Subunit structure of rod cGMP-phosphodiesterase.
The rod cGMP phosphodiesterase (PDE) is the G-protein-activated effector enzyme that regulates the level of cGMP in vertebrate photoreceptor cells. Rod cGMP PDE is generally viewed as a heterotrimeric protein composed of catalytic alpha and beta subunits ( approximately90 kDa each) and two copies of the inhibitory subunit gamma ( approximately 10 kDa). However, the possibility that rod PDE could exist as distinct isoforms, such as alphaalphagamma2 and betabetagamma2 has not been ruled out. We have studied this question using cross-linking of PDE subunits with maleimidobenzoyl-N-hydroxysuccinimide ester and para-phenyldimaleimide. The cross-linking resulted in major products with molecular mass of 100 and 150 kDa, a doublet at approximately 180-190 kDa, and a doublet at approximately 210-220 kDa. Cross-linked products were analyzed using polyclonal-specific anti-PDEalphabeta, anti-PDEalpha, anti-PDEbeta, or anti-PDEgamma antibodies. The anti-PDEalpha and anti-PDEalphabeta antibodies recognized all the cross-linked products, whereas anti-PDEbeta and anti-PDEgamma antibodies did not interact with the 150-kDa band, indicating that the composition of this band is most likely alphaalpha. Similar analysis of cross-linked products of trypsin-treated PDE preparations revealed bands that are likely formed by PDEbeta subunit. The molecular size of holo-PDE and trypsin-activated PDE were studied using analytical ultracentrifugation in order to determine if oligomerization of PDE could account for the cross-linking of identical PDE subunits. The sedimentation analysis of both holo-PDE and ta-PDE revealed homogeneous samples with molecular masses of approximately220 and approximately150 kDa, respectively. These results indicate that PDE is likely a mixture of the major species alphabetagamma2, minor species alphaalphagamma2, and possibly betabetagamma2. Our data are consistent with the detection of low PDE activity in the rd mouse, which lacks any functional PDEbeta subunit.
Artemyev
N O
NO
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Surendran
R
R
Lee
J C
JC
Hamm
H E
HE
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D051379:Mice; D008817:Mice, Mutant Strains; D008970:Molecular Weight; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013388:Succinimides; D014461:Ultracentrifugation
D000906:Antibodies; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D013388:Succinimides; C013097:3-maleimidobenzoyl N-hydroxysuccinimide; D015106:3',5'-Cyclic-GMP Phosphodiesterases