Isolation of the α subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized βγ subunits

I. H. Pang, P. C. Sternweis

Research output: Contribution to journalArticlepeer-review

46 Scopus citations

Abstract

Immobilized βγ subunits of GTP-binding regulatory proteins (G proteins) were used to isolate α subunits from solubilized membranes of bovine tissues and to separate specific α subunits based on their differential affinities for βγ subunits. The βγ subunits were cross-linked to ω-aminobutyl agarose. Up to 7 nmol of α subunit could bind to each milliliter of βγ-agarose and be recovered by elution of AlF4-. This affinity resin effectively separated the α subunits of G(i1) and G(i2) from 'contaminating' α subunits of G(o), the most abundant G protein in bovine brain, by taking advantage of the apparent lower affinity of the α subunits of G(o) for βγ subunits. The βγ-agarose was also used to isolate mixtures of α subunits from cholate extracts of membranes from different bovine tissues. α subunits of 39-41 kDa (in various ratios) as well as the α subunits of G(s) were purified. The yields from extracts exceeded 60% for all α subunits examined and apparently represented the relative content of α 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 GPT-binding proteins interact with these βγ subunits, the interaction is either of low affinity or mechanistically unique from the α subunits isolated in this study.

Original languageEnglish (US)
Pages (from-to)7814-7818
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume86
Issue number20
DOIs
StatePublished - 1989

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Isolation of the α subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized βγ subunits'. Together they form a unique fingerprint.

Cite this