|Budget Amount *help
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2005: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥1,500,000 (Direct Cost: ¥1,500,000)
The aim of this study is to elucidate whether the maitake mushroom (Grifola frondosa) fibrynolytic enzyme exhibits the activity in vivo by the oral administration. Effective purification method using strong cationic exchange column, source s (Mono S, Amersham Biosciences), at pH 6.0 was newly developed. Homogeneous enzyme preparation was obtained by this method from maitake mushroom. The purified enzyme was used for the oral administration experiment to mice and the production of polyclonal antibody by using rabbit. The antibody raised was available for the western blot analysis and the immunohisotochemical analysis. Two distinct bands of the purified preparation were revealed in the SDS-PAGE analysis, of which N-terminal amino acid sequences were completely identical. The results suggested the difference in the mobility in SDS-PAGE analysis was possibly due to the post translational modification of the enzyme by glycosidation. N-terminal amino acid sequence of the both protein bands was identical with the metalloendopeptidase reported by Nonaka et al. in 1997 (J.Biol.Chem., 272(48), 30032-30039). To elucidate the in vivo fibrynolytic activity of maitake mushroom enzyme, the enzyme preparation was added to drinking water in our earlier study. However, it was difficult to estimate the exact amount of the enzyme taken by mice by this method. Thus, the administration by oral zonde was attempted. The purified enzyme preparation (200 μg) was administrated to 6 week old mice ♂C57BL/6 CrSlc (n=7). After 5 days administration, mice were sacrificed and the blood was collected from the heart. No apparent fibrynolytic activity, however, was revealed by the zymographic analysis. The amount of the enzyme and the period of administration would be reexamined in the following studies.