Characterization of particulate cyclic nucleotide phosphodiesterases from bovine brain: Purification of a distinct cGMP-stimulated isoenzyme
- National Institutes of Health, Bethesda, MD (USA)
In the absence of detergent, {approx}80-85% of the total cGMP-stimulated phosphodiesterase (PDE) activity in bovine brain was associated with washed particulate fractions; {approx}85-90% of the calmodulin-sensitive PDE was soluble. Particulate cGMP-stimulated PDE was higher in cerebral cortical gray matter than in other regions. Homogenization of the brain particulate fraction in 1% Lubrol increased cGMP-stimulated activity {approx}100% and calmodulin-stimulated {approx}400-500%. Although 1% Lubrol readily solubilized these PDE activities, {approx}75% of the cAMP PDE activity (0.5 {mu}M ({sup 3}H)cAMP) that was not affected by cGMP was not solubilized. This cAMP PDE activity was very sensitive to inhibition by Rolipram but not cilostamide. Thus, three different PDE types, i.e., cGMP stimulated, calmodulin sensitive, and Rolipram inhibited, are associated in different ways with crude bovine brain particulate fractions. The brain enzyme exhibited a slightly greater subunit M{sub r} than did soluble forms from calf liver or bovine brain, as evidenced by protein staining or immunoblotting after polyacrylamide gel electrophoresis under denaturing conditions. Incubation of brain particulate and liver soluble cGMP-stimulated PDEs with V{sub 8} protease produced several peptides of similar size, as well as at least two distinct fragments of {approx}27 kDa from the brain and {approx}23 kDa from the liver enzyme. After photolabeling in the presence of ({sup 32}P)cGMP and digestion with V{sub 8} protease, ({sup 32}P)cGMP in each PDE was predominantly recovered with a peptide of {approx}14 kDa. All of these observations are consistent with the existence of at least two discrete forms (isoenzymes) of cGMP-stimulated PDE.
- OSTI ID:
- 5393765
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 29:22; ISSN 0006-2960; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
AMP
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
BRAIN
CATTLE
CENTRAL NERVOUS SYSTEM
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ELECTROPHORESIS
ENZYMATIC HYDROLYSIS
ENZYME INHIBITORS
ENZYMES
ESTERASES
GLANDS
HYDROGEN COMPOUNDS
HYDROLASES
HYDROLYSIS
ISOENZYMES
ISOTOPES
LIGHT NUCLEI
LIVER
LYSIS
MAMMALS
MOLECULAR STRUCTURE
NERVOUS SYSTEM
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOSPHODIESTERASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PURIFICATION
RADIOISOTOPES
RUMINANTS
SOLVOLYSIS
TRITIUM COMPOUNDS
VERTEBRATES