Identification and partial purification of DnaK homologue from extremely halophilic archaebacteria, Halobacterium cutirubrum.
The levels of synthesis of six proteins were increased at elevated growth temperature of the extremely halophilic archaebacterium Halobacterium cutirubrum. One of these proteins, with an apparent molecular mass of 97 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), bound to an ATP-agarose column in the presence of 4 M NaCl, but not in the absence of salt, indicating that this protein retained its ATP-binding activity only at high salt concentration. The NH2-terminal sequence of this protein and the internal sequences of the tryptic peptides covering 1/3 of the total number of residues coincided with that deduced from the nucleotide sequence of the dnaK gene isolated from H. cutirubrum. The results strongly suggest that this apparent 97-kDa protein is the gene product of dnaK, although the molecular mass calculated from the nucleotide sequence is only 68,495, much smaller than the value of this protein determined by SDS-PAGE. Ferguson plot analysis indicated that this protein showed anomalous mobility on SDS-PAGE. We have purified DnaK homologue to greater than 90% homogeneity with stepwise elution from an ATP-agarose column.
Tokunaga
H
H
Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, Japan.
Hara
S
S
Arakawa
T
T
Ishibashi
M
M
Gupta
R S
RS
Tokunaga
M
M
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002847:Chromatography, Agarose / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006217:Halobacterium / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism
D019843:Archaeal Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D014357:Trypsin; C063842:dnaK protein, E coli