Switch to
| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
5
|
| pubmed:dateCreated |
1994-3-18
|
| pubmed:abstractText |
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.
|
| pubmed:grant | |
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical | |
| pubmed:status |
MEDLINE
|
| pubmed:month |
Feb
|
| pubmed:issn |
0021-9258
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
4
|
| pubmed:volume |
269
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
3265-71
|
| pubmed:dateRevised |
2008-11-21
|
| pubmed:meshHeading |
pubmed-meshheading:8106363-3',5'-Cyclic-GMP Phosphodiesterases,
pubmed-meshheading:8106363-Amino Acid Sequence,
pubmed-meshheading:8106363-Animals,
pubmed-meshheading:8106363-Baculoviridae,
pubmed-meshheading:8106363-Base Sequence,
pubmed-meshheading:8106363-Blotting, Western,
pubmed-meshheading:8106363-Cell Line,
pubmed-meshheading:8106363-Cell Membrane,
pubmed-meshheading:8106363-DNA Primers,
pubmed-meshheading:8106363-Electrophoresis, Polyacrylamide Gel,
pubmed-meshheading:8106363-Gene Expression,
pubmed-meshheading:8106363-Kinetics,
pubmed-meshheading:8106363-Macromolecular Substances,
pubmed-meshheading:8106363-Mice,
pubmed-meshheading:8106363-Molecular Sequence Data,
pubmed-meshheading:8106363-Moths,
pubmed-meshheading:8106363-Mutagenesis, Site-Directed,
pubmed-meshheading:8106363-Point Mutation,
pubmed-meshheading:8106363-Protein Binding,
pubmed-meshheading:8106363-Protein Prenylation,
pubmed-meshheading:8106363-Protein Processing, Post-Translational,
pubmed-meshheading:8106363-Retinal Rod Photoreceptor Cells,
pubmed-meshheading:8106363-Rod Cell Outer Segment,
pubmed-meshheading:8106363-Transfection
|
| pubmed:year |
1994
|
| pubmed:articleTitle |
Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase.
|
| pubmed:affiliation |
Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
|
| pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
|