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1.
Certain phospholipase A2 enzymes (E.C.3.1.1.4) selectively inhibit neurotransmitter release from cholinergic nerve terminals. Both specific acceptor proteins and the physical state of nerve terminal phospholipids have been implicated in studies of the mechanism of phospholipase neurotoxin action. Here we have examined the effects of charge on a micellar phospholipid substrate by comparing the enzyme activity and binding of two neurotoxic phospholipases (beta-bungarotoxin and crotoxin) with other non-neurotoxic phospholipases. This has been achieved by altering either the phospholipid or the ionic charge of the detergent in the mixed phospholipid micelle. The neurotoxic phospholipases were only active on negatively charged micelles, whereas the non-neurotoxic enzymes were equally active in hydrolyzing neutral micelles. This distinction was also reflected in binding studies; the non-neurotoxic phospholipases bound to both types of substrate, whereas beta-bungarotoxin and crotoxin selectively bound to negatively charged micellar structures. These experiments suggest that, in addition to the existence of any specific acceptor proteins, neurotoxin binding is also governed by the charge on the lipid phase of the nerve terminal membrane. 相似文献
2.
The effect of phospholipases and proteases on the membrane-bound and solubilized A1 adenosine receptor has been studied. Phospholipids modulate the [3H]N6-(R)-phenylisopropyladenosine binding to A1 adenosine receptors in crude membranes and in soluble preparations, because changes in the phospholipid environment decrease both the binding capacity and the affinity for the ligand. It has become clear that 1) there is co-solubilization of receptor and phospholipids; 2) the phospholipid requirements are different for the coupled and the uncoupled receptor; 3) a net charge in the polar head produced by phospholipase D prevents the agonist binding to the receptor-G protein complex; alternatively, when the whole polar head is removed by phospholipase C the uncoupled receptor is altered; and 4) the protease action upon the receptor suggests that receptor coupled to G protein is more protected by the membrane than the uncoupled receptor. In kinetic experiments performed on membranes it was demonstrated that phospholipase C and trypsin increased the Kd value of the high-affinity state by modifying both k1 and k-1. In contrast they only modified the dissociation constant of the low-affinity state. In conclusion it should be noted that phospholipids play a key role for the binding of R-PIA to A1 adenosine receptor. Also, a different disposition within the membrane of the coupled and uncoupled receptor is encountered. 相似文献
3.
The role of phospholipids in the binding of 125I-choriogonadotropin to bovine corpus luteum plasma membranes has been investigated with the use of purified phospholipase A and phospholipase C to alter membrane phospholipids. The phospholipase C-digested plasma membrane preparation showed 85 to 90% inhibition of 125I-choriogonadotropin binding activity when 70% of the membrane phospholipid was hydrolyzed. Similarly treatment of plasma membranes with phospholipase A resulted in 45 to 55% hydrolysis of membrane phospholipid and almost 75% inhibition of receptor activity. Both these enzymes hydrolyzed membrane-associated phosphatidylcholine to a greater extent than phosphatidylethanolamine and phosphatidylserine. Phosphorylaminoalcohols of phospholiphase C end products were completely released into the medium, while phospholipase A by-products remained associated with plasma membranes. Addition of a phospholipids suspension or liposomes to plasma membranes pretreated with phospholipase A and C did not restore gonadotropin binding activity. Soluble phosphorylcholine, phosphorylethanolamine, and phosphorylserine and insoluble diglyceride products of phospholipase C action had no effect on receptor activity. In contrast, end products of the phospholipase A action, such as lysophosphatides and fatty acids, inhibited both on the membrane-associated and solubilized receptor activity. Lysophosphatidylcholine was the most effective end product inhibiting the binding of gonadotropin to the receptor, followed by lysophosphatidylethanolamine and lysophosphatidylserine. The inhibitory effects of phospholipase A or lysophosphatides were completely reversed upon removal of membrane-bound phospholipid end products by washing the membranes with defatted bovine serum albumin. However, phospholipase C inhibition could not be overcome by defatted albumin washings. Solubilization of plasma membranes with detergents which had been pretreated with phospholipase C partially restored the inhibited activity. It is concluded that the phospholipase-mediated inhibition of gonadotropin binding activity was due to hydrolysis and alterations of the phospholipid environment in the case of phospholipase C and by direct inhibition by end products in the case of phospholipase A. 相似文献
4.
The susceptibility of partially peroxidized liposomes of 2-[1-14C] linoleoylphosphatidylethanolamine ([14C]PE) to hydrolysis by cellular phospholipases was examined. [14C]PE was peroxidized by exposure to air at 37 degrees C, resulting in the formation of more polar derivatives, as determined by thin-layer chromatographic analysis. Hydrolysis of these partially peroxidized liposomes by lysosomal phospholipase C associated with cardiac sarcoplasmic reticulum, and by rat liver lysosomal phospholipase C, was greater than hydrolysis of non-peroxidized liposomes. By contrast, hydrolysis of liposomes by purified human synovial fluid phospholipase A2 or bacterial phospholipase C was almost completely inhibited by partial peroxidation of PE. Lysosomal phospholipase C preferentially hydrolyzed the peroxidized component of the lipid substrate which had accumulated during autoxidation. The major product recovered under these conditions was 2-monoacylglycerol, indicating sequential degradation by phospholipase C and diacylglycerol lipase. Liposomes peroxidized at pH 7.0 were more susceptible to hydrolysis by lysosomal phospholipases C than were liposomes peroxidized at pH 5.0, in spite of greater production of polar lipid after peroxidation at pH 5.0. Sodium bisulfite, an antioxidant and an inhibitor of lysosomal phospholipases, prevented: (1) lipid autoxidation, (2) hydrolysis of both non-peroxidized and peroxidized liposomes by sarcoplasmic reticulum and (3) loss of lipid phosphorus from endogenous lipids when sarcoplasmic reticulum was incubated at pH 5.0. These studies show that lipid peroxidation may modulate the susceptibility of phospholipid to attack by specific phospholipases, and may therefore be an important determinant in membrane dysfunction during injury. Preservation of membrane structural and functional integrity by antioxidants may result from inhibition of lipid peroxidation, which in turn may modulate cellular phospholipase activity. 相似文献
5.
Evidence for involvement of guanine nucleotide-binding regulatory proteins in the activation of phospholipases by hormones 总被引:19,自引:0,他引:19
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. 相似文献
6.
Hydrophobic interactions of brush border alkaline phosphatases: the role of phosphatidyl inositol 总被引:1,自引:0,他引:1
Tissue-specific (intestinal) and tissue-nonspecific (kidney) rat alkaline phosphatases are released from their respective brush border membranes by different enzymes. To elucidate the mechanism underlying their membrane attachment, we tested the ability of these enzymes to partition into lipid or aqueous phases both before and after treatment with phospholipases and proteases. Interaction with Triton X-114 micelles was eliminated or decreased by treatment of intestinal enzyme with phospholipase A2 or papain, while only phosphatidylinositol (PI)-specific phospholipase C (PIPLC) and subtilisin were effective with the kidney enzyme. Binding to octyl Sepharose for the intestinal enzyme was decreased by phospholipase A2 more than by PIPLC, whereas the reverse was true for the kidney enzyme. Treatment with phospholipases decreased the apparent mass of the phosphatases by 50-80 kDa, presumably due to loss of bound lipid and detergent. PIPLC treatment of the kidney, but not the intestinal enzyme, prevented binding of the phosphatase to phospholipid vesicles. These results show that both enzymes are bound to respective membranes by hydrophobic anchor peptides to which phospholipids are bound. However, their sensitivity to phospholipases is different. The data are consistent with the hypothesis that, in the kidney enzyme, the PI is bound covalently, while with the intestinal enzyme, binding of PI appears to be tight but not covalent. 相似文献
7.
The ability of bovine corpus luteum plasma membranes to bind 125I-choriogonadotropin has been examined after prior treatment of the membranes with phospholipases A, C, and D. Treatment of the purified membranes with low concentrations of phospholipases A and C resulted in the inhibition of the binding of 125I-choriogonadotropin to its receptors, whereas phospholipase D had no effect. Receptor activity was decreased by low concentrations of phospholipase A from either bee venom, Vipera russelli or Crotalus terrificus terrificus. Similarly, low concentrations of phospholipase C from Clostridium perfringens and Clostridium welchii also inhibited the binding activity while comparatively higher concentrations of phospholipase C from Bacillus cereus were required to achieve comparable inhibition. The time required to produce 50% inhibition of in vitro binding by phospholipases A and C was found to be 6 and 23 min, respectively. Upon either removal or chelation of calcium ions by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) both enzymes were completely inhibited as evidenced by the complete retention of the membrane binding activity. The decrease in the specific binding of choriogonadotropin to membranes after phospholipase digestion resulted in a decrease in the number of binding sites and was not accompanied by a change in the affinity of the hormone-receptor complex. The rates of association and dissociation of the 125I-choriogonadotropin-receptor complex and the equilibrium dissociation constant (Kd) were nearly identical in untreated and phospholipase-treated membranes. Phospholipases did not have any effect on the preformed hormone-receptor complex or on solubilized receptor. Filtration through Sepharose 6B of solubilized 125I-choriogonadotropin-receptor complex from untreated membranes or membranes which had been pretreated with phospholipase C prior to carrying out hormone binding did not alter the profile (Kav 0.38). Gel filtration of membranes treated with phospholipase A showed two peaks of bound radioactivity with distribution coefficients (Kav) of 0.08 and 0.35, respectively. 相似文献
8.
Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state 总被引:2,自引:0,他引:2
Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases. 相似文献
9.
George K. Chacko 《The Journal of membrane biology》1979,47(3):285-301
Summary The role of phospholipids in the binding of [3H] tetrodotoxin to garfish olfactory nerve axon plasma membrane was studied by the use of purified phospholipases. Treatment of the membranes with low concentrations of either phospholipase A2 (Crotalus adamanteus andNaja naja) or phospholipase C (Bacillus cereus andClostridium perfringens) resulted in a marked reduction in tetrodotoxin binding activity. A 90% reduction in the activity occurred with about 45% hydrolysis of membrane phospholipids by phospholipase A2, and with phospholipase C the lipid hydrolysis was about 60–70% for a 70–80% reduction in the binding activity. Phospholipase C fromB. cereus andCl. perfringens had similar inhibitory effects. Bovine serum albumin protected the tetrodotoxin binding activity of the membrane from the inhibitory effect of phospholipase A2 but not from that of phospholipase C. In the presence of albumin about 25% of the membrane phospholipids remained unhydrolyzed by phospholipase A2. It is suggested that these unhydrolyzed phospholipids are in a physical state different from the rest of the membrane phospholipids and that these include the phospholipids which are directly related to the tetrodotoxin binding component. It is concluded that phospholipids form an integral part of the tetrodotoxin binding component of the axon membrane and that the phospholipase-caused inhibition of the binding activity is due to effects resulting from alteration of the phospholipid components. 相似文献
10.
Structural studies of Fc receptors. IV. Structure required for phospholipids for reconstitution of the delipidated Fc receptor of macrophages 总被引:1,自引:0,他引:1
To analyze the interaction of the macrophage Fc receptor with phospholipids, we established an experimental system for delipidation of Fc receptor fraction and reconstitution of the Fc receptor activity in phospholipid vesicles. The separation of FcR from membrane phospholipids was achieved by ion exchange chromatography on DEAE-cellulose of the anionic detergent-lysate of the crude membrane fraction of guinea pig macrophages in the presence of detergent. The separation was based on the difference in charge between the complex of FcR and the anionic detergent and that of phospholipids and the detergent. The FcR fraction free of phospholipids showed no FcR activity as assessed in terms of its ability to inhibit the binding of labeled soluble immune complex of IgG2 antibody to macrophages, but the same fraction showed a definite activity when associated with phospholipids. This fraction was shown to contain a component of 44,000 daltons that is susceptible to surface-labeling and binds to IgG2-Sepharose in the affinity chromatography, indicating this component to be the Fc receptor. Reconstitution experiments with this fraction showed that phosphatidylcholine is the most effective phospholipid to reconstitute the FcR activity among those tested. Phosphatidylserine, phosphatidylinositol, and sphingomyelin were ineffective, while phosphatidylethanolamine showed a moderate effect. The inactivating effect of phospholipase C treatment on the Fc receptor activity of the membrane was shown to be due to the cleavage of phospholipids in the membrane but not due to modification of the Fc receptor molecule itself. 相似文献
11.
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 X 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 an apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similarly, the free receptor also showed higher sedimentation profile with an 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 preformed 125I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex. 相似文献
12.
A N Erin N V Gorbunov V I Brusovannik V A Tiurin L L Prilipks 《Biokhimii?a (Moscow, Russia)》1985,50(6):998-1004
Using fluorescent and EPR spin probing techniques, the effects of phospholipases A2, C and D on rat brain synaptosomal membranes were investigated. It was shown that treatment of synaptosomal membranes with phospholipases A2, C and D results in their depolarization and increase of their surface negative charge. In case of phospholipases A2 and C, these changes are also accompanied by a decrease of the microviscosity of the synaptosomal membrane lipid bilayer. alpha-Tocopherol protects synaptosomal membranes against the damaging action of phospholipases. The stabilization of synaptosomes by vitamin E consists in the reconstitution of the transmembrane potential and in an increased microviscosity of phospholipase-treated membranes. The stabilizing effect of alpha-tocopherol is due to the binding of phospholipid hydrolysis products rather than to the inhibition of phospholipases. The observed stabilization of synaptosomal membranes by alpha-tocopherol is interpreted as a feasible mechanism of biological effects of vitamin E on biological membranes. 相似文献
13.
Effect of Phospholipase Digestion and Lysophosphatidylcholine on Dopamine Receptor Binding 总被引:4,自引:4,他引:0
[3H]Spiperone specific binding by microsomal membranes isolated from sheep caudate nucleus is decreased by trypsin and phospholipase A2 (Vipera russeli), but is insensitive to neuraminidase. The inhibitory effect of phospholipase A2 is correlated with phospholipid hydrolysis. After 15 min of phospholipase (5 micrograms/mg protein) treatment, a maximal effect is observed; the maximal lipid hydrolysis is about 56% and produces 82% reduction in [3H]spiperone binding. Equilibrium binding studies in nontreated and treated membranes showed a reduction in Bmax from a value of 388 +/- 9.2 fmol/mg protein before phospholipase treatment to a value of 52 +/- 7.8 fmol/mg protein after treatment, but no change in affinity (KD = 0.24 +/- 0.042 nM) was observed. Albumin washing of treated membranes removes 47% of lysophosphatidylcholine produced by phospholipid hydrolysis without recovering [3H]spiperone binding activity. However, the presence of 2.5% albumin during phospholipase A2 action (1.5 micrograms/mg protein) prevents the inhibitory effect of phospholipase on [3H]spiperone binding to the membranes, although 28% of the total membrane phospholipid is hydrolysed. Lysophosphatidylcholine, a product of phospholipid hydrolysis, mimics the phospholipase A2 effect on receptor activity, but the [3H]spiperone binding inhibition can be reversed by washing with 2.5% defatted serum albumin. Addition of microsomal lipids to microsomal membranes pretreated with phospholipase does not restore [3H]spiperone stereospecific binding. It is concluded that the phospholipase-mediated inhibition of [3H]spiperone binding activity results not only from hydrolysis of membrane phospholipids, but also from an alteration of the lipid environment by the end products of phospholipid hydrolysis. 相似文献
14.
Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers. 总被引:17,自引:0,他引:17
R A Demel W S Geurts van Kessel R F Zwaal B Roelofsen L L van Deenen 《Biochimica et biophysica acta》1975,406(1):97-107
The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes. The phospholipases which cannot hyorolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A2 from pig pancreas and Crotalus adamanteus and phospholipase D from cabbage, can hydrolyse phospholipid monolayers at pressure below 31 dynes/cm only. The phospholipases which can hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Clostridium welchii phospholipase A2 from Naja naja and bee venom and sphingomyelinase from Staphylococcus aureus, can hydrolyse phospholipid monolayers at pressure above 31 dynes/cm. It is concluded that the lipid packing in the outer monolayer of the erythrocyte membrane is comparable with a lateral surface pressure between 31 and 34.8 dynes/cm. 相似文献
15.
A Muga J L Arrondo J I Gurtubay F M Go?i 《The Journal of biological chemistry》1990,265(11):5956-5959
When the major polar lipid of purple membrane, a dialkyl analogue of phosphatidyl glycerophosphate, is treated with phospholipase D under the usual assay conditions for this enzyme, the reaction yields dialkylglycerol and glycerol bisphosphate, i.e. the kind of products that would be expected from a phospholipase C reaction. The effect is seen both in native purple membranes and with the pure phospholipid in the form of liposomes. The specific activity and kinetic parameters Km and Vmax of phospholipase D for the purple membrane phospholipid are similar to those for egg phosphatidylcholine. The presence of phospholipase C impurities in the phospholipase D preparations has been ruled out as an explanation for the above observations. A hypothesis is suggested, taking into account the peculiar headgroup structure of the bacterial lipid, to explain the seemingly anomalous enzyme behavior. 相似文献
16.
S Forst J Weiss J M Maraganore R L Heinrikson P Elsbach 《Biochimica et biophysica acta》1987,920(3):221-225
Exposure of Escherichia coli to the bactericidal/permeability-increasing protein (BPI) of neutrophils renders the bacterial phospholipids susceptible to hydrolysis by only a few of numerous phospholipases A2 tested. To explore further the determinants of hydrolysis we measured the binding of 125I-labeled phospholipase A2 to E. coli in the presence and absence of BPI. Phospholipases A2 from Aqkistrodon piscivorus piscivorus venom and pig pancreas neither degraded nor bound to BPI-treated E. coli. In contrast, the phospholipases A2 from Aqkistrodon halys blomhoffii and Aqkistrodon halys palas venoms actively hydrolyzed the phospholipids of BPI-treated E. coli: they also bound to E. coli in the presence but not in the absence of BPI. Carbamylation of lysines of the A.h. blomhoffii phospholipase A2 progressively reduced binding in parallel with reduced phospholipid hydrolysis. Both binding and hydrolysis increased with increasing BPI dose. However, maximal binding occurred at 25% of the BPI dose that produced optimal hydrolysis. Thus, binding may be necessary but is not sufficient for maximal BPI-mediated phospholipid hydrolysis. Comparison of the NH2-terminal amino sequences of the active and inactive phospholipase A2 suggests that this portion of the phospholipase A2 molecule plays a role in BPI-independent binding and hydrolysis. 相似文献
17.
Highly purified mitochondria from rat liver contain a phospholipase A that catalyzes removal of 2-fatty acids, with a pH optimum above pH 8.0. Lysosomal preparations appeared to have two phospholipases A associated with them, one with a pH optimum at about pH 4.0, the second between pH 6.0 and 7.0. Mitochondrial phospholipase A hydrolyzed exogenous phospholipid as fast as or faster than endogenous phospholipid. The difference in specific radioactivity of (14)C-ethanolamine-labeled endogenous mitochondrial phospholipid before and after incubation indicates that a fraction of mitochondrial phosphatidyl ethanolamine is hydrolyzed more rapidly than the mitochondrial phospholipids as a whole. Acyl bond hydrolysis of exogenous and endogenous phospholipid by mitochondria was stimulated by free fatty acid, Ca(++), or in certain cases, monoacyl phospholipids or by treatments that disrupt the mitochondrial membrane. Of various fatty acids tested, lauric, myristic, oleic, and linoleic were most effective. ADP and ATP inhibited mitochondrial phospholipase, probably because they compete for Ca(++). Mg(++) also behaved as a competitive inhibitor; the effect was overcome by relatively little Ca(++). 相似文献
18.
Günther F. E. Scherer 《Plant Growth Regulation》1996,18(1-2):125-133
Phospholipid signalling is mediated by phospholipid breakdown products generated by phospholipases. The enzymes from animals and plants generating known or potential lipid-derived second messengers are compared. Plants possess a phospholipase C and a phospholipase A2 both of which are agonist-activated. These agonists (auxin, elicitors, perhaps others) bind to the external surface of the plasma membrane. The target enzyme for potential plant lipid-derived second messengers is lipid-activated protein kinase but the possibility that other enzymes may be also lipid-modulated should not be precluded.Abbreviations DAG
diacylglycerol
- CDPK
calmodulin-like domain protein kinase
- PLA2
phospholipase A2
- PLC
phospholipase C
- PLD
phospholipase D
- PKC
protein kinase C
- PS
phosphatidylserine 相似文献
19.
Because receptors, G proteins, and phospholipases all exist within a membrane lipid environment, it is not unreasonable to assume that an enzyme capable of changing the lipid environment can affect the coupling relationship among these signal transducing components. Our previous study showed that a muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D via a G protein in brain. We demonstrate here that phosphatidylinositol phospholipase C and phosphatidylcholine phospholipase D are simultaneously activated within 15 s by muscarine in the presence of 1 microM GTP gamma S. More important, inhibition of phospholipase D by zinc attenuated carbamylcholine-induced activation of phospholipase C by 30%. Our additional evidence strongly indicates that the receptor-regulated phospholipase D plays an important modulatory role in agonist-stimulated phosphatidylinositol breakdown. This modulatory effect may be achieved by changing the membrane microenvironment in which phospholipase C and phosphoinositol lipids reside, consequently amplifying the inositol phospholipid signaling process. Our results lead us to postulate that the potential interaction between two different signaling pathways may provide a cell with intracellular coordination and enable the cell to achieve functional responses. 相似文献
20.
Action of cobra venom phospholipase A2 on the gel and liquid crystalline states of dimyristoyl and dipalmitoyl phosphatidylcholine vesicles 总被引:1,自引:0,他引:1
The activity of phospholipase A2 from cobra venom toward phospholipid in single-walled, sonicated vesicles was analyzed, particularly with respect to its activity toward the saturated phosphatidylcholines in the gel and liquid crystalline states. When egg phosphatidylcholine vesicles are used as substrate, the phospholipase has an apparent Km of 4.4 mM, an apparent Vmax of 100 mumol min-1 mg-1 of protein, and a pH optimum of 5.0 at 40 degrees C. The phospholipase hydrolyzed the gel state of dimyristoyl phosphatidylcholine vesicles and dipalmitoyl phosphatidylcholine vesicles at a rate 2 to 3 times greater than the liquid crystalline state, taking into account temperature effects on the enzymatic reaction itself. The results suggest that, toward sonicated vesicles, there is no specific enhancement of the rate when the both liquid crystalline and gel states are present together, as has been suggested to occur for multibilayers studied with other phospholipases. An apparent stimulation of activity as the reaction proceeded was observed above the phase transition temperature. This might be attributed to an increase in the phase transition temperature caused by free fatty acids so that, in the presence of reaction products, the enzyme is actually hydrolyzing gel state phospholipid which was found to be the preferred lipid state for phospholipase activity. 相似文献