Details

Autor(en) / Beteiligte

• BIRNBAUM, ROBERT J

Titel

PHOSPHODIESTERASES OF VERTEBRATE SMOOTH MUSCLE (CALMODULIN, PHENOTHIAZINE, VERAPAMIL)

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

1985

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Changes in cyclic nucleotide levels in smooth muscle influence the functioning of this tissue. Intracellular levels of cyclic nucleotides are controlled by a balance between synthesis, catalyzed by a cyclase, and degradation, catalyzed by a phosphodiesterase (PDE). This thesis describes the separation and characterization of four isoenzymic variants of the enzyme 3':5'-cyclic nucleotide phosphodiesterase (E.C. #3.1.4.17), from vertebrate smooth muscle (chicken gizzard). These isoforms were separated from one another by ion-exchange chromatography on DEAE-cellulose and by calmodulin-Sepharose affinity chromatography. Each form migrated as a single discrete band when electrophoresed on alkaline non-denaturing polyacrylamide gels and stained for PDE activity. Each form also eluted as a single peak on gel-permeation chromatography, giving apparent Mr values of 114,000, 116,000, 122,000, and 59,000. All four enzymes had apparent Km values in the 0-20 (mu)M range, although their relative specificities for cyclic AMP and cyclic GMP differed. Two of the forms bound to a calmodulin-Sepharose affinity column in a calcium-dependent manner and could be dissociated from the column by the antipsychotic trifluoperazine. However, only one of these calmodulin binding forms was activated by calmodulin. The interaction of the other calmodulin-binding form with calmodulin could be disrupted by the papaverine derivative verapamil or by ethylene glycol. The effects of verapamil, nicardipine, theophylline, papaverine, RO 20-1724, and trifluoperazine on the activity of the four isoforms are reported. One of the isoenzymic variants that displayed no interaction with calmodulin exhibited a calcium dependent interconvertibility between an origin staining form and a rapidly migrating form on alkaline non-denaturing gels stained for enzyme activity. The significance of this interconversion is considered with respect to possible in vivo redistribution of the enzyme from membrane bound to cytosolic compartments. Studies aimed at determining whether any of the four isoforms might be interrelated by aggregation, proteolytic degradation, or phosphorylation are also described. The characteristics of each of the isoforms are discussed in relation to the properties described by other investigators for isoforms of the enzyme isolated from other smooth muscle sources and other tissues.