Publication Type |
Journal Article |
School or College |
School of Medicine |
Department |
Ophthalmology |
Creator |
Baehr, Wolfgang |
Other Author |
Qin, N |
Title |
Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase |
Date |
1994 |
Description |
Using recombinant baculovirus vectors, the three subunits of mouse rod photoreceptor cGMP phosphodiesterase (PDE) (alpha beta gamma 2) have been expressed in insect cells. The recombinant alpha,beta subunits accumulate to 5 mg/liter culture, but most (98%) of the expressed polypeptides are insoluble. In the soluble fraction, individually expressed alpha and beta subunits showed insignificant PDE activity, but coexpression (by coinfection) of alpha beta subunits elevated PDE activity 7-fold and coexpression of alpha beta gamma up to 15-fold. The soluble expressed holoenzyme associated with ROS membranes under isotonic, but not hypotonic, conditions. The Km of the soluble holoenzyme was 11-16 microM both for coexpressed alpha beta subunits and for alpha beta gamma subunits, similar to the Km (6-80 microM) of native PDE. Site-directed mutagenesis of cysteine to serine in the C-terminal CAAX box of both alpha and beta subunits substantially decreased the protein expression level, abolished post-translational isoprenylation, and prevented subunit binding to the rod outer segment (ROS) membranes. The mutant holoenzyme, however, showed a cGMP hydrolytic activity comparable with that of the normal recombinant enzyme. These results suggest that both alpha and beta subunits are required for the formation of a functional enzyme and that isoprenylation of the subunits is essential for membrane association and stability of PDE. |
Type |
Text |
Publisher |
American Society for Biochemistry and Molecular Biology (ASBMB) |
Volume |
269 |
Issue |
5 |
First Page |
3265 |
Last Page |
3271 |
Subject |
Electrophoresis, Polyacrylamide Gel; Macromolecular Substances; Protein Processing, Post-Translational |
Subject MESH |
Rods (Retina); Point Mutation; 3',5'-Cyclic-GMP Phosphodiesterase |
Language |
eng |
Bibliographic Citation |
Qin N, Baehr W. (1994). Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase. J Biol Chem, 269(5), 3265-71 |
Rights Management |
(c)American Society for Biochemistry and Molecular Biology (ASBMB) |
Format Medium |
application/pdf |
Identifier |
ir-main,1718 |
ARK |
ark:/87278/s6qr5fg5 |
Setname |
ir_uspace |
ID |
704695 |
Reference URL |
https://collections.lib.utah.edu/ark:/87278/s6qr5fg5 |