Abstract
Soluble guanylate cyclase (sGC) is a heterodimeric hemoprotein that catalyzes the conversion of GTP to cGMP. Upon binding NO to its heme cofactor, purified sGC was activated 300-fold. sGC was only activated 67-fold by nitroglycerin (GTN) and Cys; and in the absence of Cys, GTN did not activate sGC. Electronic absorption spectroscopy studies showed that upon NO binding, the Soret of ferrous sGC shifted from 431 to 399 nm. The data also revealed that activation of sGC by GTN/Cys was not via the expected ferrous heme-NO species as indicated by the absence of the 399 nm heme Soret. Furthermore, EPR studies of the reaction of GTN/Cys with sGC confirmed that no ferrous heme-NO species was formed but that there was heme oxidation. Potassium ferricyanide is known to oxidize ferrous sGC to the ferric oxidation state. Spectroscopic and activity data for the reactions of sGC with GTN alone or with K(3)Fe(CN)(6) were indistinguishable. These data suggest the following: 1) GTN/Cys do not activate sGC via GTN biotransformation to NO in vitro, and 2) in the absence of added thiol, GTN oxidizes sGC.
Highlights
From the Departments of ‡Chemistry and ¶Molecular and Cell Biology, University of California, Berkeley, California 94720-1460 and the §Department of Chemistry, Michigan State University, East Lansing, Michigan 48824
Soluble guanylate cyclase1 is a hemoprotein that catalyzes the conversion of guanosine 5Ј-triphosphate (GTP) to the second messenger, cyclic guanosine 3Ј:5Ј-monophosphate [1, 2]
SGC Activity—At 37 °C and pH 7.4, DEA/nitric oxide (NO) is known to have a half-life of ϳ2 min releasing 1.5 eq of NO [25]
Summary
Materials—Unless otherwise indicated, all reagents were purchased from Sigma. 2-(N,N-Diethylamino)-diazenenolate-2-oxide (DEA/NO) was purchased from Cayman Chemical Company, Ann Arbor, MI. Assays were incubated with either DEA/NO, GTN, or potassium ferricyanide (K3Fe(CN)6) for 1 min, initiated with 0.2– 0.4 g of sGC, and quenched after 2 min with 400 l of 125 mM Na2CO3 and 500 l of 125 mM Zn(CH3CO2). Activity of sGC was measured in 100-l assays in 50 mM HEPES, pH 7.4, 1.5 mM GTP, 5.0 mM MgCl2, and 2.0 mM Cys at 37 °C. Assays were incubated with either 100 M DEA/NO or 1.0 mM GTN for 1 min, initiated with 0.2– 0.4 g of sGC, and quenched after 2 min with 400 l of 125 mM Na2CO3 and 500 l of 125 mM Zn(CH3CO2). After centrifugation of the assay mixtures and appropriate dilution, cGMP was measured as directed using a cGMP enzyme immunoassay kit
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