Association of guanylate cyclase with the axoneme of retinal rods.

Axonemes were isolated from purified bovine retinal rod outer segments by dissolving the outer segment membranes in detergent and separating the axonemes by centrifugation on a linear detergent-containing sucrose density gradient. Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.61.2) activity was concentrated in the axoneme fraction. Guanylate cyclase eluted in the void volume when detergent-solubilized rod outer segments were subjected to exclusion chromatography on Sepharose 4B. Attempts to extract guanylate cyclase from isolated axonemes with salt, EDTA, base and other reagents were successful.

Guanylate cyclase of isolated bovine retinal rod axonemes.

The guanylate cyclase activity of axoneme--basal apparatus complexes isolated from bovine retinal rods has been investigated. The Mg2+ and Mn2+ complexes of GTP4- serve as substrates. Binding of an additional mole of Mg2+ or Mn2+ per mole of enzyme is required. Among cations which are ineffective are Ca2+, Ni2+, Fe2+, Fe3+, Zn2+, and Co2+. The kinetics are consistent with a mechanism in which binding of Mg2+ or Mn2+ to the enzyme must precede binding of MgGTP or MnGTP. The apparent dissociation constants of the Mg--enzyme complex and the Mn--enzyme complex are 9.5 x 10(-4) and 1.1 x 10(-4) M, respectively. The apparent dissociation constants for binding of MgGTP and MnGTP to the complex of the enzyme with the same metal are 7.9 x 10(-4) and 1.4 x 10(-4) M, respectively. The cyclase activity is maximal and independent of pH between pH 7 and 9. KCl and NaCl are stimulatory, especially at suboptimal concentrations of Mg2+ or Mn2+. Ca2+ and high concentrations of Mg2+ and Mn2+ are inhibitory. Ca2+ inhibition appears to require the binding of 2 mol of Ca2+ per mol of enzyme. The dissociation constant of the Ca2--enzyme complex is estimated to be 1.4 x 10(-6) M2. The axoneme--basal apparatus preparations contain adenylate cyclase activity whose magnitude is 1--10% that of the guanylate cyclase activity.