Ostrich intestinal glycohydrolases: distribution, purification and partial characterisation.
Intestinal glycohydrolases are enzymes involved in assimilating carbohydrate for nutrition. The avian forms of these enzymes, in particular the maltase-glucoamylase complex (MG), are not well characterised. This study encompassed characterisation of these enzymes from ostrich intestines, and the first kinetic analysis of an avian MG. Proteolytically solubilised MG from ileal brush border membrane vesicles was purified by Sephadex G-200 gel filtration and Tris-affinity-chromatography, while jejunal sucrase-isomaltase (SI) and MG were purified by Toyopearl-Q650 and phenyl-Sepharose chromatography. Amino acid sequences and compositions of enzyme subunits, resulting from SDS-PAGE, were determined. Kinetics of hydrolysis of linear oligosaccharides was studied. Ostrich MG and SI showed the highest activity in the jejunum, followed by the ileum and duodenum. No lactase or trehalase activity could be detected. The jejunal MG and SI, resulting from brush-border membrane vesicles, could not be separated during purification. However, a minor form 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. Although 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 amino acid composition. The pH optimum of maltose-, starch- and isomaltose-hydrolysing activity was 6.5 and that of sucrose-hydrolysing activity 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-hydrolysing activities. Kinetic parameters (K(m), kcat and kcat/K(m)) for the hydrolysis of maltooligosaccharides, starch and glycogen are reported for ileal MG. Maltotriose and maltotetraose displayed partial inhibition of ileal MG. The study revealed large similarities between ostrich SI and MG in charge, size, shape and hydrophobicity, based on their inseparability by several methods. Measurement of the specificity constants for maltooligosaccharide hydrolysis by ileal MG revealed less efficient hydrolysis of longer substrates as compared to maltose and maltotriose.
Oosthuizen
V
V
Department of Biochemistry and Microbiology, University of Port Elizabeth, South Africa.
Weldrick
D P
DP
Naudé
R J
RJ
Oelofsen
W
W
Muramoto
K
K
Kamiya
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001717:Birds / Q000378:metabolism*; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000201:enzymology*; D007700:Kinetics; D008871:Microvilli / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013394:Sucrase-Isomaltase Complex / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine; D014018:Tissue Distribution
D000596:Amino Acids; D006026:Glycoside Hydrolases; D013394:Sucrase-Isomaltase Complex; D005087:Glucan 1,4-alpha-Glucosidase