Purification of bovine cathepsin B: proteomic characterization of the different forms and production of specific antibodies.
Cathepsin B (EC 3.4.22.1) has been highly purified (14,225 fold) from bovine kidney by a rapid procedure that included the preparation of an enriched lysosomal extract, a selective fractionation with ammonium sulphate, size-exclusion chromatography, two cation-exchange chromatographies, and anion-exchange chromatography on diethylaminoethyl-Sephacel. After the last purification step, two hydrolytic peaks against Z-Phe-Arg-AMC were separated from each other, a minor peak corresponding to the cathepsin B single-chain form and a major one representing the double-chain form of cathepsin B. The single-chain form showed a molecular mass of 31 kDa on sodium dodecyl sulphate - polyacrylamide gel electrphoresis (PAGE) under reducing conditions, whereas the heavy chain of the double-chain form appeared as a doublet with molecular masses of 23.4 and 25 kDa, respectively. The identity of the different cathepsin B isoforms and the quality of the final enzyme preparation were confirmed by using two types of antibodies, one against a synthetic peptide sequence and one against purified cathepsin B. The proteomic study confirmed the identity of the different SDS-PAGE protein bands as cathepsin B isoforms and allowed the comparison and study of some structural differences between them at the level of their primary structures.
Sentandreu
M A
MA
Muscle Biochemistry Group, SRV, INRA-Theix, 63122 Saint Genès Champanelle, France.
Aubry
L
L
Ouali
A
A
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002401:Cathepsin B / Q000737:chemistry / Q000302:isolation & purification*; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007668:Kidney / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D020033:Protein Isoforms; D002401:Cathepsin B