Subunit stoichiometry of retinal rod cGMP phosphodiesterase.
The cyclic GMP phosphodiesterase of the retinal rod is composed of three distinct types of polypeptides: alpha (90 kDa), beta (86 kDa), and gamma (10 kDa). The gamma subunit has been shown to inhibit phosphodiesterase activity associated with alpha and beta. To investigate the subunit stoichiometry of the retinal phosphodiesterase, we have developed a panel of monoclonal and peptide antibodies that recognize individual phosphodiesterase subunits. By quantitative and immunochemical analysis of the purified subunits, we have shown that each phosphodiesterase molecule contains one copy each of alpha and beta subunit and two copies of gamma subunit. Moreover, gamma can be chemically cross-linked to both alpha and beta, but not to itself, suggesting that alpha and beta may each bind one gamma. The phosphodiesterase is fully activated when both copies of gamma were removed by proteolysis with trypsin. Upon recombination of the purified gamma subunit with the trypsin-activated phosphodiesterase containing alpha beta, the alpha beta gamma 2 stoichiometry is once again restored, with concomitant total inhibition of activity. Our results suggest that at least two activated transducin molecules are required to fully activate one molecule of phosphodiesterase in retinal rods.
Fung
B K
BK
Jules Stein Eye Institute, UCLA School of Medicine 90024.
Young
J H
JH
Yamane
H K
HK
Griswold-Prenner
I
I
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001001:Anura; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D051379:Mice; D010786:Photoreceptor Cells / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D014357:Trypsin