Rapid purification of human lysosomal membranes, characterisation of the detergent resistent microdomains, purification and reconstitution of the vacuolar proton pump (V-ATPase)

The lysosomal membrane is a dynamic environment where specific interactions among proteins and between proteins and lipids occur. These interactions are necessary for the proper functioning of the lysosomal apparatus that allows the passage of molecules into and out of the lysosomes for the degradat...

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Gespeichert in:
1. Verfasser: Chandramohanadas, Rajesh
Beteiligte: Hasilik, Andrej (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2006
Physiologische Chemie
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Zusammenfassung:The lysosomal membrane is a dynamic environment where specific interactions among proteins and between proteins and lipids occur. These interactions are necessary for the proper functioning of the lysosomal apparatus that allows the passage of molecules into and out of the lysosomes for the degradation and recycling. From the previous studies on human placental lysosomes, occurrence of detergent resistant microdomains, in which special proteins including the acetyl-CoA: α-glucosaminide N-acetyl transferase are localised has been reported by Taute et al. (2002). In an attempt to characterise the proteins that are part of such microdomains from the lysosomal membranes, the V-ATPase was found to be enriched in fractions containing the DRMs. This protein complex catalyses an ATP dependent proton translocation and is responsible for the acidification of the lysosomal lumen. It is known that lipids and lipid peroxidation products influence the biological activity of the V-ATPase that may contribute to age-related macular degeneration, a major cause of vision loss as reported by Kopitz et al. (2004). In this work a method was established for the isolation and reconstitution of the human V-ATPase which should allow further studies on the role of special lipids and lipid derivatives on the macular degeneration. To isolate the V-ATPase, a simple and efficient system for purifying the lysosomal membrane proteins was sought as the commonly used procedures yield lysosomal preparations that are contaminated with mitochondria. To reduce the contamination, a substrate-induced selective disruption of the lysosomal vesicles followed by aggregating and sedimenting the bulk of contaminating membranes, especially those from the mitochondria, was developed. A purification of the lysosomal membrane proteins up to 300 fold as compared to the placental homogenate, based on the specific activity of the membrane-associated ß-glucosidase was achieved using this novel and convenient procedure. The lysosomal membranes thus prepared may also be used for the selective isolation and characterisation of lysosomal membrane proteins that are not yet studied in detail, like the acetyl-CoA: α-glucosaminide N-acetyl transferase and Niemann- Pick C1 protein known to be involved in the degradation of heparan sulphate and in cholesterol transport, respectively. From the purified lysosomal membranes, the V-ATPase was extracted using Triton X-100 and CHAPS and further purified by gel filtration. This is the first report on the purification of V-ATPase from a human tissue. Methyl-ß-cyclodextrin was included during the extraction step to deplete cholesterol and thereby to disrupt the microdomains. Since a number of the V-ATPase subunits possess a basic isoelectric point, and as such are difficult to be analysed by the common two-dimensional electrophoretic systems, a novel CETAB/SDS-PAGE system was used for the proteomic characterisation of the purified V-ATPase. The V-ATPase activity fractions were devoid of all the major proteins present in the lysosomal preparation used for gel filtration, though two other DRM-associated proteins, placental alkaline phosphatase and stomatin were identified pointing towards the possibility of different types of DRMs on the lysosomal or the contaminating membranes, that were not removed during the purification steps. The purified protein complex was successfully reconstituted into unilamellar vesicles. When the vesicles containing lecithin and cholesterol were used for the reconstitution, a recovery of upto 30 % was observed in the incorporated fraction, as compared to 14 % when the experiment was performed with cholesterol-free vesicles. Finally, the dose-dependant inhibitory effects of the lipofuscin componenent, A2-E were confirmed with the reconstituted V-ATPase. The ATP hydrolysis by the enzyme was completely inhibited at an A2-E concentration of 10 µM. This result extends the previous findings using cell cultures on the inhibition of V-ATPase by A2-E described by Bergmann et al. suggesting a role of A2-E in the pathogenesis of age-related macular degeneration. The inhibition of ATP hydrolysis and thereby of the lysosomal acidification may cause an accumulation of lipofuscin within the lumen of the lysosomes and thus contribute to the degeneration of the retinal epithelium. The purified and reconstituted enzyme may facilitate further studies on its inhibitory and protective agents.