Ribonucleotide reductase from Rhizobium meliloti grown on the cobalt-deficient medium containing methionine was purified about 37-fold by streptomycin treatment, ammonium sulfate fractionation and column chromatographies on 1st DEAF-cellulose, 2nd DEAE-cellulose and Sepharose 6B. In agar gel electrophoresis, the purified enzyme moved as a single protein band. In sedimentation velocity experiments and analytical gel filtration, the purified enzyme was proved to aggregate and disaggregate. The s20, w of the enzyme at high concentration was about 27.1. From the intercept of the extrapolated curve line, the s20, w of approximately 20 was obtained. In analytical gel filtration, the location of elution peak varied with the protein concentration placed on the column. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme exhibited two bands, the molecular weights of which were estimated to be approximately 130, 000 (a major band) and 110, 000 (a minor band). The proportion of the major band to the minor band was 3:1. The pH optimum for the enzyme activity was 9.0_??_10.0. The activity was maximum at 45_??_50°C. The Km value for adenosyl-B12 was 0.77×10-6M. The concentrations of ADP and reduced lipoic acid necessary for saturation of the activity were 0.75×10-3M and 30×10-3M, respectively. Ribonucleoside diphosphates were most effective as substrates.
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Inukai et al. (1979) studied this question.