Uu/Mamiya/YssunoO, eds. Fontiers in Protein Chemistry 297 PHOSPHOLIPHASES A2: STRUCTURE, FUNCTION AND EVOLUTION

As part of an ongoing study of crotalid venom phospholipases A2, the amino acid sequence Las been determined for the enzyme from western diamondback rat-tlesnake, C.ko.a2wS CCX. The C. tttitOX phospholipase sequence shows strong homol-ogy to that of the enzyme from the venom of the eastern diamondback rattlesnake (Cto.tiw3 adarntxn.tEw3) determined earlier in our laboratory [Heinrikson, R.L., Krueger, E.T. and Keim, P.S., J. Biol. Chem. (1977) 252, 4913]. In fact, the C. a.ttox phospholipase A2 differs in only six amino acid substitutions from C. adwnan.teit6 a and in five substitutions from the B form. The fact that all of the substitutions involving ionizable residues lead to increased negative charge in C. attox phospholipase A2 accounts for the observed chromatographic and electrophoretic properties of the enzyme as contrasted with those of the C. adwr2euA a and 0 forms. The single methionyl residue at position 10 in the C. a.tAOx phospholipase A2 has been alkylated at pH 2.6 by reaction with a 50-fold molar excess of iodoacetsmide. The resulting derivative was separated from unmodified enzyme by ion-exchange chromatography and, like the native phospho-lipase, is a fully active diner. Cleavage with cyanogen bromide at Met-ID yields two fraaments, an amino-terminal decapeptide and a 112-residue C-terminal fragment which contains all of the seven disulfide bonds in the native molecule. Neither the individual fragments, nor a reconstituted mixture of the two showed enzyme activity. These findings are discussed relative to those obtained in studies of enzymes from several sources in an attempt to elucidate structure-function and evolutionary relationships among phospholipases A2 in general