Purification and properties of a pantetheine-hydrolyzing enzyme from pig kidney.

A microsomal glycoprotein that catalyzes the hydrolysis of pantetheine to pantothenate and cysteamine was solubilized and purified to homogeneity as determined by sodium dodecyl sulfate electrophoresis. The enzyme from pig kidney cortex was solubilized on exposure to butanol and purified by heat treatment, ammonium sulfate fractionation, hydrophobic chromatography, and hydroxyapatite chromatography. The purified enzyme (Mr = 57,000) has a specific activity of 14 mumol of pantothenate produced per min/mg of protein, a value 35 times that previously reported. A method for localizing enzymatic activity on polyacrylamide gels is presented, and enzyme activity, protein, and carbohydrate are shown to migrate identically by electrophoresis on nondenaturing polyacrylamide gels. Amino acid analysis indicated an absorbance index E1%1cm (280 nm) of 11.3, and carbohydrate analysis revealed the presence of galactose, mannose, fucose, glucose, galactosamine, and sialic acid for a total carbohydrate composition of 11.8%. The enzymatic hydrolysis of various pantetheine analogs indicated the enzyme had a high specificity for the pantothenate moiety but a low specificity for the cysteamine portion.