Isolation and characterization of cGMP phosphodiesterase from bovine rod outer segments

A simple purification method has been developed for isolation of bovine cGMP phosphodiesterase from photoreceptor rod outer segments. The enzyme is peripherally membrane bound in its native state and is present in relatively high concentrations. In the bovine photoreceptors its molar ratio to rhodopsin can be estimated to be as great as 1:40 and not lower than 1: 170. The isolated enzyme is purified to homogeneity as demonstrated by gel electrophoresis under native and denaturing conditions and analytical ultracentrifuga- Con. The core enzyme has a molecular weight of approximately 170,000, as demonstrated by sucrose gradient centrifugation and analytical ultracentrifugation, and is composed of two major subunits, 88,000 (CY) and 84,000 (p), augmented by a small subunit of 13,000 (y). In its purified state, the enzyme is activated neither by light nor GTP in contrast to the native membranebound enzyme. As isolated, the molar activity of the enzyme is 45 mol of cGMP hydrolized s-' mol-' with a Km = 150 PM (cGMP) and Km > 4 XnM (CAMP). Protamine activates the enzyme to 360 mol of cGMP see-' mol-' @-fold) and limited digestion by trypsin activates it to as great as 2100 mol of cGMP s-l mol-' @O-fold).