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AD+-Dependent Glutamate Dehydrogenase Which Is Subject to Allosteric Regulation

Chung-Dar Lu and
Ahmed T. Abdelal*

+ Author Affiliations

Department of Biology, Georgia StateUniversity, Atlanta, Georgia 30303

ABSTRACT

The NAD+-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence wasdetermined. This sequence information was used in identifying and cloning the encodinggdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the nativeNAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptidesfrom Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, andCaulobacter crescentus. Amoderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) fromSaccharomyces cerevisiae or Neurospora crassa. Comparison withthe yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and forcatalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from anarginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a...
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