Preparation of an enzymatically active cross-linked complex between brain cyclic nucleotide phosphodiesterase and 3-(2-pyridyldithio)propionyl-substituted calmodulin.
Cyclic nucleotide phosphodiesterase (0.07 nM) was activated by near stoichiometric concentrations of [3-(2-pyridyldithio)propionyl]calmodulin (PDP-CaM) after initial incubation of these proteins at 200-fold higher concentrations; activity in assays with EGTA was 80% of that in the presence of Ca2+. The enzyme incubated with native calmodulin under identical conditions required approximately 1 nM for half-maximal activation, and no activation was observed in the absence of calcium. These data suggested formation of a covalent complex between phosphodiesterase and PDP-CaM. On high-performance gel-permeation chromatography in the presence of metal chelators, the complex appeared considerably larger than the native enzyme. Incubation of phosphodiesterase with the thiolated (inactivated) form of PDP-CaM did not change its chromatographic behavior, indicating that reactive sulfhydryl groups were involved in complex formation. Although the total activities recovered from chromatography were not significantly different, maximal activation of PDP-CaM-phosphodiesterase complex was only approximately 20%, whereas the control enzyme was activated 6-8-fold by Ca2+ plus calmodulin. Kinetics of cGMP hydrolysis in the presence of EGTA by the isolated complex differed from those of control enzyme but were indistinguishable from those of control enzyme assayed with saturating Ca2+ and CaM. The calmodulin antagonists W-7 and trifluoperazine had relatively little effect on activity of the PDP-CaM-phosphodiesterase complex. Incubation of the complex with dithiothreitol dramatically increased its Ca2+ and calmodulin responsiveness, suggesting that reduction of the disulfide cross-link released phosphodiesterase from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002147:Calmodulin / Q000031:analogs & derivatives* / Q000378:metabolism; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004533:Egtazic Acid; D006868:Hydrolysis; D007700:Kinetics; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*
C037852:3-(2-pyridyldithio)propionylcalmodulin; D002147:Calmodulin; D004533:Egtazic Acid; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP; D004229:Dithiothreitol