Molecular cloning of the dnaK locus, and purification and characterization of a DnaK protein from Bacillus brevis HPD31.
Using part of the dnaK gene from Bacillus subtilis as a probe, a 4. 4-kbp SacI-BglII fragment of chromosomal DNA of Bacillus brevis, a protein-hypersecreting bacterium, was cloned. Nucleotide sequencing revealed 3 open reading frames in the order of grpE-dnaK-dnaJ homologues. We purified DnaK protein to homogeneity from B. brevis HPD31 harboring a multi-copy dnaK expression plasmid. Purified DnaK showed ATPase activity which was synergistically stimulated 14-fold by the addition of glutathione S-transferase-DnaJ and glutathione S-transferase-GrpE fusion proteins. DnaK hydrolyzed not only ATP but also CTP, UTP, and GTP at about 40% of the efficiency of ATP. The specific activity of DnaK-ATPase was 7.25x10-3 unit/mg protein (the turnover number against ATP was 0.47 min-1) under our assay conditions. The DnaK dimers dissociated into monomers on addition of ATP, GTP, CTP, UTP and ATPgammaS, but not ADP or AMP. DnaK formed a stable complex with permanently unfolded carboxymethylated alpha-lactalbumin but not with native alpha-lactalbumin, and this complex was dissociated by addition of ATP/Mg. Formation of this complex was inhibited in the presence of inorganic phosphate.
Tokunaga
H
H
Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890, Japan. tokunaga@chem.agri.kagoshima-u.ac.jp
Yamakawa
M
M
Mizukami
M
M
Takagi
H
H
Tokunaga
M
M
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D029968:Escherichia coli Proteins; D005982:Glutathione Transferase / Q000235:genetics; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D006360:Heat-Shock Proteins / Q000494:pharmacology; D007768:Lactalbumin / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D001426:Bacterial Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009711:Nucleotides; D010710:Phosphates; D011993:Recombinant Fusion Proteins; D007768:Lactalbumin; D005982:Glutathione Transferase; D010450:Endopeptidases; D000251:Adenosine Triphosphatases; C115251:DnaK protein, Brevibacillus choshinensis; C063842:dnaK protein, E coli