Structural change of bovine retinal cGMP phosphodiesterase by release of its gamma subunit: direct imaging by improved low angle rotary shadowing

Cyclic GMP phosphodiesterase (PDE), a key enzyme for phototransduction, con tains two catalytic subunits, Pa and PP, and two identical regulatory subun its, Pys. Neither the structure of the subunits of PDE nor their changes in structure during PDE regulation have been known. Here, improved low angle rotary shadowing was applied to depict the three-dimensional structure of b ovine PDE (P alpha beta gamma gamma) and its changes by P gamma release. P alpha beta gamma gamma and P alpha beta gamma were isolated from photorecep tor membranes after treatment with a hydrolysis-resistant GTP analogue, and P alpha beta was prepared from P alpha beta gamma gamma by tryptic digesti on. Images of P alpha beta gamma gamma consisted of two crooked strands. Th ese two strands faced each other to make a ring shape, but this ring struct ure was bent at the centre Line between the two strands. In P alpha beta ga mma, one of these strands changed its shape toward reducing the central spa ce of the ring structure. This ring appeared to be more bent at the centre line. In P alpha beta, both strands changed their shape such that the ring structure appeared to be a twisted quasi ring shape. These observations sug gest that in P alpha beta gamma gamma each P gamma is complexed with a cata lytic subunit, and that the shapes of P alpha and P beta are drastically ch anged by the P gamma release. These shape changes are no doubt: crucial for various PDE regulations, such as activation of cGMP hydrolysis by P alpha beta, interaction of P alpha beta with GARP2 and a GARP2-like protein and c GMP binding to non-catalytic sites on P alpha beta.