Purification and characterization of bradykinin-hydrolyzing enzyme from 2-day-old rat epidermis.
Bradykinin-hydrolyzing enzyme was purified 200-fold from a soluble fraction of cornified cells from 2-day-old rat epidermis. The enzyme has an Mr of 80,000 as identified by SDS polyacrylamide gel electrophoresis and HPLC gel filtration. The isoelectric point of the enzyme is 5.05. The enzyme hydrolyzed Phe5-Ser6 of bradykinin and seven bradykinin-related peptides, and Tyr5-Ser6 of Tyr5-bradykinin. Production of bradykinin fragments, Arg-Pro-Pro-Gly-Phe and Ser-Pro-Phe-Arg, proceeded in a stoichiometric fashion. Km and Vmax values for bradykinin were 33 microM and 22.2 mumol/min per mg, respectively. The enzyme did not hydrolyze azocasein, denatured hemoglobin or synthetic substrates for other epidermal proteinases. The enzyme activity was enhanced by reducing agents and inhibited by sulfhydryl-blocking agents and divalent cations. Diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride had no effects. The enzyme has a pH optimum of 7.0-7.5 and is stable at 4 degrees C for 1 month, but loses activity completely at 60 degrees C for 10 min. The epidermal endopeptidase differs in several properties from endooligopeptidase A purified from brain which hydrolyzes Phe5-Ser6 of bradykinin.
Kikuchi
M
M
Department of Dermatology, University of California, San Francisco 94143-0536.
Fukuyama
K
K
Epstein
W L
WL
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism; D001920:Bradykinin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004817:Epidermis / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D015853:Cysteine Proteinase Inhibitors; D003546:Cysteine Endopeptidases; C055858:bradykinin hydrolyzing enzyme, epidermal; D001920:Bradykinin