Inactivation of a cytosolic phospholipase A2 by thiol-modifying reagents: cysteine residues as potential targets of phospholipase A2.
The cytosolic phospholipase A2 (cPLA2) from the human monocytic cell line U937 contains nine cysteine residues and is subject to oxidation. 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 reducing agents such as DTT, cPLA2 takes up only 2.8 equiv of [1-14C]iodoacetamide at pH 8.03/37 degrees C. With DTT present, cPLA2 is in its fully reduced form, and 4-5 equiv of acetamide are taken up without altering enzyme activity to give IA-cPLA2. A single equivalent of DTNB suffices to inactivate IA-cPLA2, giving a TNB-labeled enzyme, with the loss of activity correlating with release of an equivalent of 5-thio-2-nitrobenzoate. The TNB-labeled enzyme is quite stable up to 33 degrees C; enzyme activity is recoverable with DTT, even after this disulfide-enzyme adduct is incubated with iodoacetamide at pH 9.5, conditions that inactivate the free enzyme. At pH 9.5/37 degrees C, a single equivalent of 14C-labeled iodoacetamide is incorporated by IA-cPLA2 concomitant with complete loss of enzyme activity. Amino acid analysis of the 14C-labeled enzyme indicates that only cysteine residues are 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 the label resides primarily in Cys324, although Cys331 is also labeled. These results identify a region of the enzyme that is susceptible to labeling by group modification reagents and may represent a suitable target for small molecule inhibitors.
Syntex Inc., Mississauga, Ontario, Canada.
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Li
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Copp
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Castelhano
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Feng
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Stahl
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Yuan
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Krantz
D000595:Amino Acid Sequence; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry*; D003600:Cytosol / Q000201:enzymology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000737:chemistry; D054467:Phospholipases A2; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D003545:Cysteine; D004229:Dithiothreitol; D007460:Iodoacetamide