1. dATP hydrolysis is only required for a short time in Apaf‐1‐mediated procaspase‐9 activation.
dATP hydrolysis is only required for a short time in Apaf‐1‐mediated procaspase‐9 activation. Two micrograms of Apaf‐1‐containing extract was mixed with 1 μl [35S]methionine‐labeled procaspase‐9 on ice in the presence of 0.1 mM dATP and 0.2 μg of cytochrome c. One millimolar ATP‐γS was added to the reaction mixture at different time intervals after the tubes containing reaction mixtures were transferred into 30°C waterbath. The reaction was incubated for a total of 30 min at 30°C. (A) Procaspase‐9 activation profile in the presence of ATP‐γS when added at different times after the beginning of the reactions. (B) Time course of inhibition of procaspase‐9 processing by ATP‐γS. The amount of unprocessed and processed caspase‐9 (p35) forms were scanned and quantitated using a phosphoimager. First, the amount of p35 processed forms was normalized to full‐length procaspase‐9 and added to the corresponding unprocessed procaspase‐9 to obtain the total amount of procaspase‐9 used in each lane. Then, p35 bands were normalized relative to total procaspase‐9 in each lane. Finally, normalized p35 fragments were divided by the p35 fragment without ATP‐γS (positive control).
Yuanming Hu et al. EMBO J. 1999;18:
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