INACTIVATION OF A CYTOSOLIC PHOSPHOLIPASE A(2) BY THIOL-MODIFYING REAGENTS - CYSTEINE RESIDUES AS POTENTIAL TARGETS OF PHOSPHOLIPASE A(2)

The cytosolic phospholipase A(2) (cPLA(2)) from the human monocytic ce ll line U937 contains nine cysteine residues and is subject to oxidati on. Iodoacetamide and 5,5'-dithiobis(2-nitrobenzoic acid) were used to explore the susceptibility of cysteine residues to thiol modification agents as outlined in Schemes 2 and 3. In the absence of thiol reduci ng agents such as DTT, cPLA(2) takes up only 2.8 equiv of [1-C-14]iodo acetamide at pH 8.03/37 degrees C. With DTT present, cPLA(2) is in its fully reduced form, and 4-5 equiv of acetamide are taken up without a ltering enzyme activity to give IA-cPLA(2). A single equivalent of DTN B suffices to inactivate IA-cPLA(2), giving a TNB-labeled enzyme, with the loss of activity correlating with release of an equivalent of 5-t hio-2-nitrobenzoate. The TNB-labeled enzyme is quite stable up to 33 d egrees C; enzyme activity is recoverable with DTT, even after this dis ulfide-enzyme adduct is incubated with iodoacetamide at pH 9.5, condit ions that inactivate the free enzyme. At pH 9.5/37 degrees C, a single equivalent of C-14-labeled iodoacetamide is incorporated by IA-cPLA(2 ) concomitant with complete loss of enzyme activity. Amino acid analys is of the C-14-labeled enzyme indicates that only cysteine residues ar e labeled. Lys-C digestion of labeled enzyme with 2 M guanidine at pH 8.0 yields a 40-mer peptide. Amino acid sequencing establishes that th e label resides primarily in Cys(324), although Cys(331) is also label ed. These results identify a region of the enzyme that is susceptible to labeling by group modification reagents and may represent a suitabl e target for small molecule inhibitors.