An antibody-induced enhancement of the transducin-stimulated cyclic GMP phosphodiesterase activity.
In this work we have characterized the ability of a carboxyl peptide-specific antibody (AS/7), raised against the alpha subunit of transducin (alpha T), to potentiate the stimulation of the cyclic GMP phosphodiesterase (PDE) by transducin. The complexation of the purified guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-bound form of alpha T (alpha T.GTP gamma S) with AS/7 results in a 2-5-fold enhancement in the total levels of cyclic GMP hydrolysis measured after 1 min. This potentiation by AS/7 cannot be attributed simply to an increase in the apparent affinity of alpha T.GTP gamma S for the effector enzyme, nor to an increased affinity of the enzyme for the substrate cyclic GMP. The AS/7-induced potentiation is specific for alpha T.GTP gamma S-PDE interactions; this antibody has no effect on the activity of the trypsin-activated PDE nor on the ability of the GDP-bound form of alpha T to inhibit the trypsin-activated enzyme (Kroll, S., Phillips, W. J., and Cerione, R. A. (1989) J. Biol. Chem. 264, 4490-4497). Phosphatidylcholine vesicles also will enhance the alpha T.GTP gamma S-stimulated PDE activity (1.5-2-fold) relative to that measured in the absence of a lipid milieu. However, the potentiations of alpha T-stimulated cyclic GMP hydrolysis elicited by AS/7 and lipids represent separate events. Titration profiles describing the AS/7-induced potentiation, as a function of the amount of antibody added to the assay mixtures, indicate that maximal activity occurs when there is one molecule of AS/7 per two molecules of alpha T.GTP gamma S; the AS/7-induced potentiation is lost when AS/7 much greater than alpha T. GTP gamma S, i.e. conditions which favor the formation of monovalent AS/7-alpha T.GTP gamma S complexes. When the AS/7 is papain-treated to yield monovalent antibody molecules, complexation between these monovalent antibodies and alpha T still occurs (as reflected by the ability of these antibodies to block rhodopsin-alpha T coupling); however, the potentiation of the alpha T.GTP gamma S-stimulated PDE activity is lost. Taken together, these results suggest that the AS/7-induced potentiation of alpha T-stimulated activity is dependent on the bivalent nature of the antibody, and maximal stimulation of PDE activity is achieved by the interactions of two activated-alpha T molecules with a single molecule of PDE.
Phillips
W J
WJ
Department of Pharmacology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853-6401.
Trukawinski
S
S
Cerione
R A
RA
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000936:Antigen-Antibody Complex; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007140:Immunoglobulin Fab Fragments; D007700:Kinetics; D046911:Macromolecular Substances; D010786:Photoreceptor Cells / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D015293:Transducin / Q000276:immunology / Q000378:metabolism*
D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D046911:Macromolecular Substances; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D012243:Rhodopsin; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin