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Purification and molecular cloning of chondroitin 4-sulfotransferase

软骨素4-磺基转移酶的纯化及分子克隆

基本信息

批准号：
09680587
负责人：
HABUCHI Osami
金额：
$ 2.05万
依托单位：
Aichi University of Education
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680587/
关键词：
CHONDROITIN SULFATE SULFOTRANSFERASE CHONDROSARCOMA DERMATAN SULFATE PROTEOGLYCANS グリコサミノグリカン

项目摘要

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

软骨素4-磺基转移酶可将硫酸根从3 ′-磷酸腺苷5 ′-磷酸硫酸根转移到软骨素中N-乙酰半乳糖胺的4位，通过肝素-琼脂糖CL-6 B、Matrex凝胶红A-琼脂糖、3 ′，5 ′-ADP-琼脂糖、纯化后的酶经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示两条蛋白带。这些蛋白质的分子量在还原条件下为60 kDa和64 kDa，在非还原条件下为50 kDa和54 kDa。这两条蛋白条带与来自Toyopolysis HW-55的软骨素4-磺基转移酶活性在50 kDa位置附近共洗脱，表明软骨素4-磺基转移酶的活性形式是单体。二硫苏糖醇激活纯化的软骨素4-磺基转移酶。纯化的酶将硫酸盐转移到软骨素和硫酸皮肤素。硫酸软骨素A和硫酸软骨素C为弱受体。鱿鱼软骨硫酸软骨素B、硫酸皮肤素、硫酸乙酰肝素和完全去乙酰化的N-硫代肝素几乎不能作为磺基转移酶的受体。硫酸盐转移到去乙酰化硫酸皮肤素优先发生在N-乙酰半乳糖胺残基的4位，在还原侧和非还原侧两侧都有葡萄糖醛酸残基。纯化后的酶蛋白经SDS-PAGE分离后，印迹于PVDF膜上，依次用胰蛋白酶、Lys-C和Asn-N消化。用毛细管反相高效液相色谱法分离释放肽，用蛋白质测序仪测定肽的氨基酸序列。结果，我们获得了6个氨基酸序列。在这些氨基酸序列的基础上，我们正在进行cDNA的克隆