Inhibition of deactivation of NO-sensitive guanylyl cyclase accounts for the sensitizing effect of YC-1.
Many of the physiological effects of the signaling molecule nitric oxide are mediated by the stimulation of the NO-sensitive guanylyl cyclase. Activation of the enzyme is achieved by binding of NO to the prosthetic heme group of the enzyme and the initiation of conformational changes. So far, the rate of NO dissociation of the purified enzyme has only been determined spectrophotometrically, whereas the respective deactivation, i.e. the decline in enzymatic activity, has only been determined in cytosolic fractions and intact cells. Here, we report on the deactivation of purified NO-sensitive guanylyl cyclase determined after addition of the NO scavenger oxyhemoglobin or dilution. The deactivation rate corresponded to a half-life of the NO/guanylyl cyclase complex of approximately 4 s, which is in good agreement with the spectrophotometrically measured NO dissociation rate of the enzyme. The deactivation rate of the enzyme determined in platelets yielded a much shorter half-life indicating either partial damage of the enzyme during the purification procedure or the existence of endogenous deactivation accelerating factors. YC-1, a component causing sensitization of guanylyl cyclase toward NO, inhibited deactivation of guanylyl cyclase, resulting in an extremely prolonged half-life of the NO/guanylyl cyclase complex of more than 10 min. The deactivation of an ATP-utilizing guanylyl cyclase mutant was almost unaffected by YC-1, indicating the existence of a special structure within the catalytic domain required for YC-1 binding or for the transduction of the YC-1 effect. In contrast to the wild type enzyme, YC-1 did not increase NO sensitivity of this mutant, clearly establishing inhibition of deactivation as the underlying mechanism of the NO sensitizer YC-1.
Russwurm
Michael
M
Pharmakologie und Toxikologie, Medizinische Fakultät MA N1, Ruhr-Universität Bochum, Germany.
Mergia
Evanthia
E
Mullershausen
Florian
F
Koesling
Doris
D
D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007191:Indazoles / Q000494:pharmacology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism*
D007191:Indazoles; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D009569:Nitric Oxide; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase