Isolation of the alpha subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized beta gamma subunits.

Abstract

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 fo r .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 AlF4-. 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.