Glucocorticoid regulation of two serine hydrolases in rat splenic lymphocytes in vitro.
A quantitative assay employing binding of [3H] diisopropylfluorophosphate ([3H]DFP) and SDS-polyacrylamide gel electrophoresis was used to measure serine hydrolases in cell-free extracts from rat splenic lymphocytes. After labeling with [3H]DFP at pH7, six major serine hydrolases are detected on 10% gels, having molecular weights of 78, 55, 34, 30, 28, and 17 (.10(-3)). When labeled at pH4, only four activities are measured, with Mr of 79, 55, 33 and 17(.10(-3)). Incubation of splenic lymphocytes for 8 h in vitro with 1 microM dexamethasone followed by [3H]-DFP labeling at pH 7 produces a 91% increase in the 17000 [3H]DFP. Hormone treatment for 8 h with subsequent labeling at pH 4 results in a 15% increase in the largest (78000) species, as well as 73% increase in the 17000 enzyme, compared with lysates from cells incubated without steroid. These effects are not observed after only 4 h of glucocorticoid exposure. Dexamethasone treatment for 8 h does not produce a decrease in any of these serine hydrolases, nor is there an apparent induction of new enzymes (i.e., having a molecular weight different from the preexisting species). Studies examining the effect of protease inhibitors on the [3H]DFP capacity of these proteins, show that the 17000 enzyme is sensitive to the protease inhibitor, pepstatin A, as well as the sulfhydryl reagents dithiothreitol and N-ethylmaleimide. These results suggest that this dexamethasone-responsive enzyme is a protease which requires a free thiol group for optimal activity. These findings are discussed with regard to the mechanism of glucocorticoid action in lymphocytes.
D000818:Animals; D003907:Dexamethasone / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000097:blood*; D007531:Isoflurophate / Q000378:metabolism; D008214:Lymphocytes / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D013154:Spleen; D013379:Substrate Specificity
D011480:Protease Inhibitors; D007531:Isoflurophate; D003907:Dexamethasone; D010450:Endopeptidases