A. de Vries

HEMOLYSIS AND SPLITTING OF HUMAN ERYTHROCYTE PHOSPHOLIPIDS BY SNAKE VENOMS.

1. 1. Lysis of washed human erythrocytes induced by Cobra snake venoms (Naja naja, Hemachatus haemachate) was accompanied by splitting of the red-cell phospholipids to lysophosphatides. 2. 2. Phospholipase A fractions (phosphatide acyl-hydrolase, EC 3.1.1.4) isolated from the Cobra venoms were devoid of hemolytic activity and caused no significant breakdown of phospholipids in the intact erythrocytes. Another protein component derived from the same venoms (the direct lytic factor) was weakly hemolytic but showed no phospholipase activity. The two fractions, when combined, produced strong hemolysis and concomitant hydrolysis of erythrocyte phospholipids to lysophosphatides. 3. 3. Heparin or dextran sulfate, which were found to precipitate the direct lytic factor, abolished both the hemolytic activity of the Cobra venoms and their capacity for hydrolyzing the phospholipid constituents of the intact red blood cells. 4. 4. Viper venoms (Vipera palestinae and Vipera russellii) containing phospholipase A but no direct lytic factor induced neither lysis nor substantial phospholipid splitting of washed erythrocytes unless fortified with the direct lytic factor fraction derived from Cobra venom. 5. 5. Treatment of the red blood cells with surface-active agents or sonic vibrations imitated the effect of the direct lytic factor in rendering the erythrocyte phospholipids susceptible to hydrolysis by phospholipase A. 6. 6. The various phospholipase A fractions studied could be differentiated into two types according to their abiltiy of hydrolyzing phospholipids in osmotic hemolyzates: the Cobra phospholipases which were active per se and the V. palestinae enzyme which required for its activity the addition of either the direct lytic factor or detergents or pretreatment of the osmotic ghosts by sonication. The Russells viper venom was found to contain phospholipases of either type 7. 7. Some aspects, bearing on the mode of action of the various factors instrumental in rendering the membrane-bound phospholipids available to splitting by snake venom phospholipases A, are discussed. The role of these factors in the mechanism underlying the hemolytic activity and cell toxicity of the snake venoms is considered.

SUSCEPTIBILITY OF ERYTHROCYTES OF VARIOUS ANIMAL SPECIES TO THE HEMOLYTIC AND PHOSPHOLIPID SPLITTING ACTION OF SNAKE VENOM.

Abstract A parallelism was found between hemolysis and erythrocyte phospholipid splitting induced by the action of cobra venom on washed erythrocytes of various species: guinea-pig, dog, human, rabbit. No significant phospholipid splitting was produced by cobra venom in camel and sheep erythrocytes, which are resistant to the hemolytic action of the venom. Isolated cobra venom phospholipase A (phosphatide acylhydrolase, EC 3.1.1.4) had no or slight hemolytic and phospholipid splitting action on the various erythrocytes, including those of the guinea-pig and dog, which are the most sensitive to the action of the whole venom. The different sensitivity of the various erythrocytes to the cobra venom is a reflection of their susceptibility to the action of venom direct lytic factor, a basic protein, which has hemolytic but no phospholipase activity. Isolated cobra venom phospholipase readily hydrolyzed the phospholipids of osmotic ghosts derived from both sensitive and resistant erythrocytes. Vipera palestinae venom which did not lyse the erythrocytes of any of the species tested, was also unable to hydrolyse the phospholipids in their osmotic ghosts. Cobra-venom direct lytic factor rendered the phospholipids of osmotic ghosts derived from sensitive erythrocytes available to the action of V. palestinae venom, but was not able to do so in the case of osmotic ghosts derived from resistant erythrocytes.

Human erythrocyte phosphoribosylpyrophosphate synthetase mutationally altered in regulatory properties

Abstract A mutant phosphoribosylpyrophosphate synthetase was found in the erythrocytes of a gouty subject with excessive purine production. The mutant enzyme exhibited normal catalytic properties but decreased sensitivity to inhibition by guanosine-5′-diphosphate, adenosine-5′-diphosphate, adenosine-5′-monophosphate and 2,3-diphosphoglyceric acid at physiological phosphate concentration. Selective alteration by mutation of the regulatory properties shows the enzyme to be allosteric. To our knowledge this is the first demonstration in man of an overproduction disease due to excess activity of a regulatory enzyme as a direct effect of mutation.

Serum xanthine oxidase in jaundice.

Serum xanthine oxidase activity was measured by a radiochemical method in 137 consecutive patients with jaundice of varying etiology and in 40 non-jaundiced patients with liver or other disease. Serum xanthine oxidase was markedly increased, up to 50 times the upper normal limit (mean + 2 S.D.), in 32 out of 34 patients with infectious hepatitis. A slight elevation of serum xanthine oxidase, up to twice the upper normal limit, was found in 2 out of 49 patients with extrahepatic obstructive jaundice and in 4 out of 20 patients with chronic renal failure. In comparison to serum glutamic-oxaloacetic transaminase and lactate dehydrogenase serum xanthine oxidase appeared to be the more sensitive and specific indicator of acute hepatocellular damage.

Hypouricemia, Hypercalciuria, and Decreased Bone Density: A Hereditary Syndrome

Genetically determined hypouricemia in man due to increased renal urate clearance is usually associated with additional renal tubular defects (1–3). Renal hypouricemia due to an isolated renal tubular defect has also been described but appears to be a rare condition (4,5). We report a new genetically determined syndrome in man, in which the renal hypouricemia is associated with idiopathic hypercalciuria and decreased bone density.

The effect of ribose 5-phosphate and 5-phosphoribosyl-1-pyrophosphate availability on de novo synthesis of purine nucleotides in rat liver slices.

The effect of increasing cellular ribose 5-phosphate (ribose-5-P) availability by methylene blue-induced acceleration of the oxidative pentose phosphate pathway on the rate of 5-phosphoribosyl-1-pyrophosphate (P-ribose-PP) generation, was studied in slices of rat liver at varying Pi concentration. It was found that at Pi concentration prevailing in the tissue of extracellular physiological Pi concentration, ribose-5-P availability is saturating for P-ribose-PP generation, as gauged by the rate of adenine incorporation into tissue nucleotides. The effect of altering P-ribose-PP availability on the rate of de novo purine production gauged by the rate of formate incorporation into purines, was also studied. It was found that the physiological P-ribose-PP concentration in rat liver tissue is limiting for purine synthesis de novo. Depletion of cellular P-ribose-PP, achieved by increase of P-ribose-PP consumption, decelerated purine synthesis, while increase of P-ribose-PP availability, achieved by activation of P-ribose-PP synthetase occurring at elevated Pi concentration, resulted in acceleration of purine synthesis.

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.

Familial Hypouricemia Due to Isolated Renal Tubular Defect

A 37-year-old female was found to have hypouricemia (1.1-1.9 mg%) with markedly increased uric acid clearance (24.7039.5 ml/min). Uric acid excretion was only slightly affected by pyrazinamide, a drug which suppresses renal tubular uric acid secretion, and by probenecid, a drug which inhibits tubular uric acid reabsorption. The attenuated response in this subject to both drugs suggest a renal tubular defect in the proximal high capacity-high affinity uric acid reabsorption mechanism. No other renal tubular or metabolic abnormalities were detected. A survey of the family-three sisters and two brothers, revealed two similarly affected sisters. The abnormality described in this family is defined as familial renal hypouricemia due to an isolated renal tubular defect with attenuated response of uric acid clearance to probenecid and pyrazinamide.

Isolation and characterization of kinin-releasing enzyme of Echis coloratus venom.

Abstract Echis coloratus venom releases bradykinin upon incubation with a kininogn-enriched equine plasma fraction. A procedure is described for the purification of kinin-releasing enzyme from the venom. Two kinin-releasing enzyme preparations were obtained: one, having in addition slight kininase activity, electrophoretically heterogeneous, the other, devoid of kininase activity, electrophoretically homogeneous. The electrophoretically homogeneous enzyme preparation possessed capillary permeability increasing activity, but was devoid of the proteolytic, hemorrhagic and fibrinolytic activities of the venom. This preparation, having an estimated molecular weight of 22,000, hydrolysed arginine and tyrosine esters and synthetic lysine peptides, but not arginine amide nor lysine esters. It was thermostable, sensitive to DFP, insensitive to trasylol and to soybean trypsin inhibitor. The properties of the enzyme are compared with other kallikreins and a possible mode of its action on kininogen is discussed.