Islet-activating Protein Substrate Research Articles

Effect of pertussis toxin on noradrenaline-and lithium-induced melanosome aggregation in fish melanophores

1. The effects of pertussis toxin, islet-activating protein (IAP), and the Ca ionophore A23187 on denervated melanophores of the medaka were investigated to characterize the melanosome-aggregating receptors for noradrenaline. 2. Responses of cells to noradrenaline were markedly suppressed when the cells were exposed to IAP, during a 14 hr culture. 3. This IAP-induced suppression disappeared when the cells were incubated with both IAP and noradrenaline. 4. In contrast to the responses to noradrenaline, similar responses of cells to lithium ion were not suppressed by the prior treatment of the cells with IAP. 5. Neither the action of noradrenaline nor the action of lithium was mimicked by A23187. 6. It is proposed that in melanophores of the medaka the IAP substrate, which is known to be involved in adenylate cyclase inhibition and Ca 2+-mobilization in a variety of other cell types, plays an essential role in the cyclic AMP-dependent signal transduction system to mediate inhibition of adenylate cyclase.

Enhancement of bradykinin-induced prostacyclin synthesis in porcine aortic endothelial cells by pertussis toxin: Possible implication of lipocortin I

Bradykinin-stimulated prostacyclin synthesis in porcine aortic endothelial cells was enhanced by pretreatment of the cells with pertussis toxin or islet-activating protein (IAP) for 5 hr or longer. Although ADP-ribosylation of a protein with a molecular weight of 41–42 kD in the cell membranes was completed by 3 hr after the addition of IAP into the incubation medium, there was good correlation between enhancement of bradykinin-induced prostacyclin synthesis and ADP-ribosylation of the IAP substrate over a wide range of IAP concentrations. Furthermore, even if IAP was removed from the incubation medium at 3 hr, bradykinin-induced prostaglandin synthesis at 24 hr was still potentiated. Cycloheximide and actinomycin D enhanced bradykinin-induced prostacyclin synthesis and apparently blocked the effect of IAP. Since this result suggested the involvement of an inhibitor protein(s) of prostacyclin synthesis in the IAP effect, we studied the effect of IAP on the level of lipocortin I which is known to inhibit phospholipase A 2. Western and Northern blot analyses revealed that IAP decreased the amounts of protein and mRNA of lipocortin I. These results suggest that the enhancement of bradykinin-induced prostacyclin synthesis by IAP is associated with a decrease in the level of lipocortin I.

Purification of GTP-binding proteins from bovine brain membranes: Identification of heterogeneity of the α-subunit of G o proteins

Using high-resolution Mono Q column chromatography, we purified 6 distinct peaks of GTP-binding proteins from bovine brain membranes. Five of them consisted of 3 polypeptides with αβγ-subunits and served as the substrate of islet-activating protein (IAP), pertussis toxin. The other one was purified as α-subunit alone and was also ADP-ribosylated by IAP in the presence of βγ-subunits. When each α-subunit was characterized by immunoblot analysis using various antibodies with defined specificity, the two of them were identified as g i-1 and G i-2, and other 4 appeared to be G o or G o-like G proteins. The α-subunits of immunologically G o-like proteins were apparently distinguishable from one another on elution profiles from the Mono Q column. Thus, there was a heterogeneity of the α-subunit of G o in the brain membranes. ADP-ribosylation; GTP-binding protein; Islet-activating protein

A Permissive Role of Pertussis Toxin Substrate G-protein in P2-purinergic Stimulation of Phosphoinositide Turnover and Arachidonate Release in FRTL-5 Thyroid Cells: Cooperative mechanism of signal transduction systems

Abstract Extracellular ATP and other purinergic agonists were found to inhibit cAMP accumulation by depressing adenylate cyclase as an and/or to stimulate arachidonate release in association with phospholipase C or A2 activation and Ca2+ mobilization as in FRTL-5 cells. The stimulatory actions of a group of P2-agonists represented by ATP were partially inhibited by the pretreatment of the cells with islet-activating protein (IAP), pertussis toxin, even when an about 41-kDa membrane protein(s) was completely ADP-ribosylated. Only the IAP-sensitive part of the stimulatory actions was antagonized by 1,3-diethyl-8-phenylxanthine (DPX), an adenosine antagonist. GTP and 8-bromoadenosine 5'-triphosphate (Br-ATP) at two to three orders of higher concentrations than ATP also exerted the stimulatory actions, although they were entirely insensitive to both IAP and DPX. Ligand binding experiments with, [35S]ATP gamma S and [3H]DPX showed that ATP occupies both DPX-sensitive and insensitive receptor sites, whereas GTP does only ATP-displaceable DPX-insensitive sites. Thus, lack of sensitivity of GTP action to DPX was associated with its inability to occupy the DPX-sensitive sites. Adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) and P1-agonists such as AMP and N6-(L-2-phenylisopropyl-adenosine (PIA) did not show any stimulatory action. Nevertheless, the agonists remarkably enhanced the stimulatory actions of GTP or Br-ATP. Such permissive actions of PIA and others were sensitive to both IAP and DPX, as were shown for a part of the stimulatory actions of ATP as well as the of both PIA and ATP. We conclude that an IAP substrate G-protein(s) which mediates the inhibitory action of purinergic agonists via a DPX-sensitive purinergic receptor(s) may not directly link to the phospholipase C or A2 system but enhance the system which links to a DPX-insensitive P2-receptor, in an indirect or permissive manner.

Extracellular Adenosine Triphosphate Completely Reverses the Thyrotropin-Induced Morphological Change in FRTL-5 Cells

We quantified the TSH-induced morphological change in FRTL-5 thyroid cells according to a morphological index corresponding to the mean cell area measured from microscopic photographs. Within 15 min, TSH induced, at 10 pM and higher concentrations, a decrease in morphological index together with a rise in cAMP levels in a TSH dose-dependent manner. Forskolin, 3-isobutyl-1-methylxanthine, and RO 20-1724, the latter two being phosphodiesterase inhibitors, mimicked these TSH effects, indicating that the rise in cAMP levels is responsible for the TSH effect. Extracellular ATP and its derivatives, known as purinergic receptor agonists, decreased cAMP levels and caused a complete reversal of the TSH morphological effect. Prior exposure of the cells to islet-activating protein (pertussis toxin), the depletion of extracellular Ca2+, or the addition of low doses of protein kinase-C inhibitors completely abolished the inhibitory action of ATP on the TSH effect, whereas phorbol 12-myristate 13-acetate, which activates protein kinase-C, mimicked the ATP action to some extent. Thus, although the TSH-induced change in cell morphology seems to be dependent on cAMP levels, the inhibition of TSH action by ATP seems to be mediated by at least two signal transduction pathways involving islet-activating protein substrate G-proteins: one inhibiting adenylate cyclase and the other involving Ca2+ and protein kinase-C.

Stimulation of adenosine receptor enhances α1 -adrenergic receptor-mediated activation of phospholipase C and Ca2+ mobilization in a pertussis toxin-sensitive manner in FRTL-5 thyroid cells

Norepinephrine (NE) stimulated FRTL-5 thyroid cells via an α1-adrenergic receptor, resulting in cytosolic Ca2+ ([Ca2+]i) mobilization and activation of phospholipase C. Adenosine and its receptor agonist, phenylisopropyladenosine (PIA), although not exerting a direct effect, markedly enhanced the NE-induced changes. Basal NE action was not totally abolished whereas the permissive action of adenosine and PIA was completely abolished by pretreatment of the cells with islet-activating protein (IAP), pertussis toxin. The decrease in cAMP level induced by adenosine or PIA is not the cause of their permissive effect, since this effect was not reversed by the addition of cAMP-increasing agents. We conclude that an IAP substrate GTP-binding protein(s) plays a novel role in forming a stimulatory coupling between an adenosine receptor and an α1-adrenergic receptor-coupled phospholipase C system.

Tonic inhibition of renal response to vasopressin by a pertussis toxin substrate

The putative role of the inhibitory guanine nucleotide binding protein (Gi) in modulating the renal response to vasopressin was investigated using islet activating protein (IAP). IAP treatment in rats in vivo abolished the capacity of alpha 2-adrenoceptors to reverse vasopressin-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in microdissected cortical collecting tubule (CCT) segments. IAP pretreatment also caused a marked upward shift in the dose-response curve of vasopressin (10(-10) to 10(-4) M)-induced cAMP accumulation. Augmentation of the response to vasopressin in rat CCT was dependent on the in vivo dose of IAP and paralleled the loss in alpha 2-adrenoceptor responsiveness. In the isolated perfused kidney the antinatriuretic and antidiuretic effects of the V2-receptor agonist desamino-8-D-arginine vasopressin (DDAVP) (1 pM) were enhanced following IAP pretreatment. alpha 2-Adrenoceptor stimulation (30 nM epinephrine) inhibited the renal effects of DDAVP (1 pM) in kidneys from control but not IAP-pretreated rats. Interestingly, IAP pretreatment alone caused increased urine flow rate and enhanced excretion of sodium and chloride without affecting potassium excretion or renal hemodynamics in vitro. Our results suggest that an IAP substrate, probably Gi, 1) is required for signal transduction by renal alpha 2-adrenoceptors, 2) may tonically modulate the response to vasopressin in the CCT but not of parathyroid hormone in the proximal convoluted tubule, and 3) participates in renal water and electrolyte reabsorption independent of exogenous adenylate cyclase stimulation.

Multiple pertussis toxin substrates as candidates for regulatory G proteins of adenylate cyclase coupled to the somatostatin receptor in primary rat astrocytes.

The involvement of G proteins in receptor mediated astroglial cAMP formation was studied. Isoproterenol or prostaglandin E2 stimulated adenylate cyclase of primary astroglial cells was inhibited by somatostatin. Preincubation of cells with increasing concentrations of islet activating protein (IAP) diminished somatostatin inhibition of adenylate cyclase. At an IAP concentration of 50 ng/ml somatostatin inhibition was completely abolished. Studies on IAP catalyzed 32P-ADP-ribosylation of astroglial cell particulate material revealed an incorporation of radiolabel into three polypeptides in the molecular weight range of 41,000-39,000 Dalton. Pretreatment of intact cells with IAP reduced radiolabeling of this molecular species in a concentration dependent manner. No further radiolabeling above background level was detectable after pretreatment of cultures with 10 ng IAP/ml or more. At present, the occurrence of at least three IAP substrates (G proteins) does not permit an identification of the somatostatin receptor coupled G protein. Rather, the finding reveals that astrocytes are endowed with multiple variants of GTP binding proteins likely to be coupled to different receptors.

GTP-binding proteins in human platelet membranes serving as the specific substrate of islet-activating protein, pertussis toxin

Two GTP-binding proteins serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, were purified from human platelet membranes as heterotrimers with an αβγ-subunit structure. The α of the major IAP substrate had a molecular mass of 40 kDa and differed from that of G i 1 or G o previously purified from brain membranes. The partial amino acid sequences of the 40 kDa α completely matched with the sequences which were deduced from the nucleotide sequences of the human G i 2 α gene. On the other hand, the α of the minor IAP substrate purified from human platelets was about 41 kDa and cross-reacted with an antibody raised against α of brain G i 1 (G i 1 α). These results indicate that the major IAP substrate present in human platelet membranes is a product of the G i 2 α gene.

Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells.

Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

An islet activating protein-sensitive G protein is involved in dopamine inhibition of angiotensin and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells.

In primary culture of anterior pituitary cells, dopamine inhibited the angiotensin (AII)-stimulated inositol phosphate production by 28 +/- 2.5% (n = 14), with an EC50 of 660 +/- 228 nM (n = 8). This effect was blocked by (+)-butaclamol, a specific dopamine receptor antagonist. RU 24926, a D2 specific agonist, but not SKF 38393, a specific D1 agonist, inhibited AII-stimulated inositol phosphate production, suggesting that this dopamine effect is mediated through a dopamine receptor of the D2 subtype. Dopamine also partially inhibited (25%) inositol phosphate production stimulated by thyrotropin-releasing hormone (TRH). Our results suggest that the dopamine-mediated inhibition of hormonally stimulated inositol phosphate production is probably not mediated through the known inhibitory effects of dopamine on cAMP and Ca2+ intracellular concentrations. Although unknown, the mechanism by which dopamine inhibited the AII and TRH-stimulated inositol phosphate production implicates a GTP binding protein sensitive to the islet activating protein (IAP) since dopamine effects were blocked by this toxin. The alpha subunit of the GTP binding protein involved could be one of the three ADP-ribosylated proteins found in anterior pituitary cells in primary cultures, the alpha o (39 kDa), the alpha i (41 kDa), and an alpha subunit of 40 kDa. Indeed, we show here that this 40-kDa IAP substrate, already described in a few tissues, is present in anterior pituitary cells. The negative coupling between dopamine receptors and the AII or TRH inositol phosphate production systems, could be implicated in the dopamine inhibition of the AII- and TRH-stimulated prolactin release since such an inhibition is blocked by IAP. Our results suggest that the negative regulation of inositol phosphate production is one of the mechanisms by which dopamine controls hormonally stimulated prolactin release.

Possible involvement of a GTP-binding protein, the substrate of islet-activating protein, in receptor-mediated signaling responsible for cell proliferation.

Serum-induced DNA synthesis, as measured by increases in [3H]thymidine incorporation, in Swiss mouse 3T3 fibroblasts was markedly inhibited by exposure of the cells to islet-activating protein (IAP), pertussis toxin. The inhibition was well correlated with the toxin-induced ADP-ribosylation of a membrane GTP-binding protein with Mr = 41,000. The IAP-induced inhibition of cell growth was characterized by the following two features. First, the inhibition was selective to certain growth factors. DNA synthesis in 3T3 cells was supported by a combination of one of the competence factors and a progression factor such as insulin or epidermal growth factor. IAP was inhibitory when thrombin, fibroblast growth factor, prostaglandin F2 alpha, or phosphatidic acid was employed as a competence factor, but was not inhibitory when DNA synthesis was induced by combined addition of cholera toxin or phorbol ester with insulin. Second, IAP-induced inhibition was still observed when the toxin was added to cell culture 1-6 h later than the addition of the IAP-sensitive competence factors, which triggered rapid cellular responses such as adenylate cyclase inhibition, releases of inositol trisphosphate and arachidonic acid, and 45Ca influx within several minutes (Murayama, T., and Ui, M. (1985) J. Biol. Chem. 260, 7226-7233; Murayama, T., and Ui, M. (1987) J. Biol. Chem. 262, 5522-5529). Thus, IAP substrate GTP-binding protein(s) appears to be involved in the duration of rapid signals or the occurrence of new slow signals which are responsible for growth factor-induced cell proliferation. The site of the involvement may be proximal to protein phosphorylation by phorbol ester-activated and cAMP-dependent kinases.

A new GTP-binding protein in differentiated human leukemic (HL-60) cells serving as the specific substrate of islet-activating protein, pertussis toxin.

A GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was partially purified from human leukemic (HL-60) cells that had been differentiated into neutrophil type. The partially purified protein, referred to as GHL, predominantly consisted of at least two polypeptides with molecular masses of 40,000 daltons (alpha) and 36,000 or 35,000 daltons (beta). The structure was similar to Gi or Go previously purified from rat brain as an alpha beta gamma-heterotrimeric IAP substrate (Katada, T., Oinuma, M., and Ui, M. (1986) J. Biol. Chem. 261, 8182-8191), although the existence of the gamma of GHL was unclear. The 40,000-dalton polypeptide contained the site for IAP-catalyzed ADP-ribosylation and the binding site for guanine nucleotide with a high affinity. The 36,000- and 35,000-dalton polypeptides were cross-reacted with the affinity-purified antibody raised against the beta of brain Gi and Go. Limited proteolysis with trypsin and immunoblot analyses with the use of the affinity-purified antibodies raised against the alpha of brain Gi or Go indicated that the alpha of GHL was different from the alpha of Gi or Go. Kinetics of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding to GHL was also quite different from that to brain Gi or Go. Incubation of GHL with GTP gamma S resulted in a resolution into GTP gamma S-bound alpha and beta(gamma) thus purified had abilities to inhibit a membrane-bound adenylate cyclase activity and to associate with the alpha of brain IAP substrate in a fashion similar to the beta gamma of brain IAP substrates, suggesting that there were no significant differences in the biological activities between the beta(gamma) of GHL and those of Gi or Go. Physiological roles of the new GTP-binding protein, GHL, purified from the neutrophil-like cells in receptor-mediated signal transduction are discussed.

Inhibitory effects of pertussis toxin on a depolarization-evoked Ca 2+ influx in NG108-15 cells

Depolarized stimulation 1.5-fold increased Ca 2+ influx which was inhibited by pretreatment with verapamil or LaCl 3. Treatment with pertussis toxin, islet-activating protein (IAP), induced a reduction in 50 mM K +- induced Ca 2+ influx and stimulated adenylate cyclase (AC) activity in NG108-15 cells. However, addition of dibutyryl cAMP or forskolin treatment elevating cAMP level exerted no effects on a depolarization-induced Ca 2+ influx. Dissociated B-oligomer of IAP after treatment with dithiothreitol and ATP increased a depolarization-evoked Ca 2+ influx. It is suggested that inhibitory GTP-binding protein (G i) or other IAP substrate proteins could directly be involved in Ca 2+ influx via voltage-sensitive Ca 2+ channel.

A new GTP-binding protein in brain tissues serving as the specific substrate of islet-activating protein, pertussis toxin

A new GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was purified from porcine brain membranes as an αβγ-heterotrimeric structure. The α-subunit of the purified protein (α 40βγ) had a molecular mass of 40 kDa and differed from that of G i(α 41βγ) or G o(α 39βγ) previously purified from brain tissues. The fragmentation patterns of limited tryptic digestion and immunological cross-reactivities among the three α were different from one another. However, the βγ-subunit resolved from the three IAP substrates similarly inhibited a membrane-bound adenylate cyclase and their β-subunits were immunologically indistinguishable from one another. Thus, the α 40βγ is a new IAP substrate protein different from G i or G o, in the α-subunit only.

Guanine nucleotide-binding protein in sea urchin eggs serving as the specific substrate of islet-activating protein, pertussis toxin

A GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was partially purified from Lubrol extract of sea urchin egg membranes. The partially purified protein possessed two polypeptides of 39 and 37 kDa; the 39 kDa polypeptide was specifically ADP-ribosylated by IAP and the 37 kDa protein cross-reacted with the antibody prepared against purified βγ-subunits of αβγ-heterotrimeric IAP substrates from rat brain. Incubation of this sea urchin IAP substrate with a non-hydrolyzable GTP analogue resulted in a reduction of the apparent molecular mass on a column of gel filtration as had been the case with purified rat brain IAP substrates, suggesting that the sea urchin IAP substrate was also a heterooligomer dissociable into two polypeptides in the presence of GTP analogues. Thus, the 39 and 37 kDa polypeptides of the sea urchin IAP substrate correspond to the α- and β-subunits, respectively, of mammalian IAP substrates which are involved in the coupling between membrane receptor and effector systems.

Islet-activating protein inhibits leukotriene D4- and leukotriene C4- but not bradykinin- or calcium ionophore-induced prostacyclin synthesis in bovine endothelial cells.

Incubation of the bovine endothelial cell line, CPAE, with leukotriene D4, leukotriene C4, bradykinin, or the calcium ionophore A23187 results in the release of arachidonic acid metabolites including 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. Pretreatment of these cells with the pertussis toxin islet-activating protein (IAP) results in a dose-dependent inhibition of the release of arachidonic acid metabolites and prostacyclin in response to leukotriene D4 and leukotriene C4. In contrast, similar responses evoked by bradykinin or ionophore were not significantly altered by the IAP pretreatment of the cells. IAP in the presence of [32P]NAD specifically [32P]ADP-ribosylates a 41-kDa protein in membranes prepared from CPAE cells. Pretreatment of the intact cells with IAP resulted in a dose-dependent inhibition of subsequent 32P labeling of the toxin substrate in the membranes and correlates with the uncoupling of the leukotriene responses. These results suggest that the 41-kDa IAP substrate, presumably a guanine nucleotide regulatory protein, mediates the response of CPAE cells to leukotriene D4 and leukotriene C4, but not to bradykinin or the calcium ionophore.

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.

Two guanine nucleotide-binding proteins in rat brain serving as the specific substrate of islet-activating protein, pertussis toxin. Interaction of the alpha-subunits with beta gamma-subunits in development of their biological activities.

Two proteins serving as substrates for ADP-ribosylation catalyzed by islet-activating protein (IAP), pertussis toxin, and binding guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with high affinities were purified from the cholate extract of rat brain membranes. The purified proteins had the same heterotrimeric structure (alpha beta gamma) as the IAP substrates previously purified from rabbit liver and bovine brain and differed from each other in alpha only; the molecular weight of alpha was 41,000 (alpha 41 beta gamma) and 39,000 (alpha 39 beta gamma). Both were further resolved into alpha (alpha 41 or alpha 39) and beta gamma which were also purified to homogeneity to compare the activities of alpha-monomers with the original trimers. The maintenance of the rigid trimeric structure by combining alpha 41 or alpha 39 with beta gamma in the absence of Mg2+ was essential for the alpha-subunit to be ADP-ribosylated by IAP. The alpha-subunit was very stable but displayed the only partial GTP gamma S-binding activity under these conditions. Isolated alpha-monomers exhibited high GTPase activities when assayed in the presence of submicromolar Mg2+ but were very unstable at 30 degrees C and not ADP-ribosylated by IAP. The most favorable conditions for the GTP gamma S binding to alpha-subunits were achieved by combining alpha 41 or alpha 39 with beta gamma in the presence of millimolar Mg2+, probably due to the increase in stability and unmasking of the GTP-binding sites. There was no qualitative difference in these properties between alpha 41 beta gamma (alpha 41) and alpha 39 beta gamma (alpha 39). But alpha 39 beta gamma (or alpha 39) was usually more active than alpha 41 beta gamma (or alpha 41), at least partly due to its higher affinity for Mg2+ and lower affinity for beta gamma. Relation of these differences in activity between alpha 41 beta gamma and alpha 39 beta gamma to their physiological roles in signal transduction is discussed.

Mechanisms for inhibition of the catalytic activity of adenylate cyclase by the guanine nucleotide-binding proteins serving as the substrate of islet-activating protein, pertussis toxin.

Two GTP-binding trimeric proteins (referred to as alpha 41 beta gamma and alpha 39 beta gamma based on the kilodalton molecular weights of their alpha-subunits) were purified from rat brain as the specific substrates of the ADP-ribosylation reaction catalyzed by islet-activating protein, pertussis toxin, and resolved irreversibly into alpha- and beta gamma-subunits by incubation with guanosine 5'-O-(thiotriphosphate) (GTP gamma S). Some of these resolved subunits interacted directly with the adenylate cyclase catalyst partially purified from rat brain in a detergent-containing solution, resulting in inhibition of the cyclase activity as follows. 1) GTP gamma S-bound alpha 41 inhibited the catalyst, but GTP gamma S-bound alpha 39 did not; the inhibition was competitive with GTP gamma S-bound alpha-subunit of Ns, the GTP-binding protein involved in activation of adenylate cyclase. 2) beta gamma from either alpha 41 beta gamma or alpha 39 beta gamma inhibited the catalyst in a manner not competitive with the activator such as forskolin or the alpha-subunit of Ns. 3) The ADP-ribosylation of alpha 41 beta gamma by islet-activating protein did not exert any influence on the subsequent GTP gamma S-induced resolution and the ability of the resolved GTP gamma S-bound alpha 41 to inhibit the catalyst. 4) The beta gamma-induced inhibition of the catalyst was additive to the inhibition caused by GTP gamma S-bound alpha 41. Thus, the direct inhibition of the catalyst by beta gamma or GTP gamma S-bound alpha 41 is a likely mechanism involved in receptor-mediated inhibition of adenylate cyclase, in addition to the previously proposed indirect inhibition due to the reduction of the concentration of the active alpha-subunit of Ns by reassociation with beta gamma.