Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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We examined the mechanisms by which the phospholipid-sensitive, calcium-dependent protein kinase (protein kinase C) regulates prostacyclin synthesis by ovarian cells. In monolayer cultures of swine granulosa cells, specific phorbol esters significantly augmented production of the stable immunoreactive metabolite of prostacyclin, 6-keto-prostaglandin F1 alpha by 3- to 8-fold. These stimulatory actions were dose (0.03-30 ng/ml) and time (24-96 h) dependent, could be reproduced by non-diterpene activators of protein kinase C, and were corroborated by high performance liquid chromatography and mass spectrometry. The rank order of potency of phorbol esters was 12-O-tetradecanoylphorbol 13-acetate (TPA) greater than phorbol 12,13-dibenzoate greater than phorbol 12,13-dibutyrate greater than pure phorbol base. TPA enhanced de novo synthesis of prostacyclin, and synergized with the divalent cation ionophore, A23187. Although prostacyclin synthetase activity was not induced, microsomal cyclooxygenase activity was significantly increased by phorbol treatment. Moreover, TPA doubled the intracellular accumulation of free arachidonic acid. An inhibitor of phospholipase A2 (quinacrine 100 microM) impeded, whereas melittin (0.01 microM), an activator of cellular phospholipase A2, and purified bacterial phospholipase A2 (5 and 50 mU/ml) both augmented prostacyclin production. RH 59022 (30 microM), an inhibitor of diacylglyceride lipase, also suppressed prostacyclin synthesis. We conclude that the protein kinase C effector pathway is functionally coupled to de novo prostacyclin production in the swine granulosa cell. Increased eicosanoid synthesis can be accounted for by enhanced phospholipase A2 and diacylglyceride lipase-mediated availability of arachidonic acid substrate and an activated cyclooxygenase enzyme without a change in prostacyclin synthetase activity.
Mol Cell Endocrinol 1989 May
PMID:Mechanism(s) by which activation of protein kinase C is coupled to prostacyclin synthesis in granulosa cells. 275 27

Cis-unsaturated fatty acids, but not saturated fatty acids, inhibited phospholipase A2 activity (PLA2) in vitro, and may function as endogenous suppressors of lipolysis. To probe the possible role of lipid peroxidation in the regulation of myocardial lipid catabolism, a neutral-active and Ca2+-dependent PLA2 was extracted from rat heart and was partially purified by sulfopropyl cation exchange chromatography. Myocardial PLA2 activity was inhibited in a dose-dependent manner by oleic, linoleic, linolenic, and arachidonic acids; the IC50 for arachidonic acid was approx 65 microM. Palmitic acid was not inhibitory. When arachidonic acid was incubated at 37 degrees C, exposed to air, there was a time- and pH-dependent peroxidation of the arachidonic acid as monitored by turbidity, thiobarbituric acid reactivity, and thin layer chromatography. Peroxidation was increased as the pH was lowered from 7.5 to 4.5, and was accompanied by a decrease in PLA2 inhibitory potency. Thus, arachidonate incubated for 24 hours at pH's 4.5, 6.0 and 7.5 lost 84%, 32%, and 20% respectively, of its inhibitory potency. Therefore, in vitro acidosis promotes the oxidation of cis-unsaturated fatty acids and relieves their inhibitory or suppressive activity toward PLA2s. Increased lipid peroxidation of unesterified unsaturated fatty acids during acidosis may therefore promote lipolysis observed during myocardial ischemia and reperfusion injury.
Mol Cell Biochem
PMID:Fatty acid oxidation and myocardial phospholipase A2 activity. 277 34

The degradation of lipids by endogenous hydrolytic activity has been studied in rat cardiac tissue deliberately damaged by freezing and thawing prior to storage under anoxic conditions. Aliquots of the freeze-thawed material were kept at 37 degrees C under an atmosphere of N2 up to 120 minutes. Triacylglycerol was hydrolyzed at a rate of 0.14 mumol fatty acids per minute per gram dry weight of tissue. Hydrolysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was associated with proportional production of lyso PC and lyso PE, respectively. This finding indicates that the activity of lysophospholipase is negligible in autolyzing cardiac tissue. The rate of hydrolysis of PC and PE was found to be 0.10 and 0.06 mumol per minute per gram dry weight of tissue. The observation that lyso PC and lyso PE mainly contained saturated and mono-unsaturated fatty acids indicates that phospholipase A2 rather than A1 is active in autolyzing cardiac tissue. The accumulation of fatty acids corresponded with the loss of triacylglycerol and phospholipids from the tissue during 120 minutes of autolysis.
Mol Cell Biochem
PMID:Degradation of phospholipids and triacylglycerol, and accumulation of fatty acids in anoxic myocardial tissue, disrupted by freeze-thawing. 278 32

Two chemically characterized peptides, arginine vasopressin (AVP) and corticotrophin-releasing factor-41 (CRF-41), known to stimulate ACTH secretion by interaction with their respective specific receptors on the corticotroph, were shown to cause the accumulation of phosphate esters of inositol (IP) and adenosine 3',5'-monophosphate (cAMP) respectively when added to rat anterior pituitary fragments incubated in vitro. The former 'second messenger' response (IP production) was unaffected in tissues removed from animals treated with prednisolone in the drinking water (1035 mumol/1) for 14 days. On the other hand, the cAMP response, whilst still present, was inhibited by some 50% in tissues taken from such animals. In contrast, pituitary glands from steroid-treated rats failed to respond to challenge with a variety of substances expected to cause the release of ACTH by mimicking or provoking the production of IP or cAMP. Indeed, of the wide range of ACTH secretagogues tested, only the phospholipase A2 activator melittin was able to cause attenuated ACTH release from tissues removed from treated rats. The failure to provoke ACTH release from tissues removed from steroid-treated animals was also seen when submaximal concentrations of CRF-41 or AVP, or hypothalamic extract or 48 mmol K+/1 were used as the stimuli. The staged recovery of the ACTH secretory response and IP and cAMP accumulation in vitro following the withdrawal of prednisolone treatment was also investigated. A cAMP response that did not differ significantly from that of control tissue and a normal ACTH response to K+ and to melittin were all recovered by 3 days after withdrawal, and the response to cholera toxin showed a partial recovery. Responses to all stimuli of ACTH secretion which cause their effect by entering the corticotrophs were normal by 5 days after withdrawal, when the response to CRF-41 was still significantly, and that to AVP still slightly, reduced compared with controls. Surprisingly, restoration of the ACTH response was most delayed when the expectedly most potent extracellular stimulus (hypothalamic extract) was used. In this case, release was still significantly impaired 7 days after steroid withdrawal. The results show that the glucocorticoid acts to compromise several distinct steps in the process whereby extracellular signals such as CRF-41 and AVP cause the secretion of ACTH. The only step that appears to be spared is the generation of IP by AVP.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Endocrinol 1988 Nov
PMID:Influence of prolonged glucocorticoid treatment on intracellular mechanisms involved in ACTH secretion in the rat. 285 96

The channel protein in the mitochondrial outer membrane of Neurospora crassa aggregates laterally into crystalline arrays by the action of phospholipase A2. When mitochondrial outer membranes are reacted with filipin and examined by negative-stain electron microscopy, filipin-sterol complexes are found everywhere on the membranes except on the crystalline channel arrays. This suggests that the channel-rich membrane domains may have a relatively low content of accessible sterol. It is proposed that in vitro segregation of protein and lipid membrane components by phospholipase A2 may reflect a mechanism by which the endogenous enzyme organizes the native mitochondrial membrane into functional domains.
J Ultrastruct Mol Struct Res 1988 Feb
PMID:Lateral segregation of sterol and channel proteins in the mitochondrial outer membrane induced by phospholipase A2: evidence from negative-stain electron microscopy using filipin. 296 38

Inositol trisphosphate (IP3) formed by phospholipase C-mediated breakdown of triphosphoinositide (PIP2) may be a ubiquitous second messenger for a number of Ca2+-mobilizing receptor agonists. Using [3H]inositol-labeled rabbit peritoneal neutrophils, we report that radiolabeled inositol phosphates are generated in response to the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). fMet-Leu-Phe-stimulated formation of [3H]IP3 occurs with a rapid time course and a concentration dependence which closely parallels that of stimulated lysosomal enzyme secretion. The synthetic peptide methionyl-leucyl-phenylalanine, which is unable to promote secretion, failed to elevate [3H]IP3 accumulation, and the competitive antagonist t-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe depressed the stimulant action of fMet-Leu-Phe on [3H]IP3 levels and secretion. The Ca2+ ionophore ionomycin, which promotes secretion, was unable to enhance IP3 levels, confirming that polyphosphoinositide hydrolysis is a specific receptor-mediated event that precedes calcium mobilization during neutrophil activation. The ability of leukotriene B4 to also promote a rapid accumulation of [3H]IP3 suggests that there exists in the neutrophil an interaction between phospholipase A2 and C-mediated events. These findings support the hypothesis that IP3 may be a pivotal messenger for signal transfer by Ca2+-mobilizing receptor agonists.
Mol Pharmacol 1985 Jan
PMID:Characterization of formylmethionyl-leucyl-phenylalanine stimulation of inositol trisphosphate accumulation in rabbit neutrophils. 298 3

A subcellular fraction enriched 12 times in glycosomes (NAD+-linked alpha-glycerophosphate dehydrogenase) and devoid of detectable contamination from other subcellular components, was prepared from bloodstream Trypanosoma rhodesiense. Using a method employing exposure to toluene as a means of studying normally latent glycosomal enzymes, and phospholipase A2 as a membrane probe, the association of adenylate kinase and alpha-glycerophosphate dehydrogenase with the glycosome was studied. The normally latent glycerophosphate dehydrogenase (NAD+ linked), it is proposed, is an intraglycosomal enzyme having no membrane association, but bound to the core by weak ionic linkages. As such it is possible to release the enzyme from permeable (toluene treated) glycosomes using Cl-, with a resulting 4-fold increase in the Km for dihydroxyacetone phosphate. The presence of Cl- also stimulates an increase in specific activity, but this is observed before any release of enzyme. In contrast adenylate kinase, a non-latent glycosomal enzyme, is clearly membrane associated, the use of phospholipase A2 revealing an absolute dependence on phospholipid for activity. Restoration of activity appears to specifically require phosphatidyl choline and to be co-operative in nature (nH = 1.56). It is proposed that adenylate kinase is an integral glycosomal membrane enzyme, probably affecting the control of intra-glycosomal ADP/ATP levels.
Mol Biochem Parasitol 1985 Feb
PMID:The presence of alpha-glycerophosphate dehydrogenase (NAD+-linked) and adenylate kinase as core and integral membrane enzymes respectively in the glycosomes of Trypanosoma rhodesiense. 298 83

Parenchymal cells (hepatocytes) are the sites at which the principal metabolic functions of the liver are located. In the perfused liver, responses (e.g. vasoconstriction and glycogenolysis) to stimulating agents such as zymosan, platelet-activating factor and arachidonic acid, are inhibited by indomethacin and bromophenacyl bromide, inhibitors of cyclo-oxygenase and phospholipase A2, respectively. Since cultured Kupffer and endothelial cells but not hepatocytes, produce eicosanoids, and since eicosanoids and especially prostaglandins induce similar patterns of responses when added directly to the perfused liver, an involvement of these non-parenchymal cells in mediating the above responses is considered likely. We propose that in most situations the responses induced by these stimulating agents are mediated through a combination of pathways that include interaction of the agents directly with hepatocytes or with vasoactive cells (endothelial and/or smooth muscle cells), or interaction of agents initially with non-parenchymal cells to produce and release eicosanoids, which then subsequently interact with hepatocytes or with vasoactive cells.
Mol Cell Biochem 1988 Sep
PMID:Non-parenchymal cells as mediators of physiological responses in liver. 306 13

A phospholipase A2 was isolated from the venom of the mexican beaded lizard (Heloderma horridum horridum) by phenyl-Sepharose chromatography followed by Sephadex G-75 gel filtration and two additional steps on ion exchange resins (DE-32 cellulose). The affinity chromatographic method (PC-Sepharose 4B) reported for the isolation of other phospholipases [Rock, Ch. O., & Snyder, F. (1975) J. Biol. Chem. 250, 2564-2566; King, T. P., Alagon, A. C., Kwan, J., Sobotka, A. K., & Lichteinstein, L. M. (1983) Mol. Immunol. 20, 297-308; King, T. P., Kochoumian, L., & Joslyn, A. (1984) Arch. Biochem. Biophys. 230, 1-12] was uneffective for the separation of this enzyme. The monomeric form of the Heloderma phospholipase has an apparent Mr of 18 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 19 060 as calculated from amino acid analysis. It also contains on the order of 7% carbohydrates per mole of enzyme. The N-terminal amino acid sequence was shown to be very different from that of phospholipases isolated from mammalian pancreas and crotalids and elapids snake venoms. The first 39 amino acid residues at the N-terminal region have 56% homology with bee venom phospholipase but differ from the bee phospholipase in that its isoelectric point is acidic (pI = 4.5), instead of basic, and it has approximately 50 amino acid residues more in the molecule. The specificity of the enzyme is mainly A2 type with possible residual B-type activity. The enzymatic activity is Ca2+-dependent. Half-cystine alignment of the Heloderma phospholipase sequence with those of other known phospholipases shows the lack of an octadecapeptide at the N-terminal region, the existence of an extra hexapeptide at positions 42-47, and an exact correspondence of Heloderma Gly-12, Gly-14, His-36, and Asp-37 with Gly-30, Gly-32, His-48, and Asp-49 from other phospholipases shown to be important for Ca2+ binding (( Dijkstra, B. W., Drenth, J., Kalk, K. H., & Vandermaalen, P. J. (1978) J. Mol. Biol. 124, 53-60 )).(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Biochemical characterization of the phospholipase A2 purified from the venom of the Mexican beaded lizard (Heloderma horridum horridum Wiegmann). 308 12

Gonadotropin release in rat pituitary monolayer cultures was stimulated by phospholipase A2, as well as by its activator melittin. A dose-dependent stimulation of luteinizing hormone secretion by melittin was observed in a dose range of 10(-8) to 10(-4) M. A higher dose (1 mM) melittin had a sub-optimal effect. The stimulatory action of melittin was calcium-dependent and blocked by phospholipase A2 inhibitors, chloroquine and quinacrine. Similar to melittin, phospholipase A2 enhanced the effect of LH release in a dose range of 0.1-100 units/ml. The effect of this enzyme was also calcium-dependent with optimal calcium concentrations at 1.5 mM, as obtained also for melittin. In superfusion experiments, the stimulatory action of melittin and phospholipase A2 was reproducible in their effects on LH release in gonadotrophs. In addition, melittin (10(-7) M) stimulated LH and 3H-arachidonic acid efflux in superfused pituicytes following prelabelling with radiolabelled arachidonate. These data suggest that phospholipase A2, which releases arachidonic acid from phospholipids, may participate in controlling gonadotropin secretion in gonadotrophs, since arachidonic acid and its metabolites have previously been found to enhance gonadotropin release.
Mol Cell Endocrinol 1987 May
PMID:Stimulation of luteinizing hormone release by melittin and phospholipase A2 in rat pituitary cells. 310 76


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