Figure 1.

Purification and adenylyltransferase activity. (A) Purification of MthRnl. Aliquots (10 µl) of the Ni–agarose preparation of His-tagged MthRnl were resolved by SDS–PAGE followed by Coomassie blue staining. The MthRnl polypeptide is denoted by an arrow. The positions and sizes (kilodalton) of marker polypeptides are indicated on the left. (B) Adenylyltransferase activity. Aliquots (1 µl) of indicated Ni–agarose fractions were assayed for adenylyltransferase activity. A ³²P-labeled ligase–AMP intermediate, denoted by an asterisk, was visualized by autoradiography of the dried gel. (C) Sedimentation analysis. MthRnl was centrifuged in a 15–30% glycerol gradient as described in Materials and methods section. Aliquots (1 µl) of the gradient fractions indicated were assayed for adenylyltransferase activity, gauged by the signal intensity of the radiolabeled MthRnl polypeptide (PSL, photo stimulatable luminescence). The peaks of the marker proteins, catalase (11.3 S), BSA (4.3 S) and cytochrome c (1.8 S), are indicated.