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

Richard G MacDonald, John A. Cidlowski

Research output: Contribution to journalArticle

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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

Fingerprint

Isoflurophate
Lymphocytes
Hydrolases
Serine
Glucocorticoids
Rats
Labeling
Dexamethasone
Enzymes
Protease Inhibitors
Molecular Weight
Molecular weight
Sulfhydryl Reagents
Enzyme Induction
Ethylmaleimide
Dithiothreitol
Cell Extracts
Electrophoresis
Sulfhydryl Compounds
Polyacrylamide Gel Electrophoresis

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Glucocorticoid regulation of two serine hydrolases in rat splenic lymphocytes in vitro. / MacDonald, Richard G; Cidlowski, John A.

In: BBA - General Subjects, Vol. 678, No. 1, 18.11.1981, p. 18-26.

Research output: Contribution to journalArticle

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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.",
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