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 Mr 190,000 by HPLC gel filtration and Mr 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 mM diisopropyl fluorophosphate and Diprotin A. The pI 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, beta-casomorphin, and their related peptides. A stoichiometric reaction of the enzyme on substance P was observed. The epidermal DPP IV had a Km of 0.3 mM and a kcat of 50.3 s-1 for substance P and the Km value decreased by shortening the peptide from the carboxyl-terminal amino acids. The enzyme hydrolyzed human and bovine beta-casomorphin with Km values of 0.025 and 0.05 mM, respectively. Shortening the bovine beta-casomorphin peptide chain did not affect enzyme affinity.
Kikuchi
M
M
Department of Dermatology, University of California, San Francisco 94143-0536.
Fukuyama
K
K
Epstein
W L
WL
D000818:Animals; D002417:Cattle; D002454:Cell Differentiation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000302:isolation & purification*; D004723:Endorphins / Q000032:analysis; D004817:Epidermis / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D012995:Solubility; D013373:Substance P / Q000032:analysis
D003902:Detergents; D004723:Endorphins; D010446:Peptide Fragments; D013373:Substance P; C021968:beta-casomorphins; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4