Purification of the G-protein G13 from rat brain membranes.
Significant amounts of G13, a member of the recently described G12-subfamily of heterotrimeric G-proteins, have been detected in rat brain membranes by specific antisera. The alpha-subunits of G13 (G alpha 13) were purified by using a combination of conventional and subunit-exchange chromatography. Purification was facilitated by the fact that the initial anion-exchange chromatography separated G13 from most of the other G-proteins, including Gq/11. Moreover, G alpha 13-enriched fractions obtained from this chromatographic step were devoid of beta gamma-dimers, despite the absence of G-protein-activating agents. Nevertheless, the purified G alpha 13 retained its ability to interact with beta gamma-dimers under appropriate conditions, i.e. the addition of Lubrol PX instead of cholate as detergent and the omission of ethylene glycol routinely used as a protecting additive. The interaction was demonstrated by (i) the binding of G alpha 13 to immobilized beta gamma-complexes and (ii) the formation of stable heterotrimers during sucrose-density-gradient centrifugation. Furthermore, our studies on G alpha 13 provide evidence for an extremely slow basal GDP/GTP exchange rate. The purified protein showed negligible binding of guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]). Accordingly, dissociation of G alpha 13 from immobilized beta gamma-complexes was achieved by AlF4-/Mg2+, but not by GTP[S]. These data indicate that G13 exhibits properties highly distinct from those of other G-proteins.
Harhammer
R
R
Institut für Pharmakologie, Universitätsklinikum Rudolf Virchow, Freie Universität Berlin, Germany.
Nürnberg
B
B
Spicher
K
K
Schultz
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D051379:Mice; D009243:NAD / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D051381:Rats; D013462:Sulfur Radioisotopes
D000906:Antibodies; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D009243:NAD; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins