Untersuchung lysosomaler Membranproteine mit dem Schwerpunkt der Charakterisierung der Acetyl-Coenzym A: a-Glucosaminid N-Acetyltransferase aus lysosomalen Membranpräparationen aus humaner Plazenta

Die Acetyl-CoA-Glucosaminid-N-Acetyltransferase ist ein lysosomales Protein, dessen Ausfall das Sanfilippo C-Syndrom (Mukopolysaccharidose Typ IIIC, OMIM 252930) bedingt. Dabei handelt es sich um eine von elf durch distinkte Enzymdefekte bedingten Mukopolysaccharidosen. Durch den Ausfall der lysosom...

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Bibliographic Details
Main Author: Wätzig, Kristin Irene
Contributors: Hasilik, Andrej (Prof.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2007
Online Access:PDF Full Text
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Table of Contents: Acetyl-CoA-Glucosaminid-N-Acetyltransferase is a lysosomal protein whose deficiency causes the Sanfilippo C-syndrome (mucopolysaccharidosis type III C, OMIM 252930). This is one out of eleven mucopolysaccharidoses caused by distinct enzyme-deficiencies. The missing enzyme function causes defective heparansulfate degradation leading to accumulation of undegraded material in the lysosome. The underlying gene sequence was described by Fan et al and Hrebicek et al in 2006. One major aim in this work war enrichment and solubilisation of the lysosomal acetyltransferase. The enzyme was isolated from human placenta. After enrichment of lysosomal membrane proteins by immunoaffinity absorption the membranes were solubilized and the extracted membrane proteins fractioned by various methods. The extraction methods were optimised and gelfiltration as well as ionexchange chromatography were used. In the hereby gained fractions the activity of acetyltransferase was measured and the fractions containing the highest amount of activity were subjected to further characterization using SDS-PAGE and 2D-electrofocussing/SDS-PAGE. An isolation of radioactive labelled acetyltransferase was not successful due to the loss of the labelling during the separation process. Later it was shown that the labelling also appeared not to be very specific. After one dimensional separation in SDS-PAGE we detected alkaline phosphatase in the possible Acetyltransferase containing regions (according molecular weight). We could demonstrate for the first time the association of acetyltransferase to rafts. Experiments with beta-cyclodextrin showed that the activity of acetyltransferase is dependent on cholesterol, a typical part of detergent resistant membrane domains. The in this work described solubilisation and fractioning methods as well as the first described role of cholesterol for the activity of Acetyltransferase could provide a ground for further isolation and reconstitution of the membrane protein in liposomes.