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 Citations (Scopus)

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

Fingerprint

Lymphocytes
Electrophoresis
Sodium Dodecyl Sulfate
Glucocorticoids
Proteolysis
Polyacrylamide Gel Electrophoresis
Degradation
Proteins
Leucine
Dexamethasone
Gels
Hormones
Molecular Weight
Steroids
Radioactivity
polyacrylamide gels
Acrylamide
Molecular weight
Cells
Phenylalanine

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

@article{048df6fb225e481a9f708ab7b1bcffc2,
title = "Glucocorticoid-stimulated protein degradation in lymphocytes: Quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis",
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.",
author = "{Mac Donald}, {Richard G.} and Cidlowski, {John A.}",
year = "1981",
month = "12",
doi = "10.1016/0003-9861(81)90381-7",
language = "English (US)",
volume = "212",
pages = "399--410",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Glucocorticoid-stimulated protein degradation in lymphocytes

T2 - Quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

AU - Mac Donald, Richard G.

AU - Cidlowski, John A.

PY - 1981/12

Y1 - 1981/12

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0019845381&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0019845381&partnerID=8YFLogxK

U2 - 10.1016/0003-9861(81)90381-7

DO - 10.1016/0003-9861(81)90381-7

M3 - Article

C2 - 7325668

AN - SCOPUS:0019845381

VL - 212

SP - 399

EP - 410

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 2

ER -