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At least 19 recordsLinked to original sources
PubMed1975

Studies on phospholipases from Streptomyces. III. Purification and properties of Streptomyces hachijoensis phospholipase C.

1. Phospholipase C [EC 3.1.4.3] found in the growth medium of Streptomyces hachijoensis was purified about sixty-fold by dialysis and column chromatography on Sephadex G-50. 2. The active fraction was separated by isoelectric focusing into two fractions, phospholipase C-I (pI 6.0) and phospholipase C-II (pI 5.6). 3. Both purified phospholipases C were homogeneous by immunodiffusion and were not differentiated as regards antigencity. 4. Phospholipase C-I had maximal activity at pH 8.0 and the optimal temperature was 50degree. Phospholipase C-I was stable at 50degrees for 30 min and was stable at neutral pH. 5. The activity of phospholipase C-I was inhibited by high concentrations of various detergents such as Triton X-100, sodium, cholate, SDS and was also inhibited by Ca2+, Ba2+, Al3+, and EDTA, but was stimulated by Mg2+, and ethyl ether. 6. The Km value of phospholipase C-I was 0.9 mM, using phosphatidylcholine as a substrate. 7. By the gel filtration procedure, the molecular weights of phospholipase C-I and -II were both determined to be 18,000. 8. Phosphatidylcholine, phosphatidylinositol, cardiolipin, sphingomyelin, and lysophosphatidylcholine were hydrolyzed by phospholipase C-I, but phosphatidylethanolamine and phosphatidylserine were hydrolyzed with difficulty under the same conditions, Phospholipase C-I also hydrolyzed phosphatidic acid.

Aluminum↗
PubMed1977

The use of a phospholipase A-less Escherichia coli mutant to establish the action of granulocyte phospholipase A on bacterial phospholipids during killing by a highly purified granulocyte fraction.

Phospholipase A2 present in a highly purified, potently bactericidal, fraction from rabbit graulocytes produces net bacterial phospholipid degradation during killing of a phospholipase A-less strain of Escherichia coli. In the wild-type parent strain phospholipid breakdown is caused not only by the action of phospholipase A2 but also by phospholipase A1, indicating activation of the most prominent phospholipase of E. coli. This activation occurs as soon as the bacteria are exposed to the granulocyte fraction. Phospholipid breakdown by both phospholipases A is dose dependent but reaches a plateau after 30-60 min and at higher concentrations of the fraction. Phospholipid degradation is accompanied in both strains by an increase in permeability to actinomycin D that is also dose dependent. Even though net hydrolysis of phospholipids is greater in the parent strain than in the mutant, the increase in permeability is the same in the two strains. The addition of 0.04 M Mg2+, after the effects on phospholipids and permeability have become manifest, initiates in both strains the restoration of insensitivity to actinomycin D, the net resynthesis of phospholipids, and the disappearance of monoacylphosphatides and the partial disappearance of free fatty acids that had accumulated. Loss of ability to multiply is not reversed by Mg2+ in either strain. Less than 5 micrograms of granulocyte fraction causes loss of viability of from 90 to 99% of 1 X 10(8) microorganisms of both strains. However, at lower concentrations the parent strain is considerably more sensitive to the bactericidal effect of the granulocyte fraction than the mutant strain.

Animals↗
PubMed1976

Studies on phospholipase A in Trimeresurus flaoviridis venom. III. Purification and some properties of phospholipase A inhibitor in Habu serum.

Phospholipase A [EC 3.1.1.4] inhibitor was purified from Habu (Trimeresurus flavivurudls) serum by gel filtration on Sephadex G-200, chromatography on DE-23 cellulose and affinity chromatography on a Sepharose 4B-phospholipase A column. By these procedures, a 31-fold increase in specific activity was attained with a yield of 15%. The purified material was homogeneous as judged by cellulose acetate and polyacrylamide gel electrophoresis. It had an apparent molecular weight of 100,000 as measured by gel filtration on Sephadex G-200. The purified inhibitor was stable for 20 min at 80 degrees and was unstable below pH 6. It migrated before albumin in cellulose acetate electrophoresis and did not form any precipitin line with the crude venom or with purified phospholipase A in immunodiffusin tests. An 8-fold excess of the purified inhibitor by weight was required to inhibit completely both the egg yolk clearing action and the hemolytic action of phospholipase A.

Animals↗
PubMed1975

Phospholipases. III. Effects of ionic surfactants on the phospholipase-catalyzed hydrolysis of unsonicated egg lecithin liposomes.

Apparent values of Km and Vmax have been measured for catalysis of hydrolysis of unsonicated egg lecithin liposomes, activated through addition of 0.4 M n-hexanol, by phospholipases A2 from bee and snake venoms and by phospholipase C from Clostridium welchii as a function of the concentration of three surfactants: hexadecylamine, hexadecyltrimethylammonium bromide, and dihexadecyl phosphate. For all three enzymes, values of Km and Vmax show little or no dependence on the concentration of these ionic surfactants, demonstrating that the liposomal surface charge is not a crucial factor in determining susceptibility to phospholipase-catalyzed hydrolysis.

Amines↗
PubMed1975

Studies on phospholipases from Streptomyces. II. Purification and properties of Streptomyces hachijoensis phospholipase D.

1. Phospholipase D [EC 3.1.4.4] from Streptomyces hachijoensis was purified about 570-fold by column chromatography on DEAE-cellulose and Sephadex G-50 followed by isoelectric focusing. 2. The purified preparation was found to be homogeneous both by immunodiffusion and polyacrylamide disc gel electrophoresis. 3. The isoelectric point was found to be around pH 8.6 and the molecular weight was about 16,000. 4. The enzyme has maximal activity at pH 7.5 at 37 degrees. The optimal temperature is around 50 degrees at pH 7.5, using 20 min incubation. 5. The enzyme was stable at 50 degrees for 90 min. At neutral pH, between 6 and 8, the enzyme retained more than 95% of its activity on 24 hr incubation at 25 degrees. However, the enzyme lost 80% of its activity under the same conditions at pH 4.0. 6. The enzyme was stimulated slightly by Ca2+, Mn2+, and Co2+, and significantly by Triton X-100 and ethyl ether. It was inhibited by Sn2+, Fe2+, Fe3+, Al3+, EDTA, sodium dodecyl sulfate, sodium cholate, and cetylpyridinium chloride. 7. This phospholipase D hydrolyzes phosphatidylethanolamine, phosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylserine, and lysophosphatidylcholine, liberating the corresponding bases. 8. The Km value was 4mM, determined with phosphatidylethanolamine as a substrate.

Aluminum↗
PubMed1977

Net activity of phospholipase A2 in brain and the lack of stimulation of the phospholipase A2-acylation stem.

Certain observations reported previously from this laboratory have not proved reproducible. These are (1) the relatively rapid hydrolysis of added phosphatidylcholine by phospholipase A2 of tissue from the cerebral cortex of the guinea pig and (2) the stimulation by 10 micron-noradrenaline and by 1.0nM-cyclic AMP of the phospholipase A2-acylation system of isolated synaptic membranes.

Retraction Notice↗
PubMed1975

Studies on phospholipase C from Pseudomonas aureofaciens. I. Purification and some properties of phospholipase C.

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Pseudomonas aureofaciens was purified 3600-fold from the culture filtrate with a recovery of 1.6%. Purification was performed with the useof (NH4)2SO4 precipitation, Sephadex G-100 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. The purified enzyme appeared to be homogeneous as revealed by polyacrylamide disc gel electrophoresis at pH 9.3. The molecular weight was estimated to be 35 000 by gel filtration on Sephadex G-75. Under our experimental conditions, phosphatidylethanolamine was more rapidly hydrolysed than phosphatidylcholine. Lyso forms of these two phosphatides were poor substrates. Phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and sphingomyelin were not hydrolysed. The enzyme activity with phosphatidylcholine as substrate was slightly stimulated by Ca2+, Mg2+, and Mn2+. However, these cations inhibited the activity with phosphatidylethanolamine as substrate. An anionic detergent, sodium deoxycholate, slightly enhanced the activity when phosphatidylcholine and phosphatidylethanolamine were used as substrates. A cationic detergent, cetyltrimethylammonium bromide, inhibited enzyme activity. EDTA and o-henanthroline inhibited the activity of the enzyme to a marked degree.

Calcium↗
PubMed1976

Gonadotropin receptors in plasma membranes of bovine corpus luteum. I. Effect of phospholipases on the binding of 125I-choriogonadotropin by membrane-associated and solubilized receptors.

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.

Animals↗
PubMed1976

Phospholipases A1 and A2 of rat liver plasma membranes; mechanism of action.

While V/S plots of phospholipase A1 show a phase transition, kinetic behaviour of phospholipase A2 acting in the same concentration range is hyperbolic. However after phospholipase A2 has been solubilized from the plasma membranes by 1 M NaCl, the V/S curve shows a phase transition. Membrane-bound phospholipase A1 shows a narrow optimum pH at 8.5 -9, while phospholipase A2 activity presents only small variations between pH 7 and 9.5. Towards exogenous phospholipids at the optimum pH 8.5 of phospholipase A1, the specific activity of the latter is 3-fold higher than phospholipase A2 specific activity. On the contrary towards endogenous phospholipids, phospolipase A2 activity is higher than phospholipase A2 activity. Moreover labeled endogenous PE hydrolysis by phospholipase A2 is decreased by addition of non labeled exogenous PE into the incubation medium. All these data suggest that the active site of phospholipase A1 is turned to the outside and acts only on exogenous substrates: for phospholipase A2 it would be inside, and exogenous phospholipids could be hydrolyzed only after penetrating the membrane.

Animals↗
PubMed1979

Effect of purified phospholipases on the binding of tetrodotoxin to axon plasma membrane.

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 and Naja naja) or phospholipase C (Bacillus cereus and Clostridium 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 from B. cereus and Cl. 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.

Animals↗
PubMed1978

Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.

Animals↗