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1.
Freeze-thawing of microsome-enriched membranes from living bark tissues of black locust trees, especially those from less hardy tissues, caused a drastic increase in sensitivity to Ca 2+ and a complete loss of the regulatory action of Mg 2+ in membrane-bound phospholipase D activity with endogenous (membrane-bound) substrates. Also, the freeze-thaw cycle made phospholipase D in these membranes more resistant to digestion by proteases. Thus, the regulatory properties of the membrane-bound phospholipase D seem to be dependent on the nature of the membranes and on the interaction between the enzyme and membranes as well. The alteration of regulatory properties by freezing was protected by sucrose, at lower concentrations, and more effectively for membranes from hardy tissues than for membranes from less hardy tissue. Addition of partially purified soluble phospholipase D to the reaction system containing membranes caused only a slight stimulation of the degradation of endogenous phospholipids. Phospholipid degradation in vivo during freezing of less hardy tissue may be catalyzed mainly by the bound enzyme. Disintegration of the tonoplast, however, besides releasing soluble phospholipase D into the cytosol, would release organic acids (lowering the pH) and free Ca 2+. Both factors would stimulate drastically the membrane-bound phospholipase D, causing degradation of membrane phospholipids. 相似文献
2.
Activity levels of UDP-glucose: (1,3)-β-glucan (callose) synthase in microsomal membranes of pericarp tissue from tomato fruit ( Lycoperisicon esculentum Mill, cv Rutgers) were determined during development and ripening. Addition of the phospholipase inhibitors O-phosphorylcholine and glycerol-1-phosphate to homogenization buffers was necessary to preserve enzyme activity during homogenization and membrane isolation. Enzyme activity declined 90% from the immature green to the red ripe stage. The polypeptide composition of the membranes did not change significantly during ripening. The enzyme from immature fruit was inactivated by exogenously added phospholipases A 2, C, and D. These results suggest that the decline in callose synthase activity during ontogeny may be a secondary effect of endogenous lipase action. 相似文献
3.
There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca 2+-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphatidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca 2+ sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se. 相似文献
4.
Phosphatidylcholine, in addition to the widely studied inositol phospholipids, is cleaved to produce second messengers in neuronal signal transduction processes. Because of the difficulty in labelling and measuring the metabolism of endogenous phosphatidylcholine in brain tissue, we investigated the utility of measuring the hydrolysis of exogenous labelled substrate incubated with rat cerebral cortical cytosol and membrane fractions as has been successful in studies of phosphoinositide hydrolysis. In the cytosol [ 3H]phosphatidylcholine was hydrolyzed at a linear rate for 60 min of incubation and GTPS stimulated hydrolysis by 63%. The products of phospholipase C and phospholipase D, phosphorylcholine and choline, contributed only 44% of the [ 3H]phosphatidylcholine hydrolytic products in the cytosol, with phospholipase D activity slightly predominating. GTPS stimulated cytosolic phospholipase C and reduced phospholipase D activity. [ 3H]Phosphatidylcholine was hydrolyzed much more slowly by membranes than by cytosol. In membranes the production of [ 3H]phosphorylcholine and [ 3H]choline were approximately equal, contributing 27% of the total [ 3H]phosphatidylcholine hydrolysis, and GTPS only caused a slight stimulation of phospholipase C activity. Chronic lithium treatment (4 weeks) appeared to slightly reduce [ 3H]phosphatidylcholine metabolism in the cytosol and in membranes, but no statistically significant reductions were achieved. Cytosol and membrane fractions from postmortem human brain metabolized [ 3H]phosphatidylcholine slowly, and GTPS had no effects. In summary, exogenous [ 3H]phosphatidylcholine was hydrolyzed by brain cytosol and membranes, and this was stimulated by GTPS, but the complex contributions of multiple metabolic pathways complicates the application of this method for studying individual pathways, such as phospholipase D which contributes only a fraction of the total processes hydrolyzing exogenous [ 3H]phosphatidylcholine. 相似文献
5.
Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [ 3H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [ 3H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [ 3H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTPγS and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [ 3H]alkylacyl-GP and [ 3H]alkylacyl-GPethanol, phospholipase D breakdown products from [ 3H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [ 3H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes. © 1996 Wiley-Liss, Inc. 相似文献
6.
Sterol UDPglucose glucosyltransferase was located predominantly in the axis tissue of etiolated pea seedlings. During the first 11 days of growth the activity reached a peak in the axis tissue after seven days. Centrifugation of tissue homogenates showed the cell fraction sedimenting between 13000 nd 25000 g to have the highest specific activity and also the bulk of the total activity. Sitosterol is the major free sterol of this fraction and cholesterol is a trace component. The composition of the aglycones of the isolated steryl glycosides shows cholesterol to be the major sterol. Although exhibiting no metal ion requirement, the enzyme is stimulated by Ca 2+ and Mg 2+, partially inhibited by EDTA and EGTA and completely inhibited by Zn 2+. The membranous nature of the enzyme is manifested by its stimulation by the addition of phosphatidyl -ethanolamine, -choline and -serine. After brief treatment with phospholipases A, C and D, enzyme activity is partially lost. After phospholipase A treatment the activity may be completely restored by the addition of phosphatidyl ethanolamine but phosphatidyl-choline and -serine are without effect. After phospholipase C and D treatment, each phospholipid brings about a partial recovery of activity but phosphatidyl ethanolamine is again superior. 相似文献
7.
A membrane preparation from rat brain catalyzed the hydrolysis of [2- 3H]glycerol-labeled lysophosphatidylinositol (lysoPI) to yield monoacylglycerol (MG) and inositolphosphates. This phospholipase C activity had an optimal pH of 8.2. The membrane preparation did not require the addition of Ca 2+ for its maximum activity, but the activity was inhibited by addition of 0.1 mM EDTA to the assay mixture and was restored by simultaneous addition of 0.2 mM Ca 2+. The activity was found to be localized in synaptic plasma membranes prepared by Ficoll and Percoll density gradients. The phospholipase C was highly specific for lysoPI; diacylglycerol formation from phosphatidylinositol, and MG formation from lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine were below 5% of that observed with lysoPI under the conditions used. We concluded that there is a pathway for phosphatidylinositol metabolism in brain synaptic membranes which is different from the well-characterized phosphoinositide-specific phospholipase C pathway.Abbreviations PI
phosphatidylinositol
- lysoPI
lysophosphatidylinositol
- lysoPI-PLC
lysophosphoinositide-specific phospholipase C
- PI-PLC
phosphoinositide-specific phospholipase C
- MG
monoacylglycerol
- PLC
phospholipase C
To whom to address reprint requests. 相似文献
8.
Evidence for the involvement of Ca 2+ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U- 14C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U- 14C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca 2+, whereas lipolytic acyl hydrolase proved to be insensitive to Ca 2+. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca 2+. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC 50 values ranging from 10 to 15 micromolar. Thus the Ca 2+-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca 2+ on phospholipase D is independent of calmodulin. The role of Ca 2+ as a second messenger in the initiation of membrane lipid degradation is discussed. 相似文献
9.
Activity of membrane-bound phospholipase D in microsomes from bark tissues of black locust tree ( Robina pseudoacacia L.) was demonstrated to be regulated by a competitive binding of divalent cations. Binding of Ca 2+ at high concentrations (1 to 50 millimolar) modified the pH activity profile, shifting the optimum pH by 0.5 unit toward neutral and increasing the activity in the neutral pH. Mg 2+, on the other hand, inhibited the reaction of membrane-bound phospholipase D without added Ca 2+, and competitively inhibited the Ca 2+ stimulation. The regulatory effects of those ions were dependent on pH. Reduction in pH resulted in a decrease in the apparent dissociation constant for Ca 2+ and an increase in that for Mg 2+. From Lineweaver-Burk double reciprocal plots of Ca 2+ and the initial velocity, it was suggested that the binding of Ca 2+ in the higher concentration resulted in nearly the same conformational change of enzyme as reduction in pH. Mg 2+, on the other hand, counteracted those effects of Ca 2+ and lower pH on the enzyme conformation in such a manner as to inactivate. The membrane-bound phospholipase D because more sensitive to Ca 2+ and less sensitive to Mg 2+ as the hardiness of the tissues decreased. This fact may indicate that some qualitative changes in membranes are involved in the hardiness changes and also in the susceptibility of phospholipid to degradation by phospholipase D in plant cells. 相似文献
10.
In order to examine the role of phospholipids in the activation of membrane bound Ca 2+/Mg 2+ ATPase, the activities of Ca 2+ ATPase and Mg 2+ ATPase were studied in heart sarcolemma after treatments with phospholipases A, C and D. The Mg 2+ ATPase activity was decreased upon treating the sarcolemmal membranes with phospholipases, A, C and D; phospholipase A produced the most dramatic effect. The reduction in Mg 2, ATPase activity by each phospholipase treatment was associated with a decrease in the V max value without any changes in the K a value. The depression of Mg 2+ ATPase in the phospholipase treated preparations was not found to be due to release of fatty acids in the medium and was not restored upon reconstitution of these membranes by the addition of synthetic phospholipids such as lecithin, lysolecithin or phosphatidic acid. In contrast to the Mg 2+ ATPase, the sarcolemmal Ca 2+ ATPase was affected only slightly by phospholipase treatments. The greater sensitivity of Mg - ATPase to phospholipase treatments was also apparent when deoxycholate-treated preparations were employed. These results indicate that glycerophospholipids are required for the sarcolemmal Mg 2+ ATPase activity to a greater extent in comparison to that for the Ca 2+ ATPase activity and the phospholipids associated with Mg 2+ ATPase are predominantly exposed at the outer surface of the membrane. 相似文献
11.
Cytidine-diphospho-choline diacyl-glycerol phosphorylcholine phosphotransferase activity was demonstrated in potato ( Solanum tuberosum L.) microsomes and the incorporation of cytidine-diphospho[ 14C]choline into phosphatidylcholine was characterized by the time course of 14C incorporation and the effect of microsomal protein concentration on choline incorporation. Potato microsomes were progressively delipidated by treatments (2 min at 0°C) with increasing amounts of phospholipase C from Bacillus cereus. A decrease in choline phosphotransferase activity was observed in parallel with the progressive hydrolysis of membrane phospholipids. A 70% (or more) phospholipid hydrolysis provoked the total inactivation of the enzyme. Adding back exogenous phospholipids (in the form of liposomes) to phospholipase C-treated membranes restored the enzymic activity. Restoration could be obtained with egg yolk phospholipids as well as with potato phospholipids. Restoration was time dependent and completed after 10 minutes; restoration was also dependent on the quantity of liposomes added to lipid-depleted membranes: the best restorations were obtained with 1 to 2.5 milligrams of phospholipid per mg of microsomal protein; higher phospholipid to protein ratios were less efficient or inhibitory. These results clearly demonstrate the phospholipid dependence of the cytidine-diphospho-choline phosphotransferase from potato microsomes. 相似文献
12.
The purification of highly homogeneous, phospholipase-free α-bungarotoxin (α-Bgt) from the venom of the elapid Bungarus multicinctus or from commercial samples of α-Bgt is described. The method combines a conventional procedure for the purification of α-Bgt [ D. Mebs, K. Narita, S. Iwanaga, Y. Samejima, and C. Y. Lee (1972) Hoppe-Seyler's Z. Physiol. Chem.353, 243–262] with high-resolution gel-filtration and cation-exchange chromatography steps to remove membrane-damaging, contaminating phospholipase activity. The procedure also removes contaminating radioactive peptides from commercial preparations of 125I-α-Bgt. Apparent homogeneity of the purified α-Bgt (referred to as fraction D in the text), as well as the absence of contaminating phospholipase A 2 activity, is assessed by (i) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, (ii) gel-filtration and cation-exchange high-performance liquid chromatography, (iii) direct measurements of phospholipase A 2 activity under conditions where very low enzymatic levels should be detected, (iv) lack of interference with the passive cation permeability properties of acetylcholine receptor membranes, (v) competitive inhibition of 125I-α-Bgt binding to the acetylcholine receptor membranes, and (vi) amino acid analysis and end-group (C- and N-terminus) determination. α-Bgt preparations subjected to these criteria do not exert the increase in membrane passive permeability to cations detected with other laboratory or commercial samples of α-Bgt. Availability of the new α-Bgt preparation allows for an assessment of the inertness of α-Bgt on lipid membrane properties while preventing cholinergic ligand binding to nicotinic acetylcholine receptor-rich membranes. These conditions are necessary for experiments requiring maintenance of the physical and phospholipid integrity of membranes. 相似文献
13.
High levels of phospholipase activity were measured in potato ( Solanum tuberosum L. cv. Kennebec or Russett Burbank) leaf extracts using a new fluorometric phospholipase assay based on 1-acyl-2-[6-[(7-nitro-2, 1, 3 benzoxadiazol-4-yl)amino]-caproyl] phosphatidylcholine (C 6-NBD-PC). Time-course studies revealed that phospholipase activity could be stimulated for a brief time by the addition of calmodulin or the catalytic subunit of cyclic AMP-dependent protein kinase. The short-lived calmodulin stimulation or protein kinase stimulation of phospholipase activity could be prolonged by either conducting the time-course reactions in the cold (5°C) or adding sodium fluoride (a phosphatase inhibitor) to the reaction mixtures. Centrifugation studies revealed that calmodulin-stimulated or protein kinase-stimulated phospholipase activities were soluble and not associated with membranes. When potatp leaves were homogenized in the presence of either of two phosphatase inhibitors, the levels of phospholipase activity in the corresponding high-speed supernatant fractions were 36–47% higher than in controls. These experiments suggest a possible protein phosphorylation-dephosphorylation mechanism for the regulation of phospholipase activity in potato leaves. 相似文献
14.
We have shown previously that the phospholipase A (PLA) activity specific for phosphatidic acid (PA) in porcine platelet membranes is of the A 1 type (PA-PLA 1) [J. Biol. Chem. 259 (1984) 5083]. In the present study, the PA-PLA 1 was solubilized in Triton X-100 from membranes pre-treated with 1 M NaCl, and purified 280-fold from platelet homogenates by sequential chromatography on blue-Toyopearl, red-Toyopearl, DEAE-Toyopearl, green-agarose, brown-agarose, polylysine-agarose, palmitoyl-CoA-agarose and blue-5PW columns. In the presence of 0.1% Triton X-100 in the assay mixture, the partially purified enzyme hydrolyzed the acyl group from the sn-1 position of PA independently of Ca 2+ and was highly specific for PA; phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) were poor substrates. The enzyme exhibited lysophospholipase activity for l-acyl-lysoPA at 7% of the activity for PA hydrolysis but no lipase activity was observed for triacylglycerol (TG) and diacylglycerol (DG). At 0.025% Triton X-100, the enzyme exhibited the highest activity, and PA was the best substrate, but PE was also hydrolyzed substantially. The partially purified PA-PLA 1 in porcine platelet membranes was shown to be different from previously purified and cloned phospholipases and lipases by comparing the sensitivities to a reducing agent, a serine-esterase inhibitor, a PLA 2 inhibitor, a Ca 2+-independent phospholipase A 2 inhibitor, and a DG lipase inhibitor. 相似文献
15.
Two inhibitors of thrombin-stimulated arachidonic acid release from platelets, p-bromophenacyl bromide and mepacrine, were examined for their ability to inhibit the phospholipase C-diglyceride lipase pathway. This pathway involves hydrolysis of phosphatidylinositol to diglyceride, followed by release of arachidonate from diglyceride, and has been proposed as an alternative or addition to phospholipase A 2 as a mechanism for arachidonate release. p-Bromophenacyl bromide, a potent alkylating agent, was shown to cause a time-dependent inhibition of phosphatidylinositol-specific phospholipase C activity in crude platelet extracts; the inhibition was >90% after 15 min incubation with 100 μm p-bromophenacyl bromide. However, p-bromophenacyl bromide was also shown to destroy about one-half of the titratable sulfhydryl groups in whole platelets under similar conditions. The lack of specificity of p-bromophenacyl bromide was further demonstrated by our finding that thrombin-stimulated serotonin release was also inhibited by conditions inhibiting arachidonate release and that diglyceride lipase activity was decreased by higher levels of p-bromophenacyl bromide. Mepacrine was found to inhibit the activity of phosphatidylinositol-specific phospholipase C and had a greater effect at low substrate concentrations. The loss of [ 14C]arachidonate from both endogenous phosphatidylinositol and phosphatidylcholine in intact platelets was also inhibited. Thrombin-stimulated serotonin release was impaired by mepacrine also but only at a concentration 10-fold greater than that required to prevent arachidonate release. Thus we have shown that these two agents which inhibit arachidonate release are inhibitors of the phosphatidylinositol-specific phospholipase C-diglyceride lipase pathway. The multiple effects produced by both compounds limit their utility as agents to examine the source and mechanism of arachidonate release. 相似文献
16.
Purified mitochondria from germinating castor bean ( Ricinus communis L.) endosperm was treated with hydrogen peroxide (H 2O 2), active oxygen form, in order to investigate the extent of membrane degradation. Incubation of mitochondria with micromolar
concentrations (50–200 μM) of H 2O 2 resulted in a concentration-dependent loss of membrane proteins. During this process extensive loss of lipid-phosphate content
was also observed in mitochondrial membranes. When L-3-phosphatidyl[2- 14C]ethanolamine was added to the mitochondrial membranes as an exogenous substrate, the level of radioactivity in the water-soluble
fraction was markedly enhanced with increasing concentration of H 2O 2. Analysis of the water-soluble products formed during the metabolism of ethanolamine-labelled phosphatidylethanolamine by
mitochondrial membranes from castor bean indicates that this loss of lipid-phosphate is attributable to action of phospholipase
D. Direct measurement of mitochondrial phospholipase D indicated that the activity of enzyme was remarkably stimulated by
calcium ion or sodium dodecylsulfate (SDS). The optimum concentrations for enzyme stimulation were 25 and 0.5 mM for calcium
ion and SDS in the reaction mixture, respectively. The substrate specificity of phospholipase D was determined by comparing
various classes of exogenous phospholipids, added in the form of sonicated vesicles, as substrates. The phospholipase D exhibited
preference for phosphatidylethanolamine. Taken together, our results suggest that increase of mitochondrial phospholipase
D activity may be a key event leading to accelerated membrane deterioration following active oxygen attack. 相似文献
17.
The emission maximum of the single tryptophan residue of melittin was measured in the presence of phosphatidylethanolamine liposomes and Escherichia coli cytoplasmic membranes. In both cases, the fluorescence maximum was shifted to shorter wavelengths indicating a transfer of the indole ring to an apolar environment. E. coli membranes were labelled in position 2 of their phospholipids with [ 14C]oleic acid. These membranes were used for measuring the activity of an endogenous phospholipase A 2. A slow hydrolysis is observed, which can be accelerated by adding melittin. The extent of the stimulation depends on the molar ratio of melittin to membrane phospholipid. Under suitable conditions, the initial rate of hydrolysis is six to seven times higher in the presence than in the absence of melittin. The action of the phospholipase A 2 from bee venom is also stimulated by melittin. An identical stimulation was observed with either E. coli membranes or pure phosphatidylethanolamine liposomes as substrate. 相似文献
18.
An auxin-binding protein can be solubilized from microsomal membranes of Zea mays using either Triton X-100 extraction of the membranes or buffer extraction of the acetone-precipitated membranes. This paper describes the properties of the binding protein solubilized by these two methods. The binding is assayed by gel filtration chromatography in the presence of naphthalene [2- 14C]acetic acid. Binding is rapid and reversible with an optimum at pH 5. Both preparations show similar molecular weights by gel filtration (80,000 daltons) at pH 7.6 and 0.1 molar NaCl, and both aggregate at low ionic strength. They appear to be the same active molecular species. The binding activity is destroyed by trypsin, pronase or para-chloromercuribenzoic acid, but not significantly reduced by phospholipase C, DNase, RNase, or dithioerythritol. Since saturating amounts of naphthalene acetic acid protect the molecule from inhibition by para-chloromercuribenzoic acid, it is concluded that the binding protein has a sulfhydryl group at the binding site, or protects such a group in its binding conformation. The dissociation constant of the protein for naphthalene acetic acid is 4.6 × 10 −8 molar with 30 picomoles of sites per gram of tissue fresh weight. Binding constants were estimated for 13 other natural and synthetic auxins by competition with naphthalene[2- 14C]acetic acid. Their dissociation constants are in general agreement with published values for their binding to intact membranes and their biological activity, although several exceptions were noted. A supernatant factor from the same tissue changes the apparent affinity of the protein for naphthalene acetic acid. This factor may be the same one as has been previously reported to alter the affinity of intact microsomes for auxin. 相似文献
19.
Isolated Golgi apparatus, highly purified from rat liver, were found to contain an acyl transfer activity capable of restoring the acyl chains of the lysophospholipid products of the action of phospholipase A 2 on phosphatidylcholine. The activity was located primarily in cis and medial Golgi apparatus fractions, had a pH optimum of 6.0 to 7.5 and was stimulated by various acyl-CoA derivatives but not by fatty acids plus ATP. The activity, determined from the conversion of [ 14C]lysophosphatidylcholine to [ 14C]phosphatidylcholine, was unaffected by EGTA, inhibited by manoalide at high concentrations (0.2 mM), and temperature-dependent. Temperature dependency, however, showed no definite transition temperature over the range 15 to 37°C. The results demonstrated that cis Golgi apparatus membranes have the enzymatic capacity to restore fatty acids lost from phospholipids through the action of phospholipase A. The latter has been previously suggested to occur at the cis Golgi apparatus membranes based on analyses of cell-free transfer of radiolabeled phosphatidylcholine. 相似文献
20.
The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with 125I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 × g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with a apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similary, the free receptor also showed higher sedimentation profile with a apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of 125I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI.U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the performed 125I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex. 相似文献
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