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Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase

Lindbladh, C; Rault, M; Hagglund, C; Small, W C; Mosbach, K LU ; Bülow, L LU ; Evans, C and Srere, P A (1994) In Biochemistry 33(39). p.8-11692
Abstract

We have expressed the DNA of the fusion of CS1 to MDH1 in Escherichia coli gltA-. The fusion protein (CS1/MDH1) is the C-terminus of CS1 linked in-frame to the N-terminus of MDH1 with a short linker of glycyl-seryl-glycyl. The fusion protein produced was isolated and purified. Gel filtration studies indicated that CS1/MDH1 had a M(r) of approximately 170,000. Western blotting analysis with SDS gel indicated a M(r) of approximately 90,000-95,000 (theoretical M(r) = 87,000). This is the expected M(r) for the fusion protein subunit. The kinetics of CS1 and MDH1 activities of the fusion protein were compared to those of the free enzymes. In addition, the effect of AAT reaction, as a competitor for the intermediate OAA of the coupled MDH-CS... (More)

We have expressed the DNA of the fusion of CS1 to MDH1 in Escherichia coli gltA-. The fusion protein (CS1/MDH1) is the C-terminus of CS1 linked in-frame to the N-terminus of MDH1 with a short linker of glycyl-seryl-glycyl. The fusion protein produced was isolated and purified. Gel filtration studies indicated that CS1/MDH1 had a M(r) of approximately 170,000. Western blotting analysis with SDS gel indicated a M(r) of approximately 90,000-95,000 (theoretical M(r) = 87,000). This is the expected M(r) for the fusion protein subunit. The kinetics of CS1 and MDH1 activities of the fusion protein were compared to those of the free enzymes. In addition, the effect of AAT reaction, as a competitor for the intermediate OAA of the coupled MDH-CS reaction, was examined. It was observed that AAT was a less effective competitor for OAA when the CS1/MDH1 fusion protein is used than when the separate enzymes are employed. In addition, the transient time for the coupled reaction sequence was less for the fusion protein than for the free enzymes.

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published
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keywords
Aspartate Aminotransferases, Base Sequence, Citrate (si)-Synthase, Escherichia coli, Genes, Fungal, Malate Dehydrogenase, Malates, Mitochondria, Models, Molecular, Molecular Sequence Data, Multienzyme Complexes, Oxaloacetates, Recombinant Fusion Proteins, Saccharomyces cerevisiae
in
Biochemistry
volume
33
issue
39
pages
7 pages
publisher
The American Chemical Society
external identifiers
  • scopus:0028151319
ISSN
0006-2960
DOI
10.1021/bi00205a004
language
English
LU publication?
yes
id
14386c3b-3474-47ef-8421-1d1665ebd8ae
date added to LUP
2016-04-18 16:03:11
date last changed
2017-07-30 05:09:51
@article{14386c3b-3474-47ef-8421-1d1665ebd8ae,
  abstract     = {<p>We have expressed the DNA of the fusion of CS1 to MDH1 in Escherichia coli gltA-. The fusion protein (CS1/MDH1) is the C-terminus of CS1 linked in-frame to the N-terminus of MDH1 with a short linker of glycyl-seryl-glycyl. The fusion protein produced was isolated and purified. Gel filtration studies indicated that CS1/MDH1 had a M(r) of approximately 170,000. Western blotting analysis with SDS gel indicated a M(r) of approximately 90,000-95,000 (theoretical M(r) = 87,000). This is the expected M(r) for the fusion protein subunit. The kinetics of CS1 and MDH1 activities of the fusion protein were compared to those of the free enzymes. In addition, the effect of AAT reaction, as a competitor for the intermediate OAA of the coupled MDH-CS reaction, was examined. It was observed that AAT was a less effective competitor for OAA when the CS1/MDH1 fusion protein is used than when the separate enzymes are employed. In addition, the transient time for the coupled reaction sequence was less for the fusion protein than for the free enzymes.</p>},
  author       = {Lindbladh, C and Rault, M and Hagglund, C and Small, W C and Mosbach, K and Bülow, L and Evans, C and Srere, P A},
  issn         = {0006-2960},
  keyword      = {Aspartate Aminotransferases,Base Sequence,Citrate (si)-Synthase,Escherichia coli,Genes, Fungal,Malate Dehydrogenase,Malates,Mitochondria,Models, Molecular,Molecular Sequence Data,Multienzyme Complexes,Oxaloacetates,Recombinant Fusion Proteins,Saccharomyces cerevisiae},
  language     = {eng},
  month        = {10},
  number       = {39},
  pages        = {8--11692},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase},
  url          = {http://dx.doi.org/10.1021/bi00205a004},
  volume       = {33},
  year         = {1994},
}