Chaya Klibansky

Enhancing action of synthetic and natural basic polypeptides on erythrocyte-ghost phospholipid hydrolysis by phospholipase A

Abstract The natural basic polypeptide, Gramicidin S, and the synthetic basic α-amino acid copolymers, poly-ornithine-lencine, poly-ornithine-leucine-alanine and poly-lysine-leucine, similarly to the direct lytic factor of Ringhals cobra venom, render the phospholipids in osmotic erythrocyte ghosts susceptible to the hydrolytic action of Vipera palestinae phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4). The synthetic basic α-amino acid polymers, poly-ornithine, poly-lysine and the copolymer poly-ornithine-valine were inactive in this respect. The basicity of the polypeptides, by promoting electrostatic attraction, is held responsible for their attachment to the membrane, whereas the lipophilic side chains are invoked in the facilitation of the approach of the phospholipase to the phospholipid substrate situated inside the membrane. Gramicidin S and the synthetic basic copolymers active in facilitating the splitting of phospholipids are strongly hemolytic. The basic polyamino acids, which are inactive in enhancement of phospholipid splitting, and the Ringhals cobra direct lytic factor have little hemolytic activity.

Phospholipase isoenzymes from Naja naja venom —I. Purification and partial characterization

Abstract Six phospholipase A-containing fractions were isolated from cobra ( Naja naja ) venom by ion-exchange and gel filtration chromatography. Each fraction was homogeneous by immunological criteria and comprised more than 1 phospholipase isoenzyme, their total number amounting to 14. The molecular weights of the isoenzymes ranged from 11000 to 24000. None of them contained free SH groups. The six fractions exhibited similar specific activities.

Effects of detergents and choline-containing phospholipids on human spleen glucocerebrosidase.

1. Glucocerebrosidase, extracted from human spleen lysosomal membrane by sodium cholate and recovered in a high speed centrifugation supernatant, aggregated following removal of the detergent. 2. Re-solubilization of the enzymatic activity from the aggregate was achieved by treatment with the non-ionic detergents Triton X-100 and Tween 20. The anionic detergents sodium cholate and sodium taurocholate and the cationic detergents cetyltrimethylammonium bromide and cetylpyridinium chloride were also effective. The solubilizing capacity of the anionic detergents was smaller than that of the nonionic detergents. Quantitative evaluation of the solubilizing capacity of the cationic detergents was not feasible because of their being potent inhibitors of glucocerebrosidase activity. 3. Treatment of the enzyme aggregate with acetone rendered it buffer-soluble. 4. In addition to the above cationic detergents some choline-containing and highly hydrophobic phospholipids were found to inhibit the glucocerebrosidase activity.

RHEOLOGICAL CONSIDERATIONS OF THE HAEMOLYSING RED BLOOD CELL

The present study is concerned with the gradual haemolysis of erythrocytes, i.e. with the liberation of haemoglobin from red blood cells, suspended in an isotonic solution, the ionic strength of which is gradually diminished by dialysis. The curves for percentage haemolysis versus external salt concentration, in gradual haemolysis, were compared with those for ordinary osmotic fragility test. The curves for gradual haemolysis are shifted towards lower salt concentrations, indicating that in this case the cells undergo a higher degree of swelling before haemolysis occurs. The explanation proposed is that when the water influx is slow (in gradual haemolysis) the inflation of the cell membrane follows the rate of swelling and no stresses develop in the membrane until a critical volume is reached; when the rate of water influx is high (in drastic haemolysis), stresses develop in the membrane, already at higher salt concentration, which lead to haemolysis or to ultimate rupture of the membrane. The cell membrane behaves as a visco-elastic body of the type of a Kelvin body. A preliminary calculation of Youngs modulus, and of the coefficient of solid viscosity of the membrane, was carried out. Serum albumin has a pronounced protective action on the cell membrane under conditions of drastic haemolysis. In the presence of serum albumin the curves of percentage haemolysis for the osmotic fragility test coincide with those obtained in gradual haemolysis.

Phospholipase isoenzymes from Naja naja venom —II. Phospholipase A and B activities

Abstract Each of six cobra (Naja naja) venom fractions possesses phospholipase A2 activity—hydrolysing 1,2 diacyl-sn-glycerophosphorylcholine, e.g. lecithin, in the 2-position, as well as phospholipase B activity, hydrolyzing 1-monoacyl-sn-glycerophosphorylcholine, e.g. lysolecithin. For all fractions the pH optimum was 9 for lecithin hydrolysis and above 10 for lysolecithin hydrolysis. The enzymatic activity of all fractions on lecithin was several orders of magnitude higher than that on lysolecithin. The Km values for lysolecithin hydrolysis by the various Naja naja phospholipase isoenzymes at pH 10 and 37° were about 1 mM.

Action of Naja naja and Vipera palestinae venoms on cat brain phospholipids in vitro

Abstract The action in vitro of Naja naja and Vipera palestinae venoms on phospholipids in cat brain slices, homogenates and mitochondria has been studied. Both venoms have no or little phospholipid splitting activity when applied to brain slices. Naja naja venom as well as its electrophoretically separated phopholipase A are able to hydrolyze lecithin, phosphatidyl ethanolamine, phosphatidyl serine and plasmalogen in cat brain homogenates and mitochondria, but Vipera palestinae venom is ineffective.

Isolation of three different neurotoxins from indian cobra (Naja naja) venom and the relation of their action to phospholipase a

Abstract Six fractions of the venom of the Indian cobra (Naja naja) were separated by paper electrophoresis of which four (fractions 6, 3, 2 and 1) were toxic in anaesthetized cats or in mice, or in both species. Of the four toxic fractions only fraction 6 contained phospholipase ; it was toxic to cats but not to mice. Fraction 3 was toxic to cats and mice and fractions 1 and 2 were toxic to mice but not to cats. Thus three different neurotoxic fractions were separated. In the anaesthetized cat the intravenous injection of fraction 6 caused the same effect as the injection of whole or boiled venom, i.e., a diphasic circulatory shock—an initial rapid fall in arterial blood pressure followed after partial recovery by a delayed gradual fall—with depression of cerebral cortical activity. The central vasoregulating mechanisms were apparently not affected during the shock since records taken from the cervical sympathetic showed increased activity. Fraction 3 did not produce the initial fall in arterial blood pressure but only the delayed phase of shock. In mice, the toxic effects of an intraperitoneal injection of whole venom resulted in convulsions, excitement, motor impairment and respiratory arrest; the injection of fractions 1 and 2 resulted in apathy, motor depression and convulsions before death, and the injection of fraction 3 in excitement, convulsions, repeated laboured respiration, and curling of the tail.

Prenatal diagnosis of Gaucher's and Niemann-Pick diseases. Assays of glucocerebrosidase and sphingomyelinase in tissue cultures using natural substrates.

Prenatal diagnosis has been successfully achieved by enzyme assays in cultured amniocytes in three high risk pregnancies for Gauchers disease and three for Niemann-Pick disease type A. [14C]Stearic acid glucocerebroside and [3H]-dihydrosphingomyelin were used as substrates for glucocerebrosidase and sphingomyelinase activity measurements, respectively. Values for the above two enzyme levels in cultured amniotic fluid cells of normal controls and in the pregnancies at risk are presented. The diagnosis made in utero in one fetus affected with Niemann-Pick disease was subsequently confirmed following abortion (at 19 weeks gestation), by the specific biochemical and pathological features of various organs of the afflicted fetus (to be published). Confirmation of diagnoses made in utero for heterozygous and healthy fetuses was obtained by post-partum examination of glucocerebrosidase and spingomyelinase activity levels in leukocytes of the appropriate infants.

Accumulation of lactosyl ceramide in leukocytes of patients with adult Gaucher's disease

Glycosphingolipids were isolated from the leukocytes of 10 patients with Type I, chronic nonneuronopathic (adult) Gauchers disease and 12 normal subjects, by silicic acid column chromatography and thin-layer chromatography. Quantitation of the individual glycosyl ceramides was achieved by the determination of hexose and sphingosine content, using colorimetric and fluorometric procedures. Lactosyl ceramide, which is the main glycolipid in leukocytes of normal subjects, was significantly increased in the leukocytes of patients with Gauchers disease. On the other hand, the amount of glycosyl ceramide, which is the main glycolipid accumulating in the reticuloendothelial cells of patients with Gauchers disease, was similar in Gaucher and in control leukocytes.

LYSOPHOSPHOLIPID BREAKDOWN IN COBRA VENOM-TREATED RABBIT ERYTHROCYTES.

Abstract Rabbit erythrocyte phospholipase B (lysolecithin acyl-hydrolase E.C. 3.1.1.5) hydrolyses the lysophospholipids produced in Ringhals cobra venom—treated rabbit erthrocytes and osmotic ghosts by the action of the venom phospholipase A (phosphatide acyl-hydrolase E.C. 3.1.1.4). Raising the incubation temperature of the osmotic hemolysate-venom mixture to 60° leads to inactivation of the erythrocyte phospholipase B with preservation of the lysocompounds formed due to the action of the heat-stable venom phospholipase A. Subjecting the rabbit erythrocytes to various hemolytic agents, such as hypotonic medium, saponin, digitonin or cobra venom-direct lytic factor (DLF), causes liberation of phospholipase B into the particle-free supernatants, as indicated by hydrolysis of added lysolecithin.