TAILIEUCHUNG - Báo cáo khoa học: Characterization of structural and catalytic differences in rat intestinal alkaline phosphatase isozymes

To understand the differences between the rat intestinal alkaline phospha-tase isozymes rIAP-I and rIAP-II, we constructed structural models based on the previously determined crystal structure for human placental alkaline phosphatase (hPLAP). Our models of rIAP-I and rIAP-II displayed a typ-ical a⁄btopology, but the crown domain of rIAP-I contained an additional b-sheet, while the embracing arm region of rIAP-II lacked the a-helix, when each model was compared to hPLAP. | ềFEBS Journal Characterization of structural and catalytic differences in rat intestinal alkaline phosphatase isozymes Tsuyoshi Harada1 Iwao Koyama1 Toshiyuki Matsunaga1 Akira Kikuno1 Toshihiko Kasahara1 Masatoshi Hassimoto1 David H. Alpers2 and Tsugikazu Komoda1 1 Department of Biochemistry Saitama Medical School Saitama Japan 2 Division of Gastroenterology Washington University Schoolof Medicine St Louis MO USA Keywords 3D modeling intestinalalkaline phosphatase isozyme rat zinc Correspondence T. Komoda Department of Biochemistry Saitama Medical School 38 Morohongo Moroyama-machi Iruma-gun Saitama 350-0451 Japan Fax 81 492 76 1155 Tel 81 492 76 1155 E-mail tkalp1lp@ Received 4 December 2003 revised 1 5 February 2005 accepted 17 March 2005 doi To understand the differences between the rat intestinal alkaline phosphatase isozymes rIAP-I and rIAP-II we constructed structural models based on the previously determined crystal structure for human placental alkaline phosphatase hPLAP . Our models of rIAP-I and rIAP-II displayed a typical a b topology but the crown domain of rIAP-I contained an additional b-sheet while the embracing arm region of rIAP-II lacked the a-helix when each model was compared to hPLAP. The representations of surface potential in the rIAPs were predominantly positive at the base of the active site. The coordinated metal at the active site was predicted to be a zinc triad in rIAP-I whereas the typical combination of two zinc atoms and one magnesium atom was proposed for rIAP-II. Using metal-depleted extracts from rat duodenum or jejunum and hPLAP we performed enzyme assays under restricted metal conditions. With the duodenal and jejunal extract but not with hPLAP enzyme activity was restored by the addition of zinc whereas in nonchelated extracts the addition of zinc inhibited duodenal IAP and hPLAP but not jejunal IAP. Western blotting revealed that nearly all of the rIAP in the jejunum extracts .

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