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

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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; Bülow, L; Evans, C; Srere, P A.

In: Biochemistry, Vol. 33, No. 39, 04.10.1994, p. 11692-8.

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Lindbladh, C ; Rault, M ; Hagglund, C ; Small, W C ; Mosbach, K ; Bülow, L ; Evans, C ; Srere, P A. / Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase. In: Biochemistry. 1994 ; Vol. 33, No. 39. pp. 11692-8.

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TY - JOUR

T1 - Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase

AU - Lindbladh, C

AU - Rault, M

AU - Hagglund, C

AU - Small, W C

AU - Mosbach, K

AU - Bülow, L

AU - Evans, C

AU - Srere, P A

PY - 1994/10/4

Y1 - 1994/10/4

N2 - 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.

AB - 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.

KW - Aspartate Aminotransferases

KW - Base Sequence

KW - Citrate (si)-Synthase

KW - Escherichia coli

KW - Genes, Fungal

KW - Malate Dehydrogenase

KW - Malates

KW - Mitochondria

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Multienzyme Complexes

KW - Oxaloacetates

KW - Recombinant Fusion Proteins

KW - Saccharomyces cerevisiae

U2 - 10.1021/bi00205a004

DO - 10.1021/bi00205a004

M3 - Article

C2 - 7918385

VL - 33

SP - 11692

EP - 11698

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 39

ER -