Isoelectric-focusing patterns of cyclic nucleotide phosphodiesterase from rat heart

1. Isoelectric focusing on a flat gel bed of the rat heart cytosolic fraction resolved cyclic nucleotide phosphodiesterase activity into several forms, characterized by their substrate specificity, kinetic constants and dependence towards Ca2+ and calmodulin. A peak of pI 4.9 displayed 20 times more...

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Bibliographic Details
Published in:Biochemical journal 1981-10, Vol.199 (1), p.113-119
Main Authors: Némoz, G, Prigent, A F, Pageaux, J F, Pacheco, H
Format: Article
Language:English
Subjects:
2',3'-Cyclic-Nucleotide Phosphodiesterases - metabolism
3':5'-Cyclic-nucleotide phosphodiesterase
Animals
Calcium - metabolism
Cytosol - enzymology
heart
Isoelectric Focusing
Isoenzymes - metabolism
isolation
Kinetics
Male
Myocardium - enzymology
Phosphoric Diester Hydrolases - metabolism
Rats
Rats, Inbred Strains
Substrate Specificity
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Summary:1. Isoelectric focusing on a flat gel bed of the rat heart cytosolic fraction resolved cyclic nucleotide phosphodiesterase activity into several forms, characterized by their substrate specificity, kinetic constants and dependence towards Ca2+ and calmodulin. A peak of pI 4.9 displayed 20 times more affinity for cyclic GMP than for cyclic AMP and was markedly inhibited by EGTA. A less substrate-specific form, only slightly sensitive to EGTA inhibition, focused at pH 5.45. Several overlapping peaks detected between pH 5.55 and pH6 specifically hydrolysed cyclic AMP, with non-Michaelian kinetics; these peaks were insensitive to Ca2+ chelation. 2. Isoelectric focusing did not dissociate enzyme-calmodulin complexes, as none of the resulting peaks was activatable by calmodulin plus Ca2+. 3. Some new information on rat cardiac phosphodiesterase is obtained with this technique, which is convenient for routine analytical studies of phosphodiesterase, as well as for preparative purposes.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj1990113