Effect of phospholipases A2 from bee and cobra venom on the phospholipid composition and Na+-,K+-ATPase activity of synaptosomes

The bee and cobra venom phospholipases A2 as well as partially acetylated cobra venom phospholipase A2 are studied for their effect on phospholipid composition of synaptosomes and their Mg2+- and Na+,K+-ATPase activity. It is established that these phospholipases induce the splitting of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine, inhibition of the Na+,K+-ATPase activity and activation of Mg2+-ATPase. Bee venom phospholipase A2 is more effective than cobra venom phospholipase A2, the both phospholipases splitting phosphatidylethanolamine most intensively. The ATPase activity may be partially or completely restored by exogenic phosphatidylcholine and phosphatidylserine; exogenic phosphatidylethanolamine is not efficient in this respect.     

Inhibitory action of phosphatidylinositol on synaptosomal (Na+ + K+)-ATPase activity

Phosphatidylinositol and several other phospholipids were tested for their ability to influence the (Na+ + K+)-ATPase activity of the cortical synaptic membrane from rats at various levels of free Ca2+. Phosphatidylinositol, but not phosphatidylethanolamine, phosphatidylcholine nor phosphatidylserine, markedly inhibited this enzyme activity, when the free Ca2+ concentration in the incubation media was less than 2.5 X 10(-6) M. This result suggests that phosphatidylinositol may play a role in the depolarization and/or the release of neurotransmitters or intracellular substances in the brain.     

Some properties of hog thyroidal membrane-bound adenosine tri-phosphatase: proteolytic activation of Mg-dependent activity.

The ATPase preparations from the hog thyroid was preincubated with various amounts of trypsin. The activity of Mg-ATPase was consistently elevated. On the contrary, the Na, K-ATPase activity decreased with increasing amounts of trypsin. The effects were similar to those which were observed in the enzyme preparations treated with basis polyamino acids as previously reported. This phenomenon seemed to be specific in the preparations from the thyroid. The Mg-dependent activity was increased after pretreatment with trypsin or poly-L-lysine (PLL) when CTP, ITP and UTP were used as substrate. Thus the substrate specificity of Mg-ATPase was low. The enzyme-kinetics using ATP as substrate showed that the increase in activity was due to an increase in Vmax and not to a change in Km. The activity of Mg-ATPase was increased even after 30 min of preincubation with trypsin, while the Na, K-ATPase activity was almost diminished. These results suggest that the activity of Mg-ATPase in the preparation from the thyroid is specifically changed by the modification of the molecular environment of the enzyme with trypsin or basic polyamino acids.     

Action of phospholipids and gangliosides on tumor cell sensitivity to the cytostatic and membrane-toxic action of splenic effectors

The authors attempted to study which parts of the lipid molecules are most responsible for an increase in the sensitivity of the tumor cell to the cytostatic and membrano-toxic action of natural spleen effectors. Use was made of different combinations of egg phosphatidylcholine, phosphatidylethanolamine, cardiolipin, dipalmitoyl phosphatidylcholine and a mixture of bovine brain gangliosides. Introduction of phosphatidylethanolamine into the membrane of tumor cells increased, that of phosphatidylcholine and cardiolipin did not change whereas introduction of a mixture of brain gangliosides reduced their sensitivity to spleen effectors. Introduction of brain gangliosides together with egg phosphatidylcholine into the membrane of the target cell increased whereas that together with dipalmitoyl phosphatidylcholine reduced its sensitivity to spleen effectors. It is concluded that the increase in the sensitivity of the tumor cell primarily depends on changes in the lipid template of its membrane, induced by unsaturated fatty acids of egg phosphatidylcholine, and to a less degree on the properties of carbohydrate heads of gangliosides.     

Effects of 5-phenyloxazolidinedione on sodium-potassium-magnesium-activated adenosine triphosphatase activity in mouse brain

—10 μm 5-Phenyloxazolidinedione (PKO) stimulated Na–K–Mg-ATPase activity in mouse brain homogenates by 45%. This activation effect was also observed for trimethadione and dimethadione. Inhibition of Na–K–Mg-ATPase by 0.1 mm ouabain was reduced in the presence of PKO. When PKO (4 mg/kg) was administered to mice intravenously, maximum levels of 2 μg (10 nmol)/g were attained in the brain within 15 min. PKO had an ld ₅₀ of 872 mg/kg and an ed ₅₀ of 375 mg/kg for protection against metrazol-induced seizures in mice.     

Characterization of Mg-ATPase and (Na+ + K+)-stimulated Mg-ATPase in smooth muscular cells of the sheep's common carotid artery.

The Mg-ATPase and (Na+ + K+)-stimulated Mg-ATPase in the mitochondrial and microsomal fraction of smooth muscular cells of the sheep's common carotid artery have been characterized in more detail. Optimal enzyme activities were found for all ATPases to be at pH 7.5-8.0 and 45 degrees C-50 degrees C. The energies of activation were found to be at 5-9 kcal/mole for both ATPases. Two-thirds of the (Na+ + K+)-stimulated Mg-ATPase were found to be ouabain-sensitive and thus attributed to the coupled (Na, K)-transport system. The pI 50 values of ouabain for microsomal and mitochondrial fractions are 6.3 and 6.0, respectively. The highest activity of (Na+ + K+)-stimulated Mg-ATPase is at 5-10 mM K+ and more than 50 mM Na+. One-third of the (Na+ + K+)-stimulated Mg-ATPase activity was found to be due to a stimulation of Mg-ATPase by Na+ alone, which is not inhibited by ouabain. The relationship of this activity to the ouabain-sensitive part of the (Na+ + K+)-stimulated Mg-ATPase and to Na+-transport is discussed. For the Mg-ATPases apparent KM(ATP) values were determined to be 1.4 and 1.0 mM, resp., and for the (Na+ + K+)-stimulated Mg-ATPases 0.15 and 0.14 mM, resp.     

Evidence for the influence of the protein-phospholipid interface on sarcoplasmic reticulum Ca++ Mg++ ATPase activity.

Sarcoplasmic reticulum from the white hind leg muscle of the rabbit was examined with 31P nuclear magnetic resonance as a nonperturbing probe of phospholipid-protein interactions in the intact membrane. The phospholipids of the sarcoplasmic reticulum appear to inhabit two distinct environments: one very similar in behavior to pure phospholipid lamellar dispersions and the other immobilized by the protein in the membrane. Measurement of the population of the latter environment suggests that it is dependent on salt concentration and probably not due to the Ca++ Mg++ ATPase of the sarcoplasmic reticulum. This immobilization can be removed completely by papain proteolysis of the membrane protein, but only partially by trypsin treatment. The phospholipid composition of recombinants with the Ca++ Mg++ ATPase was varied in order to look for effects of the phospholipid-protein interface on enzymatic activity of the Ca++ Mg++ ATPase. Both transphosphatidylated phosphatidylethanolamine (from egg phosphatidylcholine) and bovine brain phosphatidylserine readily partitioned into the putative boundary layer, whereas under the same conditions soybean phosphatidylethanolamine was excluded. Only phosphatidylserine affected the activity of the enzyme, causing an inhibition that was proportional to the phosphatidylserine content, relative to phosphatidylcholine.     

Reconstitution of the sodium-calcium exchanger from cardiac sarcolemmal vesicles

The Na+-Ca2+ exchanger was extracted from cardiac sarcolemmal vesicles and reconstituted into phospholipid vesicles by a cholate-dialysis method. Reconstitution was attempted with different phospholipids. Phosphatidylcholine alone was ineffective, whereas phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) showed high activity, but a significant Ca2+ uptake in the absence of Na+ gradient. Optimal reconstitution was obtained with a mixture of phosphatidylcholine and phosphatidylserine (9:1, mol/mol). The reconstituted proteoliposomes showed an ouabain-sensitive (Na+ + K+)-ATPase activity and a Na+-Ca2+ exchange with a specific activity comparable to that of the original vesicles. The specificity toward Na+ was also recovered. A partial purification of the exchanger was obtained by the method of transport-specificity fractionation ( Goldin , S.M. and Rhoden , V. (1978) J. Biol. Chem. 253, 2575-2583). When proteoliposomes were reconstituted with sodium oxalate inside and incubated with calcium in the presence of an outwardly directed Na+ gradient, the vesicles containing the Na+-Ca2+ exchanger specifically accumulated calcium which precipitated inside as calcium oxalate. The resulting increase in density allowed separation of the proteoliposomes containing the Na+-Ca2+ exchanger from the rest of the vesicles on a sucrose density gradient.     

Phosphatidylethanolamine methyltransferase: evidence for influence of diet fat on selectivity of substrate for methylation in rat brain synaptic plasma membranes

Male weanling rats were fed diets containing 20% (w/w) fat differing in fatty acid composition for 24 days. Synaptic plasma membranes were isolated from the brain and the fatty acid composition of phosphatidylethanolamine and phosphatidylcholine was determined. In vitro assays of phosphatidylethanolamine methyl-transferase activity were performed on fresh membrane samples to assess effect of dietary fat on the rate of phosphatidylethanolamine methylation for phosphatidylcholine synthesis via the phosphatidylethanolamine methyltransferase pathway. Dietary level of n-6 and ratio of n-6 to n-3 fatty acids influenced membrane phospholipid fatty acid composition and activity of the lipid-dependent phosphatidylethanolamine methyltransferase pathway. Rats fed a diet rich in n-6 fatty acids produced a high ratio of n-6/n-3 fatty acids in synaptosomal membrane phosphatidylethanolamine, and elevated rates of methylation of phosphatidylethanolamine to phosphatidylcholine by phosphatidylethanolamine methyltransferases, suggesting that the pathway exhibits substrate selectivity for individual species of phosphatidylethanolamine containing long-chain homologues of dietary n-6 and n-3 fatty acids (20:4(n-6), 22:4(n-6), 22:5(n-6) and 22:6(n-3). It may be concluded that diet alters the membrane content of n-6, n-3 and monounsaturated fatty acids, and that change in phosphatidylethanolamine species available for methylation to phosphatidylcholine alters the rate of product synthesis in vivo by the phosphatidylethanolamine methyltransferase pathway.     

Phenobarbital-induced alterations in phosphatidylcholine and triglyceride synthesis in hepatic endoplasmic reticulum

Biosynthetic pathways of phosphatidylcholine and triglyceride were studied in proliferating hepatic endoplasmic reticulum of rats pretreated with phenobarbital. Phosphatidylcholine accounted for the major increment in membrane phospholipid. In vitro measurements of hepatic microsomal enzymes which catalyze phosphatidylcholine biosynthesis revealed a significant increase in specific activity of the enzyme governing phosphatidylcholine synthesis by sequential methylation of phosphatidylethanolamine. The specific activity of phosphorylcholine-glyceride transferase, which catalyzes phosphatidylcholine synthesis from d-1,2-diglyceride and CDP-choline, was not altered. Specific activity of diglyceride acyltransferase, which catalyzes triglyceride biosynthesis, was increased to a degree comparable to the increase in specific activity found in the phenobarbital-induced drug-metabolizing enzyme which oxidatively demethylates aminopyrine. In vivo incorporation of methyl-(3)H from l-methionine-methyl-(3)H into microsomal phosphatidylcholine was significantly increased, resulting in an increased methyl-(3)H to choline-1,2-(14)C incorporation ratio of more than three times that found in control animals. A comparable increase in this incorporation ratio was noted in serum phospholipids. The in vitro enzyme studies, in agreement with in vivo incorporation data, indicate that the increase in phosphatidylcholine content of phenobarbital-induced proliferating endoplasmic reticulum is related to increased activity of the pathway of phosphatidylcholine biosynthesis involving the sequential methylation of phosphatidylethanolamine.     

Electroencephalogram changes in animals with neurosis as a correlate of metabolic brain disorders

Experiments were made to correlate changes in bioelectrical activity of the brain of different animals (rats, cats, monkeys) with neurosis and its treatment to the time course of the activity of ATPase, one of the most important enzymes of energy metabolism. It was demonstrated that the electroencephalogram (EEG) taken during neurosis is marked by an increase in the total energy of quick-wave components and reduction in the total energy of slow-wave ones in all the structures under study, by the deterioration of the rhythm change response. These changes corresponded with the inhibited activity of brain Na, K-ATPase, particularly of Mg-ATPase. The EEG returned to normal and ATPases were activated after nikogamol injections.     

Activity of erythrocyte membrane Na,K-ATPase in rats with experimental botulism

The effect of type C botulinum toxin on Na, K, Mg-ATPase activities of erythrocyte membranes of white rats was studied in experiments in vivo and in vitro. The activity of Na, K, Mg-ATPase was found to be markedly inhibited in the preclinical period of poisoning, 2 hours after intraperitoneal injection of the toxin. In this case Mg-ATPase activity noticeably increased. In the presence of the development of a grave paralytic syndrome one day after intraperitoneal injection of the toxin, the activity of Na, K-ATPase of the erythrocyte membrane remained decreased as was the case in the preclinical period of poisoning, whereas the activity of Mg-ATPase returned to normal. The experiments in vitro with preincubation of erythrocyte membranes with botulinum toxin in the concentrations corresponding to the mean calculated ones in the experiments in vivo demonstrated inhibition of Na, K-ATPase. The magnitude of Mg-ATPase activity remained virtually unchanged in all the modifications of the experiments with boiled and native botulinum toxin. The in-vivo experiments with intraperitoneal injection of glutathione and unithiol to the pretreated animals attested to normalization of Na, K-ATPase in the preclinical period of poisoning, with this normalization being brought about by unithiol. In the in-vitro experiments with addition of unithiol or glutathione into the incubation medium, each of the donators of sulphhydryl groups prevented Na, K-ATPase inhibition with botulinum toxin.     

The effect of anions on Mg-ATPase in red blood cells.

Anions exert an influence on the passive permeability of Na+ and K+ in erythrocytes. THE EFFECT ON Mg-ATPase activity has been studied in human erythrocytes. 40 mM bicarbonate increased the activity as compared to the effect of 40 mM chloride; 20 mM sulphate inhibited it. Salicylate acted first as an activator then as an inhibitor of Mg-ATPase; maximum activity was reached at 60 mM CONCENTRATION. Thiocyanate inhibited saponin-stimulated Mg-ATPase, Ki = 1.85 X 10(-2)M. The probable mechanisms of action of the above anions on Mg-ATPase and possible relation to passive permeability of Na+ and K+ ions are discussed.     

The effect of low-level x-irradiation on the key enzymes of the active ion transport system in the cell

A study was made of the effect of ionizing radiation (0.013 C/kg) on Na, K- and Ca, Mg-ATPase activity in membranes of rat organs differing in radiosensitivity. It was shown that radiation mainly caused activation of enzymes that was most pronounced in brain membranes.     

Neutral Phospholipids Stimulate Na,K-ATPase Activity: A SPECIFIC LIPID-PROTEIN INTERACTION

Membrane proteins interact with phospholipids either via an annular layer surrounding the transmembrane segments or by specific lipid-protein interactions. Although specifically bound phospholipids are observed in many crystal structures of membrane proteins, their roles are not well understood. Na,K-ATPase is highly dependent on acid phospholipids, especially phosphatidylserine, and previous work on purified detergent-soluble recombinant Na,K-ATPase showed that phosphatidylserine stabilizes and specifically interacts with the protein. Most recently the phosphatidylserine binding site has been located between transmembrane segments of αTM8–10 and the FXYD protein. This paper describes stimulation of Na,K-ATPase activity of the purified human α1β1 or α1β1FXYD1 complexes by neutral phospholipids, phosphatidylcholine, or phosphatidylethanolamine. In the presence of phosphatidylserine, soy phosphatidylcholine increases the Na,K-ATPase turnover rate from 5483 ± 144 to 7552 ± 105 (p < 0.0001). Analysis of α1β1FXYD1 complexes prepared with native or synthetic phospholipids shows that the stimulatory effect is structurally selective for neutral phospholipids with polyunsaturated fatty acyl chains, especially dilinoleoyl phosphatidylcholine or phosphatidylethanolamine. By contrast to phosphatidylserine, phosphatidylcholine or phosphatidylethanolamine destabilizes the Na,K-ATPase. Structural selectivity for stimulation of Na,K-ATPase activity and destabilization by neutral phospholipids distinguish these effects from the stabilizing effects of phosphatidylserine and imply that the phospholipids bind at distinct sites. A re-examination of electron densities of shark Na,K-ATPase is consistent with two bound phospholipids located between transmembrane segments αTM8–10 and TMFXYD (site A) and between TM2, -4, -6, -and 9 (site B). Comparison of the phospholipid binding pockets in E2 and E1 conformations suggests a possible mechanism of stimulation of Na,K-ATPase activity by the neutral phospholipid.     

Phospholipid synthesis in rat liver endoplasmic reticulum after the administration of phenobarbitone and 20-methylcholanthrene

1. Phenobarbitone injection did not affect the concentration of phospholipids in the liver endoplasmic reticulum, but it increased the rate of incorporation of [(32)P]orthophosphate into the phospholipids. 20-Methylcholanthrene caused a transient increase in total phospholipid but a decrease in the turnover rate of the phospholipids. 2. Incorporation of [(32)P]orthophosphate into phosphatidylcholine, compared with that into phosphatidylethanolamine, was increased by phenobarbitone injection but decreased by 20-methylcholanthrene injection. 3. The activity of S-adenosylmethionine-phosphatidylethanolamine methyltransferase increased 12h after phenobarbitone injection, when incorporation of [(32)P]orthophosphate into phosphatidylcholine was a maximum, but at other times, and after 20-methylcholanthrene injection, the activity of the enzyme did not correlate with the rate of phosphatidylcholine synthesis. 4. [(14)C]Glycerol was incorporated more rapidly into phosphatidylcholine than into phosphatidylethanolamine, whereas [(32)P]orthophosphate and [(14)C]ethanolamine were incorporated more rapidly into phosphatidylethanolamine than into phosphatidylcholine. 5. Incorporation of [(32)P]orthophosphate into phosphatidylethanolamine of liver slices incubated in vitro was much more rapid than into phosphatidylcholine, and incorporation into phosphatidylcholine was markedly stimulated by addition of methionine to the medium. Changes in the incorporation of [(32)P]orthophosphate into phospholipids observed in vivo after injection of phenobarbitone or methylcholanthrene could not be reproduced in slices incubated in vitro. 6. It is concluded that phenobarbitone injection causes an increased rate of turnover of total phospholipids in the endoplasmic reticulum and an increased conversion of phosphatidylethanolamine into phosphatidylcholine, whereas 20-methylcholanthrene injection depresses both the turnover rate of total phospholipids and the formation of phosphatidylcholine.     

Effect of acetylcholine on the cerebral microsomal Na, K-ATPase of rats of different ages

Na, K- and Mg-ATPase activity of the cerebral cortex microsomal fraction has been studied and compared in adult and old rats. The activity of Na, K-ATPase decreases while that of Mg-ATPase increases with age. The total ATPase activity remains unchanged. The effect of acetylcholine on ATPase activity has been found to be age-dependent.     

The distribution of ATPase activities in purified transverse tubular membranes

Vesiculated fragments of transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes were purified from heterogeneous microsomal membrane fractions of chicken breast muscle by a modification of an iterative calcium-oxalate loading technique. The distribution of ATPase activities were determined for the TT and SR and were compared to enriched fractions of sarcolemma (SL) membranes. The TT membranes were characterized by high rates of magnesium-stimulated ATPase (Mg-ATPase) and 5′-nucleotidase activities but were virtually devoid of calcium-stimulated, magnesium-dependent ATPase (Ca,Mg-ATPase) activity. Moderate levels of a latent sodium and potassium-stimulated ATPase (Na,K-ATPase) were observed for TT membranes when unmasked with valinomycin and monensin. In contrast to the behavior of TT membranes, highly purified SR membranes displayed an active Ca,Mg-ATPase but negligible Na,K-ATPase, Mg-ATPase, and 5′-nucleotidase activities. High levels of Na,K-ATPase and 5′-nucleotidase activities were observed for SL membranes; however, the SL displayed no appreciable Ca,Mg-ATPase and Mg-ATPase activities. The lack of significant Mg-ATPase activity in the SR and SL fractions suggested that the Mg-ATPase was uniquely associated with the TT membranes. The TT Mg-ATPase was further characterized by its pH and temperature dependences, and its sensitivity to pharmacologic agents. The Mg-ATPase of the TT was insensitive to inhibition by sodium azide and oligomycin in concentrations shown to exert maximum inhibition on the F₁ ATPase of submitochondrial particles. The Mg-ATPase was also resistant to the effects of ouabain and orthovanadate in concentrations which abolished the Na,K-ATPase and Ca,Mg-ATPase activities of the SL and SR, respectively. The Mg-ATPase displayed temperature and pH optima (25 °C, pH 7.3) which were distinguishable from the Ca,Mg-ATPase (45 °, pH 7.0) of highly purified SR fractions but which were very similar to the temperature and pH dependencies of the mixed microsomal fractions (MMF) from which the TT membranes were derived. Similarities in the pH and temperature dependencies of the TT and MMF Mg-ATPases plus the absence of appreciable Mg-ATPase activity in highly purified SR membranes suggests that the “basic” Mg-ATPase often seen in crude SR fractions may originate from TT membrane contamination. The resistance of the TT Mg-ATPase to inhibition by the pharmacologic agents tested plus its unique temperature and pH dependences indicate that this ATPase is distinguishable from other ATPases and may, therefore, be of value as a specific biochemical marker for TT membranes.     

Dolichol induces membrane leakage of liposomes composed of phosphatidylethanolamine and phosphatidylcholine

Dolichol promotes the leakage of membranes in liposomes composed of phosphatidylethanolamine and phosphatidylcholine but not liposomes composed only of phosphatidylcholine. The membrane leakage was assayed by measuring the entrapment of TEMPOcholine, a cationic spin probe, in liposomes using ESR methods. The percent of membrane leakage induced by dolichol was found to be linearly proportional to the concentrations of dolichol. It is proposed that dolichol enhances the formation of non-bilayer configurations in liposomes containing phosphatidylethanolamine, thereby membrane leakage.