Purification of dipeptidyl-aminopeptidase IV from human kidney by anti dipeptidyl-aminopeptidase IV affinity chromatography.
Dipeptidyl-aminopeptidase IV (DAP-IV) was purified 850 fold with a yield of 16.5% from human kidney cortex. Only two chromatographic procedures of DEAE-cellulose and anti DAP-IV Sepharose were used. The purified enzyme was shown to be homogeneous by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Among various substrates with the structure of X-Y-p-nitroanilide, Lys-Pro-Gly-Pro- and Arg-Pro-p-nitroanilides were hydrolyzed very fast by DAP-IV. Optimum pH of DAP-IV in human kidney was pH 8.7, Km value for Gly-Pro-pNA was 2.51 +/- 0.01 x 10(-4) M and V max was 66.6 +/- 0.7 mumol/min/mg protein. Diisopropylfluorophosphate completely inhibited DAP-IV at 0.1 mM. However, the enzyme was almost unaffected by N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, phenylmethylsulfonylfluoride, and several metals. The amino acid composition of DAP-IV purified from human kidney was similar to that of DAP-IV purified from pig kidney, liver and intestine. These results indicate that the properties of DAP-IV purified from human kidney are almost same as those from pig kidney.
D000906:Antibodies; D002846:Chromatography, Affinity; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000276:immunology / Q000302:isolation & purification*; D010450:Endopeptidases / Q000302:isolation & purification*; D006801:Humans; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D007672:Kidney Cortex / Q000201:enzymology*
D000906:Antibodies; D010450:Endopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases