Effects of surfactants on the activity of phospholipase D.

We studied the dependence of the activity of cabbage phospholipase A on the substrate (phosphatidylcholine) the aggregated state of which is regulated by addition of either anionic (sodium dodecyl sulfate, cholate or oleate) or cationic (cetyl-trimethylammonium bromide) surfactants. Activation of the enzyme induced by anionic surfactants was shown to correlate with the size of their polar groups. The phospholipase hydrolase activity correlated with the transformation of multilayer liposomes into micelles. The relationship between the processes was of a complex character. The dependence of the amount of enzymically released choline on the calcium concentration passed through a sharp maximum in the presence of the anionic detergents and monotonically increased in the presence of the cationic detergent. In the former case, the sharp increase in the enzyme activity was suggested to be caused by precipitation of phospholipase D with the anionic detergent calcium salt, which can be considered as a specific type of immobilization.     

Effect of ethyleneoxide groups of anionic surfactants on lipase activity

The use of enzymes in laundry and dish detergent products is growing. Such tendency implies dedicated studies to understand surfactant‐enzyme interactions. The interactions between surfactants and enzymes and their impact on the catalytic efficiency represent a central problem and were here evaluated using circular dichroism, dynamic light scattering, and enzyme activity determinations. This work focuses on this key issue by evaluating the role of the ethyleneoxide (EO) groups of anionic surfactants on the structure and activity of a commercial lipase, and by focusing on the protein/surfactant interactions at a molecular level. The conformational changes and enzymatic activity of the protein were evaluated in the presence of sodium dodecyl sulfate (SDS also denoted as SLE₀S) and of sodium lauryl ether sulfate with two EO units (SLE₂S). The results strongly suggest that the presence of EO units in the surfactant polar headgroup determines the stability and the activity of the enzyme. While SDS promotes enzyme denaturation and consequent loss of activity, SLE₂S preserves the enzyme structure and activity. The data further highlights that the electrostatic interactions among the protein groups are changed by the presence of the adsorbed anionic surfactants being such absorption mainly driven by hydrophobic interactions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1276–1282, 2016     

Is phospholipase D really an enzyme? A comparison of in situ and in vitro activities.

Leaf phospholipase D activity was compared in vitro and in situ. In the in situ reaction stimulated by methanol only phosphatidylcholine and phosphatidylethanolamine were degraded until approx. 80% of these endogenous substrates had been consumed. Only then was a limited amount (approx. 20%) of endogenous phosphatidylglycerol degraded. Endogenous phosphatidylinositol was apparently not susceptible to phospholipase D in situ. In the vitro reaction the relative susceptibilities to degradation of added phospholipid substrates were (a) in the absence of "activators" phosphatidylethanolamine greater than phosphatidylglycerol greater than phosphatidylcholine, (b) in the presence of diethyl ether phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylglycerol and (c) in the presence of sodium dodecyl sulphate phosphatidylcholine greater than phosphatidylethanolamine = phosphatidylglycerol. Minimum rates calculated for the in situ reaction in cauliflower leaf were 5-fold higher than maximum in vitro rates reported for the same material. Phospholipase D activity has been demonstrated by the in situ reaction in all leaf tissue so far examined. From these data we conclude that phospholipase D may be an integral part of membranes containing phosphatidylcholine and phosphatidylethanolamine, but not of membranes containing phosphatidylglycerol. We also suggest that phospholipase D may not be a physiological enzyme, but rather a structural protein of phosphatidylcholine- and phosphatidylethanolamine-containing membranes and which, under certain non-physiological conditions, possess enzymic properties.     

Synthesis of cyclopropane fatty acids in isolated bacterial membranes

Isolated E. coli membrane vesicles can synthesize cyclopropane fatty acids from S-adenosylmethionine using endogenous membrane phospholipid as the lipidsubstrate. The major methylated products are methylenehexadecanoic acid and methyleneoctadecenoic acid esterified to phosphatidylethanolamine. The membrane system is slightly stimulated by sodium dodecyl sulfate at low concentrations but is inhibited by neutral surfactants. The reaction is inhibited by phospholipase C and phospholipase A. The Arrhenius plot for the enzyme reaction is discontinuous over the temperature range 0–35 °C.     

Characterization of purified cytochrome b-c1 complex from Rhodopseudomonas sphaeroides R-26

A highly purified cytochrome b-c1 complex from Rhodopseudomonas sphaeroides R-26 was isolated by a procedure involving Triton X-100 solubilization, calcium phosphate column chromatography, and ammonium sulfate fractionation. The purified enzyme complex contains, in nanomoles/mg of protein, cytochrome b, 8.3; cytochrome c1, 8.3; iron-sulfur protein, 15; phospholipids, 182; and ubiquinone, 5. Four major polypeptides with apparent molecular weights of 48,000, 30,000, 24,000, and 12,000 were detected in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr = 48,000 and 30,000 proteins are cytochromes b and c1, respectively. The enzyme complex catalyzes electron transfer from ubiquinol to cytochrome c with a specific activity of 12.6 mumol of cytochrome c reduced per min/mg of protein at 23 degrees C. This is lower than that of the mitochondrial enzyme, although both systems have similar essential redox components and a similar Km for ubiquinol. The activity is fully sensitive to antimycin A and 5-n-undecyl-6-hydroxy-4, 7-dioxobenzothiazole. The enzyme complex is stable at neutral pH and at lower temperatures, but became less stable when the incubation temperature was raised. At 37 degrees C, the half-life is 15 min. The enzymatic activity was insensitive to treatment with N',N'-dicyclohexylcarbodiimide. No p-chloromercuriphenylsulfonate-alkylable sulfhydryl groups were detected. The major phospholipids associated with the purified enzyme complex are phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol with molar per cent distributions of 25, 21, and 35, respectively. About 60% of the enzymatic activity was abolished upon treatment with phospholipase A2. The phospholipase A2-inactivated activity can be partially restored by the addition of EDTA followed with phospholipids prepared from either the cytochrome b-c1 complex of the same source or a mixture of phosphatidylglycerol and asolectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipase C from Clostridium perfringens: preparation and characterization of homogeneous enzyme

A new procedure for the purification of phospholipase C from Clostridium perfringens has been devised that results in essentially pure enzyme. The procedure consists of ammonium sulfate fractionation, ion-exchange chromatography on QAE-Sephadex, and affinity chromatography on phosphatidylcholine linked to Sepharose. The molecular weight of the enzyme, determined by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, and gel filtration, is 43,000; and the isoelectric point is pH 5.4. The enzyme was optimally active with phosphatidylcholine dispersed in sodium deoxycholate, although appreciable activity was observed with either phosphatidylcholine or sphingomyelin dispersed with ethanol. The requirement for metal ions in the assay could be met by a number of different ions. The pure enzyme was found to contain 2 mol zinc per mol enzyme, thus implicating it as a zinc metalloenzyme.     

Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants.

The biodegradation of polycyclic aromatic hydrocarbons (PAH) often is limited by low water solubility and dissolution rate. Nonionic surfactants and sodium dodecyl sulfate increased the concentration of PAH in the water phase because of solubilization. The degradation of PAH was inhibited by sodium dodecyl sulfate because this surfactant was preferred as a growth substrate. Growth of mixed cultures with phenanthrene and fluoranthene solubilized by a nonionic surfactant prior to inoculation was exponential, indicating a high bioavailability of the solubilized hydrocarbons. Nonionic surfactants of the alkylethoxylate type and the alkylphenolethoxylate type with an average ethoxylate chain length of 9 to 12 monomers were toxic to a PAH-degrading Mycobacterium sp. and to several PAH-degrading mixed cultures. Toxicity of the surfactants decreased with increasing hydrophilicity, i.e., with increasing ethoxylate chain length. Nontoxic surfactants enhanced the degradation of fluorene, phenanthrene, anthracene, fluoranthene, and pyrene.     

Liquid membrane elctrodes for bile salts

A liquid membrane electrode is described which can measure the activity of biological detergents in solution. The results for two bile salts, sodium taurocholate and sodium taurodeoxycholate, are compared with those for a conventional detergent, sodium dodecyl sulfate. The activity of the bile anion is also measured in a mixed micellar solution (phosphatidylcholine added).     

Several properties of cotton seed phospholipase D]

Properties of phospholipase D were studied using purified enzyme preparation from cotton seeds. The results obtained differ from those described in literature. It has been shown that the promoting action is exerted not only by diethyl ether and sodium dodecyl sulfate commonly used as initiators, but by some organic solvents in the presence of calcium ions as well. The activation of phospholipase D is also possible in the presence of other bivalent cations, e.g. Sr2+, Ba2+, Mn2+ and Mg2+. It is assumed that the enzyme activation occurs only in the presence of the stable heterogenous system: water-soluble enzyme--phospholipid--non-aqueous phase. Another important factor is the type of modification of the surface of the phospholipid phase, responsible for the enzyme adsorption and its subsequent activation. Comparison is made of the properties of phospholipases D isolated from cotton seeds and some other sources.     

Human epidermal transglutaminase. Preparation and properties.

A transglutaminase from human hair follicle-free epidermis was purified to homogeneity using gel filtration and ion exchange chromatography. The enzyme had an apparent Mr = 51,000 +/- 2,000 by sodium dodecyl sulfate electrophoresis, 100,000 +/- 5,000 by discontinuous gel electrophoresis, and 50,000 +/- 2,000 by gel filtration in Bio-Gel A-0.5m agarose. The enzyme cross-linked Factor XIII-free fibrinogen forming gamma dimers and alpha polymers. Either calcium or strontium was necessary for enzyme activity. In the presence of calcium, enzyme activity was increased by heating at 56 degrees or by treating with dimethylsulfoxide. Activation required calcium and occurred in the presence of serine protease inhibitors. The activated and native enzyme had apparently identical mobilities in acrylamide disc electrophoresis and sodium dodecyl sulfate electrophoresis. The Km values for two substrates in the reaction, casein and putrescine, were very similar for the native and the activated enzyme. The activated enzyme had a larger elution volume on Bio-Gel A-0.5m in the presence of calcium than did the native enzyme. The detailed mechanism of activation remains to be determined.     

Effect of phospholipase D on micellar phospholipids and the role of calcium ions]

The rates of the reaction products formation under simultaneous phospholipase D effect on phosphatidyl ethanolamine and phosphatidyl choline were studied. The hydrolysis of cephalin, unlike the phospholipase D effect on lecithin, does not require Ca2+ ions. Ca2+ does not affect the enzymatic degradation of lecithin and inhibits the reaction with cephalin in "inorganized" phospholipid emulsions. The hydrolysis of micellar phospholipids by phospholipase D (in the presence of the anionic detergent sodium dodecyl sulfate) is accelerated by Ca2+ ions for both substrates. The apparent Km value is equal to 1.5 mM and does not depend on the phospholipid type. In contrast, the value of kcat for lecithin is twice as high as that for cephalin. It was demonstrated that the phase state of the phospholipids and the chemical nature of the alcohol residue in the phospholipid molecule are essential for the substrate specificity of phospholipase D.     

Purification and kinetics of extracellular phospholipase a ofSalmonella newport

Attempts were made to purify and study the kinetics of extracellular phospholipase A of Salmonella newport (6,8, eb; 1,2). The enzyme was purified by salt precipitation followed by gel filtration, using different grades of Sephadex. The enzymically active purified preparation was found to be a protein, having molar mass ranging between 43 and 67 kDa. The enzyme had a pH optimum at 7.5, giving 18.2 micrograms of lysophosphatidylcholine per mg protein. Its activity was enhanced by all metal ions except potassium, by solvents and surfactants except sodium dodecyl sulfate. It hydrolyzed the membrane phospholipids of red blood cells and was inhibitory to the growth of other microorganisms.     

Phospholipase C from human melanoma: purification and characterization of a phosphatidylinositol-selective enzyme.

Phospholipase C was purified from human melanoma grown as solid tumors in nude mice. The specific activity of the pure enzyme was approx. 100 mumol/min per mg; its apparent molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 150 kDa. The enzyme required calcium for activity and was activated by deoxycholate in the presence of the substrate phosphatidylinositol. The melanoma phospholipase C has a distinctly different substrate preference than those identified from normal tissues; it prefers phosphatidylinositol to phosphatidylinositol bisphosphate. The tumor enzyme was approx. 4-5-fold more active using phosphatidylinositol than phosphatidylinositol bisphosphate as the substrate.     

Purification and characterization of rabbit platelet cytosolic phospholipase A2

A phospholipase A2 was purified from rabbit platelet cytosolic fraction to near homogeneity by sequential column chromatographies on heparin-Sepharose, DEAE-Sephacel, butyl-Toyopearl, DEAE-5PW ion-exchange HPLC, and TSK gel G3000SW gel-filtration HPLC. The final preparation with an estimated specific activity of 8630 nmol/min per mg protein, showed a single band with a molecular mass of about 88 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The 88-kDa phospholipase A2 exhibited a fatty acid preference; it hydrolyzed phospholipid bearing an arachidonoyl residue at the sn-2 position more effectively than that with a linoleoyl residue. The catalytic activity of the purified enzyme with phosphatidylcholine or phosphatidylethanolamine increased sharply in the presence of between 10(-7) and 10(-6) M calcium ion, indicating that it could be regulated by less than micromolar concentration of calcium. These characteristics differ from those of platelet secretory 14-kDa phospholipase A2 reported previously. Therefore, this 88-kDa enzyme is a novel phospholipase A2 and may participate in the stimulus-dependent release of arachidonoyl residues in rabbit platelets.     

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.     

Purification of phospholipase C from Bacillus cereus by hydrophobic chromatography on palmitoyl cellulose.

Phospholipase C (phosphatidylcholine choline-phosphohydrolase, EC 3.1.4.E) from Bacillus cereus (IAM-1208) was adsorbed to palmitoyl cellulose from a crude enzyme solution at pH 5--9. The adsorption was not influenced by ionic strength up to 2 M NaCl. The adsorbed enzyme was eluted almost completely by washing the cellulose with a suitable detergent, such as Triton X-100, Adekatol SO-120, Cation DT-205, or sodium deoxycholate. The enzyme was then purified by column chromatography on a palmitoylated textile (palmitoylated gauze) with an overall recovery of 91% and a 467-fold increase in specific activity over that of enzyme in the crude culture supernatant. Subsequent fractionation with acetone and chromatography on a Sephadex G-75 column separated two nearly homogeneous phospholipase C's. The enzyme adsorbed on palmitoyl cellulose was active, although its activity was about one-fourth that of free phospholipase C. Therefore, the enzyme appeared to be adsorbed to the cellulose through a hydrophobic site that was distinct from the catalytic site on the enzyme molecule.     

Purification of a soluble phospholipase A2 from synovial fluid in rheumatoid arthritis

A soluble phospholipase A2 (PLA2) was purified 4,500-fold from human rheumatoid synovial fluid. Preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded two bands of PLA2 activity of molecular weights 15,000 and 17,000 and pl 4.2-5.0. Purified PLA2 had absolute 2-acyl specificity, and hydrolyzed phosphatidylcholine with optimal activity at pH 7.5-8.0 and phosphatidylethanolamine with optimal activity at pH 7.0. Human synovial fluid PLA2 did not cross-react with anti-human pancreatic PLA2, as tested by radioimmunoassay.     

A shift in the optimum pH of phospholipase D produced by activating long-chain anions

1. The activity of phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) towards ultrasonically treated phosphatidylcholine or large phosphatidylcholine particles activated with ether was maximal near pH5, and there was little activity above pH6. 2. When the enzyme was activated by the addition of phosphatidic acid to large phosphatidylcholine particles the pH optimum was shifted to pH6.5 irrespective of the amount of activator added. 3. When the enzyme was activated with low concentrations of dodecyl sulphate the pH optimum was 5.5 with little activity above pH6. With higher concentrations of dodecyl sulphate the pH-activity profile was shifted upwards towards a pH optimum of 6.5-6.6, the magnitude of the shift depending on the extent of the hydrolysis. 4. The shifts in the pH-activity profiles cannot be correlated with changes in the ;surface pH' of the substrate particles calculated from the measurement of their zeta-potentials (electrophoretic mobilities).     

Partial purification and properties of a rat brain phospholipase.

At least 90% of a membrane-bound phospholipase D was solubilized by extraction of freeze-dried rat brain with 0.8% Miranol H2M and 0.5% cholate. The bulk of base exchange reaction enzymes remained firmly bound to the particulate fraction under these conditions. The phospholipase D specific activity was enriched 240-fold by a purification protocol employing ammonium sulfate precipitation, and both Sepharose 4B and DEAE-cellulose column chromatography. The approximate molecular weight of the partially purified enzyme was calculated to be 200,000 based upon the elution profile from Sepharose 4B and Sephadex G-200 columns. The optimum pH was 6.0, and Km values for phosphatidylcholine and phosphatidylethanolamine were 0.75 mM and 0.91 mM, respectively. The enzyme activity was not dependent on the presence of divalent cation although Ca2+ and Fe2+ showed stimulatory effects.     

Biochemical characterization of phospholipase D activity from human neutrophils

We have found a phospholipase D activity in the postnuclear fraction of human neutrophils, employing phosphatidylinositol as exogenous substrate. This phospholipase D activity was assessed by both phosphatidate formation and by free inositol release in the presence of 15 mM LiCl in the reaction mixture and in the absence of Mg2+ ions to prevent inositol-1-phosphate phosphatase activity. To assess further the phospholipase D activity, we studied its capacity to catalyze a transphosphatidylation reaction, as a unique feature of the enzyme. It was detected as [14C]phosphatidylethanol formation when the postnuclear fraction was incubated with [14C]phosphatidylinositol in the presence of ethanol. The phospholipase D showed a major optimum pH at 7.5 and a minor one at pH 5.0. Neutral and acid phospholipase D activities were differentially located in subcellular fractionation studies of resting neutrophils, namely in the cytosol and in the azurophilic granules, respectively. Neutral phospholipase D required Ca2+ ions to the active, whereas the acid enzyme activity was Ca2(+)-independent. The neutral phospholipase D activity showed a certain specificity for phosphatidylinositol, as it was able to hydrolyze phosphatidylinositol at a much higher rate than phosphatidylcholine, in the absence and in the presence of different detergents. This neutral phospholipase D activity behaved as a protein of high molecular mass (350-400 kDa) by gel filtration chromatography. Moreover, neutral phospholipase D activity was detected in the postnuclear fraction of human monocytes, by measuring free inositol release from phosphatidylinositol as exogenous substrate, under the same experimental conditions as those used with neutrophils. The enzyme displayed similar specific activities in both cell types as well as the same degree of activation after cell stimulation with the calcium ionophore A23187. These results demonstrate the existence of two phospholipase D activities with different pH optima and intracellular location in human neutrophils. Furthermore, these results suggest that this phospholipase D can play a role in signal-transducing processes during cell stimulation in human phagocytes.