ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin

The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.     

Microinjection of a 19-kDa guanine nucleotide-binding protein inhibits maturation of Xenopus oocytes

ADP-ribosylation factors (ARFs) are 19-21-kDa proteins purified from bovine brain that bind guanosine 5'-triphosphate (GTP). They exhibit GTP-dependent activity as activators of cholera toxin-catalyzed ADP-ribosylation of the alpha-subunit of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system (Gs alpha). ARF, which interacts directly with the catalytic subunit of cholera toxin, has no known physiologic role. Intracellular microinjection of ARF was employed to investigate the effect of ARF on progesterone- and insulin-stimulated maturation of Xenopus oocytes. Maturation was inhibited by injection of ARF 3-8 h before exposure of oocytes to progesterone or insulin. ARF inhibition was dependent on progesterone concentration but not on insulin concentration. Inhibition was enhanced by concomitant injection of GTP and to a greater extent by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) which, in the absence of ARF, inhibited somewhat at early time points. The demonstration of this effect of ARF on both progesterone- and insulin-stimulated oocyte maturation may provide a clue to the physiologic role of this guanine nucleotide-binding protein.     

Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin

Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation.     

Endogenous ADP-Ribosylation in Brain: Initial Characterization of Substrate Proteins

Cholera and pertussis toxin-mediated ADP-ribosylation has been used extensively to study regulation of guanine nucleotide binding proteins (G proteins) in the nervous system, but much less is known about possible endogenous ADP-ribosylation of G proteins in brain. The present study demonstrates endogenous ADP-ribosylation, in the absence of cholera and pertussis toxins, of four predominate proteins in homogenates of rat cerebral cortex. These proteins showed apparent molecular masses of 20, 42, 45, and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 42- and 45-kDa proteins comigrated precisely with the major cholera toxin-labeled bands. Furthermore, the endogenous ADP-ribosylated and cholera toxin-ADP-ribosylated bands yielded identical 32P-labeled peptide fragments by one-dimensional peptide mapping, indicating that they are probably the same proteins, presumably the alpha-subunits of Gs. In contrast, peptide maps of the 50-kDa protein, which migrated close to a 48-kDa cholera toxin-labeled band, demonstrated that this protein is distinct from the toxin-labeled band and from Gs alpha. Levels of endogenous ADP-ribosylation activity showed regional heterogeneity in brain, with a nearly threefold variation observed among the brain regions examined. Chronic administration (7 days) of corticosterone significantly increased overall levels of endogenous ADP-ribosylation, indicating that components of this system may be under hormonal control in vivo. Attempts to identify neurotransmitters or second messenger systems that regulate endogenous ADP-ribosylation activity in brain have so far been unsuccessful with one exception.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein

Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator.     

ADP-Ribosylation with Pertussis Toxin Modulates the GTP-Sensitive Opioid Ligand Binding in Digitonin-Soluble Extracts of Rat Brain Membranes

Pertussis toxin-catalyzed ADP-ribosylation of the guanine nucleotide-binding proteins Gi and Go is shown to proceed in Mg2+-digitonin extracts from rat brain; the Mr 41,000 and Mr 39,000 peptides are labelled there as in the membranes. The ADP-ribosylation in detergent solution retains the differential sensitivity to guanine nucleotide analogues. This reaction also removes the partial inhibition by the guanine nucleotides of the binding of opioid agonists, as does the same treatment in the membranes. The partial inhibition of agonist binding by Na+, however, is left unchanged. The binding of the antagonist naloxone is little affected by Na+ or by guanine nucleotides in the treated membranes, but the treated soluble receptors show an enhanced binding in high-Na+ medium, although still guanine nucleotide insensitive. The data suggest that the toxin reaction in the absence of guanine nucleotides and agonist stabilizes the opioid receptor in a receptor-G-protein coupled state which is no longer sensitive to guanine nucleotides but retains its sensitivity to the Na+ ions.     

Evidence for a guanine nucleotide-binding regulatory protein in invertebrate and mammalian sperm. Identification by islet-activating protein-catalyzed ADP-ribosylation and immunochemical methods

The abalone sperm adenylate cyclase does not appear to be regulated by guanine nucleotides, but has a Mg2+-supported catalytic activity similar to other hormone- and guanine nucleotide-regulated enzymes (Kopf, G. S., and Vacquier, V. D. (1984) J. Biol. Chem. 259, 7590-7596; Kopf, G. S., and Vacquier, V. D. (1985) Biol. Reprod. 33, 1094-1104). The present studies were undertaken to ascertain whether the abalone enzyme has associated guanine nucleotide-binding regulatory proteins. Membrane fractions were incubated with either islet-activating protein (IAP) or cholera toxin and analyzed by sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis for the presence of toxin-catalyzed ADP-ribosylated proteins. The supernatant from a Lubrol PX-extracted 48,000 X g pellet fraction contained a Mr = 41,000 IAP substrate. This substrate could not be ADP-ribosylated prior to detergent extraction. Lubrol PX-solubilized fractions of membrane preparations from mouse, bovine, and human sperm also contained a Mr = 41,000 IAP substrate. These proteins co-migrated on sodium dodecyl sulfate-polyacrylamide gels with the Mr = 41,000 alpha i-subunit of the inhibitory guanine nucleotide-binding regulatory protein (Gi) from transformed chicken embryo fibroblast and mouse S-49 lymphoma membrane extracts. The sperm IAP substrates displayed similar protease digest patterns to alpha i of mouse S-49 lymphoma cells. Sea urchin sperm analyzed in a similar manner contained a Mr = 39,000 IAP substrate. Cholera toxin-catalyzed ADP-ribosylation of specific sperm membrane proteins was not observed in any of the sperm preparations tested. The presence of the beta-subunit common to both the stimulatory and inhibitory guanine nucleotide-binding regulatory heterotrimers was confirmed in sperm using an antiserum directed against the purified beta-subunit of the guanine nucleotide-binding regulatory proteins from bovine brain. It is concluded that all of the sperm tested, with the possible exception of sea urchin sperm, contain a Gi-like protein. Additional properties of these proteins and their role(s) in sperm function are currently being examined.     

GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.     

ADP-Ribosylation by Cholera Toxin of Membranes Derived from Brain Modifies the Interaction of Adenylate Cyclase with Guanine Nucleotides and NaF

We have developed a method to ADP-ribosylate the stimulatory guanine nucleotide-binding protein of adenylate cyclase (GS) in brain membranes by using cholera toxin. In particular, we used isonicotinic acid hydrazide and 3-acetylpyridine adenine dinucleotide to inhibit the potent NAD-glycohydrolase activity of brain membranes, and we used the detergent Triton X-100 (at 0.1%) to improve the accessibility of the toxin and guanine nucleotides used for supporting the ADP-ribosylation. This method reveals that GS is a very abundant protein in membranes derived from calf brain (approximately 30 pmol/mg of protein). In brain, GS exists in large excess over the previously reported amount of the adenylate cyclase catalytic subunit. The modification of GS with an ADP-ribosyl residue (a) elicits a four- to fivefold activation of adenylate cyclase by GTP, (b) increases the stabilization of adenylate cyclase by GTP, and (c) reduces adenylate cyclase activation by fluoride but does not change basal activity, activation by guanosine 5'-(beta, gamma-imido)triphosphate, or the sensitivity of adenylate cyclase to heat-induced denaturation. A correlation between ADP-ribosylation and the alterations in the activation of adenylate cyclase by guanine nucleotides and by fluoride is presented.     

Regulation of Endogenous ADP-Ribosylation by Acute and Chronic Lithium in Rat Brain

Abstract: In the present study, we investigated the effects of lithium on endogenous ADP-ribosylation in rat brain. It was found that addition of lithium in vitro inhibits endogenous ADP-ribosylation activity in extracts of frontal cortex at therapeutically relevant concentrations. Inhibition is observed at concentrations as low as 0.3 m M and is maximal at 1 m M when 50% inhibition is obtained. A similar degree of inhibition of endogenous ADP-ribosylation was observed for all substrate proteins identified, including G_sα, suggesting that lithium's effect may be achieved at the level of ADP-ribosyltransferases and not specific substrate proteins. In contrast to lithium, chloride salts of sodium and potassium do not alter endogenous ADP-ribosylation activity in frontal cortex. To assess the possible in vivo relevance of this in vitro action of lithium, we studied the effect of chronic lithium administration on levels of endogenous ADP-ribosylation in frontal cortex. It was found that chronic lithium treatment, in contrast to the inhibitory effect of the drug in vitro, produced a >35% increase in endogenous ADP-ribosylation activity. A similar degree of increase was observed for all of the substrate proteins identified. These novel findings raise the possibility that certain endogenous ADP-ribosyltransferases are among the acute targets of lithium in the brain and that adaptations in these enzymes may be part of the mechanisms underlying lithium's long-term effects on brain function.     

Isolation of a Ca2+-Dependent Actin-Fragmenting Protein from Brain, Spinal Cord, and Cultured Neurones

Extracts of ox spinal cord and chicken brain were fractionated by ion-exchange chromatography and assayed for their ability to reduce the viscosity of muscle F-actin solutions. Two distinct peaks of activity were obtained, one of which was further purified by affinity chromatography on a DNAase-actin Sepharose column. Following molecular exclusion chromatography, the actin component appeared as a complex of 1 molecule of a protein with molecular weight 90,000 and 2 molecules of actin (42,000). This tightly bound complex was resistant to most methods of protein separation, but was resolvable into its component proteins by sodium dodecyl sulphate acrylamide gel electrophoresis. The protein of molecular weight 90,000 could be eluted from such a gel in a fully active form. The activity of the protein from ox spinal cord was closely similar to that of gelsolin, an actin-fragmenting protein originally isolated from rabbit lung macrophages. Like gelsolin, the protein from ox spinal cord produced fragmentation of muscle F-actin filaments at Ca2+ concentrations greater than 10(-7) M, and had a nucleating effect on the polymerisation of muscle actin; the latter was measured most easily by the enhancement of fluorescence of muscle actin conjugated to N-(1-pyrenyl)iodoacetamide. Nucleation was more effective in the presence of Ca2+, but also occurred in its absence, and the same was true of complex formation between the 90,000 protein and muscle G-actin. On the basis of its actin-fragmenting activity, we estimate that the 90,000 molecular weight protein constitutes 0.2% of the protein initially extracted from ox spinal cord. A very similar protein, indistinguishable in its action on actin but containing variable amounts of a protein of molecular weight 85,000 as well as 90,000, was isolated from chicken brain. A similar protein was also detected in pure cultures of sympathetic neurones by enrichment on a DNAase-actin affinity column and by immune blotting and by immunofluorescence. We conclude that a protein similar, if not identical to macrophage gelsolin is present in neurones and that it probably plays a part in the actin-based movements of these cells.     

Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain

Choleragen (cholera toxin) activates adenylate cyclase by catalyzing ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide-binding protein. It was recently found (Tsai, S.-C., Noda, M., Adamik, R., Moss, J., and Vaughan, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5139-5142) that a bovine brain membrane protein known as ADP-ribosylation factor or ARF, which enhances ADP-ribosylation of Gs alpha, also increases the GTP-dependent NAD:arginine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase, and auto-ADP-ribosylation activities of choleragen. We report here the purification and characterization of two soluble proteins from bovine brain that similarly enhance the Gs alpha-dependent and independent ADP-ribose transfer reactions catalyzed by toxin. Like membrane ARF, both soluble factors are 19-kDA proteins dependent on GTP or GTP analogues for activity. Maximal ARF effects were observed at a molar ratio of less than 2:1, ARF/toxin A subunit. Dimyristoyl phosphatidylcholine was necessary for optimal ADP-ribosylation of Gs alpha but inhibited auto-ADP-ribosylation of the choleragen A1 subunit and NAD:agmatine ADP-ribosyltransferase activity. It appears that the soluble factors directly activate choleragen in a GTP-dependent fashion. The relationships of the ARF proteins to the ras oncogene products and to the family of guanine nucleotide-binding regulatory proteins that includes Gs alpha remains to be determined.     

Target protein for eucaryotic arginine-specific ADP-ribosyltransferase

Among ADP-ribosyltransferases reported in eucaryotes, arginine-specific transferases from turkey erythrocytes, chicken heterophils and rabbit skeletal muscle have been purified and extensively studied. They were reported to modify a number of proteinsin vitro. ADP-ribosylation of Ha-ras-p21 and transducin by the turkey erythrocyte transferase inhibits their GTPase and GTP-binding activities. Chicken heterophil enzyme modifies several substrate proteins for protein kinases and decreases the phosphate-acceptor activity. Rabbit skeletal muscle Ca²⁺-ATPase is inhibited by ADP-ribosylation catalyzed by the muscle transferase. Three transferases all ADP-ribosylate small molecular weight guanidino compounds such as arginine, arginine methylester and agmatine and poly-L-arginine and nuclear histones. However, the observation that muscle transferase did not ADP-ribosylate casein or actin, both of which can be modified by the heterophil transferase under the same conditions indicates that substrate specificity of these two enzymes are different. Substrate-dependent effects were observed with polyions of nucleotides such that polyanions stimulate the ADP-ribosylation of possible target protein, p33 by chicken heterophil transferase but has no effect on the modification of casein by the same enzyme.     

Inhibition by Drebrin of the Actin-Bundling Activity of Brain Fascin, a Protein Localized in Filopodia of Growth Cones

Abstract: The purification of drebrin, an actin-binding protein that is specifically expressed in embryonic rat brain, was described previously. During the purification of drebrin, we found that an actin-binding protein of 54 kDa was also expressed at high levels in embryonic brain, and this protein was identified by immunoblotting as fascin. To explore the roles of fascin in brain development, we purified fascin from brains of infant rats and characterized it. We found that the actin-binding activity of fascin was strongly inhibited by drebrin. Fascin caused formation of actin bundles, a process that was inhibited in the presence of drebrin, as confirmed by electron microscopy and a low-speed centrifugation assay. In PC12 cells, fascin was localized in the filopodia of growth cones, whereas drebrin was localized in the basal region of growth cones. Our results suggest that fascin might play an important role in the organization of actin in filopodia and that this organization might be regulated by drebrin.     

Enzymatic and nonenzymatic ADP-ribosylation of cysteine

Mono-ADP-ribosylation is a protein modification that occurs at a number of different amino acids, dictated by the specificity of the individual ADP-ribosyltransferases. A specific cysteine in several guanine nucleotide-binding regulatory proteins is ADP-ribosylated by the bacterial protein pertussis toxin. Recent purification of an ADP-ribosylcysteine hydrolase and NAD:cysteine ADP-ribosyltransferase, and detection of ADP-ribose-cysteine linkages in tissue samples has raised hope that an endogenous regulatory cysteine-specific ADP-ribosylation pathway exists. A current goal is the identification of such a pathway for ADP-ribosylation of cysteine within animal cells. Interpretation of the data in this field has been complicated by recent reports that revealed several unforeseen chemical reactions of NAD and its metabolites with free cysteine and cysteine in proteins. This mini-review covers the latest understanding of the ADP-ribosylation reactions associated with cysteine, and provides a set of criteria for future research to establish positively the existence of an endogenous cysteine-specific mono-ADP-ribosyltransferase.     

The stimulatory guanine-nucleotide regulatory unit of adenylate cyclase from bovine cerebral cortex. ADP-ribosylation and purification.

Hormonal stimulation of adenylate cyclase from bovine cerebral cortex is mediated by a guanine-nucleotide regulatory protein (Gs). This protein contains at least three polypeptides: a guanine nucleotide-binding alpha s component and a beta X gamma component, which modulates the function of alpha s. The alpha s component from many tissues can be ADP-ribosylated with cholera toxin, but has been unusually difficult to modify in brain. We have improved incorporation of ADP-ribose by including isonicotinic acid hydrazide to inhibit the potent NAD glycohydrolase activity of brain. ADP-ribosylation is further improved by addition of detergent to render the substrates accessible and 20 mM-EDTA to chelate metal ions. Although Mg2+ is absolutely required for activation of adenylate cyclase by the GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), it is not obligatory for p[NH]ppG-stimulated ADP-ribosylation by cholera toxin. Under these conditions, the ADP-ribosylation of brain membranes is not enhanced by a cytosolic protein. We find that there are two major sizes of brain alpha s, which we have named 'alpha sL', with an apparent Mr of 42,000-45,000, and 'alpha sH' with an apparent Mr of 46,000-51,000 depending on the gel-electrophoretic system used. The alpha sL and alpha sH components can incorporate different amounts of ADP-ribose depending on the reaction conditions, so that one or the other may appear to predominate. Thus we show that incomplete ADP-ribosylation by cholera toxin is not a good indication of the relative amounts of alpha s units. Functionally, however, both forms of alpha s appear to be similar. Both forms associate with the catalytic unit of adenylate cyclase, but neither of them does so preferentially. There is an excess of each of them over the amount associated with catalytic unit. We have now substantially purified Gs from brain by a modification of the method of Sternweis et al. [(1981) J. Biol. Chem. 256, 11517-11526] as well as by a new, simplified, procedure. On SDS/polyacrylamide-gel electrophoresis, the purified brain Gs contains both the 45 and 51 kDa alpha s polypeptides revealed by ADP-ribosylation and a beta X gamma component. Activation of purified alpha s by guanine nucleotides or fluoride can be reversed by addition of purified beta X gamma component. The activated form of purified brain Gs has an Mr of 49,000 as determined by hydrodynamic measurements, which is consistent with the idea that the active form of brain Gs is the dissociated one.     

Evidence for a functionally active inhibitory guanine nucleotide-binding regulatory protein in the swine ovary

Incubation of particulate fractions of swine granulosa cells or luteal minces with purified pertussis toxin (islet-activating protein) and [32P]-NAD catalyzed the (32P)-ADP ribosylation of a 41,000 dalton membrane protein. ADP-ribosylation was markedly reduced by prior incubation of intact cells with toxin. The functional relevance of this presumptive inhibitory guanine nucleotide-binding protein in pig granulosa cells was indicated by the ability of prior treatment with pertussis toxin to increase cyclic AMP generation and progesterone production significantly in response to follicle stimulating hormone. Prior cellular intoxication also enhanced cyclic AMP production stimulated by luteinizing hormone and choleratoxin, but not basally or after forskolin. These results demonstrate the presence of an inhibitory guanine nucleotide-binding protein in both the follicular (granulosa cell) and luteal compartments of the mammalian ovary, and indicate its functional relevance in cyclic AMP generation and progesterone secretion.     

Mechanism of inhibition of transducin guanosine triphosphatase activity by vanadate

The visual excitation system of the retinal rod outer segments and the hormone-sensitive adenylate cyclase complex are regulated through guanine nucleotide-binding proteins, transducin in the former and inhibitory and stimulatory regulatory components, Gi and Gs, in the latter. These proteins are functionally and structurally similar; all are heterotrimers composed of alpha, beta, and gamma subunits and exhibit guanosine triphosphatase activity stimulated by light-activated rhodopsin or the agonist-receptor complex. Adenylate cyclase can be stimulated by vanadate, which, like NaF, probably acts through Gs. Effects of vanadate on the function of a guanine nucleotide-binding protein were investigated in a reconstituted model system consisting of purified transducin subunits (T alpha, T beta gamma) and rhodopsin in phosphatidylcholine vesicles. Vanadate (decameric) inhibited [3H]GTP binding to T alpha and noncompetitively inhibited GTP hydrolysis in a concentration-dependent manner with maximal inhibition of approximately 90% at 3-5 mM. Vanadate also inhibited release of bound GDP but did not affect the rate of hydrolysis of bound GTP (single turnover rate), indicating that vanadate did not interfere with the intrinsic GTPase activity of T alpha. Binding of T alpha to rhodopsin and the ADP-ribosylation of T alpha by pertussis toxin, both of which are enhanced in the presence of T beta gamma, were inhibited by vanadate. These findings are consistent with the conclusion that vanadate can cause the dissociation of T alpha from T beta gamma, resulting in the inhibition of GDP-GTP exchange and thereby GTP hydrolysis. Adenylate cyclase activation could result from a similar effect of vanadate on Gs.     

Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19 kDa protein

Cholera toxin causes the devastating diarrheal syndrome characteristic of cholera by catalyzing the ADP-ribosylation of Gs alpha, a GTP-binding regulatory protein, resulting in activation of adenylyl cyclase. ADP-ribosylation of Gs alpha is enhanced by 19 kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs. We investigated the effects of agents known to alter toxin-catalyzed activation of adenylyl cyclase on the stimulation of toxin- and toxin subunit-catalyzed ADP-ribosylation of Gs alpha and other substrates by an ADP-ribosylation factor purified from a soluble fraction of bovine brain (sARF II). In the presence of GTP, sARF II enhanced activity of both the toxin catalytic unit and a reduced and alkylated fragment ('A1'), as a result of an increase in substrate affinity with no significant effects on Vmax. Activation of toxin was independent of Gs alpha and was stimulated 4-fold by sodium dodecyl sulfate, but abolished by Triton X-100. sARF II therefore serves as a direct allosteric activator of the A1 protein and may thus amplify the pathological effects of cholera toxin.     

Human Brain Lectin: A Soluble Lectin That Binds Actin

A biotinylated probe was used for detection of endogenous ligands of a human brain lectin on blotted human brain soluble proteins. Of the various proteins from brain extract resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five reacted with the biotinylated probe. After elimination of saccharidic moieties by periodic treatment of the same extract, a single band with Mr approximately 43,000 was recognized by the lectin. This band was identified as actin using an anti-actin antibody. These results were confirmed by binding of biotinylated lectin to purified actin.