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High affinity binding proteins for the regulatory subunit of cAMP-dependent protein kinase: Cloning, characterization and expression of P150 and P75

Thesis/Dissertation ·
OSTI ID:7152133
cAMP-dependent protein kinase II-B appears to be adapted for function in the mammalian central nervous system (CNS) via the properties of its regulatory subunit (RII-B). RII-B is selectively expressed in brain, tightly associated with cerebral cortex membranes and avidly complexed by the bovine brain calmodulin-binding protein designated P75. Complexes of RII-B and P75 polypeptides can be purified to near-homogeneity from either membrane or cytosolic fractions of brain homogenates, suggested that the binding protein plays a role in determining the subcellular localization and/or other CNS-specific properties of protein kinase II-B. In general, a single high-affinity RII-B binding protein is expressed in the brains of mammals, but the size of the protein varies (e.g., cow: 75 kDa; rat: 150 kDa). To investigate these non-abundant and CNS-enriched RII-B binding proteins, cDNAs for bovine brain P75 and rat brain P150, the homologue of P75, have been cloned and characterized. The cDNAs were retrieved from bovine and rat lambda gt11 expression libraries using {sup 32}P-RII-B as a functional probe. cDNA inserts subcloned into pIN-IA2 or pET-3b expression plasmids directed the production of partial P75 and P150 polypeptides in E. coli that exhibited RII-B binding activity. P150 cDNAs hybridize with 3 rat mRNAs (7.3, 5.0, 4.2 kb) that are present in brain and lung, but not other tissues. These mRNAs are not detected in fetal brain, but are expressed during the period of post-natal synaptogenesis and in adult rats. The P75 cDNA hybridizes to a 6 kb bovine brain mRNA. Finally, 3'-deletion analysis demonstrated that the C-terminal 15-25 amino acids of P150 or P75 are essential for binding with RII-B.
Research Organization:
Yeshiva Univ., New York, NY (USA)
OSTI ID:
7152133
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOASSAY
BODY
BRAIN
CARBOXYLIC ACIDS
CATTLE
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM
CLONING
COWS
DATA
DAYS LIVING RADIOISOTOPES
DNA HYBRIDIZATION
DNA-CLONING
DOMESTIC ANIMALS
ENZYMES
EXPERIMENTAL DATA
GENES
HYBRIDIZATION
INFORMATION
ISOTOPES
LIGHT NUCLEI
MAMMALS
MESSENGER-RNA
NERVOUS SYSTEM
NUCLEI
NUCLEIC ACIDS
NUMERICAL DATA
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PLASMIDS
RADIOASSAY
RADIOISOTOPES
RATS
RNA
RODENTS
RUMINANTS
TRANSFERASES
VERTEBRATES