Simultaneous isolation of human kidney cathepsins B, H, L and C and their characterisation.
A procedure for the simultaneous isolation of four cysteine proteinases, cathepsins B, H, L and C, from human kidney is described. The method includes concentration of the acidified homogenate by ammonium sulphate precipitation. The resuspended and dialysed precipitate was chromatographed on DEAE-cellulose DE-32, to allow separation of cathepsins H and C from cathepsins B and L. The main isoform of cathepsin H was separated from cathepsin C by cation-exchange chromatography on CM-Sephadex C-50. These two enzymes were further purified by covalent chromatography on thiopropyl Sepharose and gel permeation on Sephacryl S-200. The last step allowed separation of cathepsin C and the minor isoform of cathepsin H. Purification of the other two enzymes, cathepsins B and L, was carried out on thiol Sepharose, followed by chromatography on CM-Sepharose C-50. In this step, pure cathepsin L was obtained, while two isoforms of cathepsin B had to be finally purified on Sephacryl S-200 columns. The purity of each enzyme was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, isoelectric focusing on polyacrylamide gels and N-terminal sequencing. The activities of the purified cathepsins B, H and L were determined in terms of kcat/KM for three substrates, Z-Phe-Arg-MCA, Z-Arg-Arg-MCA and Arg-MCA. The method produced 25 mg of cathepsin B, 6.5 mg of cathepsin H, 1.5 mg of cathepsin L and 3.8 mg of cathepsin C from 3.5 kg of human kidney.
Popovic
T
T
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Puizdar
V
V
Ritonja
A
A
Brzin
J
J
D000595:Amino Acid Sequence; D002401:Cathepsin B / Q000737:chemistry / Q000302:isolation & purification*; D020789:Cathepsin C; D056655:Cathepsin H; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry / Q000302:isolation & purification*; D002412:Cations; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006801:Humans; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D017421:Sequence Analysis
D002412:Cations; D010446:Peptide Fragments; D002403:Cathepsins; D010450:Endopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C534126:CTSH protein, human; D056655:Cathepsin H