Chromophoric and fluorophoric peptide substrates cleaved through the dipeptidyl carboxypeptidase activity of cathepsin B.
The action of bovine spleen cathepsin B as a dipeptidyl carboxypeptidase on newly synthesized substrates of the type peptidyl-X-p-nitrophenylalanyl (Phe(NO2))-Y (X,Y = amino acid residue) or 5-dimethylaminonaphthalene-1-sulfonyl (Dns)-peptidyl-X-Phe(NO2)-Y was investigated. The kinetic parameters of hydrolysis of the X-Phe(NO2) bond were determined by difference spectrophotometry (delta epsilon 310 = 1600 M-1 cm-1) or by spectrofluorometry by following the five- to eightfold increase of Dns-group fluorescence with excitation at 350 nm and emission at 535 nm. The substrates were moderately sensitive to cathepsin B; kcat varied from 0.7 to 4 s-1 at pH 5 and 25 degrees C; Km varied from 6 to 240 microM. The very acidic optima of pH 4-5 are characteristic for dipeptidyl carboxypeptidase activity of cathepsin B. Bovine spleen cathepsins S and H had little and no activity, respectively, when assayed with Pro-Glu-Ala-Phe(NO2)-Gly. These peptides should be a valuable tool for routine assays and for mechanistic studies on cathepsin B.
Pohl
J
J
Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague.
Davinic
S
S
Bláha
I
I
Strop
P
P
Kostka
V
V
D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D056655:Cathepsin H; D002403:Cathepsins / Q000378:metabolism; D002863:Chromogenic Compounds / Q000138:chemical synthesis*; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010455:Peptides / Q000138:chemical synthesis*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D010455:Peptides; D002403:Cathepsins; D010450:Endopeptidases; C020071:dipeptidyl carboxypeptidase; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056655:Cathepsin H; C028217:cathepsin S