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, contains two catalytic subunits, Palpha and Pbeta, and two identical regulatory subunits, Pgammas. 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 bovine PDE (Palphabetagammagamma) and its changes by Pgamma release. Palphabetagammagamma and Palphabetagamma were isolated from photoreceptor membranes after treatment with a hydrolysis-resistant GTP analogue, and Palphabeta was prepared from Palphabetagammagamma tryptic digestion. Images of Palphabetagammagamma consisted of two crooked strands. These two strands faced each other to make a ring shape, but this ring structure was bent at the centre line between the two strands. In Palphabetagamma, one of these strands changed its shape toward reducing the central space of the ring structure. This ring appeared to be more bent at the centre line. In Palphabeta, both strands changed their shape such that the ring structure appeared to be a twisted quasi ring shape. These observations suggest that in Palphabetagammagamma each Pgamma is complexed with a catalytic subunit, and that the shapes of Palpha and Pbeta are drastically changed by the Pgamma release. These shape changes are no doubt crucial for various PDE regulations, such as activation of cGMP hydrolysis by Palphabeta, interaction of Palphabeta with GARP2 and a GARP2-like protein and cGMP binding to non-catalytic sites on Palphabeta.
Kachi
S
S
Department of Ophthalmology, Nagoya University, School of Medicine, Japan.
Yamazaki
M
M
Tanaka
Y
Y
Hayashi
F
F
Miyake
Y
Y
Wakabayashi
T
T
Yamazaki
A
A
Usukura
J
J
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism / Q000648:ultrastructure*; D000818:Animals; D020134:Catalytic Domain / Q000502:physiology*; D002417:Cattle; D008854:Microscopy, Electron / Q000379:methods; D012160:Retina / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D015293:Transducin / Q000648:ultrastructure
D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin