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J Bacteriol. 1972 January; 109(1): 169-178
Copyright © 1972 American Society for Microbiology. All Rights Reserved.

Further Studies on the Binding of Vitamin B₁₂ to the Cell Wall of a B₁₂-Requiring Lactobacillus

¹ Fermentation Research Laboratories, Sankyo Co., Ltd., Hiromachi, Shinagawa-ku, Tokyo, 140, Japan

ABSTRACT

The vitamin B₁₂-binding property of Lactobacillus leichmannii ATCC 7830 has been studied. The organism could bind 0.52 µg of B₁₂ per mg of cells. With regard to the cellular site for B₁₂ accumulation, three-quarters of the B₁₂ bound to the cell was found in the crude cell wall fraction, and the remaining one-quarter was found in the particulate (ribosome) fraction. After receiving enzymatic treatments with ribonuclease, lipase, and trypsin, the wall fraction retained three-fifths of the initial B₁₂. The possibility of cross-contamination of the wall and particulate fractions was excluded by measuring the contents of ribonucleic acid and hexosamines in each fraction. The B₁₂-binding activity of the wall was destroyed by pretreatment of the wall with pepsin, Pronase, or trypsin. However, once bound to the wall, the B₁₂ was not released by the same treatments. These facts suggest that B₁₂ is bound to a polypeptide in the wall on which these enzymes act and that, once bound, B₁₂ somehow inhibits the enzymatic actions as described earlier with L. delbrueckii no. 1. A B₁₂-polypeptide complex was isolated by treatment with 0.2 N HCl from walls to which B₁₂ had been bound. The complex was then purified. The complex moves as a single band on polyacrylamide gel electrophoresis. Its molecular weight was estimated around 21,500 with microheterogeneity on a Sephadex G-75 column. The mode of B₁₂ binding was found to be similar to that of L. delbrueckii.