Chromatographic behavior of cyclic 3',5'-nucleotide phosphodiesterases on columns of immobilized inhibitors

Mohindru, A.; Chenet, A.; Rhoads, A.R.

Biochemistry 17(16): 3297-3304

1978


ISSN/ISBN: 0006-2960
PMID: 80229
DOI: 10.1021/bi00609a019
Accession: 068519823

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Abstract
The chromatographic behavior of cyclic 3',5'-nucleotide phosphodiesterase [EC 3.1.4.17] from bovine heart ventricle was studied on columns of immobilized inhibitors. Succinylated trimethylpapaveroline (STMP) and the 7-acetic acid derivative of 1-methyl-3-isobutylxanthine (MIX) were coupled to diaminodipropylamine-substituted agarose. Chromatography was performed either in the presence or absence of Ca. In the presence of 100 .mu.M Ca, the heart enzyme eluted as a single peak of activity from the STMP-agarose column. When the Ca in the elution buffer was replaced by 100 .mu.M ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), phosphodiesterase was resolved by chromatography on STMP-agarose into 2 forms of activity. The endogenous protein activator of heart phosphodiesterase eluted between the 2 activity peaks. The first activity peak (peak I) could be stimulated several fold by the addition of a Ca-dependent protein activator of bovine brain cortex. This activator-dependent form of phosphodiesterase was also subject to activation by proteolytic treatment. The second activity peak (peak II) did not respond to protein activator or to proteolytic treatment. Peak II was more sensitive to inhibition by papaverine than peak I. Additional evidence from kinetic studies, sensitivity to sulfhydryl reagents, and polyacrylamide gel electrophoresis indicate that peaks I and II are distinct forms of phosphodiesterase activity. When phosphodiesterase of heart was chromatographed on MIX-agarose in the presence of Ca or EGTA, the elution profile was similar to that observed on STMP-agarose. Two activity peaks differing in their response to the protein activator were again resolved in the presence of EGTA. The performance of the substituted agaroses in separating phosphodiesterase of bovine cerebrum was also examined and compared with results of heart tissue.