Rapid purification of human lysosomal membranes, characterisation of the detergent resistant microdomains, purification and reconstitution of the vacuolar proton pump (V-ATPase) [Elektronische Ressource] / vorgelegt von Rajesh Chandramohanadas

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• Gesundheit

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2006
• Nombre de lectures: 33
• Poids de l'ouvrage: 4 Mo

Extrait

Aus dem
Institut für Physiologische Chemie
der
Philipps-Universität Marburg
Geschäftsführender Direktor: Prof. Dr. Andrej Hasilik



Arbeitsgruppe Biochemie und Pathobiochemie des lysosomalen Apparates
Leiter: Prof. Dr. Andrej Hasilik

Rapid purification of human lysosomal membranes, characterisation
of the detergent resistant microdomains, and purification and

reconstitution of the vacuolar proton pump (V-ATPase).









INAUGURAL DISSERTATION


Zur Erlangung des Doktorgrades der Humanbiologie
(Dr. rer. physiol.)

dem Fachbereich Humanmedizin
der Philipps-Universität Marburg

vorgelegt

von

Rajesh Chandramohanadas

aus

Haripad, Kerala, Indien


Marburg 2006






































Angenommen vom Fachbereich Humanmedizin der Philipps-Universität Marburg
am 30. 03. 2006


Dekan: Prof. Dr. B. Maisch
Referent: Prof. Dr. A. Hasilik
Koreferent: Prof. Dr. Aumüller


















Dedicated to my family!




























INDEX

1 Introduction ......................................................................... 1
1.1 Lysosome-the organelle ....................................................................... 2
1.2 Synthesis and trafficking of lysosomal proteins .................................... 3
1.3 Lysosomal membrane proteins.............................................................. 5
1.3.1 The LAMPs.................................................................................. 5
1.3.2 The LIMPs .................................................................................. 6
1.3.3 Lysosomal proton pump (V-type ATPase) ................................. 6
1.3.4 Niemann-Pick C1 protein ............................................................ 9
1.3.5 Acetyl CoA: α glucosaminide N-acetyl
transferase .................................................................................... 9
1.3.6 The CLN proteins ........................................................................ 10
1.3.7 Other transport proteins in the lysosomal membrrane................. 11

2 Aims and objectives ............................................................. 12

3 Materials and Methods ........................................................ 13
3.1 Materials ................................................................................................ 13
3.1.1 Chemicals ..................................................................................... 13
3.1.2 Antibodies .................................................................................... 15
3.1.3 Apparatus ..................................................................................... 16
3.2 Methods ................................................................................................. 16
3.2.1 Purification of lysosomal proteins from
human placenta ............................................................................ 16
3.2.1.1 Tissue homogenisation and removal of
debris and nuclei ................................................................. 16
3.2.1.2 Subcellular fractionation ..................................................... 17
3.2.1.3 Isolation of membranes by ultracentrifugation ................... 18
3.2.1.4 Immuno-affinity purification .............................................. 19
3.2.1.5 Purification of dense pool by MME treatment ................... 19
3.2.1.6 Separation of membrane complexes by
gel filtration ........................................................................ 20

III 3.2.1.7 Separation of floating material in
Optiprep ™ density gradient ................................................ 21
3.2.1.8 Assays of enzyme activities ................................................ 22
3.2.1.8.1 Assay of N-acetyl-ß-D-glucosaminidase;
EC 3.2.1.52 (von Figura, 1977) ................................ 22
3.2.1.8.2 Assay of acid ß-glucocerebrosidase; EC 3.2.1.45 (Gatt, 1969) ........................................... 22
3.2.1.8.3 Assay of Acetyl-CoA: α-glucosaminide
N-acetyl transferase; EC 2.3.1.78
(Voznyi, 1991) .......................................................... 23
3.2.1.8.4 Assay of placental alkaline phosphatase;
EC 3.1.3.1 ................................................................. 24
3.2.1.8.5 Assay of tripeptidyl peptidase-1; EC 3.4.14.9 (Junaid et al., 2001) .............................. 24
3.2.1.8.6 Assay of Succinate dehydrogenase;
EC 1.3.5.1 ................................................................ 25
3.2.1.8.7 Assay of inorganic phosphate ................................... 25
3.2.1.9 Estimation of Total Protein by Bradford method .............. 26
3.2.1.10 SDS-PAGE ......................................................................... 26
3.2.1.10.1 Preparation of acrylamide gel ................................... 26
3.2.1.10.2 Sample preparation ................................................... 28
3.2.1.10.3 Electrophoresis ......................................................... 28
3.2.1.10.4 Staining of proteins by silver nitrate
(modified from Heukeshoven, 1988) ........................ 28
3.2.1.10.5 Coomassie staining of proteins ................................. 29
3.2.1.11 Two Dimensional Electrophoresis, 2DE
(O` Farrell, 1975) ...................................................... 30
3.2.1.11.1 Isoelectric focusing of proteins ................................. 30
3.2.1.11.2 Urea/CHAPS gel and solubilisation system ............. 30
3.2.1.11.3 Thiourea/ASB 14 gel and solubilisation system ....... 31
3.2.1.11.4 Performing isoelectric focusing ................................ 33
3.2.1.11.5 Equilibration prior to the second dimensional
SDS-PAGE .............................................................. 33
IV 3.2.1.11.6 The second dimension SDS-PAGE ........................... 34
3.2.1.12 Diagonal electrophoresis (CETAB/SDS-PAGE) ...... 34
3.2.2 Identification of proteins .............................................................. 36
3.2.2.1 Western blotting ................................................................. 36
3.2.2.2 Identification of proteins by mass spectrometry ................. 38
3.2.3 Reconstitution of proteins ............................................................... 39
3.2.3.1 Preparation of liposomes ..................................................... 39
3.2.3.2 Solubilisation of proteins and reconstitution ....................... 40

4 Results ........................................................................................... 41
4.1 Purification of lysosomes ...................................................................... 42
4.1.1 Isolation of lysosomal membrane by ultracentrifugation ............ 42
4.1.2 Immuno-affinity purification of lysosomes ................................. 43
4.1.3 Purification of lysosomes by MME-treatment ............................ 46
4.2 Analysis of detergent resistant microdomains
from the lysosomal membrane ............................................................. 53
4.2.1 Floatation experiments of detergent resistant
microdomains ............................................................................. 53
4.2.2 Identification of Floating components on the
lysosomal membrane .........................................................