Hormonal regulation of the phosphorylation of ATP citrate lyase in 3T3-L1 adipocytes. Effects of insulin and isoproterenol

G. D. Swergold, O. M. Rosen, C. S. Rubin

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Abstract

ATP citrate lyase was purified to homogeneity from murine liver. In vitro, the purified enzyme served as a good substrate for the catalytic subunit of cAMP-dependent protein kinase, accepting up to 1 mol of covalently bound phosphate/mol of 110,000-dalton subunit. cGMP-dependent protein kinase also catalyzed the phosphorylation of lyase. High titer anti-ATP citrate lyase serum was produced in rabbits and was employed as a specific probe for studying the hormonal regulation of the phosphorylation of ATP citrate lyase in 3T3-L1 adipocytes. Treatment of the cells with either insulin (0.18 μM) or isoproterenol (10 μM) plus methylisobutylxanthine (0.2 mM) stimulated the incorporation of 32P(i) into lyase approximately 3-fold. A combination of optimal concentrations of both hormones also produced a 3-fold elevation in the labeling of 3T3-L1 adipocyte ATP citrate lyase suggesting a lack of additivity in the effects of the lipogenic and lipolytic hormones. Sites phosphorylated in the presence and absence of hormones were compared by complete tryptic digestion of immunoprecipitated [32P]ATP citrate lyase and subsequent reverse-phase HPLC and two-dimensional mapping on thin layers of cellulose. These analyses revealed that the basal, insulin-stimulated, and isoproterenol-stimulated phosphorylations in 3T3-L1 adipocyte ATP citrate lyase all occurred on a single peptide. The M(r) of the phosphopeptide was estimated to be ~1000 by gel permeation HPLC. The same peptide was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase and cGMP-dependent protein kinase.

Original languageEnglish (US)
Pages (from-to)4207-4215
Number of pages9
JournalJournal of Biological Chemistry
Volume257
Issue number8
StatePublished - 1982

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ATP Citrate (pro-S)-Lyase
Phosphorylation
Isoproterenol
Adipocytes
Insulin
Cyclic GMP-Dependent Protein Kinases
Lyases
Hormones
Cyclic AMP-Dependent Protein Kinases
Catalytic Domain
High Pressure Liquid Chromatography
Phosphopeptides
Peptides
Permeation
Cellulose
Liver
Labeling
Digestion
Gels
Phosphates

ASJC Scopus subject areas

  • Biochemistry

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Hormonal regulation of the phosphorylation of ATP citrate lyase in 3T3-L1 adipocytes. Effects of insulin and isoproterenol. / Swergold, G. D.; Rosen, O. M.; Rubin, C. S.

In: Journal of Biological Chemistry, Vol. 257, No. 8, 1982, p. 4207-4215.

Research output: Contribution to journalArticle

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