Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog
✍ Scribed by Nicholas J. Hoogenraad; Teresa M. Sutherland; G.J. Howlett
- Publisher
- Elsevier Science
- Year
- 1980
- Tongue
- English
- Weight
- 464 KB
- Volume
- 101
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Omithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supematant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of omithine transcarbamylase, 6-N-(phosphonacetyl)-L-omithine, immobilized on epoxyactivated Sepharose 6B through the a-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 pmol min-' mg-1 at pH 8.5, 37"C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods.