Purification and characterization of kidney and intestinal brush-border membrane trehalases from the rabbit.
Trehalase (alpha, alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized from the brush-border membrane of rabbit intestine and kidney by Emulphogen BC 720. The intestinal and kidney enzymes were purified 10 400-times and 4457-times respectively, in a five-step procedure, including DEAE-Trisacyl, chromatofocusing, AcA 34 gel filtration, HA Ultrogel and preparative polyacrylamide gel electrophoresis. The purified enzymes were homogeneous on polyacrylamide gel electrophoresis. The specific activities of kidney and intestinal pure trehalase were identical. Kidney and intestinal trehalases have the same molecular weight (about 85 000 by Sephadex G-200 gel filtration and 75 000 by SDS-polyacrylamide gel electrophoresis). A Stokes radius of 38 A was determined. Detergent solubilized trehalase is not retarded by phenyl-Sepharose CL-4B chromatography. The isoelectric point, measured by chromatofocusing, is between pH 3.8 and 4.2 for kidney trehalase and between pH 4.6 and 4.8 for intestinal trehalase. The enzymic properties for both kidney and intestinal trehalases are identical. The optimum pH is between 5.5 and 6.0. Trehalase is heat stable up to 50 degrees C. The apparent Km was found to be 3.5 mM in maleate buffer pH 6.0. The activation energy of trehalase is 11.17 kcal/mol. Tris, sucrose and phloridzin are fully competitive inhibitors with Ki of 3.7 mM, 3.1 mM, 1.1 mM respectively. Schiff's staining on polyacrylamide gel and interaction with Con A-Sepharose indicate that trehalase is a glycoprotein. Trehalase accounts for 0.1% and 0.3% of total brush-border membrane protein of intestine and kidney, respectively.
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006358:Hot Temperature; D007422:Intestines / Q000648:ultrastructure*; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D007668:Kidney / Q000648:ultrastructure*; D007700:Kinetics; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D010695:Phlorhizin / Q000494:pharmacology; D011817:Rabbits; D013395:Sucrose / Q000494:pharmacology; D014198:Trehalase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
D007527:Isoenzymes; D013395:Sucrose; D010695:Phlorhizin; D014198:Trehalase