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PubMed1988

Evidence for involvement of guanine nucleotide-binding regulatory proteins in the activation of phospholipases by hormones.

Guanine nucleotide-binding regulatory proteins similar to Gs and Gi may be involved in the activation of phospholipases C and A2 by hormones and other ligands. The binding of hormones to receptors that activate phospholipase C is decreased by guanine nucleotides and these hormones also stimulate a high-affinity GTPase activity in cell membranes. Effects of hormones on phospholipase C activity in cell-free preparations are dependent on the presence of guanine nucleotides. In addition, fluoride and nonhydrolyzable GTP analogs activate phospholipases in a manner that can be blocked by GDP beta S. The putative guanine nucleotide-binding regulatory protein that appears to be involved in activation of phospholipase C is sensitive to pertussis toxin in some cells but not in others.

Animals↗
PubMed1988

Inhibitors of liver lysosomal acid phospholipase A1.

Lysosomal acid phospholipase A1, as well as other lysosomal enzymes, may be released under pathophysiological conditions into extralysosomal compartments. As shown here, several unspecific mechanisms exist which inhibit the hydrolysis of membrane diacylphospholipids by lysosomal acid phospholipase A1 and hence prevent an uncontrolled membrane destruction. These findings were obtained by employing partially purified rat liver lysosomal acid phospholipase A1 and sonicated radioactively labeled phosphatidylethanolamine or phosphatidylcholine as substrate. The inhibitory principles found include (1) pH, (2) inorganic cations, and (3) various proteins. Inorganic cations and proteins, however, inhibited lysosomal acid phospholipase A1 activity only below pH 6.0, and inhibition never exceeded 96%. Of the inorganic cations studied, the divalent species, as compared to the monovalent one, impaired lysosomal acid phospholipase A1 activity at significantly lower concentrations. Virtually all of the intracellular and extracellular proteins studied inhibited the enzyme activity, but the inhibitory potencies of the different proteins varied considerably. In general, basic and hydrophobic proteins were the most potent inhibitors, whereas glycoproteins appeared to be less inhibitory. The degree of inhibition of the enzyme activity in both proteins and inorganic cations depended on the substrate concentration and not on that of the enzyme. Binding studies provided evidence for inhibitor-substrate and against inhibitor-enzyme interactions.

Animals↗
PubMed1989

Glutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A2.

In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium-binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387-5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site-directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66----Asn, Glu71----Asn and Glu92----Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low-affinity calcium binding.

Animals↗
PubMed1982

Activation of brain guanylate cyclase by phospholipase A2.

Various pure snake venom phospholipases A2 were used for studying their effect on guanylate cyclase activity. All the phospholipases A2 tested were found to activate guanylate cyclase from a rat brain homogenate. It was shown that particulate guanylate cyclase was especially affected. Intact glial cells incubated in presence of phospholipase A2 showed also an increased guanylate cyclase activity, demonstrating that the phospholipase effect, observed in disrupted cells, occurs also at the cellular level. These results suggest that in intact cells membrane-bound phospholipase A2 activity could be involved in the modulation of the cellular cyclic GMP content.

Animals↗
PubMed1984

Selective effects of proteases and phospholipase A2 on monoamine oxidases A and B of human brain and liver.

Human brain and liver mitochondria contain membrane-bound monoamine oxidase of both A and B types. Monamine oxidase-A (MAO-A), either membrane-bound or in detergent-solubilized extracts from these tissues, was selectively inhibited during incubations with trypsin, chymotrypsin, thermolysin, or papain. MAO-A in solubilized, but not in membrane-bound, preparations was also very sensitive to the action of phospholipase A2, while MAO-B was unaffected. Membrane-bound MAO-A of rat brain mitochondria was more sensitive to phospholipases and less sensitive to proteases than was human brain enzyme, indicating that these agents may reveal species differences in MAO properties. Human brain and liver MAO-A, either solubilized or bound in mitochondrial membranes, apparently contains basic and aromatic peptide moieties that are available to proteases. Hydrolysis of these peptide bonds leads to rapid denaturation unless substrate molecules stabilize the active site. Phospholipase A2 may disrupt the phospholipid microenvironment of MAO-A, the integrity of which is essential for MAO-A activity, but not for MAO-B. No interconversion of the two activities was observed. After phospholipase A2 treatment, remaining MAO-A activity was recovered in low-molecular-weight regions of a gel filtration gradient, suggesting that MAO-A subunits were released. Although these experiments argue against the proposal that phospholipids may regulate the ratio of A/B activities of a single enzyme molecule, it is conceivable that endogenous phospholipases or proteases in mitochondrial membranes may influence MAO-A activity independently of MAO-B activity.

Animals↗
PubMed1987

Characterization and localization of phospholipase A2 activity in sympathetic ganglia.

Superior cervical ganglion phospholipase A2 activity was characterized using 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine as a substrate. The enzyme activity exhibited linearity with interval of incubation and tissue concentration; there appeared to be two pH optima of the enzyme, at pH 6.0 and 9.0. A Lineweaver-Burk plot of the reciprocal of activity versus substrate concentration yielded an apparent Km of 0.53 mM and a Vmax of 5.3 nmol/h/mg of protein. The enzyme exhibited a partial Ca2+ dependence; in the absence of Ca2+ and presence of EGTA, activity was reduced by 40%. The phospholipase A2 activity was heat sensitive and was completely inactivated after treatment at 100 degrees C for 30 min. For determination of whether the enzyme had a preference for hydrolysis of specific fatty acid substituents in the 2 position of phosphatidylcholine, several different substrates were tested. The order of preference for hydrolysis by the ganglionic enzyme was 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine = 1-palmitoyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-2-[1-14C]palmitoyl-sn-glycero-3-phosphocholine. For determination of the localization of the phospholipase A2 enzyme in sympathetic ganglia, two approaches were used. Guanethidine, which results in destruction of adrenergic cell bodies in sympathetic ganglia, was administered to rats; an approximately 50% decline in phospholipase A2 activity was observed after this treatment. In other experiments, the preganglionic nerve to the ganglion was sectioned in rats; after 2 weeks of denervation, no significant change in ganglionic phospholipase A2 activity was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Protein kinase C and phospholipase C mediate alpha 1- and beta-adrenoceptor intercommunication in rat hypothalamic slices.

These experiments examined the mechanism by which phenylephrine enhances beta-adrenoceptor-stimulated cyclic AMP formation in rat hypothalamic and preoptic area slices. To this end we manipulated phospholipase C. phospholipase A2, and protein kinase C activity in slices and assessed the effects of these manipulations on phenylephrine augmentation of isoproterenol-stimulated cyclic AMP generation. Since previous work indicated that estrogen enhances the alpha 1-component of cyclic AMP formation, we examined slices from both gonadectomized and estrogen-treated animals. The alpha 1-antagonist prazosin eliminated phenylephrine augmentation of the beta-response, suggesting that alpha 1-adrenergic receptors mediate the potentiation of cyclic AMP formation. Inhibition of protein kinase C by H7 attenuated the alpha 1-augmentation of beta-stimulated cyclic AMP formation. Staurosporine, a more potent protein kinase C inhibitor, completely abolished the alpha 1-augmenting response. In addition, phenylephrine potentiation of the isoproterenol response was not observed if protein kinase C was first stimulated directly with a synthetic diacylglycerol (1-oleoyl-2-acetyl-sn-glycerol) or phorbol ester (phorbol 12,13-dibutyrate). Neomycin, an inhibitor of phospholipase C, decreased alpha 1-receptor enhancement of beta-stimulated cyclic AMP formation, whereas quinacrine, an inhibitor of phospholipase A2, did not. The data suggest that the postreceptor mechanism involved in alpha 1-adrenergic receptor potentiation of cyclic AMP generation in hypothalamic and preoptic area slices includes activation of phospholipase C and protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1987

Effects of a toxic phospholipase A2 (AgTx) from the venom of the Pit viper (Agkistrodon halys (Pallas)) on the crayfish stretch receptor neuron.

Neurotoxicity of an isolated fraction (V) of the venom from the snake Agkistrodon halys (Pallas) was studied on the crayfish stretch receptor using a two-electrode voltage-clamp technique. The toxin was previously shown to have less phospholipase A2 activity but to be more toxic in mice compared with bee venom phospholipase A2. At 5 micrograms ml-1 (0.35 microM) the AgTx had very little effects on the electrophysiological properties of the receptor neuron whereas at 50 micrograms ml-1 (3.5 microM) the leak conductance was increased about three times and the net outward (K) current was reduced to 70% of control. The net inward (Na) current was not affected except for a small (+ 10 mV) shift in the I-V relationship. The resting membrane potential and the membrane capacitance were not changed by AgTx. These effects of AgTx were similar to those seen after exposure to bee venom phospholipase A2 at concentrations 10-20 times lower. At concentrations of AgTx which presynaptically affect the frog neuromuscular transmission (5-10 micrograms ml-1) the effects on ion channels in the stretch receptor neuron are very small and probably reflect the low phospholipase A2 activity compared with that of bee venom phospholipase A2.

Animals↗
PubMed1980

Carrageenan-induced activation of human platelets is independent of phospholipase A2 and of formation of thromboxanes.

Aggregation of washed rabbit platelets by thrombin and by carrageenan is accompanied by the activation of phospholipase A2 and by the synthesis of thromboxanes. Accordingly, aggregation, the accompanying release reaction and the activation of phospholipase are blocked by p-bromophenacyl bromide and by CB 874 (2,3-dibromo (4'-cyclohexyl-3'-chloro)-phenyl-4-oxo-butyric acid), two recognized inhibitors of the enzyme. Since these two reagents also inhibit aggregation and the release reaction induced by thrombin and by carrageenan on washed human platelets, it might have been anticipated that the mechanisms of aggregation of the platelets from the two species are similar. Nevertheless, no thromboxanes A2 or B2, nor activation of phospholipase A2 could be demonstrated with the use of carrageenan on human platelets, under conditions where thrombin was effective. It is concluded that carrageenan activates the human platelets by phospholipase A2- and thromboxane A2-independent mechanisms, and that the inhibitors of phospholipase A2 may block platelet functions by mechanisms other than inhibition of the expected enzyme.

Adenosine Diphosphate↗
PubMed1980

Stepwise degradation of membrane sphingomyelin by corynebacterial phospholipases.

The mechanism of in vitro synergistic lysis of sheep erythrocytes by Corynebacterium ovis and Corynebacterium equi was investigated. Hemolysis required (i) the action of phospholipase D from C. ovis, (ii) the action of an extracellular protein of C. equi, and (iii) Mg2+. Maximum lysis required imposition on the system of a fourth condition (step iv), such as chilling. Steps i, ii, and iv occur sequentially and in that order. Mg2+ functions in steps i and ii. The extracellular protein C. equi was purified to homogeneity and found to be a phospholipase C capable of hydrolyzing ceramide phosphate, phosphatidic acid, and all of the isolated major phospholipids of mammalian erythrocyte membranes. The principal features of the synergistic hemolytic system could be reproduced in experiments involving liposomes containing either sphingomyelin or ceramide phosphate and trapped [14C]glucose. We inferred that sphingomyelin of sheep erythrocytes is first converted to ceramide phosphate by C. ovis phospholipase D. On the basis of results with liposomes, we propose that the ceramide phosphate is then converted to ceramide by C. equi phospholipase C. We believe that the resulting in situ ceramide then undergoes dislocation by chilling and perhaps also by virtue of an affinity between ceramide and C. equi phospholipase C. The dislocation of ceramide presumably disorganizes the lipid bilayer sufficiently to result in cell lysis.

Animals↗
PubMed1987

C-reactive protein (CRP) and serum phospholipase A2 in the assessment of the severity of acute pancreatitis.

The present study examines the value of C-reactive protein (CRP) determinations in the assessment of the severity of acute pancreatitis and the correlation of CRP with serum phospholipase A2 activity and the clinical status. Fifty three patients with acute pancreatitis were studied; 17 with haemorrhagic pancreatitis and 36 with a mild form of the disease. S-phospholipase A2 activity increased significantly (p less than 0.05) in patients with fatal pancreatitis but not in those with mild disease. Phospholipase A2 concentrations were below 10 nmol FFA/ml min in mild, while they rose to 20-40 nmol FFA/ml min in haemorrhagic pancreatitis. In fatal cases very high (up to 50-60 nmol FFA/ml min) serum phospholipase A2 concentrations were recorded. The increase in CRP was greater in the patients with severe pancreatitis. One day after admission mean CRP was 280 mg/l in patients with haemorrhagic and 45 mg/l in those with the mild pancreatitis (p less than 0.001). High CRP values also correlated with the prognostic signs indicative of severe pancreatitis. CRP and S-phospholipase A2 determinations are valuable in the early assessment of the severity of acute pancreatitis, but the CRP assay is much easier to include in hospital routine.

Acute Disease↗
PubMed1985

Cone-shaped lipids increase the susceptibility of phospholipids in bilayers to the action of phospholipases.

Addition of cardiolipin or diacylglycerol to dispersions of phosphatidylcholine greatly increased hydrolysis by snake venom or pancreatic phospholipase A2, as well as by a microbial phospholipase. Monogalactosyl diglyceride which, like cardiolipin and diacylglycerol, will form nonbilayer hexagonal II structures also caused an increase in the breakdown of phosphatidylcholine. Addition of digalactosyl diglyceride, a bilayer lipid from the same source, had a much smaller effect on the three phospholipases, indicating that stimulation by the nonbilayer lipids was not due to their fatty acid compositions. Stimulation of the microbial phospholipase by cardiolipin did not require the presence of calcium, leading to the conclusion that the formation of nonbilayer structures was not necessary. The results suggest that cone-shaped lipids increase the accessibility of lipids in bilayers to phospholipases by decreasing the packing of the polar head groups.

Animals↗
PubMed1988

Increased phospholipase A2 activity of Ileal mucosa in Crohn's disease.

The activity of phospholipase A2 (EC 3.1.1.4) in endoscopic biopsy specimens of intestinal mucosa was compared in patients with Crohn's disease and controls without inflammatory bowel disease. In postresection Crohn patients there was significantly enhanced phospholipase A2 activity proximal to the anastomosis in the neoterminal ileum, whether or not the mucosa was inflamed at the time of biopsy. Highly elevated ileal phospholipase A2 activity had a predictive value for symptomatic relapse within 1 year after resection. Patients with concomitant Crohn's colitis, in whom the risk of ileal recurrence is particularly high, had greater ileal phospholipase A2 activity than noncolitis Crohn patients. Association thus was demonstrated between activity of phospholipase A2 in ileal mucosa and proneness to ileal inflammation in Crohn's disease.

Adult↗
PubMed2004

Expression of phospholipases A2 and C in human corneal epithelial cells.

PURPOSE: To achieve a better understanding of the involvement of phospholipases in the inflammation and wound-healing processes in human corneal epithelial cells (HCECs), expression of phospholipase A2s (PLA2s) and phospholipase Cs (PLCs) was examined in the human corneal epithelium. METHODS: Specific primers were designed for RT-PCR amplification of the known secreted (s)PLA2, cytosolic (c)PLA2, and PLC mRNAs. Corresponding PCR products were cloned and the DNA sequenced. Immunofluorescence of flatmounted corneal sections and Western blot analyses were used to detect the PLA2s and PLCs expressed by HCECs. RESULTS: The mRNAs for the following phospholipases were detected by RT-PCR in the HCECs: sPLA2GIII, -GX, and -GXIIA; cPLA2alpha and -gamma; PLCbeta1, -beta2, -beta3, -beta4, -gamma1, -gamma2, -delta1, -delta3, -delta4, and -epsilon. Immunofluorescence analyses conducted on corneal epithelium cryosections and Western blot on freshly isolated HCECs demonstrated the presence of sPLA2GIII, -GX, and -GXIIA; cPLA2alpha and -gamma; and PLCbeta2, -beta3, -gamma1, -gamma2, and -delta3. CONCLUSIONS: Many phospholipase isoforms are expressed by HCECs and may play a major role in signal transduction (PLCs) as well as in the release of precursors of potent mediators of inflammation, such as leukotrienes and prostaglandins (PLA2s). Moreover, the sPLA2s expressed by the corneal epithelium could be involved in the normal antibacterial activity in the tears and in wound healing.

Adult↗
PubMed1998

Isolation and properties of a novel phospholipase A from rat brain that hydrolyses fatty acids at sn-1 and sn-2 positions.

A Ca(2+)-independent phospholipase A that releases various fatty acids from sn-1 and sn-2 positions was partially purified from rat brain soluble fraction. The enzyme showed an approximate molecular mass of 300 kDa on gel filtration column chromatography. Its enzymatic properties are distinct from those of well characterized phospholipase A2 enzymes; by using a series of synthetic phosphatidylcholines, the enzyme cleaved oleic, linoleic, and arachidonic acids like phospholipase A2, and released palmitic and stearic acids like phospholipase A1. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid were hydrolysed with almost equal efficiencies by this enzyme. These results indicate that the enzyme isolated is a novel Ca(2+)-independent intracellular phospholipase A that might be responsible for production of various fatty acids from membrane phospholipids.

Animals↗
PubMed1986

Immunoelectron microscopic localization of calmodulin and phospholipase A2 in spermatozoa. I.

Using the Lowicryl K4M embedding technique, together with indirect immunoferritin or immunogold labeling on ultra-thin sections, tubulin, calmodulin and phospholipase A2 were distinctly localized in ejaculated bull spermatozoa. Calmodulin was concentrated on the plasma membrane, nucleus, post-acrosomal substance, and, in lesser amounts, between coarse fibers and axonemal microtubules of the flagellum. Phospholipase A2 was distributed evenly along the plasma membrane, nucleus, acrosome, post-acrosomal substance, and in the flagellum, on mitochondria, fibrous sheath, coarse fibers, between coarse fibers and axonemal microtubules. Antibodies to tubulin labeled only axonemal microtubules, including the central pair of microtubules. Patterns of tubulin labeling were identical when ferritin granule- or gold particle-conjugated antibodies were tested. In agreement with our previous biochemical studies demonstrating calmodulin binding to phospholipase A2, concomitant with enhancement of phospholipase A2 activity (Arch Biochem Biophys 241:413, 1985), the overlapping distribution of calmodulin and phospholipase A2 in several parts of the sperm suggests that these proteins may play a concerted role in male gamete function in preparation for or during fertilization. The distinct distribution of tubulin along flagellum microtubules indicates their special function in sperm mobility.

Animals↗
PubMed1984

Stimulation of anterior pituitary prolactin release by melittin, an activator of phospholipase A2.

Melittin, a 26-amino acid polypeptide contained in bee venom and an activator of phospholipase A2, stimulated PRL secretion from bovine anterior pituitary cells in vitro. Over the dose range 0.25-2 micrograms/ml, melittin stimulation of PRL release was dose-related, reversible, and calcium dependent. Within this same dose range melittin did not deplete cell PRL stores, nor did it alter [3H]leucine uptake or trypan blue exclusion, indicators of cell viability. The phospholipase A2 inhibitors quinacrine and dibromoacetophenone blocked stimulation of PRL release by melittin and by themselves inhibited spontaneous PRL secretion. Addition of phospholipase A2 to pituitary cell cultures was associated with increased PRL secretion. A possible product of phospholipase A2 action, arachidonic acid, also stimulated PRL release. Indomethacin, an inhibitor of arachidonic acid conversion to prostaglandins, did not block melittin-induced PRL release but instead enhanced it. These data suggest that phospholipase A2 may participate in controlling PRL secretion by causing release of arachidonic acid from membrane phospholipids. Arachidonic acid or its noncyclooxygenase metabolite may serve as an intracellular regulator of secretion in the lactotroph.

Animals↗
PubMed1991

Effect of phospholipase A2 on temperature-induced high-affinity [3H]tryptamine binding sites in rat brain.

To investigate a link between membrane phospholipids and tryptamine binding molecules, we examined the effects of phospholipases A2 and D on the temperature-sensitive high-affinity [3H]tryptamine binding sites in rat brain. When the phospholipase A2-treated membranes were exposed to 1% bovine serum albumin (BSA) before assaying for [3H]tryptamine binding, a complete dose-dependent inhibition curve was observed. At a concentration of 0.03 U, the action of phospholipase A2 resulted in the splitting of phosphatidylserine (PS), choline phosphatides (PC) and ethanolamine phosphatides (PE) by about 32, 34 and 65%, respectively, and reduced [3H]ligand binding by about 32%. On the contrary, in the case of phospholipase D (500 U), PS and PC decreased by about 8% and 33% and PE by about 29% with no significant alteration in the binding capacity. Moreover, Scatchard analysis of the [3H]tryptamine binding showed that phospholipase A2 drastically increased only the KD value of the high affinity sites, and this was accompanied by a decrement of the Bmax values of both the high and low affinity binding sites. From these results, it is inferred that certain lipids (PS) may be a modulator for the function of the temperature-induced high-affinity [3H]tryptamine binding molecules.

Animals↗