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

C. Lindbladh, M. Rault, C. Hagglund, W. C. Small, K. Mosbach, L. Bulow, C. Evans, P. Srere

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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 ~170 000. Western blotting analysis with SDS gel indicated a M(r) of ~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.

Original languageEnglish
Pages (from-to)11692-11698
Number of pages7
JournalBiochemistry
Volume33
Issue number39
DOIs
Publication statusPublished - 1994

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Citrate (si)-Synthase
Malate Dehydrogenase
Fungal Proteins
Mitochondrial Proteins
Fusion reactions
Kinetics
Proteins
Enzymes
Protein Subunits
Gels
Gel Chromatography
Western Blotting
Escherichia coli
DNA

ASJC Scopus subject areas

  • Biochemistry

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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.; Bulow, L.; Evans, C.; Srere, P.

In: Biochemistry, Vol. 33, No. 39, 1994, p. 11692-11698.

Research output: Contribution to journalArticle

Lindbladh, C, Rault, M, Hagglund, C, Small, WC, Mosbach, K, Bulow, L, Evans, C & Srere, P 1994, 'Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase', Biochemistry, vol. 33, no. 39, pp. 11692-11698. https://doi.org/10.1021/bi00205a004
Lindbladh, C. ; Rault, M. ; Hagglund, C. ; Small, W. C. ; Mosbach, K. ; Bulow, L. ; Evans, C. ; Srere, P. / Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase. In: Biochemistry. 1994 ; Vol. 33, No. 39. pp. 11692-11698.
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AU - Rault, M.

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AU - Mosbach, K.

AU - Bulow, L.

AU - Evans, C.

AU - Srere, P.

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