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Figure 1 | BMC Biochemistry

Figure 1

From: Biochemical characterization of Cdk2-Speedy/Ringo A2

Figure 1

Characterization of [unP]Cdk2, [pT160]Cdk2, Speedy/Ringo A2, and K2A2coexp. (A) Activation of [unP]Cdk2 and [pT160]Cdk2 by cyclin A. 0.05 μg of [unP]Cdk2 or [pT160]Cdk2 was preincubated with the indicated amounts of cyclin A prior to determination of its histone H1 kinase activity. (B) Activation of [unP]Cdk2 and [pT160]Cdk2 by Speedy/Ringo A2. 0.5 μg of [unP]Cdk2 or [pT160]Cdk2 was preincubated with the indicated amounts of GST-Speedy/Ringo A2 prior to determination of its histone H1 kinase activity. (C) Gel filtration analysis of K2A2coexp. Cdk2 and Speedy/Ringo A2-His6 were coexpressed in E. coli and purified on a metal affinity column. The purified K2A2coexp was loaded on a Superdex-200 column; one-ml fractions were collected. Proteins from 20 μl of the input (lane L) or from 40 μl of fractions 9–21 were resolved by SDS-PAGE, transferred to a PVDF membrane, and detected with a Cdk2-specific antibody (lower panel), or by staining with Coomassie Brilliant Blue R-250 (upper panel). (D) Time course of ATPase activity of [pT160]Cdk2-cyclin A and K2A2coexp. 16.7 μM of [pT160]Cdk2-cyclin A or K2A2coexp was incubated with [γ-32P]ATP for the indicated times. Samples were chromatographed to resolve 32Pi from [γ-32P]ATP. The rate of ATP hydrolysis was quantitated by phosphorimaging analysis. (E) The relative ATPase activities of [pT160]Cdk2-cyclin A and K2A2coexp were calculated from the slopes in panel D. The ATPase activity of [pT160]Cdk2-cyclin A was set to 100%.

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