Purification and properties of detergent-solubilized pig kidney trehalase.
Trehalase (alpha, alpha-trehalase, EC 3.2.1.28) was solubilized from the brush border membrane of pig kidney cortex by Triton X-100 and sodium deoxycholate in the presence of inhibitors of proteolytic enzymes. The kidney enzyme was purified 3060-fold using gel filtration, ion exchange chromatography, Con A-Sepharose chromatography, phenyl-Sepharose CL-4B hydrophobic interaction chromatography, Tris-Sepharose 6B affinity chromatography, and hydroxylapatite chromatography. Tris-Sepharose 6B was utilized to absorb contaminant proteins. Purity was estimated as 99% or greater, based on amino-terminal amino acid analysis. The purified enzyme had a specific activity of 278 units/mg protein, showed one major band after silver staining, and had an estimated molecular weight of 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was a glycoprotein and contained 2 mol of glucosamine per mole of trehalase. Kidney trehalase was inhibited by Tris, HgCl2, and phlorizin with Ki values of 3.8 mM, 11 microM, and 2.4 mM, respectively. Inclusion of Cl- in the reaction mixture protected the enzyme from inactivation by HgCl2. The apparent Km for trehalose was calculated to be 2.1 mM. Kidney trehalase was highly specific for trehalose and exhibited an optimal pH of 5.9. The isoelectric point was between pH 4.7 and 4.4, as measured by chromatofocusing.
D000818:Animals; D002845:Chromatography / Q000379:methods; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D008871:Microvilli / Q000201:enzymology; D012995:Solubility; D013379:Substrate Specificity; D013552:Swine; D014198:Trehalase / Q000302:isolation & purification* / Q000378:metabolism
D003902:Detergents; D014198:Trehalase