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

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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.

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