0.086 ± 0.014 N/D 0.736 0.074 0 0 6 50 075 8.6 1/100 N/D 0.097 0.003 0.319 ± 0.007 0.222 ± 0.008 3.3 a. The means and standard deviations of seven determinations are given. The incubation with substrate was for 30 min. b. The means and standard deviations of two determinations are given. The incubation with substrate was for 90 min. 37 23-lauryl ether (Brij-35) have the same deleterious effect and n-octyl-glucoside gives a false positive result (data not shown). Removal of Triton X-100 with Bio-Beads SM-2 does not remove sufficient detergent to allow activity above control levels (data not shown). Acetone precipitation of samples did remove the Triton X-100 but resulted in only a very weak signal (Table 4). X" cl S Despite these problems, I tried to detect p23 in frac tions separated by immunoaffinity chromatography with the ELISA because I thought the purified protein would give a strong enough signal to be detected with this assay. Samples contain ing Triton X-100 were acetone-precipitated before being used for the ELISA. A 0.3% Triton X-100 membrane extract was applied to a column that had been pre-equilibrated in Tris-salts (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 100 mM NaCl). In order to remove unbound protein and detergent the column was washed with Tris-salts until the O.D._ori reached zero. Several conditions were used to try to elute p23raS: 0.5 M NaCl; 0.1 N HC1; 0.1 M glycine-HCl, pH 2.5 containing 50% ethylene glycol; 1 M and 4 M guanidine-HCl; and 3 M and 6 M NaSCN. In all experiments, ]C cl S p23 was bound to the column but was not eluted by any of the elution conditions. Approximately 25% of the total protein applied to the column was never recovered. This suggested that Triton X-100 was needed during chromatography to keep the mem brane proteins solubilized. An assay was therefore required 38; Table 4. ELISA with Vegetative Membranes and Acetone-Precipitated 0.3% Triton X-100 Membrane Extract O-D.405 Normal Activity Condition Rat Serum Y13-259 - Control A/C Ratio Vegetative 0.464 0.872 0 .408 1.9 Membranes ± 0.070 ± 0.055 ± 0.089 Acetone- 0 .110 0 .192 0 .082 1.7 Precipitated ± 0.014 ± 0.113 ± 0.114 The means and standard deviations of four determinations are given. The RARIG was diluted 1/1000 for this experiment and the incubation with substrate was for 30 min. 39 that could be used in the presence of detergent, since the ELISA had poor sensitivity on acetone-treated fractions. Two immunoassays that can be performed with the antigen in the presence of detergent are the dot blot and the Western blot. The dot blot assay was insufficiently specific to be of value in that there was an equally strong activity when the antigen was incubated with Y13-2 59 antibody as when it was in cubated with normal rat serum. The Western immunoblotting Z? ci S technique would only detect p23 if the protein was immuno precipitated prior to electrophoresis and then electroblotted to nitrocellulose for detection, and this method proved to be insufficiently sensitive (data not shown). The most sensitive assay that could be performed with the antigen in the presence of detergent was immunoprecipitation of radioactively-labeled proteins followed by radioautography. This assay was there fore used to monitor the fractionation of membrane extracts by immunoaffinity chromatography. C) Detection by Immunoprecipitation 35 For these experiments, S-methionine-labeled membrane extracts were applied to immunoaffinity columns that had been pre-equilibrated under various detergent conditions. Since the immunoprecipitation of p23 with Y13-259 is done in the presence of 1% Triton and 0.5% SDS, an immunoaffinity column equili brated with the same detergents was initially used to try to purify p2 3. The membrane extract applied to this column was also solubilized with 1% Triton X-100 and 0.5% SDS. About 30% 40 of the total protein bound nonspecifically to this column but IT ci. S p23 did not bind under these conditions (Table 5) so other buffer conditions were tried. A membrane extract solubilized with 0.3% Triton X-100 was applied to a Y13-259 column that had been equilibrated in Tris-salts buffer (Table 6). Under these conditions there was lit tle nonspecific binding by total protein but again none of the p23ras was bound. 41 Table 5. Immunoaffinity Chromatography with Y13-259 Column Equilibrated in Tris-Salts Plus 1% Triton X-100 and .0.5% Sodium Dodecyl Sulphate (SDS). Fl6w- Not Through Elution 1 Elution 2 Recovered Protein (%) 68 0.8 0 . 2 31 P23ras (%) 100 0 0 0 a. The column was eluted sequentially with 0.1 M diethylamine, 1 M guanidine, and 1 M NaSCN, each containing 1% Triton X-100. b. The column was then eluted with the harsher condition of 6 M NaSCN + 1% Triton X-100. 42 Table 6. Immunoaffinity Chromatography with Y13-259 Column Equilibrated in Tris-Salts Flow- b Not Through Elution 1 Elution 2 Recovered Protein (%) 98 1.7 0.3 0 p23raS (%) 100 0 0 0 a. The column was eluted sequentially with Tris-Salts + 0.5% SDS, 0.1 N HCl, and 0.1 M diethylamine each containing 1% Triton X-100. b. The column was then sequentially eluted with 4M guanidine and 6 M NaSCN, each containing 1% Triton X-100. 43 DISCUSSION In this study, I attempted to demonstrate GTP binding by 3T cl S p23 using three different approaches: a filter assay, a Western blot assay, and photoaffinity labeling. The filter assay involved incubation of cell extracts with radiolabeled GDP or GTP followed by immunoprecipitation with anti-ras anti body. The immunoprecipitated complexes were then collected on nitrocellulose filters and counted directly. This approach has been used successfully to demonstrate GDP and GTP binding by KiMSV transformed cell lysates immunoprecipitated with anti-p21 antibody (Scolnick et al., 1979). Experiments where the immune complexes were boiled to release the protein instead of being collected on nitrocellulose filters showed guanine nucleotide binding by yeast cell extracts (Tamanoi et al., 1984). Since the same monoclonal antibody (Y13-259) specifically immunopre-X* ci S cipitates p21 and p23 (Pawson et al., 1985), this assay should detect guanine nucleotide binding with D. discoideum cell extracts if p23 does bind guanine nucleotides. One pos-i" cl S sible reason for the absence of specific GTP binding by p23 in the filter assays described in this investigation is the hydrolysis of bound GTP to GDP and its subsequent release by i" ci S the enzyme. However, since p23 did not bind a nonhydro-lyzable GTP-analogue, this explanation was rejected. For the Western blot assay proteins from cell lysates and immunoprecipitated protein were separated on polyacryla mide gels and then electro-blotted to nitrocellulose. The 44 nitro-cellulose was then incubated with radiolabeled GTP. This ^ Si S method had been used to show GTP binding by p21 that had been expressed in Escherichia coli under conditions where this protein represented 5 to 10 per cent of the total intracellular bacterial protein (McGrath et al., 1984). In vegetative cells of D. discoideum, p23 represents only 0.02 to 0.03 per cent of total protein (Pawson et al., 1985). The low cellular level 10 Si S of p23 may account for the failure of the Western blot assay to detect GTP binding to the protein. In the third approach, p. discoideum cell lysates were photoaffinity labeled with GTP and the labeled proteins were either electrophoresed directly or immunoprecipitated and then electrophoresed. In addition, cell lysates were immunoprecipi tated and then photoaffinity labeled with GTP prior to electro phoresis. This approach led to detectable labeling of the 2T cl S p21 protein from HaMSV-transformed NIH-3T3 cells in the 10 cl S presence of 1.2 yM GTP (Fig. 4), but p23 exhibited no detec table GTP binding using the same experimental conditions. One possible reason for a lack of GTP binding by cell ly sates photoaffinity labeled and then immunoprecipitated is that the bound GTP interferes with immunoprecipitation of p23 by Y13-259. However, incubation of Triton-solubilized whole cell lysate with 1 mM GTP under similar conditions to those used in the photoaffinity labeling experiments did not interfere with immunoprecipitation of p23 by Yl3<+259 (Fig. 3). There are two possible explanations for the lack of 10 Si s GDP/GTP binding by p23 : the protein from D. discoideum may 45 have a very low affinity for guanine nucleotides and binding is therefore undetectable using the experimental procedures de-cl S scribed in this thesis or p23 may not bind guanine nucleo tides at all. The first possibility could theoretically be tested by using high concentrations of GTP but this experiment is impractical in terms of the large amounts of radiolabel that would be required. The second possibility seems unlikely in view of the fact that the RASl and RAS2 proteins of Saccharo-myces cerevisiae bind GTP and GDP specifically (Temeles et al., 1985; Tamanoi et al., 1984) like the mammalian ras proteins (Scolnick et al., 1979; Shih et al., 1980; Trahey et al., 1987) although they apparently perform different physiological func tions (Beckner et al., 1985; Toda et al., 1985; Uno et al., 1985) . A number of different enzyme activities in D. discoideum cells that had been transfected with the missense mutation Dd-12 ras-Thr have been examined (Van Haaster et al., 1987). This mutation, analogous to that found in oncogenic mammalian ras genes (Newbold, 1984), results in abberant morphogenesis of D. 12 discoideum cells (Reymond et al., 1986). The Dd-ras-Thr transformants exhibit normal folate and cAMP-induced activation and desensitization of adenylate cyclase but the transient in crease in cGMP is terminated earlier in these cells than in untransformed cells. This latter phenomenon is not due to a lower activity of guanylate cyclase or to a higher activity of cGMP-stimulated phosphodiesterase, but rather to an altered 17 cl S desensitization of guanylate cyclase. Thus p23 does not 46 appear to be involved in the regulation of adenylate cyclase but to be directly or indirectly involved in the enhanced de-sensitization of guanylate cyclase. The Dd-ras gene used in the studies described above was originally detected as a homologue of the mammalian ras gene (Reymond et al., 1984). It encodes a protein of Mr of 24,000. X" cl S There are distinct differences in the expression of p23 and the messenger ribonucleic acid (mRNA) that is hybridized by the ras gene during development. Synthesis of p2 3 is most ra pid in vegetative cells and then drops during differentiation until the pseudoplasmodial stage where there is a slight in crease in the rate of synthesis before the rate continues to drop to about 10% of the original ameoboid level at the end of development (Pawson et al., 1985). In contrast ras mRNA is present in low levels in vegetative cells, but in high levels in pseudoplasmodia (Reymond et al., 1984). At the pseudoplas modial stage, newly synthesized p23 is localized in prespore cells (Weeks & Pawson, 1987) whereas p24 mRNA is localized in prestalk cells (Reymond et al., 1984). It is possible that X" cl S p23 and the product of the cloned ras gene are not identical and that each protein has a specific function at particular X* cl S points of development. Thus p2 3 may not be involved in the desensitization of guanylate cyclase like the product of the cloned ras gene and it may not act as a GTP binding regulatory protein since there is evidence from this study suggesting cl S that it does not bind guanine nucleotides. However p23 and 47 the cloned ras gene product may be identical in that the pro tein immunoprecipitated by Y13-259 is recognized by a polyclo nal antibody raised against the ras gene product. Also Y13-259 thus far has been shown to immunoprecipitate all known ras gene products. It is interesting to note that Firtel's group have not reported guanine nucleotide binding by the Dd-ras gene product and it is possible that they have attempted to demonstrate GTP binding, but have also been unsuccessful. Ras-related genes have been found in Drosopholia melanogaster (Weinberg & Shilo, 1981), in the mollusc Aplysia (Madaule & Axel, 1985), and Schizosaccharomyces pombe (Fukui & Kaziro, 1985), but there have been no reports that their gene products bind GTP or GDP. The definitive determination of nucleotide binding by p23 awaits the purification of this protein. In addition, func tional studies on the ras protein in D. discoideum will require purified protein. My attempts at purification of p23 by immu-noaffinity chromatography have not been successful but I be lieve that this protein can at least be enriched by this method because other membranous proteins have been purified or great ly enriched using immunoaffinity chromatography, including the src oncogene product (Erikson et al., 1979). There seems to be one main problem with the immunoaffi-cl S nity purification of p23: p23 does not bind to the column (Tables 5 and 6). The D. discoideum protein may be aggregating and this may prevent binding of p23 to the antibody on the 48 column. Altering the equilibration and sample buffers may allow the disaggregation of the protein and allow the binding 3T cl S of p23 to the column. Membrane-associated proteins from other organisms have been purified by immunoaffinity chromatography in combination with other chromatographic methods (Aubry et al., 1987; Shen & Tai, 1986). This approach is difficult in the case of D. discoideum membrane proteins since they nonspecifically bind and aggregate very easily and do not fractionate easily on gel filtration and ion-exchange columns (MacDonald, 1986). Other membrane proteins have been purified by passing the preparation over a column of the matrix itself (Hugues & August, 1982) or a column made with a nonspecific antibody (Suzuki et al., 1987) to remove nonspecifically binding protein before application to the immunoaffinity column. This approach was not necessary for this study because there was no problem with nonspecific binding if the column was equilibrated in the absence of detergent (Table 6) . Although it would be worthwhile to systematically vary the conditions of equilibration and binding in an attempt to obtain an enrichment of p2 3 by immunoaffinity chromatography, because the protein is found at such low levels in the cell, it may be simpler to isolate the genomic ras clone and express p23 in a suitable organism (such as E. coli) so that there is an enrichment for p23 before, application to an immunoaffinity column. This would aid in the detection of the protein during 49 chromatography and should eliminate the problems of nonspecific aggregation by D. discoideum proteins. 50 REFERENCES Aubry, M., Berteloot, A., Beaumont, A., Roques, B.P., & Crine, P. (1987). The use of a monoclonal antibody for the rapid purification of kidney neutral endopeptidase ("enkephalinase") solubilized in octyl glucoside. 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