Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase
(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.
(Less)
- author
- Lindbladh, C ; Rault, M ; Hagglund, C ; Small, W C ; Mosbach, K LU ; Bülow, L LU ; Evans, C and Srere, P A
- organization
- publishing date
- 1994-10-04
- type
- Contribution to journal
- publication status
- published
- subject
- 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 (ACS)
- external identifiers
-
- pmid:7918385
- 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
- 2024-02-18 14:23:12
@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}},
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}},
language = {{eng}},
month = {{10}},
number = {{39}},
pages = {{8--11692}},
publisher = {{The American Chemical Society (ACS)}},
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}},
doi = {{10.1021/bi00205a004}},
volume = {{33}},
year = {{1994}},
}