Facile solubilization of tumour-associated cathepsin B by acid treatment.

Cathcpsin B is a lysosomal thiol proteinase that is thought to play an important role in the destruction of the extracellular matrix and in the invasivc process of metastatic tumour cells In this study, we have investigated the cell surface expression of a cathepsin 9-like enzyme found in association with the plasma membrane of human breast tumour cells (MDA. MB. 436). These cells were grown in serum-free medium for 48 h and washed with phosphate-buffered saline. After this, the cathepsin B-like enzyme was fully dissociated by brief acid treatment [ l ] : incubation of a cell monolayer in a glycine/ HCI buffer, pH 3.0, for approximately 3 min at room temperature and neutralization t o an optimum pH 6.4 with Hcpes buffer (pH 7.5). The absence of 0-glucuronidase (a lysosomal marker enzyme) in this acid wash indicated that there was no leakage of lysosomal contents. In addition, cell viability studies demonstrated that 99% o f the tumour cells were still intact after acidification. A specific fluorometric substrate for cathepsin B, benzyloxycarbonyl-arginylarginyl-7-amino-4-methylcoumarin (Z-Arg-Arg-A MC), was used to detect activity in the acid wash 121. The rate of hydrolysis of the substrate was determined by measuring the rate of increase of fluorescence at 455 nm (excitation at 383 nm). The concentration of the product being obtained by reference to a 1 p~ solution of 7-amino-4-methylcoumarin. The rate of turnover of this substrate, found in the acid wash, was 4 x lo-' M/min per 1.0 x 1 Os cells. Substrate hydrolysis was inhibited by Z-Phe-Ala-diazomethylketone, which selectively inhibits cysteine but not serine proteinases [3].