Effects of Retinylacetate on the Kinetics of Rat Liver Guanylate Cyclase: Possible Interaction with Sulfhydryl Groups

Abstract

The effect of retinylacetate (RA) was investigated on rat liver guanylate cyclase stimulated by nitroprusside (NP). The stimulated enzyme seemed to adhere to Michaëlis‐Menten kinetics with an apparent Km for MnGTP of 0.10 mM and a Vmax, of 410 pmol cGMP/min. × mg prot. RA (0.1–1mM) dose‐dependently inhibited the enzyme stimulated by NP (0.1–10 mM). In the presence of 1 mM RA the activity was only 10% of the control activity. The inhibitory action of RA seemed to be non‐competitive since it depressed Vmax without affecting the Km for MnGTP. In contrast, however, RA was instead found to stimulate the enzyme when low concentrations of NP (0.01–0.1 mM) were used. The concentration‐activity curve for NP was bell‐shaped showing an optimum at 0.5 mM. The inhibition induced by RA could not be surmounted by increasing the NP concentration indicating that RA did not compete with NP. A bell‐shaped activity curve was also seen when the enzyme activity was measured in the presence of increasing Mn2+ concentrations and during these conditions RA also caused inhibition. In the presence of the sulphydryl reductant dithiothreitol (DTT), the NP concentration needed for optimal enzyme activation was about 100‐fold less than in the absence of DTT. The maximal enzyme activity was also slightly increased. In the presence of DTT, RA was much less effective to induce inhibition of the stimulated enzyme, than when DTT was absent. (In the presence of DTT 1 mM RA caused only 30% inhibition compared to 90% inhibition in its absence). These results suggest that sulphydryl groups are of crucial importance in regulating guanylate cyclase activity and that the inhibitory effect of RA on the enzyme might be dependent on the interaction with these sulphydryl groups.