| Co-Investigator(Kenkyū-buntansha) |
YAMAJI Ryoichi Osaka Prefecture University, Graduate School of Life and Environment Sciences, Lecturer (00244666)
WATANABE Toshiaki University of Hyogo, School of Human Science and Environment, Professor (30091846)
EBARA Syuhei University of Hyogo, School of Human Science and Environment, Associate Professor (10372856)
WATANABE Fumio Tottori University, Faculty of Agriculture, Professor (30210941)
MIURA Takumi Osaka Prefecture University, Graduate School of Life and Environment Sciences, Associate Professor (60405355)
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| Research Abstract |
In mammals, it is well-known that Cbl acts as a cofactor for two enzymes : Ado-Cbl-dependent MCM and Me-Cbl-dependent MS. Cbl deficiency results in decreases in these two enzyme activities, leading to methylmalonic aciduria and megaloblastic anemia. The biochemical mechanisms, which result in these manifestations, still remain almost unclear.
MCM activity was greatly decreased in rat liver under Cbl-deficient conditions, and MMA, which causes a decrease in the respiratory activity, was abnormally excreted into the urine. Holo-MCM activity was only 3% of total (holo + apo) MCM activity in the liver even if rats were fed a diet with a sufficient level of Cbl. The holoenzyme activity was barely detected in the liver when weanling rats were maintained on a Cbl-deficient diet for 17 weeks. Western blot analysis confirmed a significant increase in the MCM protein level in the liver due to the Cbl deficiency. However, the MCM mRNA level was rather lower in the Cbl-deficient rats than the suffi
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cient controls. These suggest that the expression level of MCM is regulated post-transcriptionally by Cbl deficiency in rat liver. To elucidate further the effects of Cbl deficiency on the expression of MCM. The cells were rendered to be Cbl deficient. Increasing the concentration of OH-Cbl supplemented into the medium elevated the holoenzyme activity; however, the holoenzyme activity was not exceeded to 30% of the total activity even though the OH-Cbl concentration was increased up to 100 μM.
Several genetic disorders concerned in the formation of Cbl coenzymes have been reported and are known as "cbl disease"; one of these diseases, cblA, develops methylmalonic aciduria in earliest infancy. Recently, gene responsible for cblA complementation group was identified as MMAA, encoding a predicted protein of 418 amino acid residues with a mitochondrial targeting sequence. In this study, it was found that the MCM holoenzyme activity was less detected in a fibroblast cell line derived from a patient with the cblA methylmalonic aciduria, even though the cells were cultured in a medium supplemented with a sufficient level of Cbl, in contrast to fibroblasts derived from a healthy subject in which the holoenzyme activity, that was about 7% of the total activity, was detected. When recombinant MMAA protein was expressed in the cbL4 fibroblasts cultured in the presence of a sufficient level of Cbl, a great increase in the MCM holoenzyme activity was observed without an increase in the total activity. This result indicates that the MMAA protein participates in the synthesis of Ado-Cbl, the coenzyme of MCM, and confirms that the MMAA gene is the causative gene of the cblA methylmalonic aciduria. Western blot analyses showed that the MMAA protein was expressed predominantly in cerebrum, cerebellum and heart in rats. In addition, this protein was also found in skeletal muscle, kidneys and liver. Submitochondrial fractionation showed that the MMAA protein was localized in the outer membrane of mitochondria, in contrast to MCM, which was found in the matrix. Less
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