FIG. 3.

Mammalian cell-derived recombinant Monarch-1ΔLRR binds and hydrolyzes ATP. (A) Diagram depicting full-length Monarch-1 and the WT and mutA/B Monarch-1ΔLRR proteins produced in HEK293EBNA cells. (B) Soluble extracts of HEK293EBNA cells expressing WT and mutA/B Monarch-1ΔLRR were enriched for Monarch-1ΔLRR using a cobalt metal affinity column (lanes 1 and 2) and further purified over an anti-Flag affinity matrix (lanes 3 and 4). The purity was evaluated by Coomassie blue staining. The sizes of molecular weight markers (in thousands) are indicated on the left. (C) The double-purified Monarch-1ΔLRR proteins were analyzed by Western blotting using anti-His and anti-Flag antibodies. (D) ATP binding activity of purified WT and mutA/B Monarch-1ΔLRR was determined by incubating 500 ng purified protein with [γ-³⁵S]ATP. Error bars represent the standard deviations of ATP binding measurements in triplicate. (E) The ATP binding affinity of WT Monarch-1ΔLRR was determined by homologous competition binding assays. (F) The nucleotide binding preference of WT Monarch-1ΔLRR was determined by incubating WT Monarch-1ΔLRR with [γ-³⁵S]ATP and increasing concentrations of unlabeled nucleotide. Significance of the differences from control samples (nucleotide concentrations of 10⁻⁹ M) was estimated by Student's t test. A double asterisk signifies a P value of <0.0005. A single asterisk signifies a P value of <0.02. (G) The ATPase activity of purified WT or mutA/B Monarch-1ΔLRR was measured by visualizing the conversion of [α-³²P]ATP to [α-³²P]ADP by TLC. HEK293EBNA lysate and purified bovine serum albumin (BSA) were used as positive and negative controls, respectively. Arrowhead, Monarch-1-NBD fusion protein.