|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1999 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1998 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Pyruvate dehydrogenase (PDH) α-subunit (E1α) deficiency is one of the most common causes of congenital lactic acidemia. The therapy of this deficiency is usually very difficult. It is expected to establish the new therapy by molecular biological analysis of the function of mutant PDH protein. Then, in this project we first try to establish the expression system of normal and mutant recommbinat PDH proteins. The expression plasmid vector containing normal or mutant E1α and normal E1β cDNA was constructed and transfected into E. coli. In transformed E. coli, massive amount of PDH protein was expressed and the recommbinat PDH proteins was purified and the enzyme activity of recommbinat PDH protein was measured. However, the enzyme activity of normal recommbinat PDH protein was very lower than the expected activity. The enzyme activities of several mutant recommbinat PDH proteins could not be detected and the dysfunction of mutant PDH proteins could not detected by this method. Therefore, it is necessary to establish the new method for constrution of recommbinat PDH protein with enough enzyme activity.
In this project, we also establish the more safe and faster new system of DNA diagnosis for female patients with PDH deficiency, who can not be diagnosed with enzymological method, than previously established system by us. With this new system, four female patients, who could not be daignased with enzymological method, could be diagnosed PDH deficiency.