|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1991 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1990 : ¥1,600,000 (Direct Cost : ¥1,600,000)
UDP-GlcNAc : lysosmal enzyme GlcNAc 1-phosphotransferase, which defects or partially expresses the activity in Mucolipidosis type II (I-cell disease) or type III, respectively, has not yet been analyzed for the biochemical property, since the enzyme is much less expressed and unstable in many mammalian tissues such as liver, kidney, etc. Furthermore the enzyme requires[beta -^<32>P]UDP-GlcNAc for assay of the activity as a donor, which is not available commercially and needs to be synthesized. These disadvantages, therefore, delay to investigate above diseases in detail. This project aims to analyze immunologically the enzyme protein and further clarify a mechanism of the defect in the Mucolipidosis after purification of the human enzyme.
(1991) 1. The activity of the enzyme was much higher in human leukemic' enzyme and the serum, and human hepatocellular carcinoma tissue as compared to their normal control. Furthermore, rat fibrosarcoma cell KMT-17, established in this university, and the ascites cells demonstrated the highest activity, suggesting better source of the enzyme to purify. 2. The enzyme has been purified from rat liver Golgi membrane by us, and poly clonal antibody against the enzyme has been prepared by rabbit. However, the antibody has not reacted immunologically to human enzyme, showing different protein between rat and human (in preparation to be submitted).
(1992) 1. The purification methods of the rat enzyme has been applied for the human enzyme, however, resulting low purification fold (10^4-fold), since fresh or unfrozen tissue from human has not been available. 2. Since the enzyme catalyzes the phosphorylation to mannan from UDP-GlcNAc, the analogues of these substrates might be employed as a photoaffinity probe. In fact, azidosalycilate derivatives of[beta -^<32>P]UDP-GlcNH_2 and mannan has been synthesized)and the derivatives are appling for detection of the enzyme in several cells.