Our long term objective is to determine why proteins with phospholipase A2 (PLA2) activity differ so markedly in their toxicological and pharmacological properties even though they have similar enzymatic activities. For example, we shall use in our studies: 1. presynaptically acting PLA2 neurotoxins (Beta bungarotoxin, notexin, caudoxin) which are very toxic and show specific effects on motor nerve terminals. 2. PLA2 from N. nigricollis snake venom which has strong direct hemolytic, anticoagulant, cardiotoxic and central effects. 3. PLA2 from N.n. atra snake venom which has relatively low toxicity with no organ specific action even though it has high enzymatic activity. To determine whether there exists (and if so its chemical nature) a region of the molecule responsible for pharmacological properties and independent from the enzymatic active site, we shall prepare known specific chemical modifications of the PLA2 enzymes and presynaptic PLA2 neurotoxins. Modifications to be performed include: 1. alkylation and methylation of histidine at the enzymatic active site, 2. carbamylation, acylation and guanidination of lysines, 3. semicarbazide addition to free carboxyls (aspartic and glutamic acids), 4. alkylation of tryptophans, 5. sulfonylation of tyrosines, 6. oxidation of methionines, 7. Cleavage of the n-terminal octapeptide (micellar recognition site). Biochemical and pharmacological properties of the native enzymes and their modified derivatives which will be tested include: enzymatic activity on substrates in various states of organization (monomers, micelles, lipoproteins, natural membranes), phospholipid substrate preferences, enzyme-phospholipid complex formation, hemolytic, and anticoagulant action, intravenous and intraventricular lethal potencies, electro-physiological effects on isolated heart tissue and the phrenic nerve-diaphragm preparation, hydrolysis of phospholipids and binding of PLA2 to heart sarcolemma and brain synaptic plasma membranes, microscope alterations of tissues. Information obtained in these studies using model PLA2 enzymes and presynaptic PLA2 neurotoxins may be relevant to the functioning of mammalian PLA2 enzymes whose activities are associated with prostaglandin formation and membrane turnover, in addition to possible roles in modulating transmitter action and membrane fluidity. Clinical disorders could be associated with abnormal enzymatic activity or toxic effects unrelated to enzymatic activity.
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