Purification and properties of leucine beta-naphthylamidase from rabbit small-intestinal mucosal cells.
Leucine beta-naphthylamidase associated with the microvilli membranes of rabbit small intestine was solubilized with papain [EC 3.4.22.2] and purified by Sephadex G-200 gel filtration, DEAE-cellulose column chromatography, passage through a column of Sepharose 4B coupled with anti-sucrase antibodies and preparative disc electrophoresis in polyacrylamide gel. The purified enzyme was homogeneous on ultracentrifugation and disc electrophoresis, but a double immunodiffusion test showed the presence of a minor component which was probably denatured enzyme. The molecular weight of the purified enzyme was estimated to be 225,000 by Sephadex G-200 gel filtration and the sedimentation coefficient (S-0-20, w) was found to be 6.90S. Purified enzyme required bovine serum albumin for maximal activity, perhaps for its protection from autodigestion. It hydrolyzed, in addition to L-leucine beta-naphthylamide, various L-amino acid beta-naphthylamides and dipeptides with a free alpha-amino group, but did not hydrolyze benzoyl-L-arginine beta-naphthylamide. Therefore, the purified enzyme is an aminopeptidase. Hg-2+ and Cu-2+ ions strongly inhibited the enzyme activity, but other metal ions and EDTA showed no or only slight effect. N-Ethylmaleimide exhibited a weak inhibition. Purified enzyme had an optimal pH and Km value for leucine beta-naphthylamide similar to those of enzymes from other sources. Antibodies against the purified enzyme were raised in guinea pigs. The antibodies obtained were found by double immunodiffusion to be specific for the enzyme. They precipitated the enzyme quantitatively and partially inhibited the enzyme activity.
D000818:Animals; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D006168:Guinea Pigs / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007413:Intestinal Mucosa / Q000201:enzymology*; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010206:Papain; D011817:Rabbits
D002413:Cations, Divalent; D007931:Leucyl Aminopeptidase; D010206:Papain