Purification of dipeptidyl-aminopeptidase IV from human kidney by anti dipeptidyl-aminopeptidase IV affinity chromatography

Summary

Dipeptidyl-aminopeptidase IV (DAP-IV) was purified 850 fold with a yield of 16.5% from human kidney cortex. Only two chromatographic procedures of DEAF-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 × 10⁴ M and Vmax was 66.6 ± 0.7 μmol/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.