Purification and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV.
Dipeptidyl aminopeptidase IV (EC 3.4.14.-) was solubilized from a particulate membrane fraction of rat intestinal mucosa with Triton X-100. The solubilized enzyme was purified to homogeneity following ammonium sulfate fractionation, chromatography on DEAE-Sepharose and hydroxyapatite, gel filtration and preparative polyacrylamide gel electrophoresis. The final enzyme preparation had a specific activity of 55 units/mg protein representing a 1373 fold purification over the starting material. Purity was judged by polyacrylamide gel electrophoresis and double immunodiffusion. The molecular weight of the native undenatured enzyme was estimated to be 230000 by gel filtration and polyacrylamide gel electrophoresis. Electrophoresis under denaturing conditions (sodium dodecyl sulfate) indicated that the protein consists of two identical 98 kDa subunits. Dipeptidyl aminopeptidase IV is a glycoprotein containing approx. 8% carbohydrate by weight. A detailed analysis of the individual sugar components demonstrated that fucose, galactose, glucose, mannose, sialic acid and hexosamine sugars were present. The nature of the constituent asparagine linked oligosaccharide side chains was further examined following cleavage from the peptide backbone by hydrazinolysis. Following high voltage paper electrophoresis approx. 80% of the isolated oligosaccharide was found with the neutral fraction while the remaining 20% consisted of a single acidic component. Gel filtration of the neutral oligosaccharide fraction indicated that it contains approx. 19 sugar residues.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000302:isolation & purification*; D010450:Endopeptidases / Q000302:isolation & purification*; D007413:Intestinal Mucosa / Q000201:enzymology*; D008297:Male; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; D002241:Carbohydrates; D010450:Endopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases