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 language | English (US) |
|---|---|
| Pages (from-to) | 18-26 |
| Number of pages | 9 |
| Journal | BBA - General Subjects |
| Volume | 678 |
| Issue number | 1 |
| DOIs | |
| State | Published - Nov 18 1981 |
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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 journal › Article
}
TY - JOUR
T1 - Glucocorticoid regulation of two serine hydrolases in rat splenic lymphocytes in vitro
AU - MacDonald, Richard G.
AU - Cidlowski, John A.
PY - 1981/11/18
Y1 - 1981/11/18
N2 - 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.
AB - 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.
KW - (Rat spleen lymphocyte)
KW - Dexamethasone
KW - Diisopropylfluorophosphate binding
KW - Glucocorticoid
KW - Protein degradation
KW - Serine hydrolase
UR - http://www.scopus.com/inward/record.url?scp=0019840651&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0019840651&partnerID=8YFLogxK
U2 - 10.1016/0304-4165(81)90043-X
DO - 10.1016/0304-4165(81)90043-X
M3 - Article
C2 - 7030406
AN - SCOPUS:0019840651
VL - 678
SP - 18
EP - 26
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
SN - 0006-3002
IS - 1
ER -