Accurate assessment and identification of naturally occurring cellular cobalamins

Hannibal, Luciana; Axhemi, Armend; Глущенко, А. В.; Moreira, E.S.; Brasch, Nicola E.; Jacobsen, Donald W.

doi:10.1515/cclm.2008.356

Cited by 56 publications

(75 citation statements)

References 31 publications

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“…In the present work we have successfully developed a [ (Hannibal et al, 2008;Mellman et al, 1978;Yamani et al, 2008;Yassin et al, 2000;Youngdahl-Turner et al, 1978) or aspects of lysosome function in cells and mice (Manunta et al, 2007;Yang et al, 2011). Our data and in mouse models of ageing and neurodegenerative disease.…”

Section: Discussionmentioning

confidence: 95%

Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue

Zhao¹,

Ruberu²,

Li³

et al. 2013

Journal of Neuroscience Methods

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Cobalamin (Cbl) utilization as a cofactor for methionine synthase and methylmalonyl-CoA mutase is dependent on the transport of Cbl through lysosomes and its subsequent delivery to the cytosol and mitochondria. We speculated that neuropathological conditions that impair lysosomal function (e.g., agerelated lipofuscinosis and specific neurodegenerative diseases) might impair lysosomal Cbl transport. To address this question, an appropriate method to quantify intracellular Cbl transport in neuronal cell types and brain tissue is required. Thus, we developed methods to measure [ 57 Co] Cbl levels in lysosomes, mitochondria and cytosol obtained from in vitro and in vivo sources. Human SH-SY5Y neurons or HT1080 fibroblasts were labeled with [ 57 Co] Cbl and homogenized using a ball-bearing homogenizer, and the lysates were separated into 10 fractions using ultracentrifugation in an OptiPrep density gradient. Lysosomes were recovered from the top of the gradient (fractions 1-5), which were clearly separated from mitochondria (fractions 7-9) on the basis of the expression of the marker proteins, LAMP2 and VDAC1. The isolated lysosomes were intact based on their colocalization with acid phosphatase activity. The lysosomal and mitochondrial fractions were free of the cytosolic markers beta-actin and methionine synthase. The relative distribution of [ 57 Co] Cbl in both neurons and fibroblasts was as follows: 6% in the lysosomes, 14% in the mitochondria and 80% in the cytosol. This technique was also used to fractionate organelles from mouse brain, where marker proteins were detected in the gradient at positions similar to those observed for the cell lines, and the relative distribution of [ 57 Co] Cbl was as follows: 12% in the lysosomes, 15% in the mitochondria and 73% in the cytosol. These methods provide a useful tool for the investigation of intracellular Cbl trafficking in a neurobiological setting. fractions using ultracentrifugation on an OptiPrep density gradient. Lysosomes were recovered at the top of the gradient (fractions 1 to 5) and were clearly separated from mitochondria (fractions 7 to 9) based on marker proteins LAMP2 and VDAC1, respectively.The isolated lysosomes were intact based on colocalisation with acid phosphatase activity.The lysosomal and mitochondrial fractions were free of cytosolic markers beta-actin and methionine synthase.

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Section: Discussionmentioning

confidence: 95%

Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue

Zhao¹,

Ruberu²,

Li³

et al. 2013

Journal of Neuroscience Methods

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“…After 48 h, cells were harvested, total cobalamins extracted with 80% aqueous ethanol and the intracellular cobalamin profile determined as recently described by Hannibal et al [15]. The cell cultures were protected from light at all times to prevent photolysis of the alkylcobalamins.…”

Section: Methodsmentioning

confidence: 99%

Processing of alkylcobalamins in mammalian cells: A role for the MMACHC (cblC) gene product

Hannibal

Kim

Brasch

et al. 2009

Molecular Genetics and Metabolism

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118

103

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The MMACHC gene product of the cblC complementation group, referred to as the cblC protein, catalyzes the in vitro and in vivo decyanation of cyanocobalamin (vitamin B 12 ). We hypothesized that the cblC protein would also catalyze the dealkylation of newly internalized methylcobalamin (MeCbl) and 5′-deoxyadenosylcobalamin (AdoCbl), the naturally occurring alkylcobalamins that are present in the diet. The hypothesis was tested in cultured endothelial cells using

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“…After a 30-min reaction at 37°C, the protein was inactivated by heating (70°C, 10 min), and the cofactors were released into solution. The supernatant was analyzed by HPLC as described previously (20). 2 Cbl by ceCblC in the presence of 4 mM GSH was carried out in 100 mM HEPES, pH 7.0, 150 mM KCl, and 10% (v/v) glycerol.…”

Section: Reduction Of Ohmentioning

confidence: 99%

Glutathione-dependent One-electron Transfer Reactions Catalyzed by a B12 Trafficking Protein

Zhu

Gherasim

Lesniak

et al. 2014

Journal of Biological Chemistry

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Background: CblC is a B 12 -processing enzyme that converts cyanocobalamin to cob(II)alamin. Results: CblC catalyzes two previously unknown reactions: GSH-dependent reductive decyanation of CNCbl and reduction of OH 2 Cbl. Conclusion: Worm and human CblC activities show different susceptibilities to oxygen. Significance: The use of GSH for single-electron transfer is unexpected and expands the range of catalytic reactions supported by CblC.

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Accurate assessment and identification of naturally occurring cellular cobalamins

Cited by 56 publications

References 31 publications

Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue

Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue

Processing of alkylcobalamins in mammalian cells: A role for the MMACHC (cblC) gene product

Glutathione-dependent One-electron Transfer Reactions Catalyzed by a B12 Trafficking Protein

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