Assay of methylmalonyl CoA mutase with high-performance liquid chromatography

Kikuchi, Masahiro; Hanamizu, H.; Narisawa, Kuniaki; Tada, Keiya

doi:10.1016/0009-8981(89)90064-8

Cited by 21 publications

(16 citation statements)

References 9 publications

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“…1, diagonally striped bar on left). Similar amounts of total holoenzyme activity have been found in human fibroblasts and glioma cells (26,27). Thus, under conditions of relatively limited cobalamin availability, about threefourths of cellular methylmalonyl-CoA mutase was present as apoenzyme, and it could be converted to holoenzyme in vitro.…”

Section: Effect Of No On Methylmalonyl-coa Mutase Activity In Vitrosupporting

confidence: 70%

“…Thus, under conditions of relatively limited cobalamin availability, about threefourths of cellular methylmalonyl-CoA mutase was present as apoenzyme, and it could be converted to holoenzyme in vitro. Other workers have also found that the enzyme in vivo is largely in the apoenzyme form, including human skin and lung fibroblasts, human glioma cells, and human, mouse, and rat liver (26,27,(33)(34)(35). When cells were incubated for 16 h in DME medium containing 10% FBS supplemented with 1 M OH-Cbl, enzyme activity in the cell extracts increased to the same level as observed when extracts were incubated with AdoCbl ( Fig.…”

Section: Effect Of No On Methylmalonyl-coa Mutase Activity In Vitromentioning

confidence: 54%

“…Acid-insoluble material was removed by centrifugation, and the supernatants were neutralized using 2.2 M KHCO 3 . The neutralized samples were injected onto a C18 reversed-phase HPLC column with methylmalonyl-CoA resolved isocratically from succinyl-CoA using 100 mM sodium phosphate, pH 4.0, with 12% methanol (26,27). The amounts of methylmalonyl-CoA and succinyl-CoA were determined by comparing the areas of their respective peaks at 254 nm to those of standards.…”

Section: Measurement Of Methylmalonyl-coa Mutase Activity In Vitromentioning

confidence: 99%

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Nitric Oxide Inhibits Mammalian Methylmalonyl-CoA Mutase

Kambo¹,

Sharma

Casteel

et al. 2005

Journal of Biological Chemistry

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Methylmalonyl-CoA mutase is a key enzyme in intermediary metabolism, and children deficient in enzyme activity have severe metabolic acidosis. We found that nitric oxide (NO) inhibits methylmalonyl-CoA mutase activity in rodent cell extracts. The inhibition of enzyme activity occurred within minutes and was not prevented by thiols, suggesting that enzyme inhibition was not occurring via NO reaction with cysteine residues to form nitrosothiol groups. Enzyme inhibition was dependent on the presence of substrate, implying that NO was reacting with cobalamin(II) (Cbl(II)) and/or the deoxyadenosyl radical (⅐CH 2 -Ado), both of which are generated from the co-factor of the enzyme, 5-deoxyadenosyl-cobalamin (AdoCbl), on substrate binding. Consistent with this hypothesis was the finding that high micromolar concentrations (>600 M) of oxygen also inhibited enzyme activity. To study the mechanism of NO reaction with AdoCbl, we simulated the enzymatic reaction by photolyzing AdoCbl, and found that even at low NO concentrations, NO reacted with both the generated Cbl(II) and ⅐CH 2 -Ado indicating that NO could effectively compete with the back formation of AdoCbl. Thus, NO inhibition of methylmalonyl-CoA mutase appeared to be from the reaction of NO with both AdoCbl intermediates (Cbl(II) and ⅐CH 2 -Ado) generated during the enzymatic reaction. The inhibition of methylmalonyl-CoA mutase by NO was likely of physiological relevance because a NO donor inhibited enzyme activity in intact cells, and scavenging NO from cells or inhibiting cellular NO synthesis increased methylmalonyl-CoA mutase activity when measured subsequently in cell extracts.In mammalian cells only two enzymes are known to require cobalamin (vitamin B 12 ) as a cofactor: methionine synthase, which uses methylcobalamin, and methylmalonyl-coenzyme A (CoA) mutase, which uses 5Ј-deoxyadenosyl-cobalamin (AdoCbl).1 During the reaction catalyzed by methionine synthase, methylcobalamin is cleaved heterolytically generating cobalamin I (Cbl(I)) and a methyl carbocation, whereas during the reaction catalyzed by methylmalonyl-CoA mutase, AdoCbl is cleaved homolytically generating cobalamin II (Cbl(II)) and a deoxyadenosyl radical (⅐CH 2 -Ado) (1, 2). The latter serves to abstract a hydrogen atom from the substrate methylmalonylCoA, and after intramolecular migration of a thioester moiety, the hydrogen atom is transferred back to generate the product succinyl-CoA. In the final step of the reaction, the deoxyadenosyl radical recombines with Cbl(II) to regenerate AdoCbl (3). It should be noted that the homolytic cleavage of AdoCbl occurs essentially only on substrate binding to the enzyme (3). Nitric oxide (NO) is produced by a variety of mammalian cell types, and has a diverse array of cellular and physiological functions including regulation of cell growth, differentiation, and apoptosis, and modulation of blood pressure, platelet aggregation, and synaptic plasticity (4 -6). At pharmacological concentrations, NO reacts with several different chemical groups, but at phys...

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Section: Effect Of No On Methylmalonyl-coa Mutase Activity In Vitrosupporting

confidence: 70%

Section: Effect Of No On Methylmalonyl-coa Mutase Activity In Vitromentioning

confidence: 54%

Section: Measurement Of Methylmalonyl-coa Mutase Activity In Vitromentioning

confidence: 99%

See 1 more Smart Citation

Nitric Oxide Inhibits Mammalian Methylmalonyl-CoA Mutase

Kambo¹,

Sharma

Casteel

et al. 2005

Journal of Biological Chemistry

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“…These 23 patients with available cell lines were confirmed to be diagnosed as mut-type MMA by remarkably reduced or undetectable intracellular MCM activity in the cultured cell lines (Table 1). MCM activity was directly determined by measuring the conversion of L-methylmalonyl-CoA to succinyl-CoA using high-performance liquid chromatography (Kikuchi et al 1989). For the remaining 19 patients, normal plasma total homocysteine (tHcy) levels were used for detection and thus rule out MMA combined with homocystinuria.…”

Section: Subjectsmentioning

confidence: 99%

Mutation Profile of the MUT Gene in Chinese Methylmalonic Aciduria Patients

Liu¹,

Liu

Yang

et al. 2012

JIMD Reports

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The mut-type methylmalonic aciduria (MMA, MIM 251000) is caused by a deficiency of mitochondrial methylmalonyl-CoA mutase (MCM, E.C. 5.4.99.2) activity, which results from defects in the MUT gene. To elucidate the mutation spectrum of the MUT gene in Chinese MMA patients, 13 exons of the MUT gene, including untranslated regions, were analyzed by PCR-based sequencing for 42 unrelated Chinese MMA patients. All the 42 patients were found to have at least one MUT mutation. A total of 41 mutations were identified. Of these mutations, 20 were JIMD Reports

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“…The diagnoses of MMA were confirmed by urinary organic acid analysis using gas chromatography and mass spectrometry. The MCM assay was performed in most cases by measuring the isomerization of L-methylmalonyl-CoA to succinylCoA by means of high-performance liquid chromatography (Kikuchi et al 1989), and MCM activity was undetectable in all of those assayed. We could not successfully subclass the patients into mut 0 or mut -by this method.…”

Section: Patientsmentioning

confidence: 99%

Mutation and haplotype analyses of the MUT gene in Japanese patients with methylmalonic acidemia

et al. 2006

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Methylmalonic acidemia (MMA) is caused by a deficiency in the activity of L-methylmalonylCoA mutase (MCM), a vitamin B12 (or cobalamin, Cbl)-dependent enzyme. Apoenzyme-deficient MMA (mut MMA) results from mutations in the nuclear gene MUT. Most of the MUT mutations are thought to be private or restricted to only a few pedigrees. Our group elucidated the spectrum of mutations of Japanese mut MMA patients by performing mutation and haplotype analyses in 29 patients with mut MMA. A sequence analysis identified mutations in 95% (55/58) of the disease alleles. Five mutations were relatively frequent (p.E117X, c.385 + 5G > A, p.R369H, p.L494X, and p.R727X) and four were novel (p.M1V, c.753_753 + 5delGGTATA, c.1560G > C, and c.2098_2099delAT). Haplotype analysis suggested that all of the frequent mutations, with the exception of p.R369H, were spread by the founder effect. Among 46 Japanese patients investigated in the present and previous studies, 76% (70/92) of the mutations were located in exons 2, 6, 8, and 13. This finding -that a limited number of mutations account for most of the mutations in Japanese mut MMA patients -is in contrast with results of a previous study in Caucasian patients.

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Assay of methylmalonyl CoA mutase with high-performance liquid chromatography

Cited by 21 publications

References 9 publications

Nitric Oxide Inhibits Mammalian Methylmalonyl-CoA Mutase

Nitric Oxide Inhibits Mammalian Methylmalonyl-CoA Mutase

Mutation Profile of the MUT Gene in Chinese Methylmalonic Aciduria Patients

Mutation and haplotype analyses of the MUT gene in Japanese patients with methylmalonic acidemia

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