INTERACTIONS OF NUCLEOTIDE ANALOGS WITH ROD OUTER SEGMENT GUANYLATE-CYCLASE

Light activation of cyclic GMP hydrolysis in rod outer segments is mediated by a G-protein which is active in the GTP-bound form. Substitution of GTP with a nonhydrolyzable GTP analogue is thought to leave the G-protein in a persistently activated state, thereby prolonging the hydrolysis of cyclic GMP. Restoration of cyclic GMP concentration in the cell also depends upon GTP since it is the substrate for guanylate cyclase, but little is known about the effects of GTP analogues on this enzyme. We report here the effects of the analogues of GTP and ATP as inhibitors and substrates of rod disk membrane guanylate cyclase. The rate of cyclic GMP synthesis from GTP in rod disk membranes was about 50 pmol min-1 (nmol of rhodopsin)-1. Analogues of GTP and adenine nucleotides competitively inhibited the cyclase activity. The order of inhibition, with magnesium as metal cofactor, was ATP > GMP-PNP > AMP-PNP almost-equal-to GTP-gamma-S; with manganese, AMP-PNP was more inhibitory than GTP-gamma-S. The inhibition constants, with magnesium as cofactor, were 0.65-2.0 mM for GTP-gamma-S, 0.4-0.8 mM for GMP-PNP, 1.5-2.3 mM for AMP-PNP, and 0.07-0.2 mM for ATP. The fraction of cyclase activity inhibited by analogues was similar at 1 and 0.03-mu-M calcium. Besides inhibition of cyclase, the analogues also served as its substrates. GTP-gamma-S substituted GTP with about 85% efficiency while GMP-PNP and ATP were about 5 and 7% as efficient, respectively. As substrates, GTP and GMP-PNP were about twice as effective at 0.03 mu-M calcium than at 1-mu-M, while GTP-gamma-S was not affected significantly by calcium. For all three substrates, cyclase activity was higher with manganese than with magnesium, but with either cofactor, the order of effectiveness remained the same, GTP > GTP-gamma-S >> GMP-PNP. These results show that in addition to prolonging the activation of G-protein and thus the hydrolysis of cyclic GMP, GTP analogues also adversely influence the synthesis of cyclic GMP. This effect, whose magnitude varies depending upon the analogue, has to be taken into account in evaluating the effects of the analogues on the light response in rod photoreceptors.