Rat intestinal brush border membrane trehalase: some properties of the purified enzyme.
Rat intestinal brush border trehalase (EC 3.2.1.28) solubilized by Triton X-100 or Emulphogen BC 720 has been purified almost to homogeneity in a five steps procedure including DEAE cellulose, Sephadex G-200, preparative flat bed electrofocusing and hydroxylapatite. The apparent molecular weight was estimated to be about 65,500 daltons by mannitol density gradient ultracentrifugation. The optimum pH of the enzyme was between 5.5 and 5.7 in phosphate, maleate or citrate buffers. The apparent Km for trehalose was found to be 10 mM in maleate buffer pH 6.0. The isoelectric point was 4.9. Tris, P-aminophenylglucoside, sucrose and maltose are fully competitive inhibitors with Kis of 2.2, 1.8, 7.7 and 170 mM, respectively. The inhibition by Phloridzin appeared to be of the mixed type with a Ki of 1.7 mM. Trehalase is heat stable up to 50 degrees C and the activation energy is 10.96 kcal/mol. Schiff's staining on polyacrylamide gel and interaction with Con-A-Sepharose indicate that rat trehalase is a glycoprotein.
D000818:Animals; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008871:Microvilli / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D013816:Thermodynamics; D014198:Trehalase / Q000302:isolation & purification* / Q000378:metabolism
D006882:Hydroxyapatites; D017886:Durapatite; D014198:Trehalase