Soluble dipeptidyl peptidase IV from terminal differentiated rat epidermal cells: Purification and its activity on synthetic and natural peptides

In terminally differentiated epidermal cells dipeptidyl peptidase IV (EC 3.4.14.5) (DPP IV) is present mainly in a soluble form. We purified the enzyme from 2-day-old rat cornified cells to homogeneity by Sephadex G-200 and Mono-Q column chromatography and finally HPLC gel filtration on G3000SW. The enzyme was estimated to be M r 190,000 by HPLC gel filtration and M r 90,000 by sodium dodecyl sulfate-electrophoresis. The enzyme showed general properties reported for detergent-solubilized DPP IV from other tissues. It was Con A binding and almost completely inhibited by 1 m m diisopropyl fluorophosphate and Diprotin A. The p I was 5.6 and the pH optimum was 7.5. The specific activity for Gly-Pro- p-nitroanilide was 31.9 units/mg. HPLC analysis demonstrated the release of dipeptides of the N-terminal of substance P, β-casomorphin, and their related peptides. A stoichiometric reaction of the enzyme on substance P was observed. The epidermal DPP IV had a K m of 0.3 m m and a k cat of 50.3 s −1 for substance P and the K m value decreased by shortening the peptide from the carboxyl-terminal amino acids. The enzyme hydrolyzed human and bovine β-casomorphin with K m values of 0.025 and 0.05 m m, respectively. Shortening the bovine β-casomorphin peptide chain did not affect enzyme affinity.