Kieler Werkstücke: Reihe C, Beiträge zur europäischen Geschichte des frühen und hohen Mittelalters
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| 003 | DE-604 | ||
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| 245 | 1 | 0 | |a Kieler Werkstücke |p Reihe C, Beiträge zur europäischen Geschichte des frühen und hohen Mittelalters |
| 246 | 1 | 3 | |a Beiträge zur europäischen Geschichte des frühen und hohen Mittelalters |
| 246 | 1 | 3 | |a Kieler Werkstücke / C |
| 264 | 1 | |a Frankfurt am Main u.a. |b Lang | |
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 655 | 7 | |0 (DE-588)4179998-7 |a Monografische Reihe |2 gnd-content | |
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| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-012167316 | |
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| _version_ | 1807501425080532992 |
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| adam_text |
I
CONTENTS
1
INTRODUCTION
AND
AIM
.
1
1.1
INTEREST
IN
ASPARAGINASE
.
1
1.2
THE
MECHANISM
OF
ACTION
OF
ASPARAGINASE
AS
A
DRUG
.
2
1.3
ESCHERICHIA
COLI
ASPARAGINASES
.
3
1.3.1
PROPERTIES
.
3
1.3.2
STRUCTURE
OF
E.
COLI
ASPARAGINASE
II
(ECA2)
.
6
1.3.3
THE
MECHANISM
OF
ACTION
OF
ECA2
.
7
1.3.4
IDENTIFICATION
OF
RESIDUES
IMPORTANT
FOR
ACTIVITY
.
9
1.3.4.1
SERINE
AND
THREONINE
.
9
1.3.4.2
TYROSINE
.
9
1.3.4.3
HISTIDINE
.
10
1.3.5
CATALYTIC
RESIDUES
.
11
1.3.5.1
ACYL-ENZYME
INTERMEDIATE
.
13
1.3.5.2
CONFORMATIONAL
FLEXIBILITY
OF
TYR
25
.
15
1.3.5.3
FLUORESCENCE
PROPERTIES
OF
ECA2
.
15
1.4
NOMENCLATURE
.
16
1.5
AIMS
OF
THIS
WORK
.
17
2.
MATERIALS
.
18
2.1
CHEMICALS
.
18
2.2
KITS
AND
ENZYMES
.
21
2.3
OLIGONUCLEOTIDES
.
21
2.4
MOLECULAR
WEIGHT
MARKERS
.
22
2.4.1
PROTEINS
.
22
2.4.2
DNA
.
22
2.5
BACTERIAL
STRAINS
AND
PLASMIDS
.
23
2.6
BUFFERS
FOR
ELECTROPHORESIS
.
23
2.7
APPARATUS
.
24
3.
METHODS.
.
26
3.1
GENERAL
PRECAUTIONS
.
26
3.2
BACTERIAL
STRAIN
.
26
3.2.1
EXPRESSION
SYSTEM
.
27
3.2.2
MUTAGENESIS
AND
EXPRESSION
.
27
3.3
CELL
GROWTH
AND
MAINTENANCE
.
28
3.3.1
TGI
CELLS
FOR
ML
3
PHAGE
.
28
3.3.1.1
CULTURE
OF
TGI
CELLS
.
28
3.3.1.2
PREPARATION
OF
COMPETENT
TGI
CELLS
.
29
II
3.3.1.3
TRANSFORMATION
OF
COMPETENT
HOST
CELLS
WITH
THE
M13MPL9DNA
.
30
3.3.2
E.COLI
CU1783
CELLS
FOR
EXPRESSION
PLASMID
.
30
3.3.2.1
CULTURE
OF
CU1783,
CU1783/PT7-7
AND
CU1783/PTWE
CELLS
.
30
3.3.2.2
PREPARATION
OF
COMPETENT
CU
1783
CELLS
.
31
3.3.2.3
TRANSFORMATION
OF
COMPETENT
CU1783
CELLS
WITH
DSDNA
.
31
3.4
DNA
PREPARATION
.
31
3.4.1
PREPARATION
OF
SINGLE
STRANDED
DNA
.
31
3.4.1.1
PRECIPITATION
OF
PHAGE
.
31
3.4.1.2
PHENOL-CHLOROFORM
EXTRACTION
.
32
3.4.1.3
DNA
PRECIPITATION
.
32
3.4.2
PREPARATION
OF
DOUBLE
STRANDED
DNA
.
33
3.4.2.1
ISOLATION
OF
CU1783/PTWE*
.
33
3.4.2.2
ISOLATION
OF
M13MPL9/ANSB*
.
34
3.5
AGAROSE
GEL
ELECTROPHORESIS
.
34
3.6
EXTRACTION
OF
DNA
FROM
THE
AGAROSE
GEL
.
35
3.7
PREPARATION
OF
OLIGONUCLEOTIDES
.
36
3.7.1
DETERMINATION
OF
DNA
CONCENTRATIONS
.
36
3.7.2
CONCENTRATION
IN
THE
SPEED-VAC
.
37
3.7.3
PREPARATION
BY
USING
NAP-5
COLUMNS
.
37
3.8
SITE-DIRECTED
MUTAGENESIS
.
37
3.8.1
PREPARATION
OF
SINGLE
STRANDED
M13MPL9/ANSB
TEMPLATE
DNA
.
37
3.8.2
5'-PHOSPHORYLATION
OF
OLIGONUCLEOTIDE
FOR
MUTAGENESIS
.
39
3.8.3
MUTAGENESIS
REACTION
(SCULPTORYY
IN
VITRO
MUTAGENESIS
SYSTEM)
.
39
3.8.4
TRANSFORMATION
OF
COMPETENT
TGI
CELLS
.
40
3.9
RESTRICTION
OF
M13MPL9
DOUBLE-STRANDED
DNA
OR
PLASMID
DNA
.
40
3.10
LIGATION
OF
INSERT
DNA
IN
PT7-7
EXPRESSION
VECTOR
.
41
3.11
TRANSFORMATION
OF
CU1783
CELLS
.
41
3.12
DNA
SEQUENCING
.
41
3.12.1
SEQUENCING
OF
SINGLE-STRANDED
DNA
.
43
3.12.2
SEQUENCING
OF
DOUBLE-STRANDED
DNA
.
43
3.13
GEL
ELECTROPHORESIS
.
44
3.14
EXPRESSION
OF
THE
ENZYME
.
45
3.14.1
BACTERIAL
CULTURE
.
45
3.14
2
OSMOTIC
SHOCK
.
46
3.14.3
PRECIPITATION
WITH
AMMONIUM
SULPHATE
.
46
3.14.4
CHROMATOFOCUSING
.
47
3.14.5
GEL
FILTRATION
.
47
3.15
SDS
POLYACRYLAMIDE
GEL
ELECTROPHORESIS
.
48
3.15.1
ISOELECTRIC
FOCUSING
.
50
3.16
DETERMINATION
OF
PROTEIN
CONCENTRATIONS
.
50
ILL
3.16.1
UV
SPECTROSCOPY
.
50
3.16.2
BRADFORD
METHOD
.
50
3.17
ASPARAGINASE
ASSAYS
.
51
3.17.1
PRINCIPLE
.
51
3.17.2
ACTIVITY
PROFILE
AFTER
CHROMATOFOCUSING
.
51
3.17.3
IDENTIFICATION
OF
CU1783/PTWE*
CLONES
.
52
3.18
DETERMINATION
OF
CONFORMATIONAL
STABILITY
.
53
3.18.1
CHEMICAL
DENATURATION
.
53
3.18.2
THERMAL
DENATURATION
.
57
3.19
EFFECT
OF
LIGAND
BINDING
ON
FLUORESCENCE
EMISSION
.
58
3.20
ENZYME
KINETICS
.
59
3.20.1
STEADY-STATE
KINETICS
WITH
MUTANTS
LYSL62CYS,
LYSL62HIS,
LYSL62TYR,
LYSL62AEC
AND
TRP66TYR/TYR25TRP
.
59
3.20.2
PRE-STEADY-STATE
KINETICS
OF
MUTANT
TRP66TYR/TYR25TR
.
60
3.21
MODIFICATION
OF
MUTANT
LYSL62CYS
WITH
P-BROMOETHYLAMINE
.
62
3.21.1
DENATURATION
OF
MUTANT
LYSL62CYS
.
62
3.21.2
DETERMINATION
OF
FREE
SH
GROUPS
WITH
DTNB
.
62
3.21
3
AMINOETHYLATION
OF
LYS
1
62CYS
.
62
4.
RESULTS
--------------------------------------------------------------------------
64
4.1
OVERVIEW
.
64
4.2
SELECTION
OF
RESIDUES
FOR
SITE-DIRECTED
MUTAGENESIS
.
65
4.3
SITE-DIRECTED
MUTAGENESIS
.
.65
4.3.1
DNA
TEMPLATE
AND
OLIGONUCLEOTIDE
PRIMERS
.
65
4.3.2
TRANSFORMATION
OF
TGI
CELLS
.
66
4.3.3
CONSTRUCTION
OF
MUTANT
PLASMIDS
AND
TRANSFORMATION
OF.CU
1783
CELLS
.
66
4.3.4
SELECTION
OF
MUTANT
SAMPLES
.
66
4.4
EXPRESSION
AND
PURIFICATION
OF
MUTANT
ENZYMES
.
68
4.4.1
EXPRESSION
.
68
4.4.2
ENZYME
PURIFICATION
.
68
4.4.3
DETERMINATION
OF
ISOELECTRIC
POINTS
.
71
4.4.4
PREPARATION
OF
THE
SEMI-SYNTHETIC
LYSL62AEC
MUTANT
.
73
4.5
CHARACTERISATION
OF
THE
MUTANTS
.
73
4.5.1
STEADY-STATE
KINETICS
.
73
4.5.1.1
KINETIC
PARAMETERS
AT
PH
7
.
.1.73
4.5.1.2
PH
DEPENDENCE
.
75
4.5.2
PRE-STEADY
STATE
KINETICS
OF
MUTANT
TRP66TYR/TYR25TRP
.
83
4.5.2.1
APPROACH
.
83
4.5.2.2
FLUORESCENCE
QUENCHING
BY
ASPARTATE
BINDING
TO
WILD
TYPE
ENZYME
AND
THE
TRP66TYR/TYR25TRP
MUTANT
.
83
IV
4.5.2.3
KINETICS
OF
CLOSURE
OF
THE
SURFACE
LOOP
OF
TRP66TYR/TYR25TRP
.
86
4.5.2.4
PRE-STEADY
STATE
KINETIC
STUDIES
OF
MUTANT
TRP66TYR/TYR25TR
.
87
4.6
DENATURATION
STUDIES
.
91
4.6.1
THERMAL
DENATURATION
.
91
4.6.2
CHEMICAL
DENATURATION
.
94
4.7
ASPARTATE
BINDING
.
95
5.
DISCUSSION
.
98
5.1
EXPRESSION
AND
PURIFICATION
OF
ECA2
MUTANTS
.
98
5.2
CATALYTIC
PROPERTIES
AND
SUBSTRATE
AFFINITY
.
98
5.3
SIGNIFICANCE
OF
MUTANT
LYSL62AEC
.
103
5.4
CONFORMATIONAL
STABILITY
.
105
5.4.1
THERMAL
DENATURATION
.
105
5.4.2
CHEMICAL
DENATURATION
.
106
5.5
ROLE
OF
THE
,
ASPARAGINASE
TRIAD
'
ASP90-LYSL62-THR89
.
108
5.6
ROLE
OF
THE
MOBILE
LOOP
12-27
.
111
5.7
ASPARTATE
BINDING
TO
MUTANT
TRP66TYR/TYR25TRP
.
114
6.
REFERENCES
_
115
7.
SUMMARY
_
127
8.
APPENDIX
.
127
8.1
ABBREVIATIONS
.
127
8.2
KINETIC
PARAMETERS
FOR
ECA2
FORMS
.
129
8.3
ACKNOWLEDGMENTS
.
132 |
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| indexdate | 2024-08-16T00:21:23Z |
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| issn | 0936-4935 |
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| spelling | Kieler Werkstücke Reihe C, Beiträge zur europäischen Geschichte des frühen und hohen Mittelalters Beiträge zur europäischen Geschichte des frühen und hohen Mittelalters Kieler Werkstücke / C Frankfurt am Main u.a. Lang txt rdacontent n rdamedia nc rdacarrier (DE-588)4179998-7 Monografische Reihe gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012167316&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Kieler Werkstücke |
| subject_GND | (DE-588)4179998-7 |
| title | Kieler Werkstücke |
| title_alt | Beiträge zur europäischen Geschichte des frühen und hohen Mittelalters Kieler Werkstücke / C |
| title_auth | Kieler Werkstücke |
| title_exact_search | Kieler Werkstücke |
| title_full | Kieler Werkstücke Reihe C, Beiträge zur europäischen Geschichte des frühen und hohen Mittelalters |
| title_fullStr | Kieler Werkstücke Reihe C, Beiträge zur europäischen Geschichte des frühen und hohen Mittelalters |
| title_full_unstemmed | Kieler Werkstücke Reihe C, Beiträge zur europäischen Geschichte des frühen und hohen Mittelalters |
| title_short | Kieler Werkstücke |
| title_sort | kieler werkstucke reihe c beitrage zur europaischen geschichte des fruhen und hohen mittelalters |
| topic_facet | Monografische Reihe |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012167316&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |