The dnaB gene product of Escherichia coli. I. Purification, homogeneity, and physical properties.
The dnaB gene product was purified to homogeneity and its physical properties were characterized. Purification was aided by the use of the Escherichia coli strain. MV12/28, which overproduced the dnaB gene product 10-fold (Wickner, S. H., Wickner, R. B., and Raetz, C. R. H. (1976) Biochem. Biophys. Res. Commun. 70, 389-396) and by taking advantage of the enzyme's high affinity for both DEAE-cellulose and phosphocellulose. The most highly purified fractions gave a single stained band on native, polyacrylamide gels and dnaB enzymatic activity was coincident with this band. On denaturing sodium dodecyl sulfate-polyacrylamide gels, a single band was observed corresponding to a molecular weight of 48,000 +/- 2,000. The native molecular weight of 290,000 +/- 12,000 was calculated from determinations of the sedimentation coefficient, which was 11.3 S, and the Stokes radius, which was 60 A. Cross-linking the protein with dimethyl suberimidate yielded six bands. We conclude that the enzyme consists of six identical subunits. The apparent pI was 4.9 and the amino acid composition was typical except for the absence of cysteine.
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007526:Isoelectric Point; D008722:Methods; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification
D000596:Amino Acids; D001426:Bacterial Proteins; D010744:Phosphoric Monoester Hydrolases