Characterization of particulate cyclic nucleotide phosphodiesterases from bovine brain: purification of a distinct cGMP-stimulated isoenzyme.
In the absence of detergent, approximately 80-85% of the total cGMP-stimulated phosphodiesterase (PDE) activity in bovine brain was associated with washed particulate fractions; approximately 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 approximately 100% and calmodulin-stimulated approximately 400-500%. Although 1% Lubrol readily solubilized these PDE activities, approximately 75% of the cAMP PDE activity (0.5 microM [3H]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. After solubilization and purification by chromatography on cGMP-agarose, heparin-agarose, and Superose 6, the brain particulate cGMP-stimulated PDE cross-reacted with antibody raised against a cGMP-stimulated PDE purified from calf liver supernatant. The brain enzyme exhibited a slightly greater subunit Mr 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 V8 protease produced several peptides of similar size, as well as at least two distinct fragments of approximately 27 kDa from the brain and approximately 23 kDa from the liver enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Murashima
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S
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Tanaka
T
T
Hockman
S
S
Manganiello
V
V
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology; D002417:Cattle; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification; D008970:Molecular Weight; D010777:Photochemistry; D011092:Polyethylene Glycols; D011760:Pyrrolidinones / Q000494:pharmacology; D020889:Rolipram; D012697:Serine Endopeptidases
D000345:Affinity Labels; D002147:Calmodulin; D007527:Isoenzymes; D011760:Pyrrolidinones; C016276:8-bromocyclic GMP; D011092:Polyethylene Glycols; C005092:lubrol; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D006152:Cyclic GMP; D020889:Rolipram; D002118:Calcium