Expression and characterization of calmodulin-activated (type I) adenylylcyclase

Wei Jen Tang, John Krupinski, Alfred G. Gilman

Research output: Contribution to journalArticle

324 Citations (Scopus)

Abstract

A complementary DNA that encodes a bovine brain, calmodulin-sensitive (type I) adenylylcyclase has been inserted into the baculovirus genome under the control of the strong polyhedron promoter. Expression of the recombinant adenylylcyclase in Sf9 cells using recombinant baculovirus increases adenylylcyclase activity in cell membranes to 10-20 nmol·min-1·mg-1 (approximately 0.1% of membrane protein). The catalytic activity of the recombinant adenylylcyclase can be stimulated by G, calmodulin, or forskolin, and it can be inhibited by adenosine analogs and by G protein βγ subunits. The specific activity of the purified recombinant protein approximates 5 μmol·min-1·mg-1. This is similar to that of the enzyme purified from bovine brain. Type I adenylylcyclase has a quasiduplicated structure. There are two membrane-spanning domains, each with six putative transmembrane helices, and there are two presumed nucleotide-binding domains that are about 55% similar to each other. No catalytic activity is detectable when each half of the adenylylcyclase molecule is expressed by itself. However, coexpression of the two halves results in considerable enzymatic activity. Interaction between the two halves of adenylylcyclase may be necessary for catalysis.

Original languageEnglish (US)
Pages (from-to)8595-8603
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number13
StatePublished - May 5 1991

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Baculoviridae
Calmodulin
Catalyst activity
Brain
Sf9 Cells
Protein Subunits
Colforsin
Cell membranes
Catalysis
GTP-Binding Proteins
Recombinant Proteins
Adenosine
Membrane Proteins
Nucleotides
Complementary DNA
Genes
Cell Membrane
Genome
Membranes
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Tang, W. J., Krupinski, J., & Gilman, A. G. (1991). Expression and characterization of calmodulin-activated (type I) adenylylcyclase. Journal of Biological Chemistry, 266(13), 8595-8603.

Expression and characterization of calmodulin-activated (type I) adenylylcyclase. / Tang, Wei Jen; Krupinski, John; Gilman, Alfred G.

In: Journal of Biological Chemistry, Vol. 266, No. 13, 05.05.1991, p. 8595-8603.

Research output: Contribution to journalArticle

Tang, WJ, Krupinski, J & Gilman, AG 1991, 'Expression and characterization of calmodulin-activated (type I) adenylylcyclase', Journal of Biological Chemistry, vol. 266, no. 13, pp. 8595-8603.
Tang, Wei Jen ; Krupinski, John ; Gilman, Alfred G. / Expression and characterization of calmodulin-activated (type I) adenylylcyclase. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 13. pp. 8595-8603.
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