Activated cGMP phosphodiesterase of retinal rods. A complex with transducin alpha subunit.

A Clerc,N Bennett

doi:10.1016/s0021-9258(19)50472-0

Abstract

Purified G-protein (transducin) activated with the nonhydrolyzable analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and cGMP phosphodiesterase (PDE) from retinal rods are added to protein-stripped disc membranes. Specific binding of the mainly soluble alpha subunit of G-protein with GTP gamma S bound (G alpha GTP gamma S, activator of the PDE) to the disc membrane in the presence of PDE is measured from gel scans or experiments with labeled G-protein alpha subunit (G alpha). Its variation as a function of G concentration matches the theoretical variation of G alpha involved in the activation of PDE calculated with previously estimated dissociation constants (Bennett, N., and Clerc, A. (1989) Biochemistry 28, 7418-7424), and the G alpha bound/PDE ratio at saturation is close to 2. No increase of G alpha binding to the membrane is observed when purified inhibitory subunit of PDE (PDE gamma) is added together with or instead of total PDE, and excess PDE gamma remains soluble. These results suggest that activated PDE is a complex with the activator G alpha GTP rather than PDE from which the inhibitory subunits have been removed. A method for purifying PDE gamma with a high yield of recovery and activity is described.

Highlights

Purified G-protein activated with the nonhydrolyzable analog guanosine 5‘-0-(thiotriphosphate) (GTPrS) andcGMP phosphodiesterase (PDE) from retinal rods are addedto protein-stripped disc membranes
Whether the two active PDEstates are complexes of GaGTP with PDEaPyy (GaPDEaPyy andGa2PDEa/3yy)or PDE from which one or two y subunits has been removed (PDEaPy and PDEaP); both hypotheses are proposed in the literature, and each is supported by different experimental data (Sitaramayya et al (1986),Hingorani et al (1988),and Kroll et al (1989)support the former, and Yamazaki et al (1983, 1990), Wensel and Stryer (1986),and Deterre et al (1986) support the latter)
Measurement of labeled GaGTPyS in the soluble and in the membrane-bound fraction is performed by mixing the isotonic super

Summary

Introduction

Purified G-protein (transducin) activated with the nonhydrolyzable analog guanosine 5‘-0-(thiotriphosphate) (GTPrS) andcGMP phosphodiesterase (PDE) from retinal rods are addedto protein-stripped disc membranes. Specific binding of the mainly soluble a subunit of G-protein with GTPrS bound (GaGTPyS, activator of the PDE) to the disc membrane in the presence of PDE is measured fromgel scans or experiments with labeled G-protein a subunit (Go).

Results

Conclusion