The identification of active forms of cysteine proteinases in Kirsten-virus-transformed mouse fibroblasts by use of a specific radiolabelled inhibitor.
The major active forms of cathepsins B and L were identified in Kirsten-virus-transformed mouse fibroblasts by the use of a specific radiolabelled inhibitor, benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2. No other proteins were labelled, demonstrating the specificity of this inhibitor for cysteine proteinases. Cathepsins B and L were distinguished by the use of specific antibodies. One active form of cathepsin B, Mr 33,000-35,000, and two active forms of cathepsin L, Mr 30,000 and 23,000, were identified. The intracellular precursors of these proteins had higher Mr values of 39,000 and 36,000 for cathepsins B and L respectively, as shown by pulse-chase experiments with [35S]methionine-labelled proteins. These did not react with the inhibitor under our culture conditions. The precursor of cathepsin L was secreted whereas the precursor of cathepsin B was not, demonstrating that secretions of the two enzymes are regulated differently. In contrast with results found previously for the purified protein [Mason, Gal & Gottesman (1987) Biochem. J. 248, 449-454], the secreted precursor form of cathepsin L did not react with the inhibitor either, indicating that it is not active and therefore, as such, cannot be directly involved in tumour invasion. The secreted protein did react with the inhibitor when incubated at pH 3.0, showing that the protein can be activated, although this did not occur under our culture conditions.
Mason
R W
RW
Department of Biochemistry, Strangeways Research Laboratory, Worts Causeway, Cambridge, U.K.
Wilcox
D
D
Wikstrom
P
P
Shaw
E N
EN
D000345:Affinity Labels; D000818:Animals; D002401:Cathepsin B / Q000032:analysis* / Q000037:antagonists & inhibitors; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis* / Q000037:antagonists & inhibitors; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D003546:Cysteine Endopeptidases; D003978:Diazomethane / Q000031:analogs & derivatives*; D004151:Dipeptides; D010450:Endopeptidases; D005347:Fibroblasts / Q000201:enzymology; D007708:Kirsten murine sarcoma virus; D051379:Mice; D011233:Precipitin Tests; D009053:Sarcoma Viruses, Murine
D000345:Affinity Labels; D004151:Dipeptides; C057752:benzyloxycarbonyltyrosylalanine diazomethane; D003978:Diazomethane; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056668:Cathepsin L; C534278:Ctsl protein, mouse