Distinct biochemical properties of the native members of the G12 G-protein subfamily. Characterization of G alpha 12 purified from rat brain.
G12 and G13 are insufficiently characterized pertussis toxin-insensitive G-proteins. Here, we describe the isolation of G alpha 12 from rat brain membranes. G alpha 12 was purified to apparent homogeneity by three steps of conventional chromatography, followed by two cycles of subunit-exchange chromatography on immobilized G subunits. Purified G alpha 12 bound guanosine 5'-[gamma-thio]triphosphate slowly and substoichiometrically. For isolation of functionally active G alpha 12, it was mandatory to use sucrose monolaurate as a detergent. Comparative studies of both rat-brain-derived members of the G12 subfamily revealed differences in the affinity of G alpha 12 and G alpha 13 for G beta gamma. G alpha 12 required a higher Mg2+ concentration for AlF4- -induced dissociation from immobilized G beta gamma than did G alpha 13. In addition, the G12 subfamily members differed in their sedimentation velocities, as determined by sucrose-density-gradient centrifugation. Analysis of sedimentation coefficients revealed a higher tendency of G12 to form supramolecular structures in comparison to G13 and other G-proteins. These G13 structures were stabilized by sucrose monolaurate, which in turn may explain the necessity for this detergent for purification of functionally active G alpha 12. Despite these distinct biochemical characteristics of G12 and G13, both purified G-proteins coupled to a recombinant thromboxane A2 (TXA2) receptor reconstituted into phospholipid vesicles. These data indicate, (1) significant differences in the biochemical properties of native members of the G12 subfamily, and (2) their specific coupling to TXA2 receptors.
Harhammer
R
R
Institut für Pharmakologie, Freie Universität Berlin, Germany.
Nürnberg
B
B
Harteneck
C
C
Leopoldt
D
D
Exner
T
T
Schultz
G
G
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000818:Animals; D001345:Autoradiography; D001923:Brain Chemistry; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000302:isolation & purification; D051381:Rats; D017482:Receptors, Thromboxane / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000494:pharmacology
D011450:Prostaglandin Endoperoxides, Synthetic; D011518:Proto-Oncogene Proteins; D017482:Receptors, Thromboxane; D011994:Recombinant Proteins; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D019204:GTP-Binding Proteins; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498017:Gnai2 protein, rat; D008274:Magnesium