Glucocorticoid-stimulated protein degradation in lymphocytes: Quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Richard G. Mac Donald, John A. Cidlowski

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

A semiquantitative method was employed to study the effect of glucocorticoids on molecular weight-dependent protein degradation in rat splenic lymphocytes in vitro. Cell proteins were pulse-labeled with [3H]leucine, followed by incubation of the cells in medium containing 5 mM leucine, with or without hormone. Molecular weight distribution of radiolabeled proteins from whole cells was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Dexamethasone, which increases the fractional protein degradative rate in splenic lymphocytes, stimulates breakdown of larger proteins proportionally more than smaller proteins. This phenomenon is manifested by a shift of the distribution of [3H]leucine-labeled proteins from dexamethasone-treated cells across the gel, causing a graded reduction in counts per minute at the high-molecular-weight (top) region of the gel and a compensatory increase of counts per minute at the gel bottom. Both the direction and degree of this shift may be quantified by mathematical analysis of the radioactivity profiles, comparing hormone-treated proteins to those from control cells. This method is considerably more sensitive than measuring protein degradation by following loss of radioactivity from acid-precipitable protein. When measured in this way, 1 μm dexamethasone increases lymphocyte proteolysis beginning 3-4 h after steroid addition. This initial stimulation continues between 4 and 7 h of hormone treatment and is followed by a secondary enhancement of degradation, which peaks after 10 h and is maintained for up to 20 h of steroid exposure. The glucocorticoid stimulation of proteolysis is specific for active glucocorticoids and depends on the hormone concentration employed (half-maximal at a dexamethasone concentration of 5-8 nm). This hormonal effect is demonstrable when either [3H]leucine or [3H]phenylalanine is used to label cell proteins, by using protein electrophoresis on gels containing 8 or 12% acrylamide. The significance of these findings is considered in relation to the mechanism of glucocorticoid-stimulated protein degradation in lymphocytes and to steroid-induced lymphocytolysis.

Original languageEnglish (US)
Pages (from-to)399-410
Number of pages12
JournalArchives of Biochemistry and Biophysics
Volume212
Issue number2
DOIs
StatePublished - Dec 1981

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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