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Fig. 5 | BMC Biotechnology

Fig. 5

From: High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Fig. 5

Steady-state kinetics for cleavage of pre-let-7a-1 by Dicer. (a) Typical Dicer cleavage reaction of 5′-[32P]-labeled pre-let-7a-1 with Dicer analyzed by denaturing gel electrophoresis. Reactions were carried for 30 min in conditions that allowed less than 10% of total substrate cleavage. (b) Normalized product formation ([let-7a-1]/[Dicer]) as a function of time for cleavage data shown in (a). The turnover frequency, expressed as vo/[E]t, was calculated by linear regression of the slope (5.8 ± 0.3 min− 1 and 6.1 ± 0.5 min− 1 for pre-let-7a-1 substrate concentration of 5.1 μM and 7.7 μM, respectively). (c) Typical steady-state kinetic analysis showing the dependence of the turnover frequency on substrate concentration (0.08 to 7.68 μM). The data shown here were fitted to the Michealis-Menten equation to yield kcat of 6.8 ± 0.8 min− 1 and KM of 1.0 ± 0.2 μM. Averages from two independent experiments yield kcat of 7.2 ± 0.5 min− 1 and KM of 1.2 ± 0.3 μM

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