Amino acid sequence of a basic Agkistrodon halys blomhoffii phospholipase A2. Possible role of amino-terminal lysines in action on phospholipids of Escherichia coli

Publisher Website
Google Scholar
Abstract
A basic (pI=10.2) phospholipase A2 of the venom of the snake Agkistrodon halys blomhoffii is one of a few phospholipases A2 capable of hydrolyzing the phospholipids of Escherichia coli killed by a bactericidal protein purified from human or rabbit neutrophil granules. We have shown that modification of as many as 4 mol of lysine per mole of the phosphlolipase A2, either by carbamylation or by reductive methylation [Forst, S., Weiss, J., and Elsbach, P. (1982) J. Biol. Chem. 257, 14055-14057], had no effect on catalytic activity toward extracted E. coli phospholipids or the phospholipids of autoclaved E. coli. In contrast, modification of 1 mol of lysine per mole of enzyme substantially reduced activity toward the phospholipids of E. coli killed by the neutrophil protein. To explore further the role of lysines in the function of this phospholipase A2, we determined the amino acid sequence of the enzyme and the incorporation of [14C]cyanate into individual lysines when, on average, 1 lysine per molecule of enzyme had been carbamylated. After incorporation of approximately 1 mol of [14C]cyanate per mole of protein, the phospholipase A2 was reduced, alkylated, and exhaustively carbamylated with unlabeled cyanate. The amino acid sequence was determined of the NH2-terminal 33 amino acids of the holoprotein and of peptides isolated after digestion with trypsin and Staphylococcus aureus V-8 protease. The protein contains 122 amino acid residues, 17 of which are lysines. The NH2-terminal region is unique among more than 30 phospholipases A2 previously sequenced because of its high content of basic residues (His-1, Arg-6, and Lys-7, -10, -11, and -15). The four NH2-terminal lysines accounted for approximately 50% (about equally distributed) of the total incorporated [14C]cyanate. On the basis of these findings and evidence that the NH2-terminal region of phospholipases A2 is a functionally important .alpha.-helix, we raise the possibility that the cluster of basic residues in the outwardly directed face of this helix is involved in the action of the basic A. h. blomhoffii phospholipase A2 on E. coli killed by the bactericidal protein of neutrophils.

This publication has 17 references indexed in Scilit: