CHEMICAL AND BIOCHEMICAL STUDIES ON VITAMIN B₁₂ AND ITS RELATED COMPOUNDS

XIV. PROPERTIES OF A VITAMIN B₁₂-GROUP COMPOUND DERIVED FROM EXOGENOUS COBALAMIN IN THE CELLS OF LACTOBACILLUS LEICHMANNII ACCOMPANYING WITH BIOSYNTHESES OF DEOXYRIBOSYL COMPOUNDS

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1. This paper deals with the study on the forms of the vitamin B₁₂ in the cells and the supernatant of Lactobacillus leichmannii incubated with cobalamin exogenously supplied. The ratio of DNA to RNA in vitamin B₁₂-starved cells, harvested from a culture on a medium containing deoxyguanosine instead of vitamin B₁₂, was significantly lower than that in normal cells. Incubation of these cells in a medium containing cyanocobalamin or hydroxocobalamin for 5 hours at 37° brought about a remarkable increase of acid-soluble deoxyribosyl compounds, followed by DNA synthesis without an increase of RNA content as well as the bacterial growth.
2. In this case the vitamin B₁₂-active substance detectable in the cells incubated with hydroxocobalamin was identical with that derived from cyanocobalamin, though hydroxocobalamin showed a grear effect than cyanocobalamin on the syntheses of acid-soluble deoxyribosyl compounds and DNA under the experimental conditions. The vitamin B₁₂-active substance present in the cells was extracted with hot 80% ethanol and separated from a contaminating yellow substance by DEAF-cellulose column chromatography. The vitamin B₁₂ contained in the effluent fraction gave a single spot identical with DBCC on paper electrophoresis performed at pH 2.7 and 3.5. Its visible absorption spectrum and the changes by illumination and by cyanide treatment were similar to those of 5, 6-dimethylbenzimidazoylcobamide coenzyme (DBCC). Furthermore; the coenzyme activity of this substance was essentially identical with that of DBCC in the propane diol-propionaldehyde intramolecular oxidation-reduction system of Abeles and Lee when the amounts were calculated on the basis of the absorbancy at 525mμ.
3. The results that DBCC was the sole vitamin B₁₂ in the cells of L. leichmannii in which the formation of deoxyribosyl compounds took place, suggest that the coenzyme is the active form participating in the biosynthesis of deoxyribosyl compounds in the bacteria associated with the finding of Blakley and Barker (9) that DBCC was an only active vitamin B₁₂ stimulating the reduction of ribotide to deoxyribotide in their enzyme system obtained from the bacteria.
DBCC added exogenously, however, showed almost the same effect with hydroxocobalamin. The reason may be attributed to the splitting of the deoxyadenosyl ligand from the coenzyme to be taken up into the bacterial cells.
4. The vitamin B₁₂-active substances in the supernatant derived from hydroxocobalamin were separated by DEAE-cellulose chromatography to an effluent fraction D-1 and a retained fraction D-2. The former behaved analogously to DBCC in paper electrophoresis and in absorption spectrophotometry. The latter, eluted with 0.1 M acetate buffer (pH 4.7), showed the analogous spectroscopic behavior to DBCC as well as D-1, but less cationic character in paper electrophoresis at several pH levels. Both D-1 and D-2 exhibited the coenzyme activity in Abeles-Lee's system, though somewhat smaller than that of DBCC. It remains unsolved whether the activities of these fractions were substantially small or lowered by the contaminating impurities, such as hydroxocobalamin. D-1 seems to be DBCC from its properties. The study on the structre of D-2 is in progress.