Lysine Carbamylation Research Articles

Dissociation of enzymatic activity from lethality and pharmacological properties by carbamylation of lysines in Naja nigricollis and Naja naja atra snake venom phospholipases A 2

Carbamylation of 9 out of 10 lysine residues in the toxic phospholipase A 2 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 A 2 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 A 2 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.

Isolation and Amino Acid Composition of Para-Kappa-Casein

One major and two minor para-κ-casein components were isolated after rennin treatment from each of two genetic variants of κ-casein (A and B) by carboxy-methyl cellulose chromatography. Homocitrullin was present only in the minor components, while the major component was richer in lysine. The minor components were not present in para-κ-casein preparations not treated with urea. These findings indicate that the minor para-κ-casein components are produced by carbamylation of lysine with the cyanate formed in the urea solution. The homocitrullin-free para-κ-caseins A and B were identical as judged by polyacrylamide gel electrophoresis and amino acid analysis. Each contained 108 amino acid residues (molecular weight of about 13,000), and no phosphorus or sialic acid.