Modulation of phospholipase A2 activity in zymogen granule membranes by GTP[S]; evidence for GTP-binding protein regulation

In membranes associated with purified pancreatic zymogen granules, GTP[S] elicited a concentration-dependent activation of phospholipase A2 (PLA2), which was converted to inhibition in the presence of added Ca2+. The GTP-binding protein inhibitor GDP[S] blocked both the stimulatory and inhibitory actions of GTP[S]. We conclude that in zymogen granule membranes GTP-binding proteins exert a dual regulation of PLA2 activity.     

Stimulation by GTP-binding proteins (Gi, Go) of partially purified phospholipase C activity from human platelet membranes

A phospholipase C exhibiting preferential hydrolytic activity for polyphosphoinositides was partially purified from the deoxycholate extract of human platelet membranes by Q-Sepharose and Heparin-Sepharose column chromatographies. The activity of this purified phospholipase C free of the GTP gamma S-binding activity was stimulated at a similar level by addition of purified rat brain Gi or Go. These results suggest that GTP-binding proteins may interact directly with a solubilized membrane phospholipase C to stimulate its activity.     

Calmodulin regulation of adenylate cyclase activity in human platelet membranes

The mechanism of calmodulin dependent regulation of adenylate cyclase has been studied in human platelet membranes. Calmodulin activated adenylate cyclase exhibited a biphasic response to both Mg2+ and Ca2+. A stimulatory effect of Mg2 on adenylate cyclase was observed at all Mg2+ concentrations employed, although the degree of activation by calmodulin was progressively decreased with increasing concentrations of Mg2+. These results demonstrate that the Vmax of calmodulin dependent platelet adenylate cyclase can be manipulated by varying the relative concentrations of Mg2+ and Ca2+. The activity of calmodulin stimulated adenylate cyclase was always increased 2-fold above respective levels of activity induced by GTP, Gpp(NH)p and/or PGE. The stimulatory influence of calmodulin was not additive but synergistic to the effects of PGE1, GTP and Gpp(NH)p. GDP beta S inhibited GTP-and Gpp(NH)p stimulation of adenylate cyclase but was without effect on calmodulin stimulation. Since the inhibitory effects of GDP beta S have been ascribed to apparent reduction of active N-protein-catalytic unit (C) complex formation, these results suggest that the magnitude of calmodulin dependent adenylate cyclase activity is proportional to the number of N-protein-C complexes, and that calmodulin interacts with preformed N-protein-C complex to increase its catalytic turnover. Our data do not support existence of two isoenzymes of adenylate cyclase (calmodulin sensitive and calmodulin insensitive) in human platelets.     

Direct activation of phospholipase A2 by GTP-binding protein in human peripheral polymorphonuclear leukocytes.

In human peripheral polymorphonuclear leukocyte (PMN), 10% of PLA2 activity was found in the particulate fraction. In the particulate fraction, the activity of phospholipase A2 was enhanced 270% by 100 microM guanosine 5'-[gamma-thio]triphosphate, a hydrolysis-resistant analog of GTP. In the soluble fraction, such enhancement was not observed. Guanosine 5'-[beta-thio]diphosphate (2 mM), which irreversibly inactivates GTP-binding protein, blocked the enhancement in the particulate fraction. Membrane-binding phospholipase A2 activity of PMN would thus appear to be regulated directly by GTP-binding protein.     

Identification of a human platelet membrane protein alkylated under conditions inhibitory of phospholipase A2 activity

Platelet membrane sulfhydryls essential for phospholipase A2 activity were alkylated by [3H]N-ethylmaleimide after the non-essential sulfhydryls were cross-linked by azodicarboxylic acid bis(di-methylamide) (DA) or alkylated by N-ethylmaleimide. A 24.5K da protein labeled under phospholipase inhibitory conditions was not labeled under non-inhibitory conditions. The polypeptide, which had neither endogenous nor DA induced disulfides, may be a platelet membrane phospholipase A2 or a lipase regulatory protein.     

G protein regulation of phospholipase A2

Many neurotransmitters and hormones activate receptors that are known to be coupled to their effectors by GTP-binding regulatory proteins, G proteins. Activation of many of these same receptors elicits arachidonate release and metabolism. During the past few years, novel experimental techniques have revealed that in many cells arachidonate release is independent of generation of other second messengers, including inositol phosphates, diacylglycerols, and elevation in free intracellular calcium. Much evidence has accumulated to implicate phospholipase A2 as the enzyme catalyzing arachidonate release, and suggesting that this effector enzyme, too, is activated by G proteins. In neural tissues as well as epithelium, endothelium, contractile and connective tissues, and blood cells, G proteins coupled to receptors for a variety of peptide and nonpeptide neurotransmitters and hormones have been shown to directly activate phospholipase A2. In retinal rod outer segments, transducin is the coupling G protein, but the G proteins coupling receptor activation to phospholipase A2 in other cell types is less clear. Some are pertussis toxin-sensitive, whereas others are not, and evidence exists that the ras gene product G protein may also be coupled to and regulate phospholipase A2.     

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 alpha beta gamma-subunit structure. The alpha of the major IAP substrate had a molecular mass of 40 kDa and differed from that of Gi 1 or Go previously purified from brain membranes. The partial amino acid sequences of the 40 kDa alpha completely matched with the sequences which were deduced from the nucleotide sequences of the human Gi 2 alpha gene. On the other hand, the alpha of the minor IAP substrate purified from human platelets was about 41 kDa and cross-reacted with an antibody raised against alpha of brain Gi 1 (Gi 1 alpha). These results indicate that the major IAP substrate present in human platelet membranes is a product of the Gi 2 alpha gene.     

Substrate-specific forms of human platelet phospholipase A2

Purification of human platelet phospholipase A2 (PLA2) from a particulate fraction by ion-exchange chromatography at 4 degrees C yielded a single peak of enzyme activity, which catalyzed the hydrolysis of arachidonic acid from the 2-position of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). The activity toward PtdCho and that toward PtdEtn differed in stability during storage, pH optimum, Ca2+ requirement, and affinity for the substrate; however, each activity preferred phospholipid with arachidonate at the 2-position. The two activities appeared to be eluted as an aggregate in a single peak from the ion-exchange column. When the column was run at 22 degrees C, an additional PLA2 activity peak specific for PtdEtn was resolved from the original PLA2 peak. But when the particulate fraction was briefly sonicated in 0.1% octylglucoside before chromatography at 22 degrees C, a different PLA2 activity peak, specific for PtdCho, was obtained. Resolution of the two specific forms of PLA2 under different conditions probably resulted from selective solubilization of the aggregate. The specific PLA2 activities thus isolated were very labile, whereas those in the aggregate were relatively stable. These findings suggest that human platelets contain at least two substrate-specific forms of PLA2, one for PtdCho and another for PtdEtn.     

Calmodulin stimulates human platelet phospholipase A2.

Calmodulin is a ubiquitous Ca²⁺-binding protein, mediating the effect of Ca²⁺ on many enzyme systems and cellular reactions. Phospholipase A₂ (phosphatide-2-acyl-hydrolase, EC 3.1.1.4) which governs the level of arachidonic acid in human platelets, requires Ca²⁺ for maximum activity. Results presented herein suggest that the stimulation of phospholipase A₂ by Ca²⁺ is also mediated through calmodulin. This finding adds to the growing list of enzymes whose activities are regulated by calmodulin.     

Tumour suppressors and the regulation of GTP-binding protein activity

GTP-binding proteins regulate a wide variety of intracellular signalling pathways in eukaryotic cells. The Ras GTP-binding proteins have received a great deal of attention since they were found to be modified by amino acid substitutions in a large number of cancers. It is now clear that Ras plays an essential role in regulating normal cell growth and differentiation, although how this is achieved biochemically is not known. The cellular concentration of Ras bound to GTP appears to be the limiting factor for signalling, and, not surprisingly, it is tightly controlled by both positive and negative regulators. There is now convincing evidence that the loss of one of these negative regulators of Ras, neurofibromin, can contribute to the development of malignancy; thus, neurofibromin behaves as a tumour suppressor gene product.     

Small molecular weight GTP-binding proteins in human platelet membranes. Purification and characterization of a novel GTP-binding protein with a molecular weight of 22,000

When guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding activity was assayed in the particulate and cytosol fractions of human platelets, most activity was found in the particulate fraction. GTP-binding proteins (G proteins) were extracted from the particulate fraction by sodium cholate and purified by several column chromatographies. At least three G proteins with Mr values of about 21,000, 22,000, and 24,000 (21K G, 22K G, and 24K G, respectively) were separated in addition to the stimulatory (Gs) and inhibitory (Gi) regulatory GTP-binding proteins of adenylate cyclase. Among them, the amount of 22K G was more than 10-fold of those of other G proteins. 22K G was purified to near homogeneity and characterized. 22K G specifically bound GTP gamma S, GTP, and GDP, with a Kd value for GTP gamma S of about 50 nM. [35S]GTP gamma S binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. 22K G hydrolyzed GTP to liberate Pi, with a turnover number of 0.01 min-1. 22K G was not copurified with the beta gamma subunits of Gs and Gi and was not recognized by the antibodies against the ADP-ribosylation factor for Gs and the ras protein. The peptide map of 22K G was different from those of the smg-25A and rho proteins, which we have purified from bovine brain membranes. 21K G was identified to be the c-ras protein, but 24K G was unidentified. These results indicate that there are multiple G proteins in platelet membranes and that a novel G protein (22K G) is a major G protein in platelets.     

A small GTP-binding protein (G protein) recognized by smg p25A GDP dissociation inhibitor (GDI) in human platelet membranes and GDI for this small G protein in human platelet cytosol

We have recently purified from bovine brain cytosol a novel type of regulatory protein for smg p25A, named smg p25A GDP dissociation inhibitor (GDI), that regulates the GDP/GTP exchange reaction of smg p25A by inhibiting the dissociation of GDP from and thereby the subsequent binding of GTP to it. This smg p25A GDI is inactive for other ras p21/ras p21-like small GTP-binding proteins (G proteins) including c-Ha-ras p21, smg p21, rhoA p21 and rhoB p20. In human platelet membranes, smg p25A was not detected but a G protein with an apparent Mr value of 24,000 (24KG) was recognized by smg p25A GDI and the dissociation of GDP from and the binding of GTP to 24KG were inhibited by smg p25A GDI. The doses of smg p25A GDI necessary for these activities for both 24KG and smg p25A were the same. This 24KG was not recognized by an anti-smg p25A monoclonal antibody. The GDI activity for human platelet 24KG and smg p25A was detected in human platelet cytosol. This human platelet GDI was recognized by an anti-smg p25A GDI polyclonal antibody. These results indicate that there is a 24KG-24KG GDI system similar to a smg p25A-smg p25A GDI system in human platelets.     

GTP-binding protein mediated phospholipase A2 activation in rat liver during the progression of sepsis

Effects of GTP-binding proteins on the activation of secretory phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2) in rat liver during two different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The results show that in the absence of G-protein modulator, hepatic sPLA2 and cPLA2 activities were activated by 40.8-46 and 91.6-105.8%, respectively, during early and late phases of sepsis. GTPS and fluoroaluminate (A1F4-) stimulated sPLA2 and cPLA2 activities within each experimental group, i.e., control, early sepsis, and late sepsis. The GTPS and A1F4--stimulated sPLA2 and cPLA2 activities remained significantly elevated during early phase (22.3-65.6% increase) and late phase (32.5-109.1% increase) of sepsis. Further analyses demonstrate that cholera toxin significantly stimulated sPLA2 and cPLA2 activities within each experimental group, and that the cholera toxin stimulated sPLA2 and cPLA2 activities remained significantly higher during early phase (23.5-37%increase) and late phase (56.7-70% increase) of sepsis. In contrast, pertussis toxin significantly inhibited sPLA2 and cPLA2 activities within each experimental group, and that the pertussis toxin-inhibited sPLA2 and cPLA2 activities remained significantly higher in early septic (57-68.5% increase) and late septic (34.6-45.5% increase) experiments. These data demonstrate that cholera toxin-sensitive Gs and pertussis toxin-sensitive Gi were both involved in the activation of sPLA2 and cPLA2 activities in rat liver during the progression of sepsis.     

Characterization of partially purified phospholipase C from human platelet membranes.

A phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-hydrolytic activity was found to be present in the human platelet membrane fraction, with 20% of the total activity of the homogenate. The membrane-associated phospholipase C activity was extracted with 1% deoxycholate (DOC). The DOC-extractable phospholipase C was partially purified approx. 126-fold to a specific activity of 0.58 mumol of PtdIns-(4,5)P2 cleaved/min per mg of protein, by Q-Sepharose, heparin-Sepharose and Ultrogel AcA-44 column chromatographies. This purified DOC-extractable phospholipase C had an Mr of approx. 110,000, as determined by Ultrogel AcA-44 gel filtration. The enzyme exhibits a maximal hydrolysis for PtdIns-(4,5)P2 at pH 6.5 in the presence of 0.1% DOC. The addition of 0.1% DOC caused a marked activation of both PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) hydrolyses by the enzyme. The enzyme hydrolysed PtdIns(4,5)P2 and PtdIns in a different Ca2+-dependent manner; the maximal hydrolyses for PtdIns(4,5)P2 and PtdIns were obtained at 4 microM- and 0.5 mM-Ca2+ respectively. In the presence of 1 mM-Mg2+, PtdIns(4,5)P2-hydrolytic activity was decreased at all Ca2+ concentrations examined, but PtdIns-hydrolytic activity was not affected.     

Evidence for a GTP-binding protein coupling thrombin receptor to PIP2-phospholipase C in membranes of hamster fibroblasts

Two different methods were used to study directly alpha-thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo-[3H]inositol prior to membrane isolation; in the other we used exogenous [3H]PIP2 with phosphatidylethanolamine (1:10) added as liposomes to freshly isolated membranes. A Ca2+-dependent PIP2 and PIP phospholipase C activity was characterized by measuring the rate of formation of inositol tris- and bisphosphate. Basal phospholipase C activity was stimulated up to 3-fold by GTP or GTP-gamma-S. Of the two mitogens, alpha-thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only alpha-thrombin is a potent activator of PIP2 breakdown in intact cells. Consistent with this observation, alpha-thrombin but not EGF potentiated GTP-gamma-S-dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP-binding protein couples alpha-thrombin receptor to PIP2 hydrolysis. Because both methods used to assay phospholipase C gave identical results, we conclude that the coupling is at the level of PIP2-phosphodiesterase activity.     

Thrombin- and nucleotide-activated phosphatidylinositol 4,5-bisphosphate phospholipase C in human platelet membranes

Thrombin, nucleotides, and chelators elicited a phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity that was associated with human platelet membranes. Both alpha- and gamma-thrombin enhanced phospholipase C activity, whereas active site-inhibited alpha-thrombin did not stimulate PtdIns-P2 hydrolysis. PtdIns-P2 phospholipase C was also activated by nucleoside triphosphates, citrate, EDTA, and NaF. Magnesium was an inhibitor of PtdIns-P2 hydrolysis stimulated by nucleotides and chelators. Only PtdIns-P2 was degraded by the phospholipase C activated by alpha-thrombin, nucleotides, and chelators. The soluble fraction phospholipase C activity was also stimulated at low protein concentrations by nucleotides; however, soluble fraction phospholipase C activity cleaved both PtdIns-P2 and phosphatidylinositol 4-phosphate and was inhibited by chelators, suggesting the presence of a different enzyme in this compartment. The pH optimum for the membrane-associated phospholipase C in the presence of alpha-thrombin or nucleotides was 6.0, and the PtdIns-P2 phospholipase C was inhibited by neomycin and high detergent concentrations. Guanine nucleotides did not synergistically activate phospholipase C in the presence of alpha-thrombin. The characteristics of the membrane-associated PtdIns-P2 phospholipase C suggest that this enzyme is involved in platelet activation by the low-affinity alpha- or gamma-thrombin-dependent pathway.