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51.
Multiple forms of phosphoinositide-specific phospholipase C of different relative molecular masses in animal tissues. Evidence for modification of the platelet enzyme by Ca2+-dependent proteinase. 总被引:7,自引:4,他引:3
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The Mr distribution of phosphoinositide-specific phospholipase C in the supernatants isolated from a variety of animal tissues was analysed by high-performance gel-filtration chromatography. In most tissues, at least four peaks of activity were resolved. However, different tissues showed quite marked differences in the distribution of activity between these peaks. In rat heart, lung and kidney, the predominant form had Mr approx. 90000, whereas the predominant form in brain had Mr approx. 290000. In liver, the Mr-90000 form predominated, but this tissue also contained relatively large amounts of a form of Mr approx. 150000. Phospholipase C in these tissues from other animal species gave similar distributions of activity between the peaks. In supernatants prepared from platelets sonicated in the presence of leupeptin (0.5 mM) or EGTA (20 mM), the Mr-290000 form predominated. However, when leupeptin or EGTA (inhibitors of Ca2+-dependent proteinase) was omitted from the sonication buffer, the Mr-290000 form appeared to be replaced by a form of Mr 100000. Similar changes in Mr were not demonstrated with the other tissues. These results may be relevant to the intracellular regulation of phospholipase C, since Ca2+-dependent proteolysis has been reported to occur during platelet activation. 相似文献
52.
Sterile peritoneal exudates produced in rabbits injected with 1% glycogen contain a phospholipase A activity in a cell-free supernatant fraction that hydrolyzed a synthetic phospholipid (1,2-diacyl-sn-glycero-3-phospho-ethanolamine) and phospholipids of autoclaved Escherichia coli. This phospholipase activity (phosphatidylacylhydrolase EC 3.1.1.4) exhibited an apparent bimodal pH optimum (pH 6.0 and pH 7.5) and was Ca(2+)-dependent; Mg(2+) and monovalent cations (Na(+) and K(+)) did not substitute for Ca(2+) in the reaction; EDTA was a potent inhibitor. The phospholipase hydrolyzed 1-[1-(14)C]palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine to form only radio-active lysophosphatidylethanolamine as the product, indicating that the enzyme had phospholipase A(2) specificity. The phospholipase A(2) was purified 302-fold by two successive chromatographic steps on carboxymethyl Sephadex. Gel filtration (Sephadex G75) of the purified enzyme resulted in a single peak of biological activity with a molecular weight of approximately 14,800. The same estimate of molecular weight was obtained by SDS-polyacrylamide gel electrophoresis, which yielded a single band. Polyacrylamide gel electrophoresis of this fraction at pH 4.3 revealed a single protein band migrating beyond lysozyme, with the dye front, suggesting that this protein was more basic than lysozyme (pI 10.5). The enzymatic and physical-chemical characteristics of this soluble enzyme were remarkably similar to a recently described phospholipase A(2) of rabbit polymorphonuclear leukocytes derived from glycogen-induced peritoneal exudates. The possible origin and physiological role of this soluble enzyme are discussed. 相似文献
53.
I T Mak A M Komarov J H Kramer W B Weglicki 《Cellular and molecular biology, including cyto-enzymology》2000,46(8):1337-1344
The potential anti-radical properties and cytoprotective effects of Mg-gluconate were studied. When microsomal membranes were peroxidized by a *O2- driven, Fe-catalyzed oxy-radical system (R* = dihydroxyfumarate + Fe2+), Mg-gluconate inhibited lipid peroxidation (TBARS formation) in a concentration-dependent manner with IC50 being 2.3 mM. For the entire range of .25-2 mM, MgSO4 or MgCl2 were < or = 20% effective compared to Mg-gluconate. When cultured bovine aortic endothelial cells were incubated with the R* for 50 min. at 37 degrees C, 56% loss of total glutathione occurred. Pre-treatment (10 min.) of the cells with 0.25-4 mM Mg-gluconate before R* exposure significantly (p<0.05) prevented the GSH loss to varying degrees; the EC50 was 1.1 mM. In separate experiments, with 30 min. of free radical incubation of endothelial monolayers (approximately 65% confluent), cell survival/proliferation determined by the tetrazolium salt MTT assay, decreased to 38% of control at 24 hrs; Mg-gluconate concentration-dependently attenuated the lost cell survival with EC50 of approximately 1.3 mM. For comparison, the effects provided by MgSO4 or MgCl2 were significantly lower and were < or = 1/3 as potent as that produced by Mg-gluconate. In a Fenton-reaction system consisting of Fe(II)+ H2O2, Mg-gluconate but not other Mg-salts, significantly inhibited the formation of OH radicals as determined by the ESR DMPO-OH signal intensity. Mg-gluconate also dose-dependently inhibited the 'Fe-catalyzed' deoxyribose degradation suggesting that Mg-gluconate could displace Fe from 'catalytic sites' of oxidative damage. These data suggest that Mg-gluconate may serve as a more advantageous Mg-salt for clinical use due to its additional anti-radical and cytoprotective activities. 相似文献
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57.
Spin trapping of oxygen and carbon-centered free radicals in ischemic canine myocardium 总被引:2,自引:0,他引:2
C.M. Arroyo J.H. Kramer R.H. Leiboff G.W. Mergner B.F. Dickens W.B. Weglicki 《Free radical biology & medicine》1987,3(5):313-316
Oxygen free radical injury has been postulated to occur during myocardial ischemia. We have used Electron Spin Resonance and Spin Trapping techniques to directly demonstrate the production of carbon-centered (R.) and oxygen-centered lipid radical (RO.) in ischemic canine heart. In addition, venous effluent from the ischemic region showed that conjugated dienes (lipid peroxidation products) increased with ischemic duration. Our results suggest that the formation of the oxygen-centered and carbon-centered lipid radical species during ischemia are a consequence of oxy-radical peroxidation of myocardial membrane lipids. 相似文献
58.
Potentiation of free radical-induced lipid peroxidative injury to sarcolemmal membranes by lipid amphiphiles 总被引:2,自引:0,他引:2
The effects of naturally occurring lipid amphiphiles on free radical-mediated peroxidative injury in isolated canine sarcolemma were studied. Highly enriched canine myocytic sarcolemmal membranes were preincubated for 10 min at 37 degrees C with or without different amphiphilic lipids before the addition of a free radical-generating system consisting of dihydroxyfumarate and Fe3+-ADP. Lipid peroxidation, assayed as malondialdehyde formation, was catalyzed linearly up to 40 min in the control samples. Pretreatment of the sarcolemma with palmitoyl-CoA, palmitoylcarnitine, or lysophosphatidylcholine accelerated the initial rates (20 min) of peroxidation in a concentration-dependent manner (10-100 microM) and achieved maximal stimulation (240%, 160%, and 210%, respectively, of controls) at 50 microM concentrations of each of these amphiphiles. However, free fatty acids, CoA, and carnitine were without effect. These promoting effects of the amphiphiles persisted over a wide pH range (pH 6.0-7.8) and exhibited additive effects when lower levels of different amphiphiles were combined together. Associated with the accelerated rates of peroxidation produced by palmitoyl-CoA and palmitoylcarnitine were greater losses in the activity of sarcolemmal (Na,K)-ATPase. Since all three kinds of amphiphilic lipids accumulate during ischemia, this study suggests a novel mechanism of potentiation of sacolemmal membrane injury when free radicals are present. 相似文献
59.
J Linden A Patel A M Spanier W B Weglicki 《The Journal of biological chemistry》1984,259(24):15115-15122
Cyclic AMP accumulation in embryonic chick heart cells and binding of the beta-adrenergic antagonist 125I-pindolol to intact cells has been examined during the first 30 min of (-)-isoproterenol-induced desensitization. Myocardial beta-adrenergic receptors exist in two states which bind agonists with high (KD congruent to 10 nM) and low (KD congruent to 10 microM) affinities. Both activation and desensitization of cyclic AMP accumulation were mediated by (-)-isoproterenol binding to high affinity receptors. (-)-Isoproterenol-induced desensitization of cyclic AMP accumulation occurred with a t1/2 of 3.8 min. Desensitization was accompanied by a decrease in the number of 125I-pindolol binding sites assessed by equilibrium radioligand binding assays conducted at 4 degrees C or short (80 s) binding assays conducted at 37 degrees C. There was an excellent temporal correlation between loss of binding and loss of (-)-isoproterenol-stimulated cyclic AMP accumulation. After (-)-isoproterenol-induced desensitization, most of the remaining receptors assayed at 4 degrees C bound (-)-isoproterenol with high affinity. A rapid (-)-isoproterenol-induced decrease in the number of 125I-pindolol binding sites also occurred in adult canine heart cells and rat adipocytes. The data suggest that agonists do not cause uncoupling of surface receptors. Receptors may be uncoupled as a consequence of rapid sequestration into a hydrophobic compartment. 相似文献
60.
Analysis of the nucleotide tightly associated with isolated erythrocyte cytoskeletons show it to be ADP, rather then ATP. This confirms that at least a major part of the erythrocyte actin is in the F-form. A re-evaluation of the stoichiometry of spectrin and actin in the erythrocyte (taking account of a gross difference between the color responses of the two proteins on staining of electrophoretic gels) leads to values of 1x10(5) and 5x10(5) for the number of molecules of spectrin tetramer and actin respectively per cell. It has been found possible to perform spectrophotometric DNAase I assays fro actin on lysed whole cells. The concentration of monomeric actin at 0 degrees C is approximately 16 μg/ml packed cells. After washing the lysed cells the monomer pool is not re-established, indicating that only a small proportion of the actin subunits are free to dissociate. The actin monomer concentration in the cytosol remains unchanged after equilibration of the cells with cytochalasin E. The ability of actin-containing complexes in the membrane to nucleate the polymerization of added G-actin was measured fluorimetrically; it was found that membranes incubated with cytochalasin E were completely inert with respect to nucleating activity under conditions that favor appreciable growth at the slowly-growing (“pointed”) ends of free actin filaments. This suggests that these ends of the actin “protofilaments” in the red cell are blocked or sterically obstructed. After treatment of the membranes with guanidine hydrochloride under conditions that dissociate F-actin, the measured concentration of actin monomer rises to approximately 180 μg/ml of packed cells, which is nearly 70 percent of the total actin content. On treatment with trypsin in the presence of DNAase, the spectrin and 4.1 are extensively degraded, but the actin remains undamaged. This treatment, followed by exposure to guanidine hydrochloride, causes a further rise in the concentration of actin responsive to the DNAase assay to 250 μg/ml of cells, compared with 270 μg/ml estimated by densitometry of stained gels. The oligomeric complex, consisting of actin, spectrin, and 4.1, that is extracted from the membrane at low ionic strength, generates no detectable actin monomer after the same treatment. From literature data on the number of cytochalasin binding sites per cell and our value for the total actin content, we obtain a number-average degree of polymerization for actin in the membrane of 12-17. The results lead to a model for the structure of the cytoskeletal network and suggest some consequences of metabolic depletion. 相似文献