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1.
We have studied the effect of choline on the activity and temperature dependency of the brush-border alkaline phosphatase isoenzymes from rat intestine (tissue-specific type), and from kidney and placenta (tissue-nonspecific type). The removal of choline with phospholipase D resulted in the loss of enzyme activity in all the membranes, whereas in situ loss in the discontinuity of Arrhenius plots occurred in the kidney and the placental membranes, but not in the intestinal membranes. The lost activity was restored either by addition of free choline or phosphatidylcholine or by the removal of the enzyme from the membrane surface. Intestinal enzyme was removed by papain, while the tissue-nonspecific enzyme was released by subtilisin and by phosphatidylinositol-specific phospholipase C. The enzyme from kidney and placental membranes aggregated (rho = 1.13) upon removal of choline, and addition of choline resulted in disaggregation (rho = 1.03). Conversion of discontinuous to continuous linear plots of alkaline phosphatase in the kidney and placental membranes paralleled the increase in membrane phosphatidic acid content, and the decrease in total phosphatidylcholines. The intestinal enzyme produced plots with break points at all phosphatidic acid/phosphatidylcholine ratios. The change brought about by treatment with phospholipidase D was not due to changes in the half-saturation kinetics (Km) for the substrate. Based on these studies we conclude that the active site of the tissue-nonspecific phosphatase is approximated to exterior membrane cholines, as in the case of the intestinal isoenzyme; that despite similar effects on the membrane content of phospholipids, phospholipase D treatment caused much greater effects on the tissue-nonspecific enzyme, as assessed by Arrhenius plots and density centrifugation; that these effects are due to different protein structures rather than to a lipid milieu unique to each brush-border membrane. 相似文献
2.
Antibodies to liposomal phosphatidylserine and phosphatidic acid 总被引:1,自引:0,他引:1
Polyclonal antisera to phosphatidylserine or phosphatidic acid were induced in rabbits by injecting liposomes containing phosphatidylserine or phosphatidic acid and lipid A. Adsorption of antisera with liposomes containing different phospholipids revealed that some degree of reactivity with one or more phospholipids other than the immunizing phospholipid was often observed. However, cross-reactivity with other phospholipids was not a universal phenomenon, and one antiserum to phosphatidylserine failed to cross-react (i.e., was not adsorbed) with liposomes containing other phospholipids. All of the antisera were inhibited by soluble phosphorylated haptens (e.g., phosphocholine but not choline), but one of the antisera to phosphatidylserine was inhibited both by phosphoserine and by serine alone. Liposomal membrane composition influenced the activity of antiserum to phosphatidylserine. Regardless of whether unsaturated (beef brain) or saturated (dimyristoyl) phosphatidylserine was used in the immunizing liposomes, the antisera reacted more vigorously with liposomes containing unsaturated than saturated phosphatidylserine. We conclude that liposomes containing lipid A can serve as vehicles for stimulating polyclonal antisera to phosphatidylserine and phosphatidic acid. Although cross-reactivity with certain other phospholipids can be observed, sera from selected animals apparently can exhibit a high degree of specific activity to the immunizing phospholipid antigen. 相似文献
3.
Alkaline phosphatase activity appears to be altered when chondrocyte cultures are incubated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study examined whether the hormone-responsive enzyme activity is associated with alkaline phosphatase-enriched extracellular membrane organelles called matrix vesicles. Confluent, third passage cultures of rat costochondral growth cartilage (GC) or resting zone chondrocytes (RC) were incubated with 1,25-(OH)2D3 or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and enzyme specific activity was assayed in the cell layer or in isolated matrix vesicle and plasma membrane fractions. Alkaline phosphatase-specific activity in the matrix vesicles was enriched at least 2-fold over that of the plasma membrane and 10-fold over that of the cell layer. Matrix vesicle alkaline phosphatase was stimulated by 1,25-(OH)2D3 in GC cultures and by 24,25-(OH)2D3 in RC cultures. The cell layer failed to reveal these subtle differences. 1,25-(OH)2D3 increased GC enzyme activity but the effect was one-half that observed in the matrix vesicles alone. No effect of 1,25-(OH)2D3 on enzyme activity of the RC cell layer or of 24,25-(OH)2D3 on either GC or RC cell layers was detected. Thus, response to the metabolites is dependent on chondrocytic differentiation and is site specific: the matrix vesicle fraction is targeted and not the cells per se. 相似文献
4.
Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding 总被引:2,自引:0,他引:2
Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid. 相似文献
5.
Extensive segregation of acidic phospholipids in membranes induced by protein kinase C and related proteins 总被引:3,自引:0,他引:3
Protein kinase C and two other proteins with molecular masses of 64 and 32 kDa, purified from bovine brain, constitute a type of protein that binds a large number of calcium ions in a phospholipid-dependent manner. This study suggested that these proteins also induced extensive clustering of acidic phospholipids in the membranes. Clustering of acidic phospholipids was detected by the self-quenching of a fluorescence probe that was attached to acidic phospholipids (phosphatidic acid or phosphatidylglycerol). Addition of these proteins to phospholipid vesicles containing 15% fluorescently labeled phosphatidic acid dispersed in neutral phosphatidylcholine resulted in extensive, rapid, and calcium-dependent quenching of the fluorescence signal. Fluorescence-quenching requirements coincided with protein-membrane binding characteristics. As expected, the addition of these proteins to phospholipid vesicles containing fluorescent phospholipids dispersed with large excess of acidic phospholipids produced only small fluorescence changes. In addition, association of these proteins with vesicles composed of 100% fluorescent phospholipids resulted in no fluorescence quenching. Protein binding to vesicles containing 5-50% fluorescent phospholipid showed different levels of fluorescence quenching that closely resemble the behavior expected for extensive segregation of the acidic phospholipids in the outer layer of the vesicles. Thus, the fluorescence quenching appeared to result from self-quenching of the fluorophores that become clustered upon protein-membrane binding. These results were consistent with protein-membrane binding that was maintained by calcium bridges between the proteins and acidic phospholipids in the membrane. Since each protein bound eight or more calcium ions in the presence of phospholipid, they may each induce clustering of a related number of acidic phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Distribution of phospholipids around gramicidin and D-beta-hydroxybutyrate dehydrogenase as measured by resonance energy transfer 总被引:1,自引:0,他引:1
A resonance energy transfer method was developed to study the distribution of phospholipids around integral membrane proteins. The method involved measuring the extent of energy transfer from tryptophan residues of the proteins to different phospholipids labeled with a dansyl moiety in the fatty acid chain. No specific interactions were observed between gramicidin and dansyl-labeled phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid. The results were consistent with a random distribution of each phospholipid in the bilayer in the presence of gramicidin. However, a redistribution of both gramicidin and dansyl-labeled phospholipids was easily observed when a phase separation was induced by adding Ca2+ to vesicles made up of phosphatidylcholine and phosphatidic acid. Polarization measurements showed that in the presence of Ca2+ a rigid phosphatidic acid rich region and a more fluid phosphatidylcholine-rich region were formed. Energy-transfer measurements from gramicidin to either dansylphosphatidylcholine or dansylphosphatidic acid showed gramicidin preferentially partitioned into the phosphatidylcholine-rich regions. Energy-transfer measurements were also carried out with D-beta-hydroxybutyrate dehydrogenase reconstituted in a vesicle composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid. Although the enzyme has a specific requirement for phosphatidylcholine for activity, the extent of energy transfer decreased in the order dansylphosphatidic acid, dansylphosphatidylcholine, dansylphosphatidylethanolamine. Thus, the enzyme reorganized the phospholipids in the vesicle into a nonrandom distribution. 相似文献
7.
Evidence has been obtained for the involvement of μ M levels of Ca2+ in phospholipid catabolism during petal senescence by following the breakdown of [U-14 C]-phosphatidylcholine by microsomal membranes from cut carnation ( Dianthus caryophyllus L. cv. White-sim) flowers. Phospholipid degradation was mediated by three membrane-associated lipases, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4) and lipolytic acyl hydrolase. The activities of phospholipase D and phosphatidic acid phosphatase were stimulated by 30 and 100%, respectively, in the presence of 40 μ M free Ca2+ , and the Ca2+ -stimulation of phosphatidic acid phosphatase was calmodulin-dependent. When L-3-phosphatidyl-[2-3 H]-inositol and L-3-phosphatidyl-[N-methyl-3 H]-choline were used as substrates, inositol and choline accounted for 95 and 99%, respectively, of the water-soluble radiolabelled products. This suggests a predominance of phospholipase D activity over phospholipase C activity in these membranes.
Breakdown of membrane phospholipids in senescing carnations is known to be accelerated by treatment of young flowers with ethylene. To determine whether this involves a specific turnover of phosphatidylinositol as observed in animal systems in response to certain agonists, young flowers pre-labelled with32 PO3- 4 were treated with 10 ppm ethylene. All phospholipids incorporated the label, but no enhanced turnover of phosphatidylinositol was observed. Inositol 1,4,5-triphosphate did not release Ca2+ from preloaded microsomal vesicles at concentrations known to be effective in animal systems (i.e. < 5 μ M ) although release of Ca2+ was observed when a higher (20 μ M ) concentration was used. 相似文献
Breakdown of membrane phospholipids in senescing carnations is known to be accelerated by treatment of young flowers with ethylene. To determine whether this involves a specific turnover of phosphatidylinositol as observed in animal systems in response to certain agonists, young flowers pre-labelled with
8.
Reaction rates of hydrophobic enzymes, aminopeptidase, and alkaline phosphatase, in microsomes prepared from the porcine brush border membrane and in vesicles pre pared from microsomes and phospholipids were measured at various temperatures. Interactions between the hydrophobic enzymes and the phospholipid layers are discussed as well as the effects of fluidity change of phospholipid layers on enzyme activity. Further, reaction properties and stabilities of the immobilized vesicles containing microsomal enzymes were studied. 相似文献
9.
Can mitochondria and synaptosomes of guinea-pig brain synthesize phospholipids? 总被引:9,自引:0,他引:9
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1. The use of ;marker' enzymes for investigating the contamination by endoplasmic reticulum of mitochondrial and synaptosomal (nerve-ending) fractions isolated from guinea-pig brain was examined. NADPH-cytochrome c reductase appeared to be satisfactory. With the synaptosomal preparation there was a non-occluded enzymic activity believed to arise from contaminating microsomes and an occluded form released by detergent, which probably was derived from some type of intraterminal smooth endoplasmic reticulum. 2. Isolated brain mitochondria, both intact and osmotically shocked, could not synthesize more labelled phosphatidylcholine from CDP-[Me-(14)C]choline or phosphoryl[Me-(14)C]choline than could be accounted for by microsomal contamination. They could synthesize only phosphatidic acid and diphosphatidylglycerol from a [(32)P]P(i) precursor and not nitrogen-containing phosphoglycerides or phosphatidylinositol. 3. The synaptosomal outer membrane and the intraterminal mitochondria could not synthesize phosphatidylcholine from CDP-[Me-(14)C]choline but the synaptic vesicles and probably the intraterminal ;endoplasmic reticulum' appeared to be capable of catalysing the incorporation of label from this substrate into their phospholipids. 4. Microsomal fractions and synaptosomes from guinea-pig brain could incorporate [Me-(14)C]choline into their phospholipids by a non-energy-requiring exchange process, which was catalysed by Ca(2+). Fractionation of the synaptosomes after such an exchange had taken place revealed that the label was predominantly in the intraterminal mitochondria and not associated with membranes containing NADPH-cytochrome c reductase. 5. On the intraperitoneal injection of [(32)P]P(i) into guinea pigs, incorporation of radioactivity into phosphatidylinositol and phosphatidic acid was much faster than into the nitrogen-containing phosphoglycerides. Mitochondria and microsomal fractions showed a roughly equivalent incorporation into individual phospholipids, and that into synaptosomes was appreciably less, whereas the phospholipids of myelin showed little (32)P incorporation up to 10h. 相似文献
10.
Promotion of acid-induced membrane fusion by basic peptides. Amino acid and phospholipid specificities 总被引:2,自引:0,他引:2
The ability of oligo- and polymers of the basic amino acids L-lysine, L-arginine, L-histidine and L-ornithine to induce lipid intermixing and membrane fusion among vesicles containing various anionic phospholipids has been investigated. Among vesicle consisting of either phosphatidylinositol or mixtures of phosphatidic acid and phosphatidylethanolamine rapid and extensive lipid intermixing, but not complete fusion, was induced at neutral pH by poly-L-ornithine or L-lysine peptides of five or more residues. When phosphatidylcholine was included in the vesicles, the lipid intermixing was severely inhibited. Such lipid intermixing was also much less pronounced among phosphatidylserine vesicles. Poly-L-arginine provoked considerable leakage from the various anionic vesicles and caused significantly less lipid intermixing than L-lysine peptides at neutral pH. When the addition of basic amino acid polymer was followed by acidification to pH 5-6, vesicle fusion was induced. Fusion was more pronounced among vesicles containing phosphatidylserine or phosphatidic acid than among those containing phosphatidylinositol, and occurred also with vesicles whose composition resembles that of cellular membranes (i.e., phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine, 50:30:20, by mol). Liposomes with this composition are resistant to fusion by Ca2+ or by acidification after lectin-mediated contact. The tight interaction among vesicles at neutral pH, resulting in lipid intermixing, does not seem to be necessary for the fusion occurring after acidification, but the basic peptides nevertheless appear to play a more active role in the fusion process than simply bringing the vesicles in contact. However, protonation of the polymer side chains and transformation of the polymer into a polycation does not explain the need for acidification, since the pH-dependence was quite similar for poly(L-histidine)- and poly(L-lysine)-mediated fusion. 相似文献
11.
L F Bonewald M B Kester Z Schwartz L D Swain A Khare T L Johnson R J Leach B D Boyan 《The Journal of biological chemistry》1992,267(13):8943-8949
Transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25D3), when added simultaneously to a human osteosarcoma cell line, MG-63, induce alkaline phosphatase activity 40-70-fold over basal levels, 6-7-fold over 1,25D3 treatment alone, and 15-20-fold over TGF beta treatment alone. TGF beta and 1,25D3 synergistically increased alkaline phosphatase specific activity in both matrix vesicles and plasma membrane isolated from the cultures, but the specific activity was greater in and targeted to the matrix vesicle fraction. Inhibitor and cleavage studies proved that the enzymatic activity was liver/bone/kidney alkaline phosphatase. Preincubation of MG-63 cells with TGF beta for 30 min before addition of 1,25D3 was sufficient for maximal induction of enzyme activity. Messenger RNA for liver/bone/kidney alkaline phosphatase was increased 2.1-fold with TGF beta, 1.7-fold with 1,25D3, and 4.8-fold with the combination at 72 h. Human alkaline phosphatase protein as detected by radioimmunoassay was stimulated only 6.3-fold over control levels with the combination. This combination of factors was tested for their effect on production of three other osteoblast cell proteins: collagen type I, osteocalcin, and fibronectin. TGF beta inhibited 1,25D3-induced osteocalcin production, whereas both factors were additive for fibronectin and collagen type I production. TGF beta appears to modulate the differentiation effects of 1,25D3 on this human osteoblast-like cell and thereby retain the cell in a non-fully differentiated state. 相似文献
12.
T Braten 《Journal of cell science》1975,17(3):647-653
Gametes, zoospores, and zygotes of the multicellular, green alga Ulva mutabilis showed acid phosphatase reaction product in Golgi vesicles and on the membrane lining the vacuole. In addition gametes and zoospores showed enzyme reaction product on the entire surface membrane including the flagellar membrane. The surface membrane enzyme activity disappears from the zygote shortly after copulation and at the same time lysosome-like bodies start to appear in the cytoplasm. No alkaline phosphatase activity could be detected. The distribution of acid phosphatase is discussed in relation to the events taking place during and shortly after fertilization. 相似文献
13.
A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521–528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes. 相似文献
14.
Phosphatidylinositol transfer protein from bovine brain. Substrate specificity and membrane binding properties 总被引:1,自引:0,他引:1
A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521-528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes. 相似文献
15.
Fourier transform infrared spectroscopy is used to characterize specific interactions between negatively charged lipids, such as phosphatidic acid, and the purified nicotinic acetylcholine receptor from Torpedo californica. The specific interaction of phosphatidic acid with acetylcholine receptor is demonstrated by the receptor-induced perturbation of the lipid ionization state, which is monitored using Fourier transform infrared bands arising from the phosphate head group. The acetylcholine receptor shifts the pKa of phosphatidic acid molecules adjacent to the receptor to a lower value by almost 2 pH units from 8.5 to 6.6. Decreased pH also leads to changes in ion channel function and to changes in the secondary structure of the acetylcholine receptor in membranes containing ionizable phospholipids. Phospholipase D restores functional activity of acetylcholine receptor reconstituted in an unfavorable environment containing phosphatidylcholine by generating phosphatidic acid. Lipids such as phosphatidic acid may serve as allosteric effectors for membrane protein function and the lipid-protein interface could be a site for activity-dependent changes that lead to modulation of synaptic efficacy. 相似文献
16.
Bovine enterokinase was incorporated into vesicles reconstituted from a soybean phospholipid mixture. A thin film hydration procedure (MacDonald, R. I., and MacDonald, R. C. (1975) J. Biol. Chem. 250, 9206-9214) produced vesicles with 40% of the enterokinase activity bound in the membrane. The highest incorporation was observed when cholesterol or dimyristoylphosphatidylethanolamine was added to the soybean phospholipids. Crude and highly purified enterokinase preparations were incorporated to the same extent suggesting that other membrane components were not required for a successful reconstitution. The properties of enterokinase in phospholipid vesicles were compared with those of alkaline phosphatase, which was also added to the reconstitution system, and with the enzyme activities present in vesicles prepared from brush-border membranes. The enzyme activities were not released by solutions of high ionic strength and remained associated with the phospholipid vesicles on gel filtration, ultracentrifugation, and sucrose density centrifugation. Enterokinase and alkaline phosphatase had their active sites exposed to substrate in the brush-border membrane vesicles. In soybean phospholipid vesicles half of the active sites of both enzymes were on the outside, since release of the enzyme with Triton X-100 almost doubled the units of enzyme present. Incubation of the soybean phospholipid and brush-border membrane vesicles with papain released the exposed molecules of enterokinase. The released enzyme molecules were fully active but could not be reincorporated into phospholipid vesicles. This suggests that the structure imbedded in the lipid bilayer was essential for a successful reconstitution. We conclude that the reconstituted soybean phospholipid vesicles are a suitable membrane system for the further study of membrane-bound enterokinase. 相似文献
17.
Final step of phosphatidic Acid synthesis in pea chloroplasts occurs in the inner envelope membrane 总被引:5,自引:0,他引:5
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The second enzyme of phosphatidic acid synthesis from glycerol-3-phosphate, 1-acylglycerophospate acyltransferase, was localized to the inner envelope membrane of pea chloroplasts. The activity of this enzyme was measured by both a coupled enzyme assay and a direct enzyme assay. Using the coupled enzyme assay, phosphatidic acid phosphatase was also localized to the inner envelope membrane, although this enzyme has very low activity in pea chloroplasts. The addition of UDP-galactose to unfractionated pea chloroplast envelope preparations did not result in significant conversion of newly synthesized diacylglycerol to monogalactosyldiacylglycerol. Thus, the envelope synthesized phosphatidic acid may not be involved in galactolipid synthesis in pea chloroplasts. 相似文献
18.
Correlation between acetylcholine receptor function and structural properties of membranes 总被引:18,自引:0,他引:18
Protein-lipid interactions were studied by using Torpedo californica acetylcholine receptor (AChR) as a model system by reconstituting purified AChR into membranes containing various synthetic lipids and native lipids. AChR function was determined by measuring two activities at 4 degrees C: (1) low to high agonist affinity-state transition of AChR in the presence of an agonist (carbamylcholine) in either membrane fragments or sealed vesicles and (2) ion-gating activity of AChR-containing vesicles in response to carbamylcholine. Sixteen samples were examined, each containing different lipid compositions including phosphatidylcholine, cholesterol, phosphatidic acid, phosphatidylethanolamine, asolectin, neutral lipid depleted asolectin, native lipids, and cholesterol-depleted native lipids. Phosphatidylcholines with different configurations of fatty acyl chains were used. The dynamic structures of these membranes were probed by incorporating spin-labeled fatty acid into AChR-containing vesicles and measuring the order parameters. It was found that both aspects of AChR function were highly dependent on the lipid environment even though carbamylcholine binding itself was not affected. An appropriate membrane fluidity was necessarily required to allow the interconversion between the low and high affinity states of AChR. An optimal fluidity hypothesis is proposed to account for the conformational transition properties of membrane proteins. In addition, the conformational change was only a necessary, but not sufficient, condition for the AChR-mediated ion flux activity. Among membranes in which AChR manifested the affinity-state transition, only those containing both cholesterol and negatively charged phospholipids (such as phosphatidic acid) retained the ion-gating activity. 相似文献
19.
Recombinant phospholipase D (PLD) from Streptomyces chromofuscus (scPLD) has been characterized using colorimetric assays, spectroscopic investigations, and site-directed mutagenesis. scPLD, which shows phosphodiesterase activity toward a wide variety of phospholipids and phosphatase activity toward p-nitrophenyl phosphate, exhibits a visible absorption band with lambda(max) at 570 nm. Metal ion analysis performed by inductively coupled plasma mass spectroscopy shows the presence of approximately 1 equivalent of iron, 0.27 equivalent of manganese, and 0.1 equivalent of zinc per mole of protein as isolated. The metal ion content coupled with the visible absorption feature is compatible with the presence of Fe(3+)-tyrosinate coordination. When scPLD was dialyzed against solutions containing Mn(2+), Zn(2+) or EDTA, the Fe(3+) content was reduced to variable extents, and the residual specific activity correlated well with the residual iron content. Sequence homology with metal ion binding motifs in known alkaline phosphatases and purple acid phosphatase from red kidney bean shows that most of the residues involved in metal ion coordination are conserved among all the sequences considered. Mutation of some of these conserved residues (C123A, D151A, Y154F, and H391A) produced enzymes lacking iron with dramatically reduced PLD activity but little change in secondary structure or ability to bind to small unilamellar vesicles of phosphatidylcholine (with Ba(2+)) or phosphatidic acid. We suggest that scPLD is a member of a family of phosphodiesterase/phosphatases with structural and mechanistic similarity to iron-dependent purple acid phosphatases. 相似文献
20.
Phosphatidic acid phosphatase is a fat-regulating enzyme that plays a major role in controlling the balance of phosphatidic
acid (substrate) and diacylglycerol (product), which are lipid precursors used for the synthesis of membrane phospholipids
and triacylglycerol. Phosphatidic acid is also a signaling molecule that triggers phospholipid synthesis gene expression,
membrane expansion, secretion, and endocytosis. While this important enzyme has been known for several decades, its gene was
only identified recently from yeast. This discovery showed the importance of phosphatidic acid phosphatase in lipid metabolism
in yeast as well as in higher eukaryotes including humans. 相似文献