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Cyclic Nucleotide Phosphodiesterase Activity in Bovine Brain Coated Vesicles
Author(s) -
Silva Walter I.,
Schook William,
Mittag Thomas W.,
Puszkin Saul
Publication year - 1986
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1986.tb00648.x
Subject(s) - phosphodiesterase , nucleotide , vesicle , cyclic nucleotide phosphodiesterase , chemistry , cyclic nucleotide , biochemistry , microbiology and biotechnology , biology , enzyme , membrane , gene
Abstract: Cyclic AMP phosphodiesterase activity in bovine brain coated vesicles displayed a K m of approximately 22 μ M for cyclic AMP, a V max of 3.2 nmol/min/mg protein, and a Hill coefficient of 1.5, suggesting positive cooperativity. The enzyme activity was stimulated by cyclic GMP with maximal indexes of stimulation ranging between 40 and 300%. Both basal and stimulated phosphodiesterase activities were immunotitrated with polyclonal antibodies against clathrin attached to heat‐inactivated, formaldehyde‐fixed Staphylococcus aureus cells. The main form of phosphodiesterase activity present in the immunoprecipitated brain coated vesicle preparation also is stimulated by cyclic GMP. The allosteric behavior was modulated by cyclic GMP. All of these properties are typical of type II or cyclic GMP‐sensitive phosphodiesterases in addition to their calcium and calmodulin independence. Competition experiments with a series of phosphodiesterase inhibitors, papaverine, 1‐methyl‐3‐isobutylxanthine, and theophylline, showed inhibition of cyclic AMP hydrolysis. Trifluoperazine was inactive at the highest concentration used, 100 μ M . These compounds also inhibited the cyclic GMP‐stimulated cyclic AMP hydrolysis with trifluoperazine practically inactive. At 5 μ M cyclic AMP none of the inhibitors was seen to stimulate the cyclic AMP hydrolytic activity. The presence of an enzyme for the breakdown of cyclic nucleotides in brain coated vesicles may suggest a role for these second messengers in the in vivo functions of this organelle.