共查询到20条相似文献,搜索用时 15 毫秒
1.
Z T Salikhova R A Akhmedzhanov T F Aripov M M Rakhimov 《Prikladnaia biokhimiia i mikrobiologiia》1989,25(6):747-751
A method of affinity chromatography was developed for purification of phospholipase A2(PL-A2) from the Central Asian cobra venon. The enzyme was covalently coupled to a polyamide sorbent with phosphatidilethanolamine (PEA) and cytotoxin (CT). The effect of CA2+ concentration and the ion strength of the solution on the enzyme adsorption was studied. The most efficient coupling of the enzyme to the sorbent was observed at pH 8--9 in case of the Ca2+ absence and a low ion strength of the solution. For desorption of the enzyme Triton X-100 at a concentration of 0.5% should be introduced in the eluting solution. The affinity adsorption chromatography enabled the isolation of two forms of phospholipase A2 with different affinity for PEA and CT. The total yield of the enzyme was 91% at a purification degree of 5.5 and 3.5, respectively. The introduction of the second ligand (CT) in the composition of the sorbent with the phospholipid ligand allowed the authors to increase its capacity and affinity for the phospholipase A2 from the snake venom. 相似文献
2.
M M Rakhimov R A Akhmedzhanov I T Iakubov B A Tashmukhamedov 《Prikladnaia biokhimiia i mikrobiologiia》1985,21(2):190-198
Methods of biospecific adsorption chromatography of phospholipase A2 obtained from porcine pancreas and Naja naja oxiana, Vipera ursini renardi, Vespa orientalis venoms were developed. Granulated polyamide with covalently linked phosphatidylethanolamine were used as an affinity adsorbent. Chemical inertness of linked phosphatidylethanolamine to the hydrolytic action of phospholipase A2 and its high affinity for biospecific complexes are shown. Forms of phospholipase A2 different in their affinity for an immobilized substrate was isolated by biospecific adsorption chromatography. The role of hydrophobic and electrostatic interactions in formation of enzyme-ligand complexes was studied. 相似文献
3.
A membrane bound phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human platelets has been purified 3500-fold, and partially characterized. Phospholipase A2 activity was assayed using [1(-14)C] oleate-labeled Escherichia coli or sonicated dispersions of synthetic phospholipids. The 2-acyl specificity of the phospholipase activity was confirmed using phosphatidylethanolamine labeled in the C-1 position as substrate. The purified enzyme was maximally active between pH 8.0 and 10.5, and had an absolute requirement for low concentrations of Ca2+. Indomethacin, but not aspirin, inhibited phospholipase A2 activity. 相似文献
4.
1. Plasma membranes were treated with phospholipase A2, phospholipase C or phospholipase D. The phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was deactivated by phospholipase C treatment, whereas phospholipase A2 and phospholipase D did not affect the enzyme. 2. Incorporation of phosphatidylethanolamine and phosphatidylglycerol into partially delipidated plasma membranes resulted in significant stimulation of the transferase, whereas inclusion of sphingomyelin and phosphatidylserine suppressed the enzyme activity. Our results suggest that phosphatidylserine is a regulator of sphingomyelin level in membranes. 3. The activity of phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was not influenced by the fluidity of its lipid environment. 相似文献
5.
Polyamide granules with high specific area were used for covalent immobilization of Trichosporon cutaneum R57. In order to increase the concentration of active (amino) groups necessary for cell immobilization, the polyamide (PA) sorbent was chemically modified. The optimal conditions for covalent immobilization of the cells were determined. Phenol degradation was studied with chemically immobilized cells. For comparison, parallel experiments were carried out with physically immobilized and free cells. Both covalently-bound and free cells fully degraded phenol at concentrations up to 1·0 g/litre. The optimal pH of phenol degradation by covalently bound cells was 6·0. The number of cycles of effective phenol degradation by immobilized cells was studied. The results obtained for covalently bound Trichosporon cutaneum R57 cells on PA granules clearly show the possibility for their application for the purification of waste water containing phenol. 相似文献
6.
The chromatography of porcine pepsin on biospecific sorbents (Sepharose-4B-epsilon-DNP-aminocapronylhydrazide and Sepharose-4B-N-DNP-N'-acetylhexamethylenediamine) was studied. The sorbents in question differ from the previously used hydrophobic sorbent Sepharose-4B-DNP-hexamethylenediamine by the lack of strongly basic groups in the site of the ligand binding to the polymeric matrix. No qualitative differences in the pepsin chromatography on the three sorbents were observed. Presumably the decrease of the pepsin binding to the sorbents, containing the dinitrophenyl group, at pH values above the isoelectric point may be due to the effects of the salt on the binding site in the enzyme molecule rather than to the screening of the positive charges of the sorbent by chlorine ions. A commercial preparation of pepsin was purified 2-fold on the sorbent Sepharose-4B-epsilon-DNP-animocapronylhydrazide. The synthesis of sorbents is described. 相似文献
7.
Phospholipase A2 activity in resting and activated human neutrophils. Substrate specificity, pH dependence, and subcellular localization 总被引:7,自引:0,他引:7
We have studied the phospholipase A2 activity in fractionated human neutrophils, employing labeled phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine as exogenous substrates. We used these phospholipid substrates labeled in the sn-1 position and measured the resulting labeled lysophospholipid forms in order to ascertain the phospholipase A2 specificity. In postnuclear supernatants from resting and A23187-activated cells, the phospholipase A2 activity showed a similar pH dependence curve with two pH optima at 5.5 and 7.5. Extracts from activated cells showed a 3-6-fold increase in enzyme activity. The subcellular distribution of phospholipase A2 activity in resting and A23187-treated human neutrophils was investigated by fractionation of postnuclear supernatants on continuous sucrose gradients. The neutral phospholipase A2 behaved as a membrane-bound enzyme and was mainly localized in the plasma membrane, the azurophilic granule, and in an ill-defined region of the gradient between the specific granules and mitochondria. The phospholipase A2 located in this undefined region showed a higher degree of activation than that located in other subcellular particulates in A23187-treated cells. This specific activation of an intracellular phospholipase A2 activity during cell stimulation indicates that cell compartmentalization may play a role in the formation of cell-activating and/or signal-transducing agents through the generation of arachidonate metabolites. Phosphatidylinositol was a better substrate for the plasma membrane enzyme, whereas phosphatidylcholine and phosphatidylethanolamine behaved as better substrates for intracellular organelle phospholipase A2 activities. The phospholipase A2 with maximal activity at pH 5.5 behaved as a soluble enzyme, and was almost completely localized in the azurophilic granules. Upon cell activation this acid enzyme activity was released in a similar way to beta-glucuronidase, a marker of azurophilic granules. These results demonstrate the different molecular properties of the phospholipase A2 activity, on the basis of its cellular location. 相似文献
8.
E Ia Sof'ina M M Rakhimov I V Shte?n I M Arends V V Dorokhov 《Prikladnaia biokhimiia i mikrobiologiia》1991,27(4):523-528
A polyamide with the covalently coupled phosphatidyl ethanolamine was used for affinity adsorption of an alkaline lipase from Pseudomonas aeruginosa. The immobilization resulted in increase of the enzyme specific activity. Some properties of native and adsorbed enzyme were compared. The temperature optima, heat and pH stability, KM and Vmax values were determined for both native and immobilized enzymes. 相似文献
9.
A nontoxic phospholipase A2 was purified from the venom of Indian krait (Bungarus caeruleus) by a four-step procedure involving electrophoresis, gel filtration and ion-exchange chromatography. The recovery of the enzyme activity was 37% and the purified preparation was 38 times as active as the crude venom. The purified enzyme had a molecular weight of 12,500 and the optimum pH of 7.2. The enzyme showed higher specificity toward phosphatidylethanolamine than phosphatidylcholine. The preparation was not very labile to heat and its activity was dependent on the presence of divalent cations, calcium ions being the most effective activators. The enzyme was completely inhibited by iodoacetic acid but showed high stability against 8 M urea. Purified phospholipase A2 was nontoxic at an iv dose of 5 microgram/g mouse. The high specific activity, the high yield and the nontoxic nature of the enzyme indicate that the major form of phospholipase A2 in Bungarus caeruleus venom is not associated with any toxicity and has properties somewhat similar to that of phospholipase A2 from some other venoms. 相似文献
10.
J M De Winter J Korpancova H van den Bosch 《Archives of biochemistry and biophysics》1984,234(1):243-252
A comparative study was made of the metal ion requirement of rat liver mitochondrial phospholipase A2 in purified and membrane-associated forms. Membrane-bound enzyme was assayed using either exogenous or endogenous phosphatidylethanolamine. Although several divalent metal ions caused increased activity of the membrane-associated enzyme, only Ca2+ and Sr2+ activated the purified phospholipase A2. The activity in the presence of Sr2+ amounted to about 25% of that found with Ca2+. When the Ca2+ concentration was varied two activity plateaus were observed. The corresponding dissociation constants varied from 6 to 20 microM Ca2+ and from 1.4 to 12 mM Ca2+ for the high- and low-affinity binding sites, respectively, depending on the assay conditions and whether purified or membrane-bound enzyme was used. A kSr2+ of 60 microM was found for the high-affinity binding site. The effect of calmodulin and its antagonist trifluoperazine was also investigated using purified and membrane-associated enzyme. When membrane-bound enzyme was measured with exogenous phosphatidylethanolamine, small stimulations by calmodulin were found. However, these were not believed to indicate a specific role for calmodulin in the Ca2+ dependency of the phospholipase A2, since trifluoperazine did not lower the activity of the membrane-bound enzyme to levels below those found in the presence of Ca2+ alone. Membrane-bound enzyme in its action toward endogenous phosphatidylethanolamine was neither stimulated by calmodulin nor inhibited by trifluoperazine. Purified enzyme was also not stimulated by calmodulin, while trifluoperazine caused small stimulations, presumably due to interactions at the substrate level. These results indicate that calmodulin involvement in phospholipase A2 activation should not be generalized. 相似文献
11.
The model enzyme alpha-chymotrypsin covalently modified by a phosphatidylethanolamine derivative has been attached to liposomal membranes in high yield. A maximal protein/lipid ratio of 5.4 X 10(-3) mol enzyme/mol lipid was achieved. 相似文献
12.
Kamejiro Yamashita James B. Field 《Biochimica et Biophysica Acta (BBA)/General Subjects》1973,304(3):686-692
Treatment of bovine thyroid plasma membranes with phospholipase A or C inhibited the stimulation of adenylate cyclase activity by thyroid-stimulating hormone (TSH). In general, basal and NaF-stimulated adenylate cyclase activity was not influenced by such treatment. When plasma membranes were incubated with 1–2 units/ml phospholipase A, subsequent addition of phosphatidylcholine or phosphatidylserine but not phosphatidylethanolamine partially restored TSH stimulation. Phosphatidylcholine was more effective than phosphatidylserine in that it caused greater restoration of the TSH response and smaller amounts of phosphatidylcholine were active. However, when the TSH effect was obliterated by treatment of plasma membranes with 10 units/ml phospholipase A, phospholipids were unable to restore any response to TSH. Lubrol PX, a nonionic detergent, inhibited basal, TSH- and NaF-stimulated adenylate cyclase activities in thyroid plasma membranes. Although phosphatidylcholine partially restored TSH stimulation of adenylate cyclase activity in the presence of Lubrol PX, it did not have a similar effect on the stimulation induced by NaF. These results indicate that phospholipids are probably essential components in the system by which TSH stimulates adenylate cyclase activity in thyroid plasma membranes. The effects do not seem to involve the catalytic activity of adenylate cyclase but the data do not permit a distinction between decreased binding of TSH to its receptor or impairment of the signal from the bound hormone to the enzyme activity. 相似文献
13.
Phospholipase A(2) from Crotalus atrox hydrolyzes all of the phospholipids that are associated with purified, detergent-solubilized cytochrome c oxidase; less than 0.05 mol cardiolipin (CL)(1) remains bound per mol enzyme. Coincident with the hydrolysis of cardiolipin is a reversible decrease of 45-50% in the electron transport activity of the dodecylmaltoside-solubilized enzyme. Full activity is recoverable (90-98%) by addition of exogenous cardiolipin, but not by either phosphatidylcholine or phosphatidylethanolamine. Unexpectedly, cleavage of cardiolipin causes the dissociation of both subunits VIa and VIb from the enzyme. These are the two subunits that form the major protein-protein contacts between the two monomeric units within the dimeric complex. Although hydrolysis of CL by phospholipase A(2) and loss of these subunits is linked, the reverse process does not occur, i.e., removal of subunits VIa and VIb does not cause dissociation of the two functionally important, tightly bound cardiolipins. Nor does addition of exogenous cardiolipin result in reassociation of the two subunits with the remainder of the complex. We conclude that cardiolipin is not only essential for full electron transport activity, but also has an important structural role in stabilizing the association of subunits VIa and VIb within the remainder of the bovine heart enzyme. 相似文献
14.
Immobilization of glycoenzymes through carbohydrate side chains. 总被引:1,自引:0,他引:1
Glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y were covalently bound to water-insoluble supports through their carbohydrate side chains. Two approaches were used. First, the carbohydrate portions of the enzymes were oxidized with periodate to generate aldehyde groups. Treatment with amines (ethylenediamine or glycyltyrosine) and borohydride provided groups through which the protein could be immobilized. Ethylenediamine was attached to glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y to the extent of 24, 20, 30, and 15 mol/mol of enzyme, respectively. These derivatives were coupled to an aminocaproate adduct of CL-Sepharose via an N-hydroxysuccinimide ester or to CNBr-activated Sepharose. Coupling yields were in the range of 37–50%. Retained activities of the bound aminoalkyl-enzymes were 41% (glucoamylase), 79% (peroxidase), 71% (glucose oxidase), 83% (carboxypeptidase Y). A glycyltyrosine derivative of carboxypeptidase Y was bound to diazotized arylamine-glass. Coupling yield was 42% and retained esterase activity was 84%. In the second approach, the enzyme was adsorbed to immobilized concanavalin A and the complex was crosslinked. Adsorption of carboxypeptidase Y on immobilized concanavalin A followed by crosslinking with glutaraldehyde was also effective. The bound enzyme retained 96% of the native esterase activity and showed very good operational stability. 相似文献
15.
Lectin-binding domains on laminin 总被引:5,自引:0,他引:5
Chicken erythrocytes have been found to have at least two kinds of phospholipase A2. The first is a soluble enzyme from the cytosole fraction and has no calcium sensitivity. The second can be extracted from the plasma membrane fraction with the nonionic detergent Triton X-100. In this study the membrane-bound enzyme was partially purified by affinity chromatography on phosphatidylcholine-Sepharose, and its specific activity was increased 1100-fold compared with that of the cell homogenate without nuclei. It has an optimum pH of 8.5 and required calcium for maximum activity. It showed the specificity for both phosphatidylcholine and phosphatidylethanolamine, but reacted preferentially on the former substrate. Analysis by concanavalin A-Sepharose affinity chromatography revealed that the membrane-bound phospholipase A2 was retained on the resin and could be eluted specifically with a haptenic sugar, methyl alpha-D-mannopyranoside. The enzyme seems to be either a concanavalin A-binding glycoprotein or a part of a complex with certain concanavalin A-binding glycoproteins. 相似文献
16.
M A Clark T M Conway R G Shorr S T Crooke 《The Journal of biological chemistry》1987,262(9):4402-4406
Antibodies prepared against the phospholipase A2 stimulatory peptide melittin were used to identify and isolate a novel mammalian protein with similar functional and antigenic properties. The mammalian protein of Mr 28,000 was isolated from cell sonicates by high performance immunoaffinity chromatography and size exclusion chromatography. This stimulatory protein was stable for several months when frozen at -70 degrees C. The purified protein selectively stimulated phospholipase A2 when phosphatidylcholine was used as a substrate but had no effect on phospholipase A2 activity when phosphatidylethanolamine was used as a substrate. Furthermore, this protein had no effect on phospholipase C activity or on pancreatic or snake venom phospholipase A2. The stimulatory activity was unaffected by RNase or DNase treatment. However, boiling or trypsin digestion inactivated the phospholipase stimulatory activity. The mechanism of phospholipase A2 stimulation appeared to result from an increase in the apparent Vmax of the enzyme. 相似文献
17.
A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7 总被引:14,自引:0,他引:14
Arachidonoyl-hydrolyzing phospholipase A2 plays a central role in providing substrate for the synthesis of the potent lipid mediators of inflammation, the eicosanoids, and platelet-activating factor. Although Ca2+ is required for arachidonic acid release in vivo and most phospholipase A2 enzymes require Ca2+ for activity in vitro, the role of Ca2+ in phospholipase A2 activation is not understood. We have found that an arachidonoyl-hydrolyzing phospholipase A2 from the macrophage-like cell line, RAW 264.7, exhibits Ca2(+)-dependent association with membrane. The intracellular distribution of the enzyme was studied as a function of the Ca2+ concentration present in homogenization buffer. The enzyme was found almost completely in the 100,000 x g soluble fraction when cells were homogenized in the presence of Ca2+ chelators and there was a slight decrease in soluble fraction activity when cells were homogenized at the level of Ca2+ in an unstimulated cell (80 nM). When cells were homogenized at Ca2+ concentrations expected in stimulated cells (230-450 nM), 60-70% of the phospholipase A2 activity was lost from the soluble fraction and became associated with the particulate fraction in a manner that was partly reversible with EGTA. Membrane-associated phospholipase A2 activity was demonstrated by [3H]arachidonic acid release both from exogenous liposomes and from radiolabeled membranes. With radiolabeled particulate fraction as substrate, this enzyme hydrolyzed arachidonic acid but not oleic acid from membrane phospholipid, and [3H]arachidonic acid was derived from phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol/phosphatidylserine. We suggest a mechanism in which the activity of phospholipase A2 is regulated by Ca2+: in an unstimulated cell phospholipase A2 is found in the cytosol; upon receptor ligation the cytosolic Ca2+ concentration increases, and the enzyme becomes membrane-associated which facilitates arachidonic acid hydrolysis. 相似文献
18.
Saccharification and adsorption characteristics of modified cellulases with hydrophilic/hydrophobic copolymers. 总被引:2,自引:0,他引:2
Saccharification and adsorption characteristics of native and modified cellulases were investigated. Copolymers, containing polyoxyalkylene and maleic anhydride (MA) were used to modify cellulase. Amino groups of the cellulase were covalently coupled with the MA. As the degree of modification (DM) increased, the activity of modified cellulase slightly decreased. At the maximum DM, the modified cellulase activity retained more than 75% of the unmodified native cellulase activity. In saccharification, native cellulase rapidly adsorbed onto the substrate at initial reaction time. Native cellulase adsorbed tightly onto the substrate surface and did not desorb as reaction time proceeded. The strong adsorption of cellulase onto the substrate can, however, be controlled by the modification. As the hydrophilicity of modified cellulase increased, free modified enzyme concentration also increased. As a result, the conversion rate of modified cellulase was higher than the native one. 相似文献
19.
A simplified procedure for the preparation of 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is described. An 80-fold purification is achieved in two steps with an overall yield of about 50%. The specific activity of the homogeneous enzyme protein is 17.7 units/mg. Compared with glycogen phosphorylase from rabbit muscle the enzyme from K. pneumoniae shows a markedly higher stability against deforming and chaotropic agents. The 1,4-alpha-glucan phosphorylase was covalently bound to porous glass particles by three different methods. Coupling with glutaraldehyde gave the highest specific activity, i.e., 5.6 units/mg of bound protein or 133 units/g of glass with maltodextrin as substrate. This corresponds to about 30% of the specific activity of the soluble enzyme. With substrates of higher molecular weight, such as glycogen or amylopectin, lower relative activity was observed. The immobilized enzyme preparations showed pH activity profiles which were slightly displaced to higher values and exhibited an increased temperature stability. 相似文献
20.
Phospholipase A2 (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A2 toward phosphatidylcholine is about 160 μmol min?1 ml?1 of glass beads, and the specific activity is about 13 μmol min?1 mg?1 of protein in this assay system. The pH rate profile and apparent pKa in 10 mm Ca2+ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A2 from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered. 相似文献