In order to elucidate the biosynthesis of the base moiety of cobalamin in Salmonella typhimurium LT2, this organism was grown in the presence of [1'-14C]riboflavin. The vitamin B12 isolated was 14C-labeled. It was shown by chemical degradation that the 14C-label was exclusively localized in carbon atom 2 of the 5,6-dimethylbenzimidazole moiety. This demonstrated the precursor function of riboflavin in the biosynthesis of 5,6-dimethylbenzimidazole in S. typhimurium.

Experiments on the incorporation of erythrose and formate into the 5,6-dimethylbenzimidazole moiety of vitamin B12 are described.In one experiment, a 1 : 1 mixture of ~-[l-~~C]erythrose and ~-[l-'~C]threose was added to a Euhucterium limosum fermentation. The vitamin B12 formed was methylated at N3 of its 5,6-dimethylbenzimidazole part and degraded to 1,5,6-trimethylbenzimidazole. The 13C-NMR spectrum of this compound exhibited a single prominent signal at 109.5 ppm due to 13C labeling in C7.This shows that C1 of erythrose or threose was originally incorporated exclusively into C4 of the 5,6-dimethylbenzimidazole moiety of vitamin B12.In another experiment, sodium [13C]formate was added to a culture of E. limosum. The vitamin B12 isolated was transformed into 1,5,6-trimethylbenzimidazole as before. The 13C-NMR spectrum also showed one prominent signal at 142.8 ppm, evoked by I3C at C2.These results demonstrate that erythrose is incorporated into the base part of vitamin B12 regiospecifically and that formate is the precursor of the C2.Aerobic microorganisms, as well as some aerotolerant anaerobes, synthesize the corrin ring of vitamin B12 from succinyl-CoA and glycine and the 5,6-dimethylbenzimidazole unit of this vitamin from riboflavin 5'-phosphate [I, 2). Anaerobic bacteria like Eubacterium limosum use glutamate as a precursor of the corrin moiety and several building blocks, e.g. methionine, glycine and glutamine, for the formation of the 5,6-dimethylbenzimidazole moiety [l, 3 -Recently we demonstrated, with E. limosum, that u-erythrose, I4C labeled in different positions, is incorporated into C4 -7 of the vitamin B12 base 161. However, due to the symmetrical structure of 5,6-dimethylbenzimidazole, the chemical degradation procedure applied for the detection of 14C could not differentiate between C4 and C7 or between C5 and C6 [6]. Therefore, we could not determine if C1 of erythrose was incorporated into C4 or into C7.The NMR experiment reported here now solves this problem, showing that CZ of erythrose is exclusively incorporated into C4 of the 5,6-dimethylbenzimidazole moiety of vitamin B12.In addition, we demonstrate that formate is the precursor of C2 of 5,6-dimethylbenzimidazole. Formate thus completes the building blocks necessary for the synthesis of the base part of vitamin B12.Correspondence to

The nuclear accessory protein in porcine intestinal nuclear extracts that activates the binding of the vitamin D receptor to its vitamin D response elements has been highly purified. It contains a protein that binds 9-cis- [H] retinoic acid, was detected on immunoblots with an anti-retinoid X receptor (RXR) peptide antibody, and supports the binding of retinoic acid receptor y to the retinoic acid receptor ,B gene response element. Most important, the two specific complexes formed by porcine nuclear extract with the vitamin D response elements from either the osteocalcin gene or the rat 24-hydroxylase gene are shifted to a larger complex by both an anti-vitamin D receptor antibody and an anti-RXR antibody, leaving no doubt that in vivo the nuclear accessory factor for the vitamin D receptor in the intestine is an RXR protein.Vitamin D regulates calcium homeostasis by the mobilization of calcium from bone, reabsorption of calcium in the kidney, and absorption of calcium from the intestine. The physiologically active metabolite of vitamin D 1,25-dihydroxyvitamin D3 [1,2D3] is known to exert these functions by binding to a transcription factor, the vitamin D receptor (VDR) (1). The VDR is a member of the steroid/thyroid receptor superfamily, which also includes nonsteroid binding receptors such as the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (2). VDR and RAR are members of a subset of this family that can bind to response elements as heterodimers and recognize response elements consisting of direct repeats (3).Several reports have demonstrated that RXRs can function in vitro as accessory factor(s) (e.g., heterodimerize) with VDR and other receptors. Heterodimerization of RXRs with VDRs, thyroid receptors, and RARs increases the affinity of these receptors for their response elements. When RXR cDNAs are cotransfected with these receptors, the transcriptional response to the respective ligand is increased (4-9). These studies provide evidence for a possible role of RXRs as a link between several ligand-induced pathways.A mammalian-derived nuclear accessory factor (NAF) required for tight binding of VDR to vitamin D response elements (DREs) has been described (10-12). The determination of the molecular weight of NAF from pig intestinal nuclear extract (PINE), a major vitamin D target tissue, revealed a molecular mass of 59-64 kDa (12). MacDonald et al. (13) MATERIALS AND METHODS Chemicals. 1,25-(OH)2D3 was purchased from Tetrionics (Madison, WI). 9-cis-Retinoic acid (RA) was synthesized by silver oxide oxidation of 9-cisretinaldehyde (Aldrich). Radioactive 9-cis-RA was synthesized from all-trans-[11,12-3H]RA (48.7 Ci/mmol; 1 Ci = 37 GBq; DuPont/NEN) by photochemical isomerization (14) and was purified by HPLC using a C18 Zorbax-ODS column as described (14). The purity of 9-cis- [11,12-3H]

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