Identification of CRAC, a cytosolic regulator required for guanine nucleotide stimulation of adenylyl cyclase in Dictyostelium

Pamela J. Lilly, Peter N Devreotes

Research output: Contribution to journalArticle

Abstract

As previously reported, guanine nucleotide regulation of adenylyl cyclase activity in the Dictyostelium mutant synag 7 can be restored in vitro by addition of a high-speed supernatant prepared from wild-type cells (Theibert, A., and Devreotes, P. N. (1986) J. Biol. Chem. 261, 15121-15125). We have designated this activity CRAC, for cytosolic regulator of adenylyl cyclase. Trypsinization of partially purified material demonstrated that this activity contains a protein. We report here a 50,000-fold purification of this protein using Q and S Sepharose fast flow and P11 cellulose chromatography, followed by sucrose gradient centrifugation and separation on SDS-polyacrylamide gel electrophoresis. Purification of wild-type and mutant supernatants in parallel allowed identification of an 88-kDa protein required for reconstituting activity. A polyclonal antibody was raised against this protein; it stains a band in unfractionated wild-type, but not mutant, supernatants. Immunoblots of fractions from each purification step show that activity and the immunostaining band cochromatograph. We have determined a short N-terminal sequence of the 88-kDa CRAC polypeptide, which matches a portion of the deduced N terminus of a gene, dagA, isolated from a mutant similar to synag 7. Expression of the dagA cDNA in synag 7 cells restores both the 88 kDa band and CRAC activity.

Original languageEnglish (US)
Pages (from-to)14123-14129
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number19
StatePublished - May 13 1994

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Dictyostelium
Guanine Nucleotides
Adenylyl Cyclases
Purification
Proteins
Centrifugation
Chromatography
Electrophoresis
Cellulose
Sepharose
Sucrose
Polyacrylamide Gel Electrophoresis
Coloring Agents
Complementary DNA
Genes
Peptides
Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of CRAC, a cytosolic regulator required for guanine nucleotide stimulation of adenylyl cyclase in Dictyostelium. / Lilly, Pamela J.; Devreotes, Peter N.

In: Journal of Biological Chemistry, Vol. 269, No. 19, 13.05.1994, p. 14123-14129.

Research output: Contribution to journalArticle

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