1998 Fiscal Year Final Research Report Summary
Purification and molecular cloning of chondroitin 4-sulfotransferase
Project/Area Number |
09680587
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Research Category |
Grant-in-Aid for Scientific Research (C)
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Allocation Type | Single-year Grants |
Section | 一般 |
Research Field |
Structural biochemistry
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Research Institution | Aichi University of Education |
Principal Investigator |
HABUCHI Osami Aichi University of Education, Education, Professor, 教育学部, 教授 (90024067)
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Project Period (FY) |
1997 – 1998
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Keywords | CHONDROITIN SULFATE / SULFOTRANSFERASE / CHONDROSARCOMA / DERMATAN SULFATE / PROTEOGLYCANS |
Research Abstract |
Chondroitin 4-sulfotransferase, which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcorna cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3', 5'-ADP-agarose, and the second heparin-Sepharose CL-6B.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 kDa and 64 kDa under reducing conditions and 50 kDa and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor accceptor. Chondroitin sulfate B from squid cartilage, dermatan sulfate, heparan sulfate and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and noreducing sides. The purified enzyme protein was separated with SDS-PAGE, blotted to PVDF membranes and digested with tpypsin, Lys-C and Asn-N successively. The released peptides were separated with capillary reversed phase HPLC.Amino acid sequence of the peptides thus obtained were sequenced with a protein sequencer. As a result, we obtained 6 amino acid sequences. On the basis of these amino acid sequences, we are now engaging in the cloning of the cDNA
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Research Products
(12 results)
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[Publications] Fukuta, M., Inazawa, J., Torii, T., Tsuzuki, K., Shimada, E.and Habuchi, O.: "Molecular cloning and characterization of human keratan sulfate Gla-6-sulfotransferase." J.Biol.Chem.272. 32321-32328 (1997)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Uchimura, K., Muramatsu, H., Kadomatsu, K., Fan, Q-W., Kurosawa, N., Mitsuoka, C., Kannagi, R., Habuchi, O., and Muramatsu, T: "Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase." J.Biol.Chem.273. 22577-22583 (1998)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Hasegawa, N., Torii, T., Nagaoka, I., Nakayasu, K., Miyajima, H., and Habuchi, O.: "Mesurement of activities of human serum sulfotransferases which transfer sulfate to galactose residue of keratan sulfate and to nonreducing end N-acetylglucosamine residue of N-acetyllactosamine trisaccharide. Comparison between normal controls and patients with macular corneal dystrophy." J.Biochem.125. 245-252 (1999)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Yamauchi, S., Hirahara, Y., Usui, H., Takeda, Y., Hoshino, M., Fukuta, M., Kimura, J.H., and Habuchi, O.: "Purification and characterization of chondroitin 4-sulfotransferase from the culture medium of a rat chondrosarcoma cell line." J.Biol.Chem.274. 2456-2463 (1999)
Description
「研究成果報告書概要(欧文)」より