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.
ASJC Scopus subject areas
- Molecular Biology