Homocysteine Methyltransferase Activity Research Articles

Intracellular binding of radioactive hydroxocobalamin to cobalamin-dependent apoenzymes in rat liver.

We identified previously an intracellular cobalamin (Cbl) binding protein(s) in cultured human fibroblasts, distinct from known Cbl "R" binders and absent from mutant cells deficient in the synthesis of the two Cbl coenzymes. In order to further characterize this binding activity, we have investigated its homologue in rat liver. After being transported to the liver by the serum protein transcobalamin II, [57Co]Cbl was bound by at least two distinct proteins, one cytosolic, the other mitochondrial. Labeled Cbl bound to cytosolic protein faster than or prior to the mitochondrial protein. With time there was a decline in radioactivity associated with the cytosolic binder and a coordinate increase in that associated with the mitochondrial binder. Although both proteins cochromatographed on Sephadex G-150 and had apparent molecular weights of 120,000, they were separated into two discrete components by polyacrylamide gel electrophoresis and by DEAE-cellulose chromatography. The cytosolic binder cochromatographed with N5-methyltetrahydrofolate:homocysteine methyltransferase activity (5-methyltetrahydropteroyl-L-glutamate:L-homocysteine S-methyltransferase, EC 2.1.1.13); the mitochondrial one with methylmalonyl CoA mutase activity (methylmalonyl-CoA CoA-carbonylmutase, EC 5.4.99.2). These proteins were distinguished further by the chemical forms of [57Co]Cbl found with them, hydroxocobalamin and methylcobalamin with the cytosolic protein and adenosylcobalamin with the mitochondrial one. These results suggest that intracellular Cbl binding activity in rat liver can be accounted for by attachment of Cbl to the two known Cbl-dependent apoenzymes, methylmalonyl CoA mutase and methyltetrahydrofolate methyltransferase. The mechanism and significance of the observered binding protein deficiency in mutant human fibroblasts must, therefore, be re-evaluated.

N 5-methyltetrahydrofolate: Homocysteine methyltransferase activity in extracts from normal, malignant and embryonic tissue culture cells

Malignant cells (J111, L1210, W-256) and human embryonic cells (FL) are unable to survive and grow when homocystine replaces methionine in tissue culture media containing excess vitamin B 12 and folic acid. Extracts of these same cells when grown in media containing methionine and more than adequate vitamin B 12 and folic acid have diminished N 5-methyltetrahydrofolate: homocysteine methyltransferase activities in the absence of added cyanocobalamin when compared with extracts of normal cells (adult rat thymus and liver fibroblasts). Extracts of human monocytic leukemia (J111) and human amnion cells (FL) have normal enzymatic activity in the presence of added cyanocobalamin whereas the rodent malignant cells (W-256 and L1210) have abnormally low activity in the absence or presence of added vitamin B 12.

S-adenosylmethionine metabolism by members of the genus Candida.

S-Adenosylmethionine and S-methylmethionine: homocysteine methyltransferase activity was measured in cell-free extracts from seven different species (15 strains) of the genus Candida. All strains were able to form methionine via a transmethylation reaction with S-adenosylmethionine and homocysteine. However, some strains were not able to use S-methylmethionine as a methyl donor. Cell-free extracts from two strains of C. guilliermondii formed no methionine when S-methylmethionine was the methyl donor and one strain (C. guilliermondii, CDC) showed methionine-forming activity with S-adenosylmethionine and homocysteine only when the growth medium was supplemented with L-methionine. In general, greater methyltransferase activity was observed in the cell-free extracts from the more pathogenic members of the genus (i.e., C. albicans and C. tropicalis). Supplementation of the growth medium with L-methionine induced homocysteine methyltransferase activity. Ten of the 15 strains had the capacity to synthesize and store S-adenosylmethionine in their vacuole and all 15 strains had S-adenosylmethionine-degrading enzymes. The activity of the latter enzyme was not increased when cells were grown in L-methionine-supplemented medium.

Regulation of 5-methyltetrahydrofolate: homocysteine methyltransferase activity by methionine, vitamin B12, and folate in cultured baby hamster kidney cells.

Rapid growth of BHK cells in methioninedeficient medium required supplementation with homocysteine, B(12), and over 40-fold greater levels of folic acid than growth in methionine-supplemented medium. The activity of the B(12)-dependent 5-methyltetrahydrofolate: homocysteine methyltransferase was studied in extracts of BHK cells grown in media containing various concentrations of the compounds of the enzyme reaction. The methyltransferase activity increased over 4-fold when B(12)-deficient deficient medium was supplemented with optimal levels of B(12); this increase was not prevented by puromycin. Addition of homocysteine to growth medium containing methionine, B(12), and folic acid was without effect. However, methyltransferase activity increased 2.5- to 4.0-fold further beyond the highest levels obtained in the presence of methionine, B(12), and folic acid when homocysteine was substituted for methionine in the growth medium. This increase was blocked by puromycin and was not due to removal of feedback inhibition of activity by the product methionine. These results suggest that methyltransferase activity may be regulated in part by derepression of the enzyme's synthesis on substitution of the substrate homocysteine for the product methionine.

Defective metabolism of vitamin B 12 in fibroblasts from children with methylmalonicaciduria

Fibroblasts from a patient with B 12 responsive methylmalonicaciduria and from a second patient with methylmalonicaciduria plus abnormal sulfur amino acid metabolism were examined for their ability to accumulate the two B 12 coenzymes, deoxyadenosyl-B 12 ∗ ∗ B 12 in used in place of cobalamin, i.e. cyane-B 12 for cyanocobalamin, etc. and methyl-B 12, and for N 5-methyltetrahydrofolate homocysteine methyltransferase activity. Cells from the first patient accumulated very little deoxyadenosyl-B 12 but accumulated methyl-B 12 normally. His cells had normal methyltransferase activity when assayed with and without added methyl-B 12 coenzyme. In contrast, cells from the second patient accumulated neither coenzyme. Methyltransferase activity was very low when assayed without added methyl-B 12 but increased markedly when coenzyme was added. We conclude that both patients have primary defects in vitamin B 12 metabolism and that these defects are biochemically distinct.

Methionine biosynthesis in Escherichia coli: Induction and repression of methylmethionine(or adenosylmethionine):Homocysteine methyltransferase

Escherichia coli Texas M, a methionine auxotroph, uses either adenosylmethionine or methylmethionine to satisfy its methionine requirement. Cell-free extracts of E. coli Texas and E. coli Texas M form methionine via a methyltransferase reaction wherein adenosylmethionine or methylmethionine serves as the methyl donor and homocysteine is the methyl acceptor. Growth of the two strains of E. coli in minimal medium supplemented with 0–40 mμmoles/ml of methionine results in a stimulation of methylmethionine:homocysteine methyltransferase activity. However, growth of E. coli Texas and E. coli Texas M in the presence of more than 50 mμmoles/ml of methionine results in a repression of the methyltransferase. Although homocysteine does not support growth of E. coli Texas M, it acts as an inducer of the methyltransferase. Product inhibition of the homocysteine methyltransferase was observed with extracts of E. coli Texas and E. coli Texas M.

Cystathionine as a precursor of methionine in Escherichia coli and Aerobacter aerogenes.

Balish, Edward (Argonne National Laboratory, Argonne, Ill.), and Stanley K. Shapiro. Cystathionine as a precursor of methionine in Escherichai coli and Aerobacter aerogenes. J. Bacteriol. 92:1331-1336. 1966.-Cystathionine has been shown to be a precursor of methionine biosynthesis in Escherichia coli and Aerobacter aerogenes. A double enzyme assay was developed to show the formation of homocysteine from cystathionine. The results obtained support the concept that cystathionine serves as a precursor of methionine via the intermediate formation of homocysteine. The latter compound is methylated by the homocysteine methyltransferase of these microorganisms. Sulfhydryl and keto acid assays were used to demonstrate cystathionase activity. Methionine represses both homocysteine methyltransferase formation and cystathionase formation. However, the presence of methionine in reaction mixtures resulted in product inhibition of homocysteine methyltransferase activity, but not of cystathionase activity.