Characterization of Guanylate cyclase of rod outer segments of the bovine retina

Abstract

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) of bovine retinal rod outer segments is almost completely particulate, i.e. associated with rod outer segment membranes. In contrast to particulate guanylate cyclase is other tissues, treatment of rod outer segments with Triton X-100 does not solubilize the enzyme but inhibits it. Enzyme activity is dependent on the presence of divalent cation, especially Mn 2+ with only poor activation by Mg 2+ (10-fold lower) and no activation seen with other cations. Expression of maximal activity required Mn 2+ and GTP in equimolar concentrations with an apparent K m of 8 · 10 −4 M and V of 10 nmol/min per mg protein. Excess of Mn 2+ over that required for the formation of the Mn · GTP complex was inhibitory , Ca 2+, Ba 2+ and Co 2+ inhibited enzyme activity when assayed with the Mn · GTP substrate complex. In the presence of a fixed concentration of 1 mM Mn 2+, the enzyme exhibited strong negative cooperative interactions with GTP, characterized by an intermediary plateau region in the substrate vs. enzyme activity curve, a curve of downward concavity in the double reciprocal plot and a Hill coefficient of 0.5. Nucleotides such as ITP, ATP and UTP at higher concentrations (1 mM) inhibit enzyme activity while ATP at lower concentrations (0.1 mM) stimulates activity by 40%. NaN 3 has no efffect on the guanylate cyclase. It is thus possible that the guanylate cyclase may be regulated in vivo by both the metal : GTP substrate ratio and the free divalent cation concentration as well as by the ATP concentration and thus play an important but yet undefined role in the visual process.