G.V. Marinetti

The action of phospholipase A on lipoproteins

Abstract Snake venom has been shown to cause a clearing of a suspension of egg yolk. This clearing is due to phospholipase A (phosphatide acylhydrolase, EC 3.1.1.4) acting on the Upoproteins to produce lysolecithin (and lysophosphatidylethanolamine). The lysolecithin produced is capable of solubilizing the egg yolk suspension. The clearing of egg yolk suspension under controlled conditions of pH, ionic strength, and type of salt used, can be the basis for a rapid assay method for phospholipase A activity. A variety of venoms were tested and a wide range of phospholipase A activity was observed. The most active venoms were Agkistrodon p.p., Naja naja, Ophiophagus hannah, Vipera russelli , and Micrurus fulvius . The activity of phospholipase A was found to be inhibited to varying degrees by certain cations (Cu 2+ , Fe 3+ , Zn 2+ ), EDTA, iodoacetate, solvents (dioxan), formaldehyde, heating, freezing, sonication, ultraviolet irradiation, and exposure to high pH. The activity of phospholipase A was enhanced by trypsin (EC 3.4.4.4) treatment and mild HCl treatment, and by addition of ethyl ether to the egg yolk suspension. Column chromatography of a variety of snake venoms on Sephadex G-75 gave different distribution profiles for each venom. Of the venoms studied only that from Vipera russelli was resolved into two distinct phospholipase A containing peaks. Paper chromatography serves as a rapid check for confirming the presence of phospholipase A. The confirmation is necessary since certain agents (Al 3+ , high pH, high salt concentration) cause a clearing of the egg yolk suspension in the absence of the enzyme.

The reaction of chemical probes with the erythrocyte membrane

SummaryTrinitrobenzenesulfonate (TNBS), fluorodinitrobenzene (FDNB) and suberimidate have been reacted with intact human erythrocytes. TNBS does not penetrate the cell membrane significantly at 23 °C in bicarbonate-NaCl buffer, pH 8.6, as estimated by the labeling of the N-terminal valine of hemoglobin. Hence, under these conditions it can be used as a vectorial probe. However, at 37 °C, especially in phosphate buffer, at pH 8.6, TNBS does penetrate the cell membrane. FDNB and suberimidate both penetrate the erythrocyte membrane. The time course reaction of TNBS with intact erythrocytes over a 24-hr period at 23 °C is complex and shows transition zones for both membrane phosphatidylethanolamine (PE) and membrane proteins. No significant cell lysis occurs up to 10 hr. The fraction of total PE or phosphatidylserine (PS) which reacts with TNBS by this time period can be considered to be located on the outer surface of the cell membrane. Under these conditions it can be shown that 10 to 20% of the total PE and no PS is located on the outer surface of the membrane and hence these amino phospholipids are asymmetrically arranged. The pH gradient between the inside and outside of the cell in our system is 0.4 pH units. Nigericin has no effect on the extent of labeling of PE or PS by TNBS. Isotonic sucrose gives a slight enhancement of the labeling of PE by TNBS. Hence, the inability of PE and PS to react with the TNBS is considered not due to the inside of the cell having a lower pH. The extent of reaction of TNBS with PE is not influenced by changing the osmolarity of the medium or by treatment of cells with pronase, trypsin, phospholipase A or phospholipase D. However, bovine serum albumin (BSA) does protect some of the PE molecules from reacting with TNBS.Cells treated with suberimidate were suspended in either isotonic NaCl or in distilled water. In both cases the suberimidate-treated cells became refractory to hypotonic lysis. Pretreatment of cells with TNBS did not prevent them from interacting with suberimidate and becoming refractory to lysis. However, pretreatment of cells with the penetrating probe FDNB abolished the suberimidate, effect. Electron-microscopic analysis of the cells showed a continuous membrane in the case of cells suspended in isotonic saline. The cells suspended in water did not lyse but their membranes had many large holes, sufficient to let the hemoglobin leak out. Since the hemoglobin did not leak out we know that the hemoglobin is cross-linked into a large supramolecular aggregate.

A rapid method for the isolation of rat liver plasma membranes using an aqueous two-phase polymer system

Abstract A simple and rapid method for the isolation of rat liver plasma membranes has been developed using an aqueous two-phase polymer (dextran-polyethylene-glycol system). Yields and purity of this preparation were determined and compared to membranes obtained by sucrose density gradient centrifugation. The technique as described in this paper provides: 1. 1. The highest reported yield of plasma membrane when compared to other methods. 2. 2. A high degree of purity determined by enzyme assays of the plasma membrane fraction, that is comparable to other separation techniques. 3. 3. A simple and rapid method, requiring only low speed centrifugations, decreasing membrane preparation time by 2–3 hr.

Analysis of human plasma phosphatides by paper chromatography

Abstract The individual phosphatides of human plasma were analyzed qualitatively and quantitatively by paper chromatography. The phosphatides which were always present were lecithin, sphingomyelin, lysolecithin phosphatidylethanolamine and a component having the properties of inositol phosphatide. Phosphatidylserine was not detected by this procedure and hence is at most a trace constituent in plasma. Very small amounts of other unidentified phosphatides also occur in plasma. One of these may be lysophosphatidylethanolamine. A study was made of the plasma phosphatides of persons having recent cardiac infarctions. There is suggestive evidence that plasma lysolecithin is significantly diminished in some of these persons. The advantages and limitations of the paper chromatographic method for the analysis of plasma phosphatides are discussed.

The enzymatic acylation and hydrolysis of lysolecithin

The present experiments show that lysolecithin is converted to lecithin by ratliver preparations which are free from cell particles. In this process lysolecithin is esterfied directly rather than being degraded to simpler compounds which are subsequently incorporated into lecithin. Two separate reactions are postulated whereby lysolecithin is converted to lecithin. These reactions are as follows: 2 lysolecithin → lecithin + glycerophosphorylcholine (1) lysolecithin + acyl-CoA → lecithin (2) Reaction 1 is from a quantitative standpoint the more important reaction although Reaction 2 has a faster rate. The Michaelis-Menten plot of lecithin synthesis vs. lysolecithin concentration gave support for Reactions 1 and 2 since the rate curve was anomalous, being composed of two separate parts which gave Km values of 0.08 mM (Reaction 2) and 3.3. mM (Reaction 1). The value of Km obtained for lysolecithin hydrolysis was 1.03 mM. When [32P]lysolecithin was used as substrate, the synthesis of lecithin as measured by either 32P-incorporation or by chemical phosphorus was the same and either method could be used to quantitate lysolecithin esterification or lysolecithin hydrolysis. The studies given here show that the rate of metabolism of lysolecithin was essentially constant at pH values between 6.2 and 6.9 but at values greater than 6.9 there was suppression of both lecithin synthesis and lysolecithin hydrolysis. Iodoacetic acid failed to inhibit either lecithin synthesis or lysolecithin hydrolysis whereas HgCl2 inhibited both. CN- inhibited lecithin synthesis but not lysolecithin hydrolysis. Stearylglycollecithin was found to be inactive as a substrate for lysolecithinase in the rat-liver supernatant fluid but inhibited lysolecithin hydrolysis and lecithin synthesis 73 and 80% respectively. Transesterification has been shown to occur by the use of doubly-labeled lysolecithin.

Differential solubilization of proteins, phospholipids, and cholesterol of erythrocyte membranes by detergents

Abstract Red cell membranes were treated with increasing concentrations of Triton X-100, sodium dodecylsulfate, or sodium deoxycholate at pH 7.5, and the solubilization of total protein, total phospholipid, cholesterol, and of individual proteins and phospholipids was determined as a function of detergent concentration. The results suggest that each detergent solubilized membrane components by a different mechanism. Sodium dodecylsulfate extracted individual protein and lipids separately, each membrane component having a sigmoid extraction curve with a different dodecylsulfate concentration at midpoint. Sodium deoxycholate solubilized all proteins and phospholipids in parallel, with little fractionation, but cholesterol was not solubilized significantly until 60% of the protein and phospholipids were extracted. The data suggest that deoxycholate intially solubilizes membrane lipoproteins by displacement of cholesterol into the residual membrane matrix, followed at higher deoxycholate concentration by removal of phospholipids from both soluble and insoluble proteins into deoxycholate-phospholipid micelles which in turn solulibilize cholesterol. Triton X-100 initially solubilizes most proteins and lipids in parallel, but proteins are released faster while sphingomyelin is preferentially retained in the pellet. At higher Triton X-100 concentration, net protein solubilization ceases while residual lipids are completely solubilized.

Calcium binding to the rat liver plasma membrane

Calcium binding to isolated rat liver plasma membranes shows saturation kinetics, has a pH optimum of 7.8–8.0, and is independent of metabolic energy. scatchard analysis shows two classes of calcium binding sites. The higher affinity sites, with an association constant of 4.0·102 M−1, accomodate 22 ± 2 (S.D.), nmoles of calcium/mg membrane protein. The lower affinity sites with an association constant of 3.2·102 M−1 accomodate 120 ± 16 (S.D.) nmoles of calcium/mg membrane protein. Mg2+ competes with Ca2+ only for the low affinity binding sites. K+ and Na+ do not inhibit calcium binding. EDTA decreases the amount of calcium bound to the membranes. The ATP effect on calcium binding depends on the calcium concentration. At 1 mM calcium, ATP at 0.3 mM inhibits binding but at 3 mM calcium, ATP stimulates biding. ADP and AMP at 0.3 mM have no effect on calcium binding at 1 mM calcium but ADP stimulates binding at 3 mM calcium. Cyclic AMP at 10−3 M increases calcium bnding at both 1 and 3 mM levesl of calcium. Phospholipases, neuraminidase and proteases were used to determine the role of phospholipids, neuraminic acid and proteins in the binding of calcium. Of the total extrapolated maximum calcium binding sites (142 + 18 (S.D.) nmoles/mg membrane proten), acidic phospholipids accounted for approximately 100 nmoles/mg membrane protein while neuraminic acid residues accounted for approximately 50 nmoles/mg membrane protein. Treatment of the membrane with a variety of functional group reagents showed that agents which react with amino groups or hydroxyl groups of proteins have a small or no effect on calcium binding. However, SH agents increase calcium biding but only when the calcium concentration in the medium was 1 mM or greater. Sodium dodecyl sulfate (0.1%) increases calcium binding very markedly, Triton X-100 (0.1%) gave a 3-fold increase in calcium binding and Lubrol (0.1%) had no influence on calcium binding. Triton may unmask certain acidic phospholipids. Sodium dodecyl sulfate is believed to become incorporated into the membrane and convert it to a highly charged form which now binds calcium. This detergent may also unmask acidic phospholipids.

The hydrolysis of lecithins by snake venom phospholipase

Abstract [ 32 P]labeled lysolecithins were prepared from rat liver by the action of snake venoms on the corresponding lecithins. The lysolecithins were oxidized under mild conditions with permanganate. Two lyco-compounds were formed after this oxidation. Acid hydrolysis of the lyso-compounds yielded at least four [ 32 P]-labeled water-soluble phosphate compounds. Similar results were obtained with egg lecithin. Beef and pig heart lecithins (mixture of the diester and plasmalogen forms) were subjected to hydrolysis with snake venom. Under conditions where egg lecithin was completely degraded to lysolecithin the beef and pig lecithins were only partially hydrolyzed (62–64%). The lecithins which resisted hydrolysis in the early time interval were essentially all the plasmalogen type. Hence the snake venom degrades the diester lecithin at a faster rate than the plasmalogen analogue. However, after several days exposure to the venom the plasmalogens are completely hydrolyzed to the corresponding lysoplasmalogens. Reduction of the lysoplasmalogens with hydrogen and subsequent acid hydrolysis gave a 70% theoretical yield of a -glycerol ether. These studies show that snake venom phospholipase A can hydrolyze either the a or β-linked fatty acid in lecithins to yield a - and β-lysolecithins.

The in vitro incorporation of 32P-labeled orthophosphate into the phosphatides of isolated rat liver mitochondria☆

Abstract 1. 1. The incorporation of 32 P i into the lipides of intact rat liver mitochondria occurs mainly into phosphatides which resemble but are not identical to phosphatidic acids and which constitute only a very small fraction of the total lipide P. Evidence is presented which indicates that one of these highly labeled phosphatides is an α,β-conjugated ester of glycerophosphate. The incorporation into the common phosphatides such as lecithin and phosphatidyl ethanolamine is extremely small in these systems. 2. 2. Glycerol, Mg, and several nucleotides such as ATP and CTP were found to stimulate the incorporation into at least 3 phosphatides one of which contains glycerophosphate. The microsomal-rich supernatant fluid also had a stimulating effect. On the other hand, cyanide, dinitrophenol, malonate, and a variety of metal ions and surface agents were found to inhibit the system. 3. 3. The identification of the mitochondrial lipides was made by chemical, infrared spectral, and paper and column chromatographic analysis. 4. 4. In contrast to intact mitochondria, the acetone powders of these particles were far less effective in incorporating 32 P i into the phosphatides. However, a striking difference was found inasmuch as in these latter systems the incorporation occurred predominantly in lecithin and phosphatidyl ethanolamine and none occured in the fast moving glycerophosphatide. 5. 5. A discussion is given of the experimental findings.

Hormone action at the membrane level. I. Properties of adenyl cyclase in isolated plasma membranes of rat liver

Abstract Membarne bound adenyl cyclase in isolated rat liver plasma membranes exhibits a requirement for Mg 2+ and has optimal activity near pH 8.0. The optimal concentration of ATP is approx. 0.3 mM at a Mg 2+ concentration of 1.0 mM. Ca 2+ at a concentration range of 0.03–0.3 mM stimulates the basal activity of this enzyme. The enzyme activity is abolished by 1 min heating at 100°. Detergents such as Triton X-100 and sodium dodecyl sulfate alter the membrane structure and slightly enhance the enzyme actively, whereas sodium deoxycholate slightly inhibits the enzyme. Glucagon and epinephrine stimulate whereas insulin inhibits adenyl cyclase activity. When studied under identical conditions on the same membrane preparation the glucagon stimulation is seen earlier and at a lover hormone concentration (1 · 10 −6 M) than is the epinephrine stimulation which requires 10 −5 M hormone. The inhibition by insulin is seen at 1 · 10 −5 M or greater. The stimulation by glucagon is inhibited by 1 · 10 −4 –1 · 10 −5 M Ca 2+ but the stimulation by epinephrine is enhanced by 1 · 10 −4 –1 · 10 −5 M Ca 2+ . Moreover, epinephrine enhances, whereas glucagon inhibits the binding of Ca 2+ to the membrane. Studies on the combined effects of the hormones show that the stimulation by glucagon plus epinephrine is not additive and that insulin antogonizes the glucagon stimulation of adenyl cyclase. Na + inhibits the basal activity of adenyl cyclase but K + stimulates the enzyme. F − inhibits the enzyme. These results point to a complex interplay of hormones and metal ions with the membrane bound adenyl cyclase.