Rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
1999
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 135 Bl. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells |c vorgelegt von: Uwe Marx |
| 264 | 1 | |c 1999 | |
| 300 | |a 135 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
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TABLE OF CONTENTS
TABLE OF CONTENTS
TABLE OF CONTENTS L
ABBREVIATIONS
. 7
I INTRODUCTION
9
1. TRANSBILAYER PHOSPHOLIPID DYNAMICS IN PLASMA MEMBRANES AND MICROSOMAL
MEMBRANES FROM ANIMAL CELLS.9
1.1 TRANSBILAYER DISTRIBUTION OF PHOSPHOLIPIDS IN PLASMA MEMBRANES AND
MICROSOMAL
MEMBRANES.9
1.2 MICROSOMAL FLIPPASE AND AMINOPHOSPHOLIPID TRANSLOCASE.10
1.3 MOLECULAR IDENTITY OF THE AMINOPHOSPHOLIPID TRANSLOCASE.12
2. TRANSBILAYER PHOSPHOLIPID DYNAMICS IN PLASMA MEMBRANES AND MICROSOMAL
MEMBRANES FROM YEAST CELLS.13
2.1 TRANSBILAYER DISTRIBUTION OF PHOSPHOLIPIDS IN PLASMA MEMBRANES AND
MICROSOMAL
MEMBRANES.13
2.2 IDENTIFICATION OF FLIPPASE PROTEINS IN THE
S. CEREVISIAE PLASMA MEMBRANE.14
3. ASSAYS FOR CHARACTERIZATION OF THE TRANSMEMBRANE DISTRIBUTION AND
MOVEMENT OF
PHOSPHOLIPIDS.17
3.1 ASSAYS FOR DETERMINATION OF THE TRANSMEMBRANE DISTRIBUTION AND
MOVEMENT OF
ENDOGENOUS PHOSPHOLIPIDS.17
3.2 ASSAYS FOR DETERMINATION OF THE TRANSMEMBRANE DISTRIBUTION AND
MOVEMENT OF
PHOSPHOLIPID ANALOGUES.19
3.3 RELIABILITY OF PHOSPHOLIPID ANALOGUES.23
3.4 DIFFICULTIES ASSOCIATED WITH MEASUREMENT OF TRANSBILAYER
DISTRIBUTION AND MOVEMENT OF
PHOSPHOLIPID ANALOGUES IN THE YEAST PLASMA MEMBRANE.25
1
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/956253253
TABLE OF CONTENTS
3.5 DIFFICULTIES ASSOCIATED WITH MEASUREMENT OF TRANSBILAYER
DISTRIBUTION AND MOVEMENT OF
PHOSPHOLIPID ANALOGUES IN RAT LIVER AND YEAST MICROSOMAL MEMBRANES.26
II
SCOPE
.28
III
MATERIALS AND METHODS
30
1. CHEMICALS.30
2. YEAST STRAINS. 30
3. TIME COURSE OF BACK-EXCHANGE OF NBD-LABELLED PHOSPHOLIPID ANALOGUES
TO BSA .30
4. LIPID EXTRACTION PROCEDURES.31
4.1
LIPID EXTRACTION ACCORDING TO THE METHOD OF BLIGH AND DYER.31
4.2 LIPID EXTRACTION ACCORDING TO THE ISOPROPANOL METHOD.31
5 ATP DEPLETION.32
6. ATP DETERMINATION.32
6.1 ATP ASSAY PROCDURE.*.32
6.2 CONTROL EXPERIMENTS.33
7. QUANTITATIVE ASSESSMENT OF INCORPORATION OF NBD-LABELLED PHOSPHOLIPID
ANALOGUES
INTO THE YEAST PLASMA MEMBRANE.34
8. DEGRADATION OF NBD-LABELLED PHOSPHOLIPID ANALOGUES IN YEAST CELLS.34
9. INTERNALIZATION OF NBD-LABELLED PHOSPHOLIPID ANALOGUES INTO YEAST
CELLS.
35
9.1 MEASUREMENT OF THE INTERNALIZED FRACTION OF ANALOGUES.35
9.2 MEASUREMENT OF THE NON-INTEMALIZED FRACTION OF ANALOGUES.36
10. PRETREATMENT OF YEAST CELLS WITH NEM, VERAPAMIL AND ORTHOVANADATC.37
11. CONSTRUCTION OF THE
S. CEREVISIAE END4 ADRS2 DELETION STRAIN.38
12. PROTEIN DETERMINATION.40
2
TABLE OF CONTENTS
13. QUANTITATION OF PHOSPHOLIPIDS BY PHOSPHATE ANALYSIS.40
14. PREPARATION OF (SUB)CELLULAR MEMBRANES FROM RAT LIVER AND YEAST.41
14.1
PREPARATION OF YEAST PLASMA MEMBRANE.41
14.2
PREPARATION OF YEAST MICROSOMES.42
34.3
PREPARATION OF SMOOTH RAT LIVER MICROSOMES. .43
15. PREPARATION OF LUV FROM THE YEAST PLASMA MEMBRANE LIPIDS.44
16. DITHIONITE ASSAY.44
17. PREPARATION OF YEAST SPHEROPLASTS. 45
18. STAINING WITH CALCOFLUOR
RM WHITE M2R.45
19. STAINING WITH ANNEXIN V-FITC .46
20. STAINING WITH DAPI. 47
21. FLUORESCENCE MICROSCOPIC ANALYSIS OF THE INTRACELLULAR DISTRIBUTION
OF INTERNALIZED
NBD-LABELLED PHOSPHOLIPID ANALOGUES.47
22. DETERMINATION OF THE OVERALL PHOSPHOLIPID COMPOSITION OF YEAST
CELLS.
47
23. STATISTICS.48
24. SYNTHESIS OF SPIN-LABELLED PHOSPHOLIPID ANALOGUES AND OF
6-O-PHENYL-ASCORBIC ACID
.'.48
25. PREPARATION OF SMALL UNILAMELLAR LIPOSOMES.48
25.1 PREPARATION OF UNLABELLED EPC SUV
.48
25.2 PREPARATION OF SPIN-LABELLED EPC SUV.48
26. PREPARATION OF UNSEALED HUMAN ERYTHROCYTE GHOSTS.49
27. STOPPED-FLOW EPR EXPERIMENTS.49
27.
1 INCORPORATION OF SPIN-LABELLED PHOSPHOLIPID ANALOGUES INTO
(BIO)MEMBRANES.50
3
TABLE OF CONTENTS
27.2 ACCESSIBILITY OF MEMBRANE-INCORPORATED SL-PC TO BSA, ASCORBATE AND
6-O-PHENYL-
ASCORBATE .50
28. REDUCTION OF SL-PC IN EPC SUV MEMBRANES BY 6-O-PHENYL-ASCORBATE AT
DIFFERENT
TEMPERATURES.51
IV RESULTS
52
1.3
TRANSBILAYER MOVEMENT OF NBD-LABELLED PHOSPHOLIPID ANALOGUES IN THE
PLASMA
MEMBRANE OF ENDOEYTOSIS-DEFICIENT YEAST CELLS.52
EFFICIENCY OF YEAST PLASMA MEMBRANE LABELLING WITH NBD-LABELLED
PHOSPHOLIPID
ANALOGUES.52
TIME COURSE OF EXTRACTION OF NBD-LABELLED PHOSPHOLIPID ANALOGUES FROM
THE PLASMA
MEMBRANE OF ENDOEYTOSIS-DEFICIENT YEAST CELLS.54
DEGRADATION OF NBD-LABELLED PHOSPHOLIPID ANALOGUES IN
ENDOEYTOSIS-DEFICIENT YEAST
CELLS.55
1.4 INTERNALIZATION OF NBD-LABELLED PHOSPHOLIPID ANALOGUES INTO
ENDOEYTOSIS-DEFICIENT YEAST
CELLS AS A FUNCTION OF INTRACELLULAR ATP CONTENT.56
1.5 EFFECT OF NEM, ORTHOVANADATE AND VERAPAMIL ON THE INTERNALIZATION OF
NBD-LABELLED
PHOSPHOLIPID ANALOGUES INTO ENDOEYTOSIS-DEFICIENT YEAST CELLS.59
1.6 DELETION OF THE
DRS2 GENE IN THE S. CEREVISIAE END4 STRAIN.60
1.7 GROWTH PHENOTYPES OF THE
S. CEREVISIAE END4 AND S. CEREVISIAE END4 ADRS2 STRAIN . 61
1.8 INTERNALIZATION OF NBD-LABELLED PHOSPHOLIPID ANALOGUES INTO
ENDOEYTOSIS-DEFICIENT
S.
CEREVISIAE END4 ADRS2
CELLS.62
1.9 TRANSBILAYER REDISTRIBUTION OF NBD-LABELLED PHOSPHOLIPID ANALOGUES
IN LUV PREPARED
FROM THE PLASMA MEMBRANE LIPIDS OF ENDOEYTOSIS-DEFICIENT YEAST CELLS.63
1.10 TRANSBILAYER ORIENTATION OF ENDOGENOUS PS IN THE PLASMA MEMBRANE OF
S. CEREVISIAE
END4 AND S. CEREVISIAE END4 ADRS2 CELLS.64
1.11 OVERALL PHOSPHOLIPID COMPOSITION OF WILD-TYPE
S. CEREVISIAE CELLS AND ENDOEYTOSIS-
DEFICIENT
S. CEREVISIAE END4 AND S. CEREVISIAE END4 ADRS2 CELLS.65
1.12 INTRACELLULAR DISTRIBUTION OF INTERNALIZED NBD-LABELLED
PHOSPHOLIPID ANALOGUES IN ATP-
DEPLETED YEAST CELLS.66
4
TABLE OF CONTENTS
1.6 RELEVANCE OF PDR PROTEINS FOR TRANSBILAYER MOVEMENT OF NBD-LABELLED
PHOSPHOLIPID
ANALOGUES IN THE PLASMA MEMBRANE OF ENDOCYTOSIS-DEFICIENT YEAST
CELLS.102
2. TRANSBILAYER PHOSPHOLIPID DYNAMICS IN RAT LIVER AND YEAST MICROSOMAL
MEMBRANES
.103
2.1 ASSAYS FOR MONITORING THE TRANSMEMBRANE MOVEMENT OF SPIN-LABELLED
PHOSPHOLIPID
ANALOGUES.103
2.2 KINETICS OF INCORPORATION OF SPIN-LABELLED PHOSPHOLIPID ANALOGUES
INTO ARTIFICIAL AND
BIOLOGICAL MEMBRANES. 104
2.3 TIME RESOLUTION OF THE ASCORBATE ASSAY. 105
2.4 REDUCTION OF SPIN-LABELLED PHOSPHOLIPID ANALOGUES BY
6-O-PHENYL-ASCORBATE.107
2.5 ASCORBATE ASSAY WITH RAT LIVER AND YEAST MICROSOMES.108
2.6 A CONTINUOUS ASSAY FOR SIMULTANEOUSLY DETERMINING TRANSMEMBRANE
DISTRIBUTION AND
MOVEMENT OF SPIN-LABELLED PHOSPHOLIPID ANALOGUES.109
2.7 STOPPED-FLOW BACK-EXCHANGE ASSAY WITH RAT LIVER MICROSOMES . . .112
2.8 STOPPED-FLOW BACK-EXCHANGE ASSAY WITH YEAST MICROSOMES.113
2.9 FLIPPASES IN THE ENDOPLASMIC RETICULUM.
.114
VI REFERENCES.116
VII
ACKNOWLEDGEMENT
128
VIII
SUMMARY
.130
IX
ZUSAMMENFASSUNG
.132
X
VEROFFENTLICHUNGEN
.134
XI
LEBENSLAUF
.135
6 |
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| id | DE-604.BV012585292 |
| illustrated | Illustrated |
| indexdate | 2024-10-09T18:14:36Z |
| institution | BVB |
| language | English |
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| physical | 135 Bl. Ill., graph. Darst. |
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| spelling | Marx, Uwe 1969- Verfasser (DE-588)12102332X aut Rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells vorgelegt von: Uwe Marx 1999 135 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Berlin, Freie Univ., Diss., 1999 Ratte (DE-588)4177011-0 gnd rswk-swf Phospholipide (DE-588)4131432-3 gnd rswk-swf Endoplasmatisches Retikulum (DE-588)4152184-5 gnd rswk-swf Proteintransport (DE-588)4273577-4 gnd rswk-swf Leberepithelzelle (DE-588)4120659-9 gnd rswk-swf Saccharomyces cerevisiae (DE-588)4178812-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Ratte (DE-588)4177011-0 s Leberepithelzelle (DE-588)4120659-9 s Endoplasmatisches Retikulum (DE-588)4152184-5 s Phospholipide (DE-588)4131432-3 s Proteintransport (DE-588)4273577-4 s DE-604 Saccharomyces cerevisiae (DE-588)4178812-6 s DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008546538&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Marx, Uwe 1969- Rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells Ratte (DE-588)4177011-0 gnd Phospholipide (DE-588)4131432-3 gnd Endoplasmatisches Retikulum (DE-588)4152184-5 gnd Proteintransport (DE-588)4273577-4 gnd Leberepithelzelle (DE-588)4120659-9 gnd Saccharomyces cerevisiae (DE-588)4178812-6 gnd |
| subject_GND | (DE-588)4177011-0 (DE-588)4131432-3 (DE-588)4152184-5 (DE-588)4273577-4 (DE-588)4120659-9 (DE-588)4178812-6 (DE-588)4113937-9 |
| title | Rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells |
| title_auth | Rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells |
| title_exact_search | Rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells |
| title_full | Rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells vorgelegt von: Uwe Marx |
| title_fullStr | Rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells vorgelegt von: Uwe Marx |
| title_full_unstemmed | Rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells vorgelegt von: Uwe Marx |
| title_short | Rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells |
| title_sort | rapid redistribution of phospholipids across cellular membranes from yeast and higher eukaryotic cells |
| topic | Ratte (DE-588)4177011-0 gnd Phospholipide (DE-588)4131432-3 gnd Endoplasmatisches Retikulum (DE-588)4152184-5 gnd Proteintransport (DE-588)4273577-4 gnd Leberepithelzelle (DE-588)4120659-9 gnd Saccharomyces cerevisiae (DE-588)4178812-6 gnd |
| topic_facet | Ratte Phospholipide Endoplasmatisches Retikulum Proteintransport Leberepithelzelle Saccharomyces cerevisiae Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008546538&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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