The purification of human enterokinase by affinity chromatography and immunoadsorption. Some observations on its molecular characteristics and comparisons with the pig enzyme.
A method is described for the purification of human enterokinase from accumulated duodenal fluid by affinity chromatography using p-aminobenzamidine as the ligand. Resolution was greatest when glycylglycine was substituted as the spacer arm. Purification was not a one-step procedure, and some contamination, principally by the alpha-glucosidases, remained. Their removal was completed by immunoadsorption using antisera raised to enterokinase-free material containing these enzymes, prepared as a by-product of the purification procedure. The final preparation had an activity of 4260 nmol of trypsin/min per mg and was free of other enzymic activity tested. Amino acid and sugar analyses of the highly purified enzyme indicated an acidic glycoprotein containing 57% sugar (neutral sugars 47%, amino sugars 10%). The apparent mol.wts. and Stokes radii of human and pig enterokinase were 296 000 and 316 000, and 5.65 and 5.78 nm respectively. Two isoenzymes were identified for human enterokinase and three for the pig enzyme. Human enterokinase demonstrated a resistance to reduction of disulphide linkages and to sodium dodecyl sulphate binding, which may be related to the need for it to retain its integrity in the digestive environment of the upper small intestine. Antisera to highly purified pig and human enterokinases specifically inhibited enterokinase activity. Immuno-inhibition of intestinal aminopeptidase, maltase and glucoamylase by homologous antisera was not observed.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001550:Benzamidines; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification*; D004765:Enteropeptidase / Q000276:immunology / Q000302:isolation & purification*; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D007106:Immune Sera; D007419:Intestinal Secretions / Q000201:enzymology; D007527:Isoenzymes / Q000032:analysis; D008970:Molecular Weight; D012967:Sodium Dodecyl Sulfate; D013552:Swine
D000596:Amino Acids; D001550:Benzamidines; D002241:Carbohydrates; D006023:Glycoproteins; D007106:Immune Sera; D007527:Isoenzymes; D012967:Sodium Dodecyl Sulfate; D010450:Endopeptidases; D004765:Enteropeptidase