skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

Abstract

We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher mass (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-foldmore » lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Institut National de la Sante et de la Recherche Medicale, Montpellier, France
OSTI Identifier:
6173792
Alternate Identifier(s):
OSTI ID: 6173792
Resource Type:
Journal Article
Journal Name:
J. Biol. Chem.; (United States)
Additional Journal Information:
Journal Volume: 262:18
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CYCLASES; BIOCHEMICAL REACTION KINETICS; ENZYME ACTIVITY; LABELLING; AMINO ACIDS; CARBON 13; ISOTOPE DILUTION; MUSCLES; NITROGEN 15; PROTEINS; TRITIUM COMPOUNDS; CARBON ISOTOPES; CARBOXYLIC ACIDS; ENZYMES; EVEN-ODD NUCLEI; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; LABELLED COMPOUNDS; LIGHT NUCLEI; LYASES; NITROGEN ISOTOPES; NUCLEI; ODD-EVEN NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; REACTION KINETICS; STABLE ISOTOPES; TRACER TECHNIQUES 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Bouhelal, R., Bockaert, J., Mermet-Bouvier, R., Guillon, G., and Homburger, V. Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line. United States: N. p., 1987. Web.
Bouhelal, R., Bockaert, J., Mermet-Bouvier, R., Guillon, G., & Homburger, V. Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line. United States.
Bouhelal, R., Bockaert, J., Mermet-Bouvier, R., Guillon, G., and Homburger, V. Thu . "Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line". United States.
@article{osti_6173792,
title = {Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line},
author = {Bouhelal, R. and Bockaert, J. and Mermet-Bouvier, R. and Guillon, G. and Homburger, V.},
abstractNote = {We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher mass (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.},
doi = {},
journal = {J. Biol. Chem.; (United States)},
number = ,
volume = 262:18,
place = {United States},
year = {1987},
month = {6}
}