Isolation of the alpha subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized beta gamma subunits.
- 1 October 1989
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 86 (20), 7814-7818
- https://doi.org/10.1073/pnas.86.20.7814
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.This publication has 27 references indexed in Scilit:
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