Purification and characterization of mouse aldehyde oxidases:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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Potsdam
[2019]
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| Beschreibung: | xiv, 125 Seiten Illustrationen 30 cm |
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| 100 | 1 | |a Küçükgöze, Gökhan |e Verfasser |0 (DE-588)1176447130 |4 aut | |
| 245 | 1 | 0 | |a Purification and characterization of mouse aldehyde oxidases |c Gökhan Kücükgöze |
| 264 | 1 | |a Potsdam |c [2019] | |
| 300 | |a xiv, 125 Seiten |b Illustrationen |c 30 cm | ||
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| 502 | |b Dissertation |c Universität Potsdam |d 2019 | ||
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|---|---|
| adam_text | TABLE OF CONTENTS
P AGE
PUBLICATIONS FROM DISSERTATION
........................................................................
III
TABLE OF
CONTENTS...........................................................................................................
V
ABBREVIATIONS..................................................................................................................
V II
LIST OF
TABLES......................................................................................................................IX
LIST OF
FIGURES...................................................................................................................
XI
SUMMARY..............................................................................................................................
X III
1.
INTRODUCTION..................................................................................................................1
1.1 THE BIOLOGICAL SIGNIFICANCE OF
MOLYBDENUM................................................1
1.2 THE MOLYBDENUM
COFACTOR...........................................................................2
1.2.1 THE FAMILY OF MONONUCLEAR MOLYBDOENZYMES
......
1.2.2 SULFURATION OF MOLYBDENUM COFACTOR IN XO FAMILY
1.3 ALDEHYDE
OXIDASE........................................................
1.3.1 THE EVOLUTION OF ALDEHYDE OXIDASE GENES
.............
.
1.3.2 REGULATION OF ALDEHYDE OXIDASE GENES
................................................
10
1.3.3 THE FOUR-ALDEHYDE OXIDASE IN MUS MUSCULUS
.....................................
11
1.3.4 CRYSTAL STRUCTURE OF MOUSE
A0X3........................................................13
1.3.5 SUBSTRATE SPECIFICITY OF
AOX................................................................16
1.3.6 COMPARISON OF STRUCTURAL DIFFERENCES AMONG MOUSE AOXS
................
17
1.3.7 THE REACTION MECHANISM OF
AOX.........................................................18
1.4 AIMS OF THE
STUDY.......................................................................................21
2. MATERIALS AND
METHODS.......................................................................................2
3
2.1
MATERIALS...................................................................................................
23
2.1.1
STRAINS.................................................................................................
23
2.1.2 MEDIA, BUFFERS, AND SOLUTIONS
..............................................................
23
2.1.3 CHEMICALS
...........................................................................................
24
2.1.4
PLASMIDS..............................................................................................24
2.1.5
PRIMERS...............................................................................................
25
2.2
METHODS.....................................................................................................
26
2.2.1 MOLECULAR BIOLOGICAL
METHODS.............................................................26
2.2.2 BIOCHEMICAL AND ANALYTICAL METHODS
..................................................
32
2.2.3 EXPRESSION AND PURIFICATION OF
PROTEINS..............................................35
2.2.4 CHEMICAL
SULFURATION...........................................................................36
2.2.5 STEADY-STATE
KINETICS...........................................................................36
2.2.6
ICP-OES...............................................................................................39
2.2.7 MEASUREMENT OF PYRIDOXAL ACTIVITY WITH HPLC
...................................
39
2.2.8 SUPEROXIDE
PRODUCTION.......................................................................39
2.2.9 DETERMINATION OF TEMPERATURE AND PH OPTIMUM
...............................
40
3.
RESULTS.............................................................................................................................
4 1
3.1 EXPRESSION AND PURIFICATION OF MAOX
ENZYMES.......................................41
3.1.1 OPTIMIZATION OF THE PURIFICATION SYSTEM FOR MA0X4
..........................
41
3.1.2 PURIFICATION OF MAOXL, MAOX3 AND MAOX2
......................................
48
3.1.3 COMPARISON OF THE PURIFICATION CHARACTERISTICS OF MAOX ENZYMES
...
50
3.1.4 EFFECT OF CHEMICAL SULFURATION ON MAOX ACTIVITY
...............................
52
3.2 CHARACTERIZATION OF MOUSE ALDEHYDE OXIDASE ENZYMES
...........................
53
3.2.1 NATIVE PAGE AND ACTIVITY STAINING
....................................................
53
M IN
N
OO
3.2.2 DETERMINATION OF MOLYBDENUM AND IRON
SATURATION..........................54
3.2.3 PH DEPENDENCY OF MAOX ENZYMES
....................................................
55
3.2.4 TEMPERATURE DEPENDENCY OF MA0X4
..................................................
56
3.2.5 MAOX MEDIATED SUPEROXIDE PRODUCTION
............................................
57
3.2.6 NADH REDUCTION OF MAOX ENZYMES
...................................................
58
3.3 DETERMINATION AND COMPARISON OF STEADY-STATE KINETIC PARAMETERS
......
60
3.3.1
BENZALDEHYDE-DERIVATIVES...................................................................60
3.3.2 N-HETEROCYCLIC COMPOUNDS
.................................................................
64
3.3.3 ALIPHATIC
COMPOUNDS..........................................................................
68
3.3.4 CINNAMOYL
ALDEHYDES..........................................................................
71
3.3.5 PHYSIOLOGICALLY IMPORTANT SUBSTRATES
.................................................
73
3.4 SITE-DIRECTED MUTAGENESIS OF MA0X4
........................................................
75
3.4.1 EXPRESSION AND PURIFICATION OF MA0X4 VARIANTS
................................
78
3.4.2 MOLYBDENUM AND IRON CONTENT OF MAOX4 VARIANTS
............................
84
3.4.3 COMPARISON OF KINETIC PARAMETERS OF VARIANTS WITH WILD
TYPE............84
4.
DISCUSSION........................................................................................................................8
9
4.1 PURIFICATION AND CHARACTERIZATION OF MOUSE ALDEHYDE OXIDASE ENZYMES.
89
4.2 NADH OXIDATION AND ROS
PRODUCTION........................................................92
4.3 DIFFERENCES IN THE ACTIVITY OF MOUSE ALDEHYDE OXIDASES
...........................
95
4.4 SITE-DIRECTED MUTAGENESIS OF AMINO ACIDS THAT DETERMINE THE
SELECTIVITY
OF
MAOX4.......................................................................................................
102
5. CONCLUSIONS AND OUTLOOK
................................................................................
1 0 5
6.
REFERENCES...................................................................................................................
1 0 9
APPENDIX..............................................................................................................................1
1 9
ACKNOWLEDGMENT.........................................................................................................1
2 3
DECLARATION OF AUTHORSHIP
................................................................................
1 2 5
|
| adam_txt |
TABLE OF CONTENTS
P AGE
PUBLICATIONS FROM DISSERTATION
.
III
TABLE OF
CONTENTS.
V
ABBREVIATIONS.
V II
LIST OF
TABLES.IX
LIST OF
FIGURES.
XI
SUMMARY.
X III
1.
INTRODUCTION.1
1.1 THE BIOLOGICAL SIGNIFICANCE OF
MOLYBDENUM.1
1.2 THE MOLYBDENUM
COFACTOR.2
1.2.1 THE FAMILY OF MONONUCLEAR MOLYBDOENZYMES
.
1.2.2 SULFURATION OF MOLYBDENUM COFACTOR IN XO FAMILY
1.3 ALDEHYDE
OXIDASE.
1.3.1 THE EVOLUTION OF ALDEHYDE OXIDASE GENES
.
.
1.3.2 REGULATION OF ALDEHYDE OXIDASE GENES
.
10
1.3.3 THE FOUR-ALDEHYDE OXIDASE IN MUS MUSCULUS
.
11
1.3.4 CRYSTAL STRUCTURE OF MOUSE
A0X3.13
1.3.5 SUBSTRATE SPECIFICITY OF
AOX.16
1.3.6 COMPARISON OF STRUCTURAL DIFFERENCES AMONG MOUSE AOXS
.
17
1.3.7 THE REACTION MECHANISM OF
AOX.18
1.4 AIMS OF THE
STUDY.21
2. MATERIALS AND
METHODS.2
3
2.1
MATERIALS.
23
2.1.1
STRAINS.
23
2.1.2 MEDIA, BUFFERS, AND SOLUTIONS
.
23
2.1.3 CHEMICALS
.
24
2.1.4
PLASMIDS.24
2.1.5
PRIMERS.
25
2.2
METHODS.
26
2.2.1 MOLECULAR BIOLOGICAL
METHODS.26
2.2.2 BIOCHEMICAL AND ANALYTICAL METHODS
.
32
2.2.3 EXPRESSION AND PURIFICATION OF
PROTEINS.35
2.2.4 CHEMICAL
SULFURATION.36
2.2.5 STEADY-STATE
KINETICS.36
2.2.6
ICP-OES.39
2.2.7 MEASUREMENT OF PYRIDOXAL ACTIVITY WITH HPLC
.
39
2.2.8 SUPEROXIDE
PRODUCTION.39
2.2.9 DETERMINATION OF TEMPERATURE AND PH OPTIMUM
.
40
3.
RESULTS.
4 1
3.1 EXPRESSION AND PURIFICATION OF MAOX
ENZYMES.41
3.1.1 OPTIMIZATION OF THE PURIFICATION SYSTEM FOR MA0X4
.
41
3.1.2 PURIFICATION OF MAOXL, MAOX3 AND MAOX2
.
48
3.1.3 COMPARISON OF THE PURIFICATION CHARACTERISTICS OF MAOX ENZYMES
.
50
3.1.4 EFFECT OF CHEMICAL SULFURATION ON MAOX ACTIVITY
.
52
3.2 CHARACTERIZATION OF MOUSE ALDEHYDE OXIDASE ENZYMES
.
53
3.2.1 NATIVE PAGE AND ACTIVITY STAINING
.
53
M IN
N
OO
3.2.2 DETERMINATION OF MOLYBDENUM AND IRON
SATURATION.54
3.2.3 PH DEPENDENCY OF MAOX ENZYMES
.
55
3.2.4 TEMPERATURE DEPENDENCY OF MA0X4
.
56
3.2.5 MAOX MEDIATED SUPEROXIDE PRODUCTION
.
57
3.2.6 NADH REDUCTION OF MAOX ENZYMES
.
58
3.3 DETERMINATION AND COMPARISON OF STEADY-STATE KINETIC PARAMETERS
.
60
3.3.1
BENZALDEHYDE-DERIVATIVES.60
3.3.2 N-HETEROCYCLIC COMPOUNDS
.
64
3.3.3 ALIPHATIC
COMPOUNDS.
68
3.3.4 CINNAMOYL
ALDEHYDES.
71
3.3.5 PHYSIOLOGICALLY IMPORTANT SUBSTRATES
.
73
3.4 SITE-DIRECTED MUTAGENESIS OF MA0X4
.
75
3.4.1 EXPRESSION AND PURIFICATION OF MA0X4 VARIANTS
.
78
3.4.2 MOLYBDENUM AND IRON CONTENT OF MAOX4 VARIANTS
.
84
3.4.3 COMPARISON OF KINETIC PARAMETERS OF VARIANTS WITH WILD
TYPE.84
4.
DISCUSSION.8
9
4.1 PURIFICATION AND CHARACTERIZATION OF MOUSE ALDEHYDE OXIDASE ENZYMES.
89
4.2 NADH OXIDATION AND ROS
PRODUCTION.92
4.3 DIFFERENCES IN THE ACTIVITY OF MOUSE ALDEHYDE OXIDASES
.
95
4.4 SITE-DIRECTED MUTAGENESIS OF AMINO ACIDS THAT DETERMINE THE
SELECTIVITY
OF
MAOX4.
102
5. CONCLUSIONS AND OUTLOOK
.
1 0 5
6.
REFERENCES.
1 0 9
APPENDIX.1
1 9
ACKNOWLEDGMENT.1
2 3
DECLARATION OF AUTHORSHIP
.
1 2 5 |
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| spelling | Küçükgöze, Gökhan Verfasser (DE-588)1176447130 aut Purification and characterization of mouse aldehyde oxidases Gökhan Kücükgöze Potsdam [2019] xiv, 125 Seiten Illustrationen 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität Potsdam 2019 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1176981862/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032737235&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Küçükgöze, Gökhan Purification and characterization of mouse aldehyde oxidases |
| subject_GND | (DE-588)4113937-9 |
| title | Purification and characterization of mouse aldehyde oxidases |
| title_auth | Purification and characterization of mouse aldehyde oxidases |
| title_exact_search | Purification and characterization of mouse aldehyde oxidases |
| title_exact_search_txtP | Purification and characterization of mouse aldehyde oxidases |
| title_full | Purification and characterization of mouse aldehyde oxidases Gökhan Kücükgöze |
| title_fullStr | Purification and characterization of mouse aldehyde oxidases Gökhan Kücükgöze |
| title_full_unstemmed | Purification and characterization of mouse aldehyde oxidases Gökhan Kücükgöze |
| title_short | Purification and characterization of mouse aldehyde oxidases |
| title_sort | purification and characterization of mouse aldehyde oxidases |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1176981862/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032737235&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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