[Purification and characterization of cytosol aminopeptidase from rat intestinal mucosa].
The isolation of cytosol aminopeptidase (CAP) from intestinal mucosa has involved difficult procedures because of low yield, instability of the enzyme and many other factors related to purification. The authors resolved these difficulties in the purification of the enzyme from intestinal mucosa of rats using hydrophobic chromatography on phenyl-TOYOPEARL 650S and ion exchange HPLC on Mono Q 5/5. In this procedure, a 60-fold purification was achieved and the purified enzyme revealed a single band with 56kDa molecular weight by SDS polyacrylamide gel electrophoresis. The purified enzyme preferentially hydrolyzed the Leucyl-containing peptides of Leu-Gly and Leu-Gly-Gly. However, it showed no activity on synthetic substrates of Leu-beta-naphthylamide, Gly-p-nitroanilide, Phe-p-nitroanilide or Met-beta-naphthylamide. The Km and Vmax for Leu-Gly were 0.91 mmol/l and 16.4 mol/min/mg protein, while for Leu-Gly-Gly they were 0.77 mmol/l and 20.6 mol/min/mg protein. The purified enzyme was heat-labile and quickly became less active in highly concentrated ammonium sulfate. The optimum pH was 6.5-10. The enzyme activity was stimulated by Mn2+ and Mg2+, while it was inhibited by EDTA, 1,10-phenanthroline, 2-mercaptoethanol and p-hydroxymercuribenzoate. The results suggested the participation of metal ions and the SH group in the enzyme activity. Furthermore the cytosol aminopeptidase was distinct from the brush-border membrane aminopeptidase in molecular weight, immunoreaction to cytosol aminopeptidase antiserum and the specificities to the substrates.
Furui
T
T
Department of Pediatrics, Nippon Medical School, Japan.
Hisayasu
S
S
D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology*; D007413:Intestinal Mucosa / Q000201:enzymology*; D007421:Intestine, Small / Q000201:enzymology; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D000626:Aminopeptidases