FIGURE 6.

Thin layer chromatographic analysis of various BirA proteins to synthesize Bio-5′-AMP and transfer the biotin moiety to AccB-87. Synthesis of biotinoyl-adenylate in reaction mixtures containing 1 μm BirA, 20 μm biotin, 5 μm ATP, 5 mm MgCl₂, 100 mm KCl, 5 mm TCEP, and 16.5 nm [α-³²P]ATP is shown. Each reaction was run in duplicate and incubated for 30 min at 37 °C, after which time point plus signs (+) purified apo-AccB-87 (50 μm) was added to one of the duplicate reactions followed by incubation for an additional 15 min. The second duplicate reaction did not receive AccB-87 (minus signs). One μl of each reaction was spotted onto a cellulose thin layer chromatography plate. The right panels with 10b BirA mutant show a separate experiment. Following plate development, the reaction products (indicated by arrows), biotinoyl-5′-AMP (Bio-AMP), ADP, AMP, and the remaining ATP were visualized by autoradiography. NE denotes the control reactions lacking BirA. The proteins assayed are given at the top of the figure, and the identities of the spots are on the left margin. A and B show the products of the BirA reaction in the presence or absence of the AccB-87 acceptor protein. The identity of each protein is given at the top of each pair of lanes. The wild type (WT) and Δwing proteins were included in both analyses as controls.