|Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1991: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1990: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1989: ¥900,000 (Direct Cost: ¥900,000)
This research has been started for diagnosis of the nutritional state of the patient with DMD. Vitamin B, (thiamin)is presence in blood as four chemical forms : thiamin monophosphate(TMP), thiamin diphosphate(TDP), thiamin triphosphate(TTP), and free thiamin. The quantitative analyses of these are performed by high-performance liquid chromatography(HPLC)according to a post column fluorescence method. In the resuits, although there is no difference of the total thiamin content between the DMD patient and control, a certain difference is observed in the proportions of TDP and TTP. On the other hand, the quantitative values by HPLC changed under the storage of normal human blood at various temperature. These facts might be due to the existence of the enzyme : TDP Kinase which catalysis the synthesis of TTPfrom TDP. The purification of TDP Kinase was performed using protein-bound TDP as a marker by a preparative isoelectric focusing apparatus(Rotofor)and TDP-ligand affinity chromatography.
The isotated protein was used as reconstitutive material for HoLoenzyme state of the TDP Kinase, but the action of the Kinase was not observed. The TDP-binding activity of the isotated protein was assessed by an equilibrium dialysis apparatus, but the attempt was unsuccessful too(Heisei 1 year). Therefore, the nature of TDP Kinase has been further investigated and has been revealed that the enzyme possessed a SH-group, existed in erythrocytes, and required a certain metal ion and a certain low-molecular component in plasma as a cofactor'(Heisei 2 year). Fractionation with ammonium sulfate between 40 and 60% saturation and DEAE -SephaceL column were newly added in purification procedure. The addition of dithiothreitol to the sample effectively introduced three proteins having molecular weight as 21OKD, 18OKD, and 125KD. For separation of the three proteins, attempts to use TDP-ligand affinity column, Dye -ligand affinity column and Rotoformn and Rotofor have been carried out. While, the relationship between TDP Kinase and DMD was investigated by using of the blood of DMD patients. The results showed that the activity of TDP Kinase was more high in DMD patients than controls, therefore, the TTP content resulted in an increase(Heisei 3 year). It is unknown that the state of the patients is due to the disease or is a result of it, this study shoud be continued. Less