| Budget Amount *help |
¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1995: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1994: ¥800,000 (Direct Cost: ¥800,000)
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| Research Abstract |
beta-galactosidase is the enzyme that catalyzes hydrolysis of the terminal beta1-4 or 1-3 galactoside linkage in the oligosuccharide chain of complex carbohydrates. Mutations of the gene coding for this enzyme result in hereditary disease in human with phenotypic variations, represented by GM1 gangliosidosis and Morquio B disease. Their manifestions are based on different mutations of the same gene, suggesting different catalytic proteins and tissue expressions of mutant enzymes. In this study, we expressed mutant cDNAs of point mutation in GM1 gangliosidosis : I51T (adult type) , R201C (juvenile type) and G123R (infantile) , and in Morquio B disease : W273L by the Baculovirus system for characterization of mutant enzymes. Ferthermore, we considered that it could clarified the relation between phenotypic variations and substrates storaged in patient body. We have failed to obtain amount of mutant protein all types of GM1 gangliosidosis, because of not secreting but remaining in Sf-9 ce
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lls. The result implied that causing abnormal processing of succharide chain in mutant protein from GM1 gangliosidosis. Only W273L mutant protein from Morquio B disease was obtained by Baculovirus system. Normal and W273L mutant protein were purified precursor (84kDa) and mature (64kDa) proteins by various column chromatographies, and assayd their catalytic activities toward MU-Gal substrate, GM1 ganglioside, oligosuccharide and keratan sulfate. 1) Normal 84kDa and 64kDa protein each has distinct character (Km, optimun pH,effect on detergent). 2) Km of W273L mutant 84kDa was same as normal, but Vmax of mutant was 1/20 of normal Vmax. 3) W273L mutant 64kDa almost lost its catalytic activity. 4) Affinity of mutant 84kDa protein toward p-aminophenyl beta-D-thiogalactopyranoside, analogue substrate, was lower than normal. 5) Mutant 84kDa has catalytic activity toward GM1 ganglioside, natural substrate, but not toward keratan sulfate which was stored in patient urea. These results suggested that the residual activity of mutant precursor protein toward beta-1,4 galactoside linkage saved Morquio B dissease patients from mental retardation which is severe in GM1 gangliosidosis. Less
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