Showing papers on "Cobalamin transport published in 2000"
TL;DR: Site-directed disulfide bonding in intact cells has been used to show that the Ton box of BtuB and residues around position 160 of TonB approach each other in a highly oriented and specific manner to form B TuB-TonB heterodimers that are stimulated by the presence of transport substrate.
Abstract: Uptake of cobalamins by the transporter protein BtuB in the outer membrane of Escherichia coli requires the proton motive force and the transperiplasmic protein TonB. The Ton box sequence near the amino terminus of BtuB is conserved among all TonB-dependent transporters and is the only known site of mutations that confer a transport-defective phenotype which can be suppressed by certain substitutions at residue 160 in TonB. The crystallographic structures of the TonB-dependent transporter FhuA revealed that the region near the Ton box, which itself was not resolved, is exposed to the periplasmic space and undergoes an extensive shift in position upon binding of substrate. Site-directed disulfide bonding in intact cells has been used to show that the Ton box of BtuB and residues around position 160 of TonB approach each other in a highly oriented and specific manner to form BtuB-TonB heterodimers that are stimulated by the presence of transport substrate. Here, replacement of Ton box residues with proline or cysteine revealed that residue side chain recognition is not important for function, although replacement with proline at four of the seven Ton box positions impaired cobalamin transport. The defect in cobalamin utilization resulting from the L8P substitution was suppressed by cysteine substitutions in adjacent residues in BtuB or in TonB. This suppression did not restore active transport of cobalamins but may allow each transporter to function at most once. The uncoupled proline substitutions in BtuB markedly affected the pattern of disulfide bonding to TonB, both increasing the extent of cross-linking and shifting the pairs of residues that can be joined. Cross-linking of BtuB and TonB in the presence of the BtuB V10P substitution became independent of the presence of substrate, indicating an additional distortion of the exposure of the Ton box in the periplasmic space. TonB action thus requires a specific orientation for functional contact with the Ton box, and changes in the conformation of this region block transport by preventing substrate release and repeated transport cycles.
88 citations
17 May 2000
TL;DR: Fluorescent analogs ofcobalamin (vitamin B12) have been developed as diagnostic markers ofcancer cells as discussed by the authors. But the results ofpreliininary studies suggest that fluorescent analogs may be a useful tool in therapeutic breast operations to define tumor margins and to distinguishneoplastic breast tissue from healthy breast tissue.
Abstract: Fluorescent analogs ofcobalamin (vitamin B12)have been developed as diagnostic markers ofcancer cells.These compounds are recognized by transcobalamin, a cobalamin transport protein, with high affinity, asshown by surface plasmon resonance. The cellular sequestration and gross distribution of fluorescentcobalamm bioconjugates in breast tissue is being examined by epifluorescence microscopy. Thedistribution ofeach compound is being evaluated in proliferative and non-proliferative tissue, ie. normaltissue and breast carcinoma. The results ofpreliininary studies suggest that fluorescent analogs ofcobalamin may be a useful tool in therapeutic breast operations to define tumor margins and to distinguishneoplastic breast tissue from healthy breast tissue.Keywords: cobalamin, vitamin B12, breast, neoplastic, fluorescent, transcobalamin, TCII 1. ENTRODUCFION Eukaryotic cells require cobalamin for metabolism and cell replication. Methionine synthase utilizesmethylcobalamin as a cofactor to methylate homocysteine in a coupled reaction with N5methyl-tetrahydrofolate. N5Methyl-tetrahydrofolate serves as a methyl group donor, thereby regeneratingtetrahydrofolate, which ultimately converts deoxyuridine monophosphate to deoxythymidinemonophosphate.' Rapidly proliferating cells have an increased demand for thymidine to support DNAreplication and thus have a need for high levels ofcobalamin. Tumor cells manifest this need forcobalamin by increasing their level ofcobalamin transport and storage.2'3In blood, cobalamin is bound to transcobalamin (TCII). Cobalamin enters cells via receptor-mediatedendocytososis ofthe TCII-cobalamin complex.4 This process allows a tumor to sequester high levels ofradiolabeled cobalamin, as seen in mice that have an implanted fibrosarcoma5 and in companion felineswith a primary mammary tumor.5 The unsaturated serum binding capacity ofvitamin B12 increases 3 to 26fold in patients with acute promyelocytic leukemia.6'7 In addition, elevated levels ofcirculating TCII havebeen observed in patients with breast carcinoma.8Breast cancer is the second most common type of cancer in women.9 Surgical therapy for breast cancerrequires the removal of a fraction of healthy breast tissue to ensure complete excision ofthe tumor. Areliable method for detecting neoplastic margins intraoperatively and differentiating it from healthy tissueis not available. Fluorescent derivatives ofcobalamin may be useful as tumor imaging agents to fill thisvoid in current medical technology. This would ensure complete excision ofneoplastic tissue whileminimizing the amount ofhealthy tissue removed. Cobalamin has been labeled with Oregon Green,naphthofluorescein, and fluorescein fluorophores. The internalization of these cobalamin bioconjugates bytumor (target) tissue is now being evaluated and compared with internalization levels in surroundingnormal (background) tissue in breast carcinoma.
4 citations