Isolation of the alpha subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized beta gamma subunits.
Immobilized beta gamma subunits of GTP-binding regulatory proteins (G proteins) were used to isolate alpha subunits from solubilized membranes of bovine tissues and to separate specific alpha subunits based on their differential affinities for beta gamma subunits. The beta gamma subunits were cross-linked to omega-aminobutyl agarose. Up to 7 nmol of alpha subunit could bind to each milliliter of beta gamma-agarose and be recovered by elution with AIF4-. This affinity resin effectively separated the alpha subunits of Gi1 and Gi2 from "contaminating" alpha subunits of Go, the most abundant G protein in bovine brain, by taking advantage of the apparent lower affinity of the alpha subunits of Go for beta gamma subunits. The beta gamma-agarose was also used to isolate mixtures of alpha subunits from cholate extracts of membranes from different bovine tissues. alpha subunits of 39-41 kDa (in various ratios) as well as the alpha subunits of Gs were purified. The yields from extracts exceeded 60% for all alpha subunits examined and apparently represented the relative content of alpha subunits in the tissues. This technique can rapidly isolate and identify, from a small amount of sample, the endogenous G proteins in various tissues and cells. So far, only polypeptides in the range of 39-52 kDa have been detected with this approach. If other GTP-binding proteins interact with these beta gamma subunits, the interaction is either of low affinity or mechanistically unique from the alpha subunits isolated in this study.
Pang
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IH
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Sternweis
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PC
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods; D019204:GTP-Binding Proteins / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009206:Myocardium / Q000032:analysis; D012685:Sepharose
D046911:Macromolecular Substances; D012685:Sepharose; D019204:GTP-Binding Proteins