Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase
- Univ. of Washington, Seattle (USA)
cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with (32P)cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments.
- OSTI ID:
- 5607548
- Journal Information:
- Journal of Biological Chemistry; (USA), Vol. 264:23; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
PHOSPHODIESTERASES
LABELLING
AMINO ACID SEQUENCE
AMP
AUTORADIOGRAPHY
CATTLE
CHYMOTRYPSIN
ELECTROPHORESIS
MOLECULAR WEIGHT
MONOCLONAL ANTIBODIES
MYOCARDIUM
NUCLEOTIDES
PEPTIDES
PROTEOLYSIS
ANIMALS
ANTIBODIES
BODY
CARDIOVASCULAR SYSTEM
CHEMICAL REACTIONS
DECOMPOSITION
DOMESTIC ANIMALS
ENZYMES
ESTERASES
HEART
HYDROLASES
MAMMALS
MOLECULAR STRUCTURE
MUSCLES
ORGANIC COMPOUNDS
ORGANS
PEPTIDE HYDROLASES
PROTEINS
RUMINANTS
SERINE PROTEINASES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques