Isolation of a detergent-solubilized maltase/glucoamylase from rat intestine and its comparison with a maltase/glucoamylase solubilized by papain.
Maltase/glucoamylase from the rat intestinal brush-border membrane was solubilized by homogenization of the intestinal mucosa in buffer containing 0.5% Triton X-100. After removal of the detergent with butan-1-ol, the enzyme was purified by chromatography on Sepharose 4B and DEAE-cellulose. The final specific activity was 70.3 units/mg of protein in six preparations, comparing favourably with the specific activity of 65.0 units/mg of protein of a pure papain-solubilized maltase/glucoamylase previously isolated and characterized by us [Flanagan & Forstner (1978) Biochem. J.173, 553-563]. The two enzymes were compared. Both migrated as single bands with the same mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were eluted at the same volume from Sepharose 4B, and had the same sedimentation pattern in mannitol gradients. The amino acid composition was similar; content of total apolar residues differed by 1.0mol%. Antibodies prepared against either enzyme gave identical precipitin lines with each. Neither enzyme bound tritiated Triton X-100. The only difference noted was the tendency of the detergent-solubilized enzyme to aggregate on storage, whereas the papain-solubilized enzyme remained unchanged. Both enzymes had two N-termini, glycine and arginine. When the two enzymes were dissociated by boiling in sodium dodecyl sulphate, each exhibited the same five species on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Single N-termini were found in the two smaller species, 1 (glycine) and 2 (arginine), whereas larger species (3-5) had both N-terminal amino acids. Both the Triton- and papain-solubilized enzymes appear to be oligomers of species 1 and 2, indicating that the native enzyme contains two subunit types. Aggregation in aqueous solutions does not depend on a proteolytically susceptible peptide fragment at the N-terminus of either subunit.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002499:Centrifugation, Density Gradient; D004356:Drug Storage; D004591:Electrophoresis, Polyacrylamide Gel; D005087:Glucan 1,4-alpha-Glucosidase / Q000302:isolation & purification*; D005959:Glucosidases / Q000302:isolation & purification*; D007413:Intestinal Mucosa / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008871:Microvilli / Q000201:enzymology; D010206:Papain; D011092:Polyethylene Glycols; D051381:Rats; D000520:alpha-Glucosidases / Q000302:isolation & purification*
D000596:Amino Acids; D046911:Macromolecular Substances; D011092:Polyethylene Glycols; D005959:Glucosidases; D000520:alpha-Glucosidases; D005087:Glucan 1,4-alpha-Glucosidase; D010206:Papain