Eleonora Condrea

Dissociation of enzymatic activity from lethality and pharmacological properties by carbamylation of lysines in Naja nigricollis and Naja naja atra snake venom phospholipases A2

Abstract Carbamylation of 9 out of 10 lysine residues in the toxic phospholipase A2 from N. nigricollis venom decreased its lethality at least 8-fold and abolished its direct hemolytic and anticoagulant activities, while the enzymatic activity, as measured on purified substrates, decreased only about 50%. Likewise, carbamylation of 3 out of 5 lysines in the relatively less toxic N. naja atra phospholipase induced detoxification and caused a loss of its blocking activity on the phrenic nerve-diaphragm preparation, while its enzymatic activity on purified substrates was unaltered. Results obtained when 7.4 out of 10 lysines in N. nigricollis phospholipase were carbamylated indicate that basicity is not an absolute requirement for high lethal potency, hemolytic activity or cardiotoxicity. The extent of phospholipid hydrolysis induced in erythrocytes, rabbit plasma, phrenic nerve-diaphragm preparation, brain minces and brain synaptic plasma membranes by incubation with the carbamylated enzymes was in agreement with their enzymatic activities as measured on purified substrates. Levels of phospholipid hydrolysis in heart, lung and kidney of mice given phospholipase intravenously, and in brain synaptic plasma membranes from rats given phospholipase intraventricularly, showed that carbamylated derivatives of N. nigricollis phospholipase A2 lost their ability to reach and/or hydrolyze substrates in vivo. However, the decrease in in vivo phospholipid hydrolysis did not correlate with the decrease in toxicity since, at comparably low levels of phospholipid hydrolysis, some phospholipases were lethal and others were not. Moreover, when intraventricularly administered, both lethal amounts of the native N. naja atra enzyme and its nonlethal carbamylated derivatives produced equally low hydrolysis of synaptic membrane phospholipids. By means of lysine carbamylation, a dissociation between hydrolytic activity and pharmacological properties of phospholipases A2 has been achieved. We suggest, therefore, that the toxicity of pure phospholipases is primarily due to a direct effect which does not correlate with levels of phospholipid hydrolysis and that this direct effect is prominent in the relatively toxic phospholipases while it is less manifest in the relatively non-toxic enzymes.

Relationship between catalysis and toxicological properties of three phospholipases A2 from elapid snake venoms

Abstract Phospholipases A 2 from three elapid snake venoms were studied in order to determine if differences in toxicity correlate with differences in pattern or level of phospholipid hydrolysis. The comparatively toxic basic phospholipase A 2 isolated from Naja nigricollis venom exhibits a cardiotoxic action following iv administration in mice that is not exhibited by the less toxic neutral phospholipase A 2 from Hemachatus haemachatus venom or the acidic phospholipase A 2 from Naja naja atra venom. This cardiotoxic action correlates with high levels of phosphatidylserine hydrolysis in heart. Levels and patterns of phospholipid hydrolysis in heart, lung and kidney following iv administration suggest that only the N. nigricollis enzyme has the ability to penetrate permeability barriers in the heart. No cardiotoxic effects are seen following intraventricular injection of a lethal dose of the phospholipases A 2 . All three phospholipases A 2 (12 μg/ml) abolish the directly and indirectly elicited muscle twitches of the rat phrenic nerve-diaphragm preparation. This block, in normal or altered bathing media, appears to correlate with the level of phospholipid hydrolysis for the N. naja atra enzyme, but not for the N. nigricollis enzyme. These results suggest that N. nigricollis phospholipase A 2 acts by another mechanism in addition to phospholipid hydrolysis.

Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachatus haemachatus snake venom—II: Pharmacological properties in relationship to enzymatic activity☆

Abstract Despite a remarkable degree of homology in amino acid sequence, the neutral phospholipase A 2 from Hemachatus haemachatus venom is much less toxic than the basic phospholipase A 2 from Naja nigricollis venom, the i.v. ld 50 in mice for the two being, respectively, 8.6 and 0.63 mg/kg. Similarly following intraventricular injection into rats, the neutral phospholipase showed convulsant and lethal dose 50 values of about 7.5 and 15 μg per rat, respectively, whereas corresponding values for the basic phospholipase were 0.5 and 0.5 μg per rat. Death appears to be due to congestion, hemorrhage and edema in the lungs. Consideration of dosages required and times until onset of action suggests that, dependent upon the route of administration, the effect is either mediated via a central action or is due to a direct effect on the cardiac and/or respiratory system in the periphery. The pattern and extent of phospholipid hydrolysis in various brain regions was similar following intraventricular injection of the two phospholipases so that no relationship between phospholipid hydrolysis and lethal potency could be established. Concentrations of 5 and 10 μmg/ml of the N. nigricollis and H. haemachatus phospholipases, respectively, were required to block electrical activity of the isolated single electroplax. The ultrastructural changes produced by both phospholipases were also similar. Parallel to the somewhat greater potency on the electroplax, N. nigricollis phospholipase produced slightly greater overall hydrolysis in the innervated and non-innervated membranes of the electroplax than did H. haemachatus phospholipase. The results suggest that these two phospholipases do not have a specific junctional effect and that the small difference in potency on the junction cannot be responsible for the large difference in lethality observed in mammalian species.

Maintenance of axonal conduction and membrane permeability in presence of extensive phospholipid splitting.

The importance of phospholipids for the maintenance of axonal conduction and membrane permeability has been investigated by treating squid giant axons with phospholipases, lysosphospholipids and fatty acids. n nIt was found that phospholipase C, from Cloistridium welchii, induces an extensive splitting of axonal phospholipids, without however blocking conduction or increasing the penetration of a lipid-insoluble compound into the axoplasm of squid giant axons. These findings are in sharp contrast with the results obtained with phospholipase A, electrophoretically separated from Ringhals venom, where a close relationship between phospholipid splitting, block of axonal conduction, and increased penetration has been reported. Phospholipase B from bovine pancreas, and D from cabbage, did not markedly hydrolyze the axonal phospholipids nor did they affect conduction or penetration. n nBoth purified lysolecithin and a mixture of lysophosphatides, prepared by the action of venom phospholipase A on the phospholipids of squid axons, blocked conduction and increased penetration into giant axons. In contrast to phospholipase A, which affects giant axons only if surrounded by adhering small nerve fibers, lysolecithin and the lysophosphatide mixture acted equally well on giant axons with or without adhering small nerve fibers. The other product of phospholipase A action, free fatty acids, was inert on the giant axon. n nOn the basis of these and earlier findings, it is concluded that extensive splitting of axonal phospholipids by phospholipases is compatible with maintenance of normal function unless the hydrolytic products are themselves active. This conclusion would explain the marked differences in action found between phospholipases A and C.

Effect of methylation of histidine-48 on some enzymatic and pharmacological activities of snake venom phospholipases A2

The effects on some pharmacological and enzymatic properties were determined following methylation of histidine at the enzymatic active site of the basic relatively toxic Naja nigricollis and the acidic relatively non-toxic Naja naja atra phospholipases A2. Following methylation a very low residual enzymatic activity (0.4-1% of control) was accompanied by a parallel loss in intraventricular lethality, anticoagulant potency, direct hemolytic action and ability to block directly and indirectly evoked contractions of the mouse phrenic nerve-diaphragm preparation. Since methylation does not impair the enzymes ability to bind monomeric or micellar substrates or Ca2+, the results suggest that the pharmacologically active region of the molecule is different from the micellular substrate binding site but strongly influenced by the invariant histidine-48 located at the enzymatic active site.

Recombination of human red cell membrane protein fractions with homologous lipids

Abstract Human red cell membrane proteins were separated from membrane lipids by butanol-water partition, solubilized in sodium dodecyl sulfate and fractionated by gel filtration on a Sepharose 4B column. the eluates were pooled in two fractions: Fraction I containing the high molecular weight proteins, around 200 000, and Fraction II comprising the proteins which migrate as 100 000 and less. Fractions I and II had different sialic acid content and pH-dependent solubility. Both protein fractions recombined with red cell lipids when mixtures in sodium dodecyl sulfate were dialyzed against aqueous buffers. The amount and composition of recombinate obtained at various pH values were different for the two fractions. The recombinates obtained at pH 3.6 from Fraction I and from Fraction II had different density and stability towards change in pH and ionic strength. The recombinates of both fractions exhibited a membrane-like structure on electron microscopy.

Exposure of phosphatidylcholine and phosphatidylinositol in plasma membranes from rat brain synaptosomes treated with phospholipase A2 toxins (β-bungarotoxin, notexin) and enzymes (Naja nigricollis, Naja naja atra)

Phospholipase A2 (PLA2) toxins act presynaptically to block acetylcholine release and are much more potent and specific in their actions than PLA2 enzymes even though they have lower enzymatic activity. Since their mechanism of action is not completely understood, it was of interest to examine the toxins effects on phospholipid asymmetry as changes in asymmetry are associated with changes in membrane functioning. Rat brain synaptosomes were treated with the PLA2 toxins beta-bungarotoxin (beta-BuTx) and notexin and with the PLA2 enzymes Naja nigricollis and Naja naja atra under relatively non-disruptive conditions as judged by leakage of lactate dehydrogenase (LDH) and levels of phospholipid hydrolysis. The exposure of phosphatidylcholine (PC) and phosphatidylinositol (PI) on the synaptosomal surface was investigated by means of a specific PC-exchange protein (PCEP) and a PI-specific phospholipase C (PI-PLC), respectively. Treatment of the synaptosomes with N. nigricollis PLA2, beta-BuTx and notexin did not affect the availability of PC to exchange by PCEP, but significantly increased the exposure of PI to hydrolysis by PI-PLC. In contrast, N. n. atra PLA2 slightly decreased the exposure of PC and did not affect that of PI. The differences between N. n. atra PLA2, on the one hand, and N. nigricollis PLA2, beta-BuTx and notexin, on the other hand, parallel differences in their pharmacological activities. Our earlier studies showed that PLA2 enzymes, and possibly PLA2 toxins, have a pharmacological site separate from the enzymatic site. Since in the present study the effect on PI was abolished by EDTA, the presence of an enzymatic site in addition to the pharmacological site may be required or alternatively divalent cations may be required for the effects on PI asymmetry independent of the inhibition of PLA2 by EDTA.