A spectrophotometric method for the assay of phospholipase D activity

Phosphatidylcholine phosphatidohydrolase (EC 3.1.4.4, phospholipase D) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. We have developed a spectrophotometric assay for phospholipase D using choline kinase, pyruvate kinase, and lactate dehydrogenase to couple the release of choline with the oxidation of NADH. The assay was linear both with time and with enzyme concentration. The assay should prove useful for continuous monitoring of enzyme activity, determination of initial rates of reaction, and detailed kinetic studies of phospholipase D. The method is limited to analysis of purified preparations of phospholipase D lacking competing activities to the coupled system.     

Radiochromatographic assay of N-acyl-phosphatidylethanolamine-specific phospholipase D activity

A radiochromatographic method has been set up to assay the activity of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), based on reversed-phase high-performance liquid chromatography (HPLC) and online scintillation counting. The anandamide (N-arachidonoylethanolamine, AEA), product released by NAPE-PLD from the N-arachidonoyl-phosphatidylethanolamine (NArPE) substrate, was separated using a C18 column eluted with methanol-water-acetic acid and was quantified with an external standard method. Baseline separation of AEA and NArPE was completed in less than 15 min, with a detection limit of 0.5 fmol AEA at a signal-to-noise ratio of 4:1. The sensitivity and accuracy of the radiochromatographic procedure allowed detection and characterization of NAPE-PLD activity in very tiny tissue samples or in samples where the enzymatic activity is very low. With this method, we could determine the kinetic constants (i.e., apparent Michaelis-Menten constant (Km) of 40.0+/-5.6 microM and maximum velocity (Vmax) of 22.2+/-3.5 pmol/min per milligram protein toward NArPE) and the distribution of NAPE-PLD activity in brain areas and peripheral tissues of mouse. In addition, we could collect unprecedented evidence that compounds widely used in studies of the endocannabinoid system (e.g., AEA and congeners, receptor a(nta)gonists and inhibitors of AEA degradation) can also affect NAPE-PLD activity.     

A continuous spectrophotometric assay for phospholipase A(2) activity

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).     

A rapid assay for activity of phospholipase A2 using radioactive substrate

A rapid method for the assay of phospholipase A2 has been developed using a radioactive substrate, L-alpha-dipalmitoyl-(2-[9,10(N)-3H]palmitoyl)-phosphatidylcholine. The substrate diluted with cold carrier (1 mM) is dissolved in 80% ethanol containing 25 mM sodium deoxycholate. The enzymatic reaction is performed in 1.0 ml 0.1 M glycine-NaOH buffer, pH 9.0, containing 2 mumol CaCl2, 10 micrograms bovine serum albumin, 2.5 mumol sodium deoxycholate, 0.01 unit (or less) phospholipase A2, and 40-100 nmol substrate. The enzymatic reaction is terminated by adding 0.2 ml 5% Triton X-100 solution containing 40 mumol EDTA. The product of the enzymatic reaction, radioactive palmitic acid, is extracted by 10 ml hexane containing 0.1% acetic acid in the presence of anhydrous sodium sulfate (0.5 g/ml). Activity of phospholipase A2 is directly determined from the radioactivity in the hexane extract. The present method achieves a quick separation of the radioactive product, [3H]palmitic acid, from the radioactive substrate, L-alpha-dipalmitoyl-(2-[3H]palmitoyl)-phosphatidylcholine, without the need of separation by TLC.     

A spectrophotometric assay for the transphosphatidylation activity of phospholipase D enzyme

We developed a specific spectrophotometric assay for the quantitative determination of phospholipase D-catalyzed transphosphatidylation activity. The assay measures p-nitrophenol liberated by phospholipase D-catalyzed reaction of phosphatidyl-p-nitrophenol and ethanol in an aqueous-organic emulsion system. The release of p-nitrophenol was linear to reaction time at an early stage of the reaction with phospholipase D from Streptomyces sp. In the spectrophotometric assay for the reaction with phospholipase D from Streptomyces chromofuscus, which has higher hydrolytic activity than transphosphatidylation activity, p-nitrophenol was not found. The advantages of this novel method for measuring the transphosphatidylation activity of phospholipase D are that (i) it does not use radioactive compounds, (ii) it can measure the initial velocity of the reaction, and (iii) it is rapid, easy, and accurate to perform.     

A subnanogram assay for phospholipase activity based on a long-chain radioiodinatable phosphatidylcholine

Here, we introduce a radioiodinatable long-chain phosphatidylcholine (BHC12PC) which serves as the base for a very sensitive phospholipase assay. This compound has a 4-hydroxyphenyl group attached at the end of the fatty acyl chain located in position sn-2. This feature enables this phospholipid to be radioiodinated. BHC12PC was tested as a substrate of Naja naja naja PLA(2) and Bacillus cereus PLC in a mixed micellar system with Triton X-100. The detection limit for the assays was 0.25ng of PLA(2) and 0.05ng of PLC, thus becoming one of the most sensitive methods described so far. A low specific radioactivity (500microCi/mmol) suffices to achieve this level of sensitivity. In both cases, the behavior of BHC12PC was indistinguishable from that shown by phospholipids with n-acyl chains of similar length. The choice of spacer prevents any unfavorable interaction of the bulky 4-hydroxyphenyl group at the active site of the enzymes. The progress of the reaction as monitored by thin-layer chromatography is compared side by side with an alternative method based on the selective adsorption of BHC12PC to silica gel, which renders identical results in a simpler fashion. An additional advantage of BHC12PC is that the cost per Ci of the radioiodinated derivative is significantly lower than that of other labeled phospholipids ((3)H, (14)C, or (32)P).     

Solubilization and assay of phospholipase A2 activity from rat jejunal brush-border membranes

The phospholipase activity of rat jejunal brush-border membranes was examined in the presence of several solubilizing agents, by measuring the hydrolysis of endogenous membrane phospholipids, as well as the hydrolysis of exogenous, radiolabelled substrates. Enzyme activity was highly stimulated by dispersion in 1% solutions of bile salts, or in a synthetic, bile-salt derivative, 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate (CHAPS). Under these conditions the endogenous membrane phospholipids were largely degraded to free fatty acids and water-soluble phosphate. In the presence of 1% CHAPS, hydrolysis of exogenous phosphatidylcholine was shown to be due to an initial phospholipase A2-type attack followed by a subsequent lysophospholipase-type attack. These activities co-purified with the brush-border membrane. Maximal phospholipase A2 hydrolysis occurred at an alkaline pH of 8-11, with bile-salt detergents present at greater than their critical micellar concentrations. Hydrolysis was completely divalent-ion independent. Phospholipase A2 activity was not stimulated by 50% diethyl ether or ethanol, or in the presence of 1% solutions of Triton X-100, Zwittergent 3-12, sodium dodecyl sulphate, or n-octylglucoside. Stimulation of phospholipase activity by detergents was not related to their effectiveness at solubilizing the membrane proteins. When assayed individually phosphatidylcholine and lysophosphatidylcholine were each hydrolyzed (at the sn-2 and sn-1 positions, respectively) at a rate of approximately 125 nmol/mg protein per min. When assayed together, the two substrates appeared to compete for the same active site over a wide range of concentrations. It was concluded that the brush-border membrane contains an integral membrane protein with phospholipase A2 and lysophospholipase activities, which is specifically stimulated by bile salts and bile salt-like detergents.     

Spin-label assay for phospholipase A2

A rapid and continuous method for measuring phospholipase A2 activity using electron spin resonance spectroscopy and a spin-labeled phospholipid as a substrate has been developed. The substrate, 1-palmitoyl-2-(4-doxylpentanoyl)glycerophosphocholine, gives rise principally to a broad ESR line in aqueous solution due to strong spin-spin interactions, probably resulting from its micellar formation. Upon addition of bee venom phospholipase A2, the water-soluble product, 4-doxylpentanoic acid, is released which brings about a sharp three-line spectrum. Thus, the kinetics of phospholipase A2 activity can be followed by monitoring the increase in the ESR signal amplitude of the three-line spectrum, which is linearly proportional to the amount of 4-doxylpentanoic acid produced; no separation of the product from the substrate is needed during the measurement. The rate of hydrolysis of 1 nmol min-1 can be accurately measured within a 5-min period of time in a sample volume of 100 microliters. This new method should be useful for assaying phospholipase A2 activities in various biological systems.     

Continuous spectrophotometric assay of phospholipase A2 activity hydrolyzing plasmalogens using coupling enzymes

We developed a continuous spectrophotometric assay of the phospholipase A2 activity specific for choline plasmalogen using rat liver lysoplasmalogenase and horse liver alcohol dehydrogenase as coupling enzymes and Naja naja venom phospholipase A2 as a source of the phospholipase A2 activity. In these coupling reactions, choline lysoplasmalogen is hydrolyzed by lysoplasmalogenase to glycerophosphocholine and free aldehyde. The free aldehyde is quantitatively converted to alcohol by alcohol dehydrogenase with the oxidation of NADH. The disappearance of NADH is measured spectrophotometrically at 340 nm. The assay is sensitive to about 0.2 nmol aldehyde produced/ml/min and also is rapid, convenient, and continuous.     

A rapid simple radiometric assay for phospholipase A2 activity

A rapid, simple radiochemical assay for phospholipase A₂ (PLA₂) activity is described in which incubations of homogenized tissue were directly applied to columns of silica gel absorbent. The metabolites and substrate were eluted from the columns with two different solvent systems so that each could be separated and quantified.     

Hemolytic assay for venom phospholipase A2

A rapid and sensitive spectrophotometric assay for venom phospholipase A₂ based on the hemolysis of guinea pig erythrocytes in the presence of decomplemented serum and cardiotoxin (direct lytic factor) is described. This assay is particularly useful for rapid multisample analyses, such as those used in monitoring chromatography fractions, and is specific for phospholipase A₂ in she presence of other potentially hemolytic venom components. The hemolytic mechanism is shown to be a combination of the action of lysophospholipids liberated from lipoproteins in the serum and the synergistic action of phospholipase A₂ and cardiotoxin on the erythrocyte membrane.     

A simplified assay for phospholipase C.

A new assay for phospholipase C activity that uses alkaline phosphatase to convert phosphorylcholine to inorganic phosphate is described. The determination of inorganic phosphate is performed in the presence of phosphatidylcholine and protein after the addition of sodium dodecyl sulfate. Phospholipase C activity determined by this coupled enzyme assay agrees well with data obtained by extracting and measuring phosphoryl[¹⁴C]choline produced from phosphatidyl[methyl-¹⁴C]choline. The assay is sensitive to 1 nmol of phosphate, requires no removal of protein or phospholipid, and will work with a variety of phospholipid substrates. The assay is faster and more sensitive than previously published procedures. Stimulation of phospholipase C from Clostridium perfringens by ammonium sulfate is also reported.     

A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts.

A radioisotope assay for the measurement of glutamine synthetase activity has been developed in which tandemly arranged ion-exchange columns of Dowex 1-acetate and Amberlite CG-50 (H⁺) are used to separate the product, [¹⁴C]glutamine, from unreacted [U-¹⁴C]glutamate and other labeled compounds, particularly γ-aminobutyrate, that are formed by competing reactions. The technique is sensitive, reproducible, and suitable for multiple determinations. The assay has been used successfully to measure glutamine synthetase activity in neural and nonneural tissues which contain appreciable amounts of glutamate decarboxylase activity.     

Kinetic characterization of phospholipase A2 modified by manoalogue

Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivate the phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, B. P., Hite, E. D., Mihelich, E. D., & Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172) by reacting with enzyme lysine residues. In the present study, the inactivation of the phospholipases A2 from pig pancreas, bee venom, and cobra (Naja naja naja) venom by manoalogue was studied in detail. Manoalogue-treated enzymes were examined in the scooting mode on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol. Here the native enzymes bound irreversibly to the vesicles and hydrolyzed all of the phospholipids in the outer monolayer without leaving the surface of the interface. All three manoalogue-treated enzymes showed reduced catalytic turnover for substrate hydrolysis in the scooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease in the fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospholipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine epsilon-amino groups have been amidinated was not inactivated by manoalogue, indicating that the modification of lysine residues and not the amino-terminus is required for the inactivation. Several studies indicated that the manoalogue-modified enzymes contain a functional active site. For example, studies that monitored the protection by ligands of the active site from attack by a alkylating agent showed that manoalogue-modified pig phospholipase A2 was capable of binding calcium, a substrate analogue, lipolysis products, and a competitive inhibitor. Furthermore, relative to native enzymes, manoalogue-modified enzymes retained significantly higher catalytic activities when acting on water-soluble substrates than when acting on vesicles in the scooting mode. Intact manoalogue had no affinity for the catalytic site on the enzyme as it did not inhibit the enzyme in the scooting mode and it did not protect the active site from alkylation. Pig pancreatic phospholipase A2 bound to micelles of 2-hexadecyl-sn-glycero-3-phosphocholine was resistant to inactivation by manoalogue, suggesting that the modification of lysine residues on the interfacial recognition surface of the enzyme was required for inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Uteroglobin inhibits phospholipase A2 activity

Although progesterone is known to produce quiescence in the mammalian uterus, the mechanism of this effect is not clearly understood. Here, we report that uteroglobin, a progesterone-induced small molecular weight (16K) protein, inhibits phospholipase A2(PLA2) derived from porcine pancreas as well as from the RAW 264.7 macrophage cell line. We speculate that progesterone may exert its antimotility effects on the uterus via uteroglobin which, by inhibiting PLA2, decreases arachidonic acid release and subsequently reduces prostaglandin levels in this organ. This may explain why progesterone is so vital for the maintenance of pregnancy in almost all mammals.     

A modified capillary assay for chemotaxis

Summary A modification is described of the capillary assay for chemotaxis. It employs a 96-well dilution plate and its cover. Capillary tubes are inserted through the cover and are supported by small rubber collars. The method is faster and less tedious and gives more precise results than earlier methods.