FIGURE 4.

Kinetic assays used to determine Michaelis-Menten constants for HCS-catalyzed biotin transfer with respect to p67, biotin, and ATP. A, incorporation of [¹⁴C]biotin into p67 was used to determine the kinetic constants with respect to p67 and ATP. The biotinylated p67 and free biotin are separated on a 16% acrylamide gel. B, biotin incorporation versus time at 150 nm 58-HCS, 50 μm p67, 10 μm [¹⁴C]biotin, and a range of ATP concentrations as follows: (■, 5 μm; ▴, 30 μm; ▾, 90 μm; ♦, 250 μm; ●, 500 μm; □, 750 μm; and ▵, 1000 μm ATP. C, incorporation of [³H]biotin into p67 used to determine the kinetic constants with respect to biotin. Biotinylated p67 is separated from unincorporated biotin by filtration through a nitrocellulose membrane. D, initial rates of biotin incorporated measured at 5 nm FL-HCS, 150 μm p67, 16.67 nm [³H]biotin and variable unlabeled biotin concentrations as follows: ▾, 20 μm; ■, 10 μm; ♦, 7 μm; ●, 3 μm; and ▴, 1 μm biotin. The inset depicts the traces obtained for the following: ▿, 400 nm; Δ, 150 nm; and 50 nm total biotin. B and D, the lines were obtained from linear regression of the measured biotin incorporation versus time.