Site-specific mutations in the regulatory subunit of cAMP-dependent protein kinase: selective screening for functional cAMP binding sites. [Escherichia coli]

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6078777

Bubis, J; Saraswat, L D; Reynolds, P; Neitzel, J; Taylor, S S

An expression vector for the type I regulatory (R) subunit was constructed in pUC7, and maximum expression (20 mg/L) was achieved in E. coli 222. The expressed R was functionally indistinguishable from the wild type protein. Site-specific mutations were introduced using oligonucleotide probes. Specific targets for mutagenesis were each of the cAMP-binding sites as well as the hinge region. Deletions also were introduced. Each mutation led to the expression of stable protein which was purified to homogeneity. Although antibodies can be used to screen for protein, immunological methods do not discriminate between active and inactive proteins. Screening for functional cAMP binding sites was achieved by photolabeling with 8-N/sub 3/-(/sup 32/P) cAMP following transfer to nitrocellulose. The R-subunit was photolabeled after SDS-gel electrophoresis and electrotransfer and also after plaque transfers to nitrocellulose. In the absence of photolysis, bound 8N/sub 3/-(/sup 32/P) cAMP was removed by washing with cold 8N/sub 3/cAMP. Better than 50% of the bound nucleotide was covalently incorporated following photolysis. In solution R/sup 1/ is labeled at 2 sites. Trp 260 is modified by 8N/sub 3/cAMP bound to site A; Tyr 381 is modified by 8N/sub 3/-cAMP bound to site B. Following transfer to nitrocellulose, photolabeling of Tyr 371 is abolished. Photolabeling of site A is retained even when mutations have been introduced into site B. The type II R-subunit is labeled at a single site in solution - Tyr 381 (site B). R/sup II/ is not photolabeled following transfer to nitrocellulose. The immobilized proteins are thus photolabeled in a site-specific manner, and photolabeling can be used to screen functional binding of 8N/sub 3/cAMP to site A.

• Research Organization: Univ. of California, San Diego, La Jolla
• OSTI ID: 6078777
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
ENZYMES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MUTATIONS
NUCLEI
ODD-ODD NUCLEI
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
RADIOISOTOPES
REACTION KINETICS
TRACER TECHNIQUES
TRANSFERASES