Glucocorticoid regulation of two serine hydrolases in rat splenic lymphocytes in vitro

Richard G. MacDonald, John A. Cidlowski

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

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 pH 7, 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 pH 4, only four activities are measured, with Mr or 79, 55, 33 and 17 (· 10-3). Incubation of splenic lymphocytes for 8 h in vitro with 1 μM 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 result 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.

Original languageEnglish (US)
Pages (from-to)18-26
Number of pages9
JournalBBA - General Subjects
Volume678
Issue number1
DOIs
StatePublished - Nov 18 1981

Keywords

  • (Rat spleen lymphocyte)
  • Dexamethasone
  • Diisopropylfluorophosphate binding
  • Glucocorticoid
  • Protein degradation
  • Serine hydrolase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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