Date of Award

1991

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Chemistry, Biochemistry.

Supervisor

Mutus, B.,

Rights

info:eu-repo/semantics/openAccess

Abstract

Three calmodulin-dependent phosphodiesterases, M$\sb{\rm r}$ 60,000 (low) and M$\sb{\rm r}$ 63,000 (high) from the bovine brain as well as M$\sb{\rm r}$ 59,000 species from heart, have been compared with respect to their steady-state kinetic parameters for the hydrolysis of cAMP and cGMP and their 2$\sp\prime$-O-anthraniloyl- and 2$\sp\prime$-O-(N-methylanthraniloyl)-derivatives. Kinetic studies with the native substrates indicate that the high molecular weight brain phosphodiesterase is cGMP specific whereas, the heart and low molecular weight brain enzymes are equally specific to cAMP and cGMP hydrolysis. These enzymes appear to be kinetically distinct from those previously isolated from the bovine brain and heart. The isoforms were indistinguishable with respect to their relative affinities for cAMP and cGMP. Using pseudosubstrates, a distinction could be made between the three enzymes. The major effect was on the maximal velocity where, the V$\sb{\rm max}$ values were 1-11% of those observed with the native substrates. A calmodulin-dependent phosphodiesterase from uterine smooth muscle has been partially purified and characterized. The uterine isoform is stimulated 2-3 fold by calmodulin with an apparent K$\sb{\rm activity}$ of approximately 10 nM. The uterine enzyme hydrolyzes cAMP and cGMP with nearly equal affinity (K$\sb{\rm m}$ = 30 $\mu$M). Comparison of inhibition profiles of the uterine enzyme with the aortic enzyme indicates that both the enzymes had identical drug sensitivities. Selective inhibitors of calmodulin-dependent phosphodiesterases, vinpocetine and 8-methoxymethyl isobutylxanthine, inhibited both the isoforms with same potency. Employing the uterine enzyme for screening new drugs would be useful. A new method has been developed for quantitating the extent of glucosylation of proteins. This assay is based on the periodate oxidation of the glucosylated protein releasing formaldehyde. The latter is complexed with the reagent 3-methyl-2-benzothiazolinone hydrazone to produce a chromogen with an absorption maxima at 630 nm. (Abstract shortened by UMI.)Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1991 .G748. Source: Dissertation Abstracts International, Volume: 53-09, Section: B, page: 4636. Supervisor: B. Mutus. Thesis (Ph.D.)--University of Windsor (Canada), 1991.

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