Purification and characterization of a soluble form of mammalian adenylyl cyclase

Carmen W. Dessauer, Alfred G. Gilman

Research output: Contribution to journalArticle

84 Scopus citations

Abstract

An engineered, soluble form of mammalian adenylyl cyclase has been expressed in Escherichia coli and purified by three chromatographic steps. The enzyme utilizes one molecule of ATP to synthesize one molecule of cyclic AMP and pyrophosphate at a maximal specific activity of 12.8 μmol/min/mg, corresponding to a turnover number of 720 min-1. Although devoid of membrane spans, the enzyme displays all of the regulatory properties that are common to mammalian adenylyl cyclases. It is activated synergistically by G(sα) and forskolin and is inhibited by adenosine (P-site) analogs with kinetic patterns that are identical to those displayed by the native enzymes. The purified enzyme is also inhibited directly by the G protein βγ subunit complex. After adenovirus-mediated expression in adenylyl cyclase-deficient HC-1 cells, the enzyme can he stimulated synergistically by G(s)-coupled receptors and forskolin.

Original languageEnglish (US)
Pages (from-to)16967-16974
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number28
DOIs
StatePublished - Jul 30 1996

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this