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1.
Br J Nutr ; 101(4): 492-8, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18710602

ABSTRACT

The aim of the present study was to examine the effects of cobalamin (Cbl) on the activity and expression of L-methylmalonyl-CoA mutase (MCM) in rat liver and cultured COS-7 cells. The MCM holoenzyme activity was less than 5% of the total (holoenzyme+apoenzyme) activity in the liver although rats were fed a diet containing sufficient Cbl. When weanling rats were maintained on a Cbl-deficient diet, the holo-MCM activity became almost undetectable at the age of 10 weeks. In contrast, a marked increase in the total-MCM activity occurred under the Cbl-deficient conditions, and at the age of 20 weeks it was about 3-fold higher in the deficient rats than in the controls (108 (SD 14.5) v. 35 (SD 8.5) nmol/mg protein per min (n 5); P<0.05). Western blot analysis confirmed that the MCM protein level increased significantly in the Cbl-deficient rats. However, the MCM mRNA level, determined by real-time PCR, was rather decreased. When COS-7 cells were cultured in a medium in which 10% fetal bovine serum was the sole source of Cbl, holo-MCM activity was barely detected. The supplementation of Cbl resulted in a large increase in the holo-MCM activity in the cells, but the activity did not exceed 30% of the total-MCM activity even in the presence of Cbl at 10 micromol/l. In contrast, the total-MCM activity was significantly decreased by the Cbl supplementation, indicating that Cbl deficiency results in an increase in the MCM protein level in COS-7 cells as well as in rat liver.


Subject(s)
Kidney/enzymology , Liver/enzymology , Methylmalonyl-CoA Mutase/analysis , Vitamin B 12 Deficiency/enzymology , Animals , Blotting, Western/methods , COS Cells , Carnitine/administration & dosage , Chlorocebus aethiops , Dietary Supplements , Gene Expression , Methionine/administration & dosage , Methylmalonyl-CoA Mutase/genetics , Methylmalonyl-CoA Mutase/urine , RNA, Messenger/analysis , Random Allocation , Rats , Reverse Transcriptase Polymerase Chain Reaction/methods , Vitamin B 12/administration & dosage , Vitamin B Complex/administration & dosage , Weaning
2.
J Dairy Sci ; 90(7): 3442-55, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17582128

ABSTRACT

The present experiment was undertaken to determine the effects of dietary supplements of folic acid and vitamin B12 given from 3 wk before to 8 wk after calving on lactational performance and metabolism of 24 multiparous Holstein cows assigned to 6 blocks of 4 cows each according to their previous milk production. Supplementary folic acid at 0 or 2.6 g/d and vitamin B12 at 0 or 0.5 g/d were used in a 2 x 2 factorial arrangement. Supplementary folic acid increased milk production from 38.0 +/- 0.9 to 41.4 +/- 1.0 kg/d and milk crude protein yield from 1.17 +/- 0.02 to 1.25 +/- 0.03 kg/d. It also increased plasma Gly, Ser, Thr, and total sulfur AA, decreased Asp, and tended to increase plasma Met. Supplementary B12 decreased milk urea N, plasma Ile, and Leu and tended to decrease Val but increased homocysteine, Cys, and total sulfur AA. Liver concentration of phospholipids was higher in cows fed supplementary B12. Plasma and liver concentrations of folates and B12 were increased by their respective supplements, but the increase in plasma folates and plasma and liver B12 was smaller for cows fed the 2 vitamins together. In cows fed folic acid supplements, supplementary B12 increased plasma glucose and alanine, tended to decrease plasma biotin, and decreased Km of the methylmalonyl-coenzyme A mutase in hepatic tissues following addition of deoxyadenosylcobalamin, whereas it had no effect when cows were not fed folic acid supplements. There was no treatment effect on plasma nonesterified fatty acids as well as specific activity and gene expression of Met synthase and methylmalonyl-coenzyme A mutase in the liver. Ingestion of folic acid supplements by cows fed no supplementary B12 increased total lipid and triacylglycerols in liver, whereas these supplements had no effect in cows supplemented with B12. The increases in milk and milk protein yields due to folic acid supplements did not seem to be dependent on the vitamin B12 supply. However, when vitamin B12 was given in combination with folic acid, utilization of the 2 vitamins seems to be increased, probably more so in extrahepatic tissues. Metabolic efficiency seems also to be improved as suggested by similar lactational performance and dry matter intake for cows fed supplementary folic acid but increased plasma glucose and decreased hepatic lipids in cows fed folic acid and vitamin B12 together.


Subject(s)
Cattle/metabolism , Dietary Supplements , Folic Acid/administration & dosage , Lactation/metabolism , Vitamin B 12/administration & dosage , 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/analysis , 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/biosynthesis , 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/genetics , Animal Feed/analysis , Animals , Diet , Female , Gene Expression/physiology , Liver/chemistry , Methylmalonyl-CoA Mutase/analysis , Methylmalonyl-CoA Mutase/biosynthesis , Milk/chemistry , Molecular Sequence Data , Pregnancy , RNA, Messenger/chemistry , Random Allocation , Time Factors , Vitamin B 12/analysis
3.
Biochem J ; 341 ( Pt 1): 133-8, 1999 Jul 01.
Article in English | MEDLINE | ID: mdl-10377254

ABSTRACT

We investigated the co-ordinate variations of the two cobalamin (Cbl)-dependent enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), and measured the levels of their respective cofactors, methylcobalamin (CH3Cbl) and adenosylcobalamin (AdoCbl) in cultured human glioma cells during nitrous oxide exposure and during a subsequent recovery period of culture in a nitrous oxide-free atmosphere (air). In agreement with published data, MS as the primary target of nitrous oxide was inactivated rapidly (initial rate of 0.06 h(-1)), followed by reduction of CH3Cbl (to <20%). Both enzyme activity and cofactor levels recovered rapidly when the cells were subsequently cultured in air, but the recovery was completely blocked by the protein-synthesis inhibitor, cycloheximide. During MS inactivation, there was a reduction of cellular AdoCbl and holo-MCM activity (measured in the absence of exogenous AdoCbl) to about 50% of pre-treatment levels. When the cells were transferred to air, both AdoCbl and holo-MCM activity recovered, albeit more slowly than the MS system. Notably, the regain of the holo-MCM and AdoCbl was enhanced rather than inhibited by cycloheximide. These findings confirm irreversible damage of MS by nitrous oxide; hence, synthesis of the enzyme is required to restore its activity. In contrast, restoration of holo-MCM activity is only dependent on repletion of the AdoCbl cofactor. We also observed a synchronous fluctuation in AdoCbl and the much larger hydroxycobalamin pool during the inactivation and recovery phase, suggesting that the loss and repletion of AdoCbl reflect changes in intracellular Cbl homoeostasis. Our data demonstrate that the nitrous oxide-induced changes in MS and CH3Cbl are associated with reversible changes in both MCM holoactivity and the AdoCbl level, suggesting co-ordinate distribution of Cbl cofactors during depletion and repletion.


Subject(s)
5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/drug effects , Cobamides/analysis , Methylmalonyl-CoA Mutase/drug effects , Nitrous Oxide/pharmacology , Vitamin B 12/analogs & derivatives , 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/analysis , Aerobiosis , Anesthetics, Inhalation/pharmacology , Female , Glioma , Humans , Methylmalonyl-CoA Mutase/analysis , Nerve Tissue/drug effects , Oxidation-Reduction , Tumor Cells, Cultured , Vitamin B 12/analysis
4.
Hum Mol Genet ; 6(9): 1457-64, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9285782

ABSTRACT

L-Methylmalonyl-CoA mutase (MUT) is an adenosylcobalamin (AdoCbl)-requiring mitochondrial matrix enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. Inherited defects in the gene encoding this enzyme result in the mut forms of methylmalonic acidemia. Expression of mature human MUT cDNA in Escherichia coli at a post-induction cultivation temperature of 12 degrees C, rather than 37 degrees C, led to the folding of the majority of the synthesized protein to a soluble form, with an activity of 0.2-0.3 U/mg protein in the cell-free extract, 10-15 times higher than that in human liver homogenate. Six missense mutations, producing the amino acid changes G94V, Y231N, R369H, G623R, H678R and G717V, were detected in MUT cDNA of patients suffering from the mut- form of methylmalonic acidemia, resulting from defective AdoCbl binding. Two (G623R and G717V) had been reported in other patients. Three (G94V, Y231N and R369H) are the first changes in the NH2-terminal part of the enzyme reported to cause the mut- phenotype. Enzymes with the mutations were individually expressed, and their kinetic parameters were generally in accord with published biochemical data from extracts of fibroblasts from these patients. The mutations increased the K(m) for AdoCbl by 40- to 900-fold, while V(max) values varied from 0.2% to nearly 100% of that of wild-type protein. In one case of a doubly heterozygous cell line, however, neither of the constituent mutant enzymes had a K(m) corresponding to the lower of the two estimated from the extract data. This finding may reflect the natural occurrence of interallelic complementation in vivo in this cell line.


Subject(s)
Escherichia coli/genetics , Methylmalonyl-CoA Mutase/genetics , Point Mutation , Cell Line , Escherichia coli/enzymology , Gene Expression , Genetic Complementation Test , Genetic Vectors , Humans , Methylmalonyl-CoA Mutase/analysis , Methylmalonyl-CoA Mutase/metabolism , Molecular Sequence Data , Phenotype , Polymerase Chain Reaction
5.
Clin Chem ; 41(8 Pt 1): 1164-70, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7628092

ABSTRACT

We constructed a fully automated assay for the cobalamin-dependent enzyme methylmalonyl coenzyme A (CoA) mutase. The assay involves preincubation of the enzyme with adenosylcobalamin, incubation with substrate, termination of the reaction by adding trichloroacetic acid, filtration to remove precipitated protein, and finally analysis of the filtrate (containing methylmalonyl CoA and the product succinyl CoA) by HPLC. These steps were carried out by an inexpensive programmable autosampler equipped with thermostated sample racks and mobile disposable extraction column racks used here as a sample filtering device. A central element in the developmental work was to measure stability of reagents, enzyme, and product against the storage conditions during unattended analysis and the time table of the program. We evaluated the performance of the method by measuring methylmalonyl CoA mutase activity in rat liver, human fibroblasts, and human glioma cells. The within-run imprecisions (CV) were 2-10% for measuring enzyme activity in 20 replicate samples of a homogenate (test of the automated assay), and 7-12% for measuring enzyme activity in homogenates from 20 culture dishes (test of the total procedure). The method allows the unattended analysis of 56 samples per 24 h. This strategy for automation may be easily adapted for other enzyme assays.


Subject(s)
Autoanalysis/methods , Methylmalonyl-CoA Mutase/analysis , Vitamin B 12/pharmacology , Acyl Coenzyme A/metabolism , Adult , Autoanalysis/statistics & numerical data , Cell Line , Chromatography, High Pressure Liquid , Enzyme Stability , Female , Fetus , Fibroblasts/enzymology , Glioblastoma/enzymology , Humans , Kinetics , Liver/enzymology , Lung , Male , Sensitivity and Specificity , Tumor Cells, Cultured
6.
J Am Vet Med Assoc ; 200(8): 1101-3, 1992 Apr 15.
Article in English | MEDLINE | ID: mdl-1351478

ABSTRACT

A 9-month-old sexually intact male longhair cat was examined because of lethargy, anorexia, cold intolerance, and failure to thrive since acquisition at an early age. Clinical signs of disease were less pronounced when the cat was fed a low-protein diet. Anemia, hypoglycemia, low total CO2 content, and hyperammonemia were detected. The cat was euthanatized. Urine obtained immediately before euthanasia contained a large amount of methylmalonic acid. Total serum cobalamin concentration was low. Hepatic methylmalonic-CoA mutase activity, with and without the addition of coenzyme adenosylcobalamin, was consistent with a cobalamin deficiency. Methylmalonic acidemia secondary to a putative defect in cobalamin absorption was diagnosed.


Subject(s)
Amino Acid Metabolism, Inborn Errors/veterinary , Cat Diseases/etiology , Methylmalonic Acid/blood , Vitamin B 12 Deficiency/veterinary , Absorption , Amino Acid Metabolism, Inborn Errors/complications , Amino Acids/blood , Animals , Cat Diseases/diet therapy , Cats , Dietary Proteins/administration & dosage , Failure to Thrive/etiology , Failure to Thrive/veterinary , Liver/enzymology , Liver/pathology , Male , Methylmalonic Acid/urine , Methylmalonyl-CoA Mutase/analysis , Sleep Stages , Vitamin B 12/blood , Vitamin B 12/pharmacokinetics , Vitamin B 12 Deficiency/diet therapy , Vitamin B 12 Deficiency/etiology
7.
Biochem Biophys Res Commun ; 177(3): 1076-81, 1991 Jun 28.
Article in English | MEDLINE | ID: mdl-1676260

ABSTRACT

In most animal species and many prokaryotes, methylmalonyl CoA mutase catalyzes isomerization between methylmalonyl CoA and succinyl CoA using adenosylcobalamin as a cofactor. We describe the absence of this enzyme in Aspergillus nidulans based on the absence of enzyme activity in vitro and the failure to metabolize methylmalonate or grow in media containing this organic acid as the sole carbon source. These data contrast previous assumptions that propionate may be metabolized through propionyl CoA and methylmalonyl CoA to the TCA cycle in this organism. This is consistent with the separate evolution of these pathways in animals and lower eukaryotes due to the distinct endosymbiotic origin of their mitochondria.


Subject(s)
Aspergillus nidulans/enzymology , Methylmalonyl-CoA Mutase/analysis , Aspergillus nidulans/growth & development , Gas Chromatography-Mass Spectrometry , Kinetics , Malonates/metabolism , Methylmalonyl-CoA Mutase/metabolism , Saccharomyces cerevisiae/physiology
9.
J Nutr ; 113(3): 531-7, 1983 Mar.
Article in English | MEDLINE | ID: mdl-6131113

ABSTRACT

The effects of vitamin B-12 deficiency on lipids and fatty acids of spinal cord myelin were studied in control and vitamin B-12-deficient fruit bats. Very low plasma and brain vitamin B-12 concentrations were present in animals fed the vitamin B-12-free, all fruit diet. Myelin was isolated from the spinal cord of control and vitamin B-12-deficient animals (n = 3 pools) by means of a flotation method in a discontinuous sucrose gradient. The molar concentration of cholineglycerophosphatide was significantly lower in the deficient bats than in the controls. The molar cholesterol: phospholipid ratio in the deficient bats was also significantly lower. The odd-chain fatty acid 15:1 was not detected in cholineglycerophosphatide from the controls, but comprised 1.4% of the total fatty acids in the deficient group. The odd-chain fatty acids 15:0, 17:1 and 19:0 were present in slightly higher amounts in cholineglycerophosphatide of deficient bats. Lipid and fatty acid differences were related to the vitamin B-12-requiring methionine synthetase and methylmalonyl CoA mutase reactions.


Subject(s)
Fatty Acids/analysis , Lipids/analysis , Myelin Sheath/analysis , Spinal Cord/analysis , Vitamin B 12 Deficiency/metabolism , Animals , Brain Chemistry , Chiroptera , Diet , Glycerylphosphorylcholine/analysis , Methylmalonyl-CoA Mutase/analysis , Phospholipids/analysis , Vitamin B 12/analysis , Vitamin B 12/blood
10.
Crit Rev Toxicol ; 10(3): 179-213, 1982 Sep.
Article in English | MEDLINE | ID: mdl-6127188

ABSTRACT

The anaesthetic gas nitrous oxide (N2O), when inhaled for longer than 6 hr, produces megaloblastic anemia in man. Longer term inhalation, as in addicts, produces a syndrome similar to that due to B12 neuropathy, and long term exposure to low concentrations results in an increased abortion rate and neuropathy, particularly in dental personnel. N2O acts by oxidizing vitamin B12 from the active reduced cob[I]alamin form to the inactive cob[III]alamin form. In turn, this inactivates the enzyme methionine synthetase which requires both B12 and folate as cofactors. In the rat, hepatic methionine synthetase is completely inactivated after 3 hr exposure to a mixture of equal parts of N2O/O2. There is an impared uptake of folate analogues by the liver so that the plasma folate level rises and thereafter there is a considerable loss of folate into the urine. Hepatic folate concentration falls to 25% within 10 days of N2O exposure. There is a failure to synthesize folate polyglutamate (the active folate coenzyme) from all other than formyltetrahydrofolate. As oxidization of the methyl of methionine is an important source of formyl, the failure of methionine synthesis in turn appears to lead to the failure in supply of formate and, hence, a lack of the formylfolate substrate.


Subject(s)
Coenzymes/analysis , Folic Acid/metabolism , Nitrous Oxide/adverse effects , Vitamin B 12/metabolism , 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/antagonists & inhibitors , Adult , Analgesia , Animals , Carbon/metabolism , Cell Division/drug effects , Female , Fetus/drug effects , Humans , Methylmalonyl-CoA Mutase/analysis , Nervous System/drug effects , Peripheral Nervous System Diseases/chemically induced , Pregnancy , Pteroylpolyglutamic Acids/biosynthesis , Rats , Tetanus/drug therapy , Thymidine/biosynthesis
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