Figure 2.

PPK does not elongate polyP oligomers to synthesize polyP₇₅₀. Activity was measured in a 70-μl assay mixture. Unlabeled ATP replaced [γ-³²P]ATP, and [³²P]polyP oligomers were added at a final concentration of 75 μM (as P_i residues). At various time intervals, 10-μl samples were removed and the reaction was stopped by addition of 25 mM EDTA. Samples were electrophoresed on a urea/15% polyacrylamide gel, and the gel was exposed to x-ray film. Unlabeled polyP was visualized by toluidene blue staining. (A) Autoradiogram of the polyacrylamide gel. The four lanes on the left represent the indicated standards: Std., polyP₇₅₀ subjected to partial acid hydrolysis. (B) Quantitation of polyP₇₅₀ in the toluidene blue-stained gel. The amount of polyP was estimated by using a standard curve plotted from known amounts of polyP₇₅₀ subjected to laser densitometry after electrophoresis and toluidene blue staining of a polyacrylamide gel.