Rapid isolation of G alpha 13 from bovine brain membranes: supportive effect of ethylene glycol.
G13 belongs to the G12-subfamily of heterotrimeric regulatory G-proteins. Employing specific antibodies, we isolated G alpha 13 from bovine brain by a four-step purification protocol combining conventional and affinity chromatography. The use of ethylene glycol as a protective agent influenced the elution properties of G alpha 13 markedly. Only in the presence of ethylene glycol (30% v/v) a clear separation of G alpha 13 from other G-proteins was achieved during the initial anion exchange chromatography. This allowed isolation of G alpha 13 by subunit exchange chromatography on beta gamma-agarose. G alpha 13 was only released from immobilized beta gamma-dimers via activation by AMF but not by GTP gamma S, pointing to a poor basal nucleotide exchange of this protein. In contrast, N-terminally truncated G alpha 13 did not bind to immobilized beta gamma-dimers.
Institut für Pharmakologie, Universitätsklinikum Rudolf Virchow, Freie Universität Berlin, Germany.
R
R
Harhammer
B
B
Nürnberg
K
K
Spicher
G
G
Schultz
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D019855:Ethylene Glycol; D005026:Ethylene Glycols / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000302:isolation & purification*; D008969:Molecular Sequence Data
D005026:Ethylene Glycols; D019204:GTP-Binding Proteins; D019855:Ethylene Glycol