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Abstract
Valine dehydrogenase (VDH) was purified to homogeneity from cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1508-fold in a 17·7% yield using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The M r of the native enzyme was determined to be 218000 and 215000, by equilibrium ultracentrifugation and size-exclusion high-performance liquid chromatography, respectively. The enzyme is composed of 12 subunits of M r 18000. Using analytical isoelectric focusing the isoelectric point of VDH was found to be 4·7. Oxidative deamination of l-valine was optimal at pH 10·6. Reductive amination of 2-oxoisovalerate was optimal at pH 8·8. The Michaelis constants (K m) were 1 mm for l-valine and 0·029 mm for NAD+. K m values for reductive amination were 0·80 mm for 2-oxoisovalerate, 0·050 mm for NADH and 22 mm for NH+ 4.
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