Large scale purification, nucleotide binding properties, and ATPase activity of the MalK subunit of Salmonella typhimurium maltose transport complex.
The malK gene, encoding a membrane-associated component of the maltose transport complex of Salmonella typhimurium was cloned into an expression vector downstream of the promoters lambda pR and lambda pL and a strong translation initiation region. Escherichia coli strain JM109 harboring the resulting plasmid pCW14 synthesized a protein of apparent molecular mass of 43 kDa upon temperature shift, as demonstrated by sodium dodecyl sulfate-gel electrophoresis. The identity of the protein was determined by N-terminal amino acid sequencing. The overproduced protein was sequestered in inclusion bodies as revealed by electron microscopy. The protein was purified to homogeneity on a large scale by disrupting the cells with a passage through a Ribi press, solubilizing the inclusion bodies with urea, and subsequent chromatography on Red Agarose. Purified MalK, as the membrane-bound MalK protein could be covalently modified by [gamma-32P]8-azido-ATP. Furthermore, the purified protein bound [gamma-32P] ATP with a dissociation constant of 150 microM and exhibited ATPase activity, which was stimulated by dimethyl-sulfoxide and inhibited by ADP.
Walter
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Universität Osnabrück, Fachbereich Biologie/Chemie, Arbeitsgruppe Mikrobiologie, Federal Republic of Germany.
Höner zu Bentrup
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Schneider
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D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008320:Maltose / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012486:Salmonella typhimurium / Q000378:metabolism*; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; C489399:MalK protein, Salmonella typhimurium; D009711:Nucleotides; D008320:Maltose; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases