Date of Award:

5-1982

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Chemistry and Biochemistry

Committee Chair(s)

R. Gaurth Hansen

Committee

R. Gaurth Hansen

Committee

Thomas Emery

Committee

Thomas Farley

Committee

Richard Olsen

Abstract

A microsomal glycoprotein hydrolyzing pantetheine to pantothenate and cysteamine has been solubilized and purified to homogeneity as determined by sodium dodecylsulfate electrophoresis. Four rapid, independent assays of pantetheine hydrolysis are described and compared along with a method for localizing enzymatic activity on polyacrylamide gels. The enzyme is solubilized on exposure to butanol and purified by heat treatment, (NH4)2SO4 fractionation, hydrophobic chromatography, and hydroxyapatite chromatography. The glycoprotein, purified 5600-fold in 22% yield, has a specific activity of 14 μmoles pantothenate produced/min/mg of protein, 35 times that previously reported. The enzyme has a pH optimum of 9.0-9.5 and a Km of 20 μM for D-pantetheine. Its molecular weight has been estimated by gel filtration (54,000) and sodium dodecylsulfate electrophoresis (60,000). Amino acid analysis indicates a high content of isoleucine (11.3 mole %) and phenylalanine (7.4 mole %) with an absorbancy index E1%1cm (280 nm) of 11 .3. Galactose, mannose, fucose, and glucose are present in decreasing concentrations totaling 90 μg of neutral hexoses per mg of protein. Galactosamine and sialic acid are also present. The enzymatic hydrolysis of various pantetheine analogs indicate the enzyme 1s specificity is high for the pantothenate moiety but low for the cysteamine portion. The enzyme should be useful in routine 11 total 11 pantothenate determinations in tissues.

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