Structural studies on microvillus aminopeptidase from pig small intestine.
Two amphiphilic forms of intestinal microvillus aminopeptidase were obtained by purification of the enzyme in the absence and in the presence of aprotinin respectively. By subsequent treatment with trypsin in vitro a hydrophilic form was obtained. These three enzyme preparations all contained three subunits. A, B and C (molecular weight 168000, 11800 and 54000 respectively). The individual subunits were purified by gel filtration on Ultrogel AcA 22 in the presence of sodium dodecyl sulphate. Subunits A and B from aminopeptidase prepared in the presence of aprotinin contained both N-terminal lysine and alanine, whereas N-terminal alanine and glycine were found for both of these subunits from the other amphiphilic form. Conversion of amphiphilic aminopeptidase to the hydrophilic form in vitro led only to a change in the N terminus of the B subunit to a valine. N-terminal serine was found in subunit C from all three forms of the aminopeptidase. The amino acid composition and peptide maps of the isolated subunits from one of the amphiphilic forms are reported. It is suggested that subunit B and C arise by cleavage of subunit A, whereas it seemed unlikely that C originates from B. Brush border membrane vesicles were cross-linked by dimethyl-3,3-dithiobispropionimidate, and the aminopeptidase subsequently isolated. Analysis of molecular weights and subunit composition of its constituent polypeptide complexes by two-dimensional polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulphate showed that AB, BBC, BCC, BB, BC and CC complexes had been formed. The subunit structures ABC and BBCC were thus proposed in agreement with the maximum molecular weight of the membrane-bound enzyme previously determined as 330000.U
D000596:Amino Acids / Q000302:isolation & purification; D000626:Aminopeptidases / Q000302:isolation & purification*; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents; D007096:Imidoesters; D007413:Intestinal Mucosa / Q000201:enzymology*; D007421:Intestine, Small / Q000201:enzymology*; D008871:Microvilli / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D013552:Swine
D000596:Amino Acids; D003432:Cross-Linking Reagents; D007096:Imidoesters; D010446:Peptide Fragments; C007471:dimethyl dithiobispropionimidate; D000626:Aminopeptidases