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pubmed:abstractText |
We investigated and characterized the ATP-dependent protease in human erythroleukemia, K562 cells. The succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarinamide hydrolytic activity in a K562 lysate at pH 9 rose more than 10-fold with the addition of 1 mM ATP. The effect of ATP on the protease activity was dose-dependent and inhibited by the addition of ADP. This activity was not inhibited by EDTA, L-3-carboxy-trans-2,3-epoxypropionyl-leucylamide-(4-guanidin o)butane or leupeptin, but was strongly inhibited by chymostatin and diisopropylfluorophosphate. The protease activity was eluted just after the void volume from a G3000SW HPLC column. The above results suggest that this protease is identical to the high-molecular-mass protease, ingensin, previously reported by us. The ATP-dependent increase in the protease activity was due to prevention of the inactivation of the protease by ATP, and not to activation of the protease itself in the reaction mixture at 37 degrees C. The depressed succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarinamide hydrolytic activity in the ATP-depleted lysate was restored to the same level by the detergent, SDS. Therefore, we conclude that the inactivation of ingensin occurring on preincubation is not irreversible.
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