The effects of protease I of Aspergillus oryzae (brinase) on membrane permeability and growth of landschütz ascites tumour cells

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Abstract
Landschütz ascites tumour cells treated in vitro with Protease I showed no changes in intracellular K and Na levels when the cells were analysed directly at the end of the 60‐min incubation period. If, however, the cells were washed twice in the saline medium before analysis significant losses of K, apparently in exchange for Na, were found to take place. Controls incubated in the absence of Protease I and similarly treated showed no such K loss. Viability tests, using lissamine green, indicated that the observed changes were not due to dead cells. These findings are interpreted as being due to an alteration in the plasma membrane associated with increased sensitivity to mechanical trauma, which in turn leads to secondary loss of K. Increases in UV absorbance in the 260 nm region were noted in the supernatant medium of treated cells.Normal mice weighing 30 g tolerated intraperitoneal injections of 0.3 mg Protease I and under, in repeated daily doses, but a single injection of 0.4 (i.e. 13 mg/kg) was lethal within 2 hours. The presence of tumour cells (7.5 mg dry wt) intraperitoneally was found to protect mice against repeated daily injections of this normally lethal dose, indicating that the enzyme is quickly adsorbed onto the tumour cells. No effect on tumour growth in vivo was found when single injections of Protease I were administered intraperitoneally at the same time as the tumour, but when additional doses of the enzyme were given on the two subsequent days, the 7‐day yield of tumour cells was significantly increased.