Liver, Pancreas, and Biliary TractLocalization of the Wilson's disease protein in human liver☆,☆☆,★
Section snippets
Materials
The C219 antibody to P-glycoproteins was purchased from Dako (Hamburg, Germany); the Golgi markers 58K and AP-1 were obtained from Sigma Chemical Co. (St. Louis, MO); rabbit polyclonal antibodies against the human WND protein8, 13 and the human sodium taurocholate–cotransporting polypeptide (NTCP) were kindly provided by Jonathan D. Gitlin (Washington University School of Medicine, St. Louis, MO) and Peter Meier (University Hospital Zürich, Switzerland), respectively. Secondary antibodies
Detection of WND protein in canalicular enriched membrane fractions
On Western blotting (Figure 1), the WND protein was detectable as a 165-kilodalton protein in both blLPM and cLPM fractions, with a clearly greater abundance in the canalicular fraction (Figure 1, lanes 1 and 2). To confirm that these fractions were enriched in
Discussion
The role of the WND protein in hepatic copper handling and its exact localization within the liver remains unclear. The present study shows endogenous hepatocellular expression of the WND protein in the human liver. Immunoblotting experiments, using material from multiple normal human livers, indicate that the WND protein is abundantly present in a canalicular membrane-enriched fraction and much less in blLPM fractions. In liver membrane fractions of a patient with WND and an unknown mutation,
Acknowledgements
The authors thank Dr. Jonathan D. Gitlin for providing the antibody against the human Wilson's Disease protein and Dr. Peter Meier for providing the antibody against human Na+/taurocholate- cotransporting polypeptide.
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Address requests for reprints to: Roel J. Vonk, M.D., Center for Liver, Digestive and Metabolic Diseases, Department of Pediatrics, University Hospital Groningen; P.O. Box 30001, 9700 RB Groningen, The Netherlands. e-mail: [email protected]; fax: (31) 50-3611746.
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Supported by German Research Council grant DFG STR 216/6-1 (to W.S.), The Netherlands Digestive Diseases Foundation grant WS96-70 (to R.J.V.), and Biomed and Health Research Program grant PL 931436 (to F.K. and W.S.). Mark Schaefer is the recipient of young scientist education grant SCHA 781/1-1 from the German Research Council/DFG.
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Drs. Schaefer and Roelofsen contributed equally to this work.