Purification of four forms of the beta gamma subunit complex of G proteins containing different gamma subunits.

T Asano,R Morishita,T Matsuda,Y Fukada,T Yoshizawa,K Kato

doi:10.1016/s0021-9258(20)80755-8

Abstract

To investigate the physiological significance of the diversity of gamma subunits of G proteins, we purified four forms of beta gamma of G proteins from bovine brain (beta gamma-B1, beta gamma-B2, beta gamma-B3), and spleen (beta gamma-S1) by the sequential chromatography on columns of DEAE-Sephacel, Ultrogel AcA 34, heptylamine-Sepharose, phenyl-5PW, and DEAE-5PW. Electrophoretic analyses showed that each beta gamma mainly contained the 36-kDa beta and a distinct but homogeneous gamma. These beta gamma complexes were subjected directly to proteolytic digestion and subsequent amino acid sequence analyses of their fragments. It was revealed that beta gamma-B1, -B2, and -B3 were identical to beta 1 gamma 7 (with a low level of beta 2 gamma 7), beta 1 gamma 2 and beta 1 gamma 3, respectively, while beta gamma-S1 was composed of beta 1 and an unidentified form of gamma. Then we examined the functional differences among these beta gamma complexes and the beta gamma of transducin (beta gamma-T, beta 1 gamma 1). Few differences were observed among all beta gamma complexes to enhance pertussis toxin-catalyzed ADP-ribosylation of the alpha subunits of G(o) and Gt. The four forms of beta gamma complexes purified from brain and spleen showed indistinguishable inhibitory effects on the release of GDP from G(o) alpha, but beta gamma-T was much less effective. Brain and spleen beta gamma complexes were equally effective in inhibiting calmodulin-stimulated adenylyl-cyclase activity, but beta gamma-T had a very weak inhibitory effect. Five forms of beta gamma facilitated metarhodopsin II-catalyzed binding of GTP gamma S to Gt alpha in a concentration-dependent manner with the following rank order of effectiveness: beta gamma-S1 > beta gamma-T > beta gamma-B1 > beta gamma-B2 > beta gamma-B3. Because the beta gamma complexes used in this study mostly contained the same beta subunit, the functional differences must be dependent on the gamma subunits. Thus, it seems likely that the receptor, the alpha subunits, and the effector are able to distinguish between the various gamma subunits.

Highlights

To investigate the physiological significance of the G proteins’ are composed of three different subunits desdiversity of y subunits of G proteins, we purified four ignated a, @a,nd y
The y2 subunit is expressed in a variety of tissues [21,22,23] and has Because the By complexes used in this study mostly contained the same j3 subunit, the functional differences must be dependent on the y subunits
When the cholate extract from bovine brain membranes was subjected to chromatography on DEAE-Sephacel, the fractions containing Py were partially separated from the main fractions that contained GTPyS-binding activity in the absence of any activating agents, such as AMF (Fig. 2 A )

Summary

RESULTS

When the cholate extract from bovine brain membranes was subjected to chromatography on DEAE-Sephacel, the fractions containing Py were partially separated from the main fractions that contained GTPyS-binding activity in the absence of any activating agents, such as AMF (Fig. 2 A ). This result is probably due, in part, to thedissociation of By from Goabecause of low affinity of Goafor Py [38].

DEAE-5PW

P J 36 kDa 35 kDa

DISCUSSION