PURIFICATION OF AN ENZYME SYNTHESIZING Z SUGAR CHAIN CHARACTERISTIC TO EGF-LIKE DOMAINS OF PROTEINS ASSOCIATED WITH BLOOD COAGULATION

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 10680582
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: OSAKA WOMEN'S UNIVERSITY
• Principal Investigator: OMICHI Kaoru OSAKA WOMEN'S UNIVERSITY, FACULTY OF SCIENCE, PROFESSOR, 理学部, 教授 (90028259)
• Co-Investigator(Kenkyū-buntansha): HASEMSYMIHIRO OSAKA WOMEN'S UNIVERSITY, GRADUATE SCHOOL OF SCIENCE, PROFESSOR, 大学院・理学研究科, 教授 (80028232)
• Project Period (FY): 1998 – 1999
• Keywords: SUGAR TRANSFERASE / XYLOSYLTRANSFERASE / BLOOD COAGULATION / EGF-LIKE DOMAIN / SUGAR CHAIN / SYNTHESIS OF SUGAR CHAIN / XYLOSYL-GLUCOSE / BLOOD COAGULATION FACTOR

Research Abstract

Xylα1-3Xylα1-3Glc is a characteristic sugar chain to EGF-like domains of proteins associated with blood coagulation. It suggested the presence of β-D-glycoside α-1, 3-xylosyltransferase (G-enzyme) to synthesize the Xylα1-3Glc structure. To test for the presence of G-enzyme, bovine liver was homogenized and centrifuged for subcellular fractionation. Each subcellular fraction was incubated with a mixture of UDP-xylose (UDP-Xyl) and the acceptor 2-[(2-pyridyl) amino] ethy1β-D-glucopyranoside (Glcβ-EPA). The product was analyzed by high-performance liquid chromatography (HPLC). A transglycosylation product, Xylα1-3Glcβ-EPA, was found in the incubation mixture of the microsomal fraction, indicating the presence of the enzyme. To solubilize the enzyme, the microsomal fraction was treated with 1% Triton X-100 at 4℃ for 1 h. The crude enzyme solution was chromatographed on a DEAE-Sephacel column and the fraction with the enzyme activity was subjected to gel-filtration on a Seahorse CL-6B column. Anion-exchange HPLC on a Mono Q column and affinity chromatography on a UDP-hexanolamine column were then successively carried out. As a result, the enzyme was purified 3800-fold. Although polyacrylamide gel electrophoresis (PAGE) showed that the enzyme preparation was not completely pure, it was usable for partial characterization. The molecular weight of the enzyme was determined to be 65,000 by gel-filtration ; the optimum pH was 7.2. The enzyme was activated with MnィイD12+ィエD1. The apparent Km values for UDP-Xyl and Glcβ-EPA were 62μM and 18 mM, respectively. It will be possible to obtain the amino acid sequence by extracting the enzyme from the PAGE enzyme-activity band, thereby enabling a G-enzyme gene-disrupted mouse to be developed. The results of the present study thus provide a basis for elucidating the biological role of the Xy1α1-3Xylα1-3Glcβ1-O-Ser structure.