Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Purification and characterization of mammalian methionine synthase and its reductive activation proteins
Abstract
Methionine synthase is one of two known mammalian enzymes that use vitamin B12 as a cofactor. It catalyzes a methyl transfer reaction from methyltetrahydrofolate to homocysteine to generate methionine and tetrahydrofolate. In humans, dysfunction of methionine synthase is associated with megaloblastic anemia, hypomethioninemia and homocystinuria. It may also be associated with heart disease and neural tube defects. We have purified methionine synthase to near homogeneity from pig liver. It has a specific activity of 1.6–1.7 μmole/min/mg protein, which is about 2-fold higher than that of the enzyme purified from pig kidney, and >100-fold higher than that reported for the human placental enzyme. It is a large monomeric protein with a molecular weight of 151–155 kDa. It binds one mole of cobalt per mole of enzyme. Its activity is dependent on AdoMet and a reducing system. Western analysis demonstrated that human methionine synthase is also a large monomeric protein with a similar molecular weight. Determination of the percent holoenzyme from pig liver samples and a human placental sample revealed that mammalian methionine synthases are predominantly cobalamin-loaded. The development of the NADPH assay led to the discovery that the activation system of mammalian methionine synthase contains two protein components. One of the protein components has been purified to homogeneity and identified as soluble cytochrome b5. The other component is microsome associated and might be cytochrome P450 reductase. The reconstitution of methionine synthase activity with cytochrome P450 reductase and purified cytochrome b5 confirmed that cytochrome P450 reductase and soluble cytochrome b5 can fully reconstitute activity of porcine methionine synthase. These results provide a basic biochemical characterization of the mammalian methionine synthase. The identification of the two components of the activation system provides new targets for the investigation of regulation of homocysteine metabolism.
Subject Area
Biochemistry|Molecular biology|Physiology
Recommended Citation
Chen, Zhiqiang, "Purification and characterization of mammalian methionine synthase and its reductive activation proteins" (1998). ETD collection for University of Nebraska-Lincoln. AAI9917824.
https://digitalcommons.unl.edu/dissertations/AAI9917824