Disruption of a regulatory system involving cobalamin distribution and function in a methionine-dependent human glioma cell line.

Cobalamin metabolism and function were investigated at the levels from transcobalamin II (TCII) receptor to the cobalamin-dependent enzymes, methionine synthase and methylmalonyl-CoA mutase, in a methionine-dependent (P60) and a methionine-independent (P60H) glioma cell line. Using P60H as reference, the P60 cells cultured in a methionine medium had slightly lower TCII receptor activity and normal total cobalamin content, a moderately reduced microsomal and mitochondrial cobalamin(III) reductase activity but only trace amounts of the methylcobalamin and adenosylcobalamin cofactors. When transferred to a homocysteine medium without methionine, P60H cells showed a slightly enhanced TCII receptor activity, but the other cobalamin-related functions were essentially unchanged. In contrast, the methionine-dependent P60 cells responded to homocysteine medium with a nearly 6-fold enhancement of TCII receptor expression and a doubling of both the hydroxycobalamin content and the microsomal reductase activity. The mitochondrial reductase and the cobalamin-related processes further down the pathway did not change markedly. In both cell lines, TCII receptor activity was further increased when growth in homocysteine medium was combined with N2O exposure. These data suggest that low methionine and/or high homocysteine exert a positive feedback control on TCII receptor activity. The concurrent increase in hydroxycobalamin content and in microsomal reductase activity are either subjected to similar regulation or secondary to increased cobalamin transport. This regulatory network is most prominent in the methionine-dependent P60 cells harboring a disruption of the network in the proximity of cobalamin(III) reductase.

Homocysteine as a risk factor for vascular disease. Enhanced collagen production and accumulation by smooth muscle cells.

An increased plasma homocysteine level is an independent risk factor for vascular disease. However, the pathological mechanisms by which homocysteine promotes atherosclerosis are not yet clearly defined. Arterial smooth muscle cells cultured in the presence of homocysteine grew to a higher density and produced and accumulated collagen at levels significantly above control values. Homocysteine concentrations as low as 50 mumol/L significantly increased both cell density and collagen production. Cell density increased by as much as 43% in homocysteine-treated cultures. Homocysteine increased collagen production in a dose-dependent manner. Smooth muscle cells treated with homocysteine at concentrations observed in patients with hyperhomocysteinemia had collagen synthesis rates as high as 214% of control values. Likewise, collagen accumulation in the cell layer was nearly doubled in homocysteine-treated cultures. Addition of aquacobalamin to homocysteine-treated cultures controlled the increase in smooth muscle cell proliferation and collagen production. These results indicate a cellular mechanism for the atherogenicity of homocysteine and provide insight into a potential preventive treatment.

Accumulation of methylmalonic acid caused by vitamin B12-deficiency disrupts normal cellular metabolism in rat liver.

To clarify the relationship between intracellular concentrations of methylmalonic acid and metabolic and growth inhibition in vitamin B12-deficient rats, hepatic methylmalonic acid levels were assayed and inhibition of glucose and glutamic acid metabolism by methylmalonic acid was studied in isolated hepatocytes. Vitamin B12-deficient rats (14 weeks old) excreted more urinary methylmalonic acid and had lower body weights than the control rats. Hepatic methylmalonic acid levels (3.6(SD 1.30)-5.3 (SD 0.51) mumol/g tissue; 7.9 (SD 2.90)-11.8 (SD 1.14) mM) were increased and correlated with the extent of the growth retardation during vitamin B12-deficiency. Isolated hepatocytes and mitochondria from normally fed rats were labelled with [14C(U)]glucose and [14C(U)]glutamic acid respectively, in the presence or absence of 5 mM-methylmalonic acid. Although methylmalonic acid did not affect the incorporation of 14C into protein and organic acid fractions in the hepatocytes, it inhibited 14CO2 formation (an index of glucose oxidation by the Krebs cycle) by 25% and incorporation of 14C into the amino acid fraction by 30%. In the mitochondria, methylmalonic acid inhibited 14CO2 formation (indicating glutamic acid oxidation by the Krebs cycle) by 70%, but not the incorporation of 14C into the protein fraction. The incorporation of 14C into the organic acid fraction was significantly stimulated by the addition of methylmalonic acid. These results indicate that the unusual accumulation of methylmalonic acid caused by vitamin B12-deficiency disrupts normal glucose and glutamic acid metabolism in rat liver, probably by inhibiting the Krebs cycle.

Identification and characterization of two enzymes involved in the intracellular metabolism of cobalamin. Cyanocobalamin beta-ligand transferase and microsomal cob(III)alamin reductase.

Two enzymes involved in the intracellular metabolism of cobalamin have been identified and characterized: cyanocobalamin beta-ligand transferase and microsomal cob(III)alamin reductase. The beta-ligand transferase is a cytosolic enzyme utilizing FAD, NADPH and reduced glutathione. The product of the reaction has been identified as glutathionyl-cobalamin. NADH-linked cob(III)alamin reductase has been found in two subcellular fractions: microsomal and inner mitochondrial membrane. The product of the reduction catalyzed by the microsomal enzyme has been identified as cob(II)alamin. In cbl C mutant fibroblasts, the specific activities of cyanocobalamin beta-ligand transferase and cob(III)alamin reductase were markedly decreased and have varied from 3%-30% and 36%-42% of normal, respectively. The specific activity of mitochondrial cob(III)alamin reductase was only 30% of normal in two cbl C mutants and normal in remaining mutant cell lines. In the cbl D cells, the specific activities were 33% and 55%. Mitochondrial cob(III)alamin reductase was not affected by cbl D mutation. Methionine synthase, L-methylmalonyl-CoA mutase and microsomal cytochrome c and b5 reductases are not affected by both mutations. The cbl E mutation affects only the activity of methionine synthase. These results support the hypothesis that the early enzymatic steps of intracellular metabolism of cobalamin are similar in the synthesis of both methylcobalamin and adenosylcobalamin and these steps are altered by the cbl C and cbl D mutations.

The inhibition of corrinoid-catalyzed oxidation of mercaptoethanol by methyl iodide: mechanistic implications.

The cobalamin coenzymes (5'-deoxyadenosyl- and methylcobalamin) and their cobinamide counterparts (5'-deoxyadenosyl- and methylcobinamide) catalyze the oxidation of 2-mercaptoethanol to its disulfide with hydrogen peroxide formation under aerobic conditions. The reactions are blocked by methyl iodide. Inhibition by methyl iodide is apparently due to the formation of the trans dialkyl corrinoids: methyl(adenosyl)cobalamin, dimethylcobalamin, methyl(adenosyl)cobinamide, and dimethylcobinamide, respectively. When the reaction system is illuminated with visible light, inhibition is released and a dramatic enhancement in the rate of oxygen consumption occurs. For reactions catalyzed by adenosyl- and methylcobalamin and then inhibited by methyl iodide, the rates observed during photolysis approach those obtained with aquacobalamin. For reactions catalyzed by adenosyl- and methylcobinamide and then inhibited by methyl iodide, the rates observed during photlysis approach those obtained with diaquacobinamide. Thus, both trans axial carbon-cobalt bonds in the putative dialkyl corrinoid are homolyzed during photolysis. In contrast to these results, the catalysis of the aerobic oxidation of 2-mercaptoethanol by aquacobalamin is only weakly inhibited by methyl iodide. This observation suggests that aquacob(II)alamin is produced during the catalysis of this reaction. Superoxide, the anticipated product of the reaction between aquacob(II)alamin and dioxygen, is formed during aquacobalamin-catalyzed 2-mercaptoethanol oxidation since superoxide dismutase decreases the rate of oxygen consumption by 50%. However, the enzyme has no effect on oxygen uptake during reactions catalyzed by cobalamin coenzymes and their cobinamide counterparts. These corrinoid catalysts apparently transfer two electrons to dioxygen from cobalt(I) intermediates formed during the reactions. Nitrogenous bases inhibit corrinoid-catalyzed thiol oxidation by competing with 2-mercaptoethanol for axial-ligand coordination sites on the catalyst. In contrast to the inhibition observed with methyl iodide, visible light has no effect on the inhibition obtained with nitrogenous bases.

Glial cells as a model for the role of cobalamin in the nervous system: impaired synthesis of cobalamin coenzymes in cultured human astrocytes following short-term cobalamin-deprivation.

The conversion of cyanocobalamin to adenosyl- and methylcobalamin is impaired in cobalamin-deficient cultured human glial cells. In contrast cultured human skin fibroblasts retained their ability to synthesize coenzyme forms when grown in cobalamin-deficient medium. Cells were pre-conditioned by growing in cobalamin-deficient media for six weeks and then subcultured in medium containing either free or transcobalamin II-bound 57Co-cyanocobalamin. Although both coenzyme levels were low in cobalamin-deficient glial cells, the decrease in methylcobalamin was more marked than that of adenosylcobalamin. Methionine synthase and Cb1 reductase activities were markedly decreased in cobalamin-deficient glial cells but were unchanged in fibroblasts cultured in cobalamin-deficient medium. Our data suggest that in glial cells, cobalamin coenzyme synthesis and function is exquisitely sensitive to short-term cobalamin deprivation. Glial cells apparently synthesize and secrete transcobalamin II since antibodies directed against the transport protein inhibit the uptake of free cobalamin.