In vivo analysis of the Walker motif mutants of Hda.
A, MK86 [Δhda ΔoriC rnhA] cells
harboring pACYC177 (Vector), pACYC/ntHda (WT),
pACYC/ntHdaA1B1 (A1B1), pACYC/ntHdaB2 (B2), or
pACYC/ntHdaA1B2 (A1B2) were grown at 37 °C in the presence of
[32P]orthophosphate. Nucleotide-bound DnaA protein was
immunochemically isolated from cell lysates, and the recovered nucleotides
were analyzed as described under “Experimental Procedures.”
Proportions (%) of ATP-DnaA included in the total amount of ATP/ADP-DnaA
molecules are shown (ATP/(ADP+ATP)). The origin and
the migration positions of ATP and ADP are indicated. The asterisk
indicates a nonspecific signal, which was seen in the samples prepared using
pre-immune serum. B, experiments shown in A were
independently repeated three times, and the results with standard deviations
are shown. C, expression levels of cells used in this experiment.
Western blotting was performed using the indicated cells
(A600 of 0.15, 67μl). The amount of ntHda was
quantified and deduced using a quantitative standard of purified Hda-cHis
(Deduced amount). ntHda is the nontagged form of Hda. The relative
level of wild-type ntHda was 180-fold increased compared with that of MG1655
(25).