OSTRICH INTESTINAL GLYCOHYDROLASES - DISTRIBUTION, PURIFICATION AND PARTIAL CHARACTERIZATION

Intestinal glycohydrolases are enzymes involved in assimilating carboh ydrate for nutrition. The avian forms of these enzymes, in particular the maltase-glucoamylase complex (MG), are not well characterised. Thi s study encompassed characterisation of these enzymes from ostrich int estines, and the first kinetic analysis of an avian MG. Proteolyticall y solubilised MG from ileal brush border membrane vesicles was purifie d by Sephadex G-200 gel filtration and Tris-affinity chromatography, w hile jejunal sucrase-isomaltase (SI) and MG were purified by Toyopearl -Q650 and phenyl-Sepharose chromatography. Amino acid sequences and co mpositions of enzyme subunits, resulting from SDS-PAGE, were determine d. Kinetics of hydrolysis of linear oligosaccharides was studied. Ostr ich MG and SI showed the highest activity in the jejunum, followed by the ileum and duodenum. No lactase or trehalase activity could be dete cted. The jejunal MG and SI, resulting from brush-border membrane vesi cles, could not be separated during purification. However, a minor for m of ileal MG was purified using Sephadex G-200 chromatography. Ileal MG contained three subunits of M-r 145 000, 125 000 and 115 000. Altho ugh the N-terminal amino acid sequences bear no homology to SI, the M- r 115 000 subunit shows homology to porcine MG in both sequence and am ino acid composition. The pH optimum of maltose-, starch- and isomalto se-hydrolysing activity was 6.5 and that of sucrose-hydrolysing activi ty 5.5. The glycohydrolases were most active at 58 degrees C, but were quickly denatured above 60 degrees C. Sucrose- and starch-hydrolysing activities were more thermostable than maltose- and isomaltose-hydrol ysing activities. Kinetic parameters (K-m, k(cat) and k(cat)/K-m) for the hydrolysis of maltooligosaccharides, starch and glycogen are repor ted for ileal MG. Maltotriose and maltotetraose displayed partial inhi bition of ileal MG. The study revealed large similarities between ostr ich SI and MG in charge, size, shape and hydrophobicity, based on thei r inseparability by several methods. Measurement of the specificity co nstants for maltooligosaccharide hydrolysis by ileal MG revealed less efficient hydrolysis of longer substrates as compared to maltose and m altotriose. (C) 1998 Elsevier Science Ltd. All rights reserved.