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G protein-effector coupling: interactions of recombinant inhibitory gamma subunit with transducin and phosphodiesterase

Griswold-Prenner, I.; Tuteja, N.; Farber, D.B.; Fung, B.K.

Biochemistry 28(15): 6145-6150

1989


ISSN/ISBN: 0006-2960
PMID: 2551365
DOI: 10.1021/bi00441a003
Accession: 040189135

A bacterial expression vector for the inhibitory gamma subunit of retinal rod phosphodiesterase has been constructed by inserting a mouse gamma cDNA into pUC19. Escherichia coli 222 transformed with this plasmid produces a 12-kDa recombinant protein consisting of 18 additional amino acids attached to the amino terminus of gamma. The fusion protein, designated beta-gal-gamma, has been refolded into an active form in formic acid and partially purified by gel filtration chromatography. Despite a large extended sequence at the amino terminus, beta-gal-gamma is able to inhibit the activity of trypsin-activated phosphodiesterase, bind tightly to the catalytic alpha beta subunits, and interact with the alpha subunit of transducin in a nucleotide-dependent manner. The availability of large quantities of active bacterial gamma, together with the ability to change its primary structure by site-directed mutagenesis, promises to provide considerable new information on the interaction between transducin and phosphodiesterase, as well as insights into the molecular mechanism of G protein-effector coupling.

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