Purification and properties of trehalase from rat intestinal mucosal cells

• Published in: Journal of biochemistry (Tokyo) Vol. 81; no. 4; pp. 1041 - 1049
• Main Authors: Nakano, M, Miyakawa, M. (Aichi Medical Coll., Nagakute (Japan)), Sumi, Y. (Nagoya Univ. (Japan). Faculty of Medicine)
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.04.1977
• Subjects: 4-Chloromercuribenzenesulfonate - pharmacology; Acetates - pharmacology; Animals; Cations, Divalent - pharmacology; Ethylmaleimide - pharmacology; Hydrogen-Ion Concentration; Intestinal Mucosa - enzymology; Kinetics; Mercury - pharmacology; Molecular Weight; Rats; Sodium Dodecyl Sulfate - pharmacology; Trehalase - isolation & purification; Trehalase - metabolism; Tromethamine - pharmacology
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a131526

Rat intestinal trehalase was solubilized from microvillous membrane and purified about 30-fold from the membrane fraction. The members fraction was digested with papain and then solubilized with a mixture of 0.5% Triton X-100 and 0.2% sodium deoxycholate. Sohibilized supernatant was precipitated with solid ammonium sulfate, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The active fraction from the DEAF-cellulose column was subjected to polyacrylamide gel electrophoresis or ultracentrifugation analysis. The apparent molecular weight of the trehalase was estimated to be 240,000 by Sephadex G-200 gel filtration. Intestinal trehalase was dissociated by sodium dodecyl sulfate into fragments with a molecular weight of 30,000. The optimum pH of the enzyme was 5.7 in phosphate buffer or histidine buffer, and shifted to 4.7 in acetate buffer. The Km value for trehalose was found to be 5.5 mM in phosphate buffer and 8.0 to 11 mM in acetate buffer. The Hill coefficient(n) was more than 1.0 in the case of acetate buffer. Then n values were 1.14 and 1.58 in 5 mM and 500 mM acetate, respectively. The n values in the presence of SH-blocking agents were larger than 1.0, and increased in the order NEM, PCMS, HgCI2 Inhibitory effects of SH-blocking agents were decreased by acetate. Thus, intestinal trehalase might undergo conformational changes in acetate. Divalent cations such as CaCl2 MgCl2 and ZnCl2 slightly stimulated the enzyme at final concentrations of 2.5 to 5.0 mM However, the activity was not activated by sodium ions, as was the case with sucrase. SH-blocking agents and phlorizin partially inhibited the activity. Tris and SDS inhibited the activity by 80–90%. It is suggested that intestinal trehalase might play a role in sugar transport in intestinal mucosal cells.