Kinetic mechanism of calcium-induced structural changes of skeletal troponin:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2012
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. und engl. Sprache |
| Beschreibung: | XIII, 147 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Kinetic mechanism of calcium-induced structural changes of skeletal troponin |c vorgelegt von Alfredo Jesus López Dávila |
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| 300 | |a XIII, 147 S. |b Ill., graph. Darst. | ||
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| 502 | |a Köln, Univ., Diss., 2012 | ||
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Datensatz im Suchindex
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| adam_text | Titel: Kinetic mechanism of calcium-induced structural changes of skeletal troponin
Autor: López Dávila, Alfredo Jesus
Jahr: 2012
1. Introduction..................................................................................................1
1.1 Preface.....................................................................................................1
1.2 Types of muscle tissue.............................................................................2
1.2.1 Striated muscle......................................................................................2
1.2.1.1 General description of the sarcomere of striated muscle....................3
1.2.1.2 Arrangement and function of the proteins of the thin filament.............4
1.2.1.3 Arrangement and function of the proteins of the thick filament...........7
1.2.1.4 The cross-bridges cycle and its regulation..........................................7
1.2.1.5 Kinetic mechanism of Ca2+-induced structural changes of sTnl in
psoas myofibrils............................................................................................12
1.2.1.6 Effect of strong-binding force-generating cross-bridges on the sTn
switch-off kinetics..........................................................................................17
1.3 Aims of the thesis....................................................................................19
2. Materials and methods...............................................................................20
2.1 Materials.................................................................................................20
2.1.1 Chemicals............................................................................................20
2.1.2 Buffers.................................................................................................20
2.1.2.1 Bufers for investigating the kinetic mechanism of Ca2+-induced
structural changes of sTnl in psoas myofibrils..............................................20
2.1.2.2 Buffers for investigating the effect of strong-binding force-generating
cross-bridges on the sTn switch-off kinetics.................................................29
2.2 Methods..................................................................................................34
2.2.1 Determination of the kinetic mechanism of Ca2+-induced structural
changes of sTnl in psoas myofibrils using stopped-flow...............................34
2.2.1.1 Description of the stopped-fiow method and the stopped-flow
apparatus......................................................................................................34
2.2.1.1.1 Principle of the stopped-flow method.............................................34
2.2.1.1.2 Stopped flow experiments in single mixing mode..........................34
2.2.1.1.3 Stopped flow experiments in double mixing mode.........................35
2.2.1.1.4 The dead time of a stopped flow apparatus...................................36
2.2.1.1.5 Settings of the stopped-flow apparatus..........................................36
2.2.1.1.6 Equipment coupled to the stopped flow apparatus in order to record
fluorescence transients.................................................................................37
2.2.1.1.7 Excitation wavelength, emission wavelength and emission filter sets
according to the selected fluorophore...........................................................37
2.2.1.1.8 Software.........................................................................................38
2.2.1.2 Purification of sTn from Chinchilla Bastard rabbits............................38
2.2.1.3 Labelling of sTn.................................................................................39
2.2.1.4 Separation of sTn from Tm...............................................................41
2.2.1.5 Preparation of sTn complexes selectively labelled either on Tnl or on
TnC...............................................................................................................41
2.2.1.5.1 Separation of sTn from Tm using hydroxyapatite-chromatography41
2.2.1.5.2 Separation of sTn in its subunits using DEAE-Sepharose-
chromatography............................................................................................42
2.2.1.5.3 Separation of contaminating proteins from Tnl using CM-
Sepharose-chromatography.........................................................................43
2.2.1.5.4 Reconstitution of the selectively labelled sTn complex..................43
2.2.1.6 Preparation of skeletal myofibrils from rabbit psoas muscle.............45
2.2.1.7 Exchange of sTn in skeletal myofibrils..............................................46
2.2.1.8 Localization of the labelled sTn in the sarcomeres of exchanged
skeletal myofibrils using immunostaining......................................................46
2.2.1.8.1 Preparation and exchange of skeletal myofibrils............................46
2.2.1.8.2 Antibodies complex formation........................................................47
2.2.1.8.3 Microscopical examination sTnIAF localization in the sarcomere.... 47
2.2.1.9 Estimation of the density of skeletal myofibrils..................................48
2.2.1.10 Measurements of the Ca2+-induced switch-on kinetics of
incorporated sTn...........................................................................................49
2.2.1.11 Measurements of the Ca2+-induced switch-off kinetics of
incorporated sTn...........................................................................................50
2.2.1.12 Measurements of the Ca2+-induced switch-on and switch-off kinetics
of isolated sTn..............................................................................................51
2.2.1.13 Measurements of Ca2+-dissociation kinetics from isolated sTn with
Quin-2...........................................................................................................51
2.2.2 Determination of the effect of strong-binding force-generating cross-
bridges on the sTn switch-off kinetics...........................................................52
2.2.2.1 Description of the apparatus.............................................................52
2.2.2.1.1 The length driver and the force transducer....................................52
2.2.2.1.2 The chamber block........................................................................54
2.2.2.1.3 Sarcomere length measurements using laser diffraction...............54
2.2.2.1.4 Components involved in the fluorescence measurements.............55
2.2.2.1.5 Software.........................................................................................57
2.2.2.1.6 Newly added components to perform the experiments for the thesis
......................................................................................................................57
2.2.2.1.6.1 Flash lamp with light guide..........................................................57
2.2.2.1.6.2 Fast reaction shutter...................................................................58
2.2.2.1.6.3 TTL pulses generator triggering the flash lamp and shutter 2.....59
2.2.2.2. Manipulation of muscle tissues used for the experiments................62
2.2.2.2.1 Isolation and storage of rabbit psoas fibers bundles......................62
2.2.2.2.2 Preparation of single rabbit psoas skinned fibers...........................63
2.2.2.2.3 Mounting a single skinned fiber in the experimental apparatus......63
2.2.2.2.4 Exchange of endogenous sTn.......................................................65
2.2.2.2.5 Measurement of Ca2+-induced thin filament activation and force
development of a single skinned fiber during steady-state isometric
contraction....................................................................................................66
2.2.2.2.6 Measurements of the Ca2+-induced sTn conformational change in
parallel with force relaxation of a single skinned fiber...................................70
2.2.3 Statistics and mathematical procedures..............................................73
2.2.3.1 Descriptive statistics and handle of data...........................................73
2.2.3.2 Inferential statistics...........................................................................73
2.2.3.3 Fluorescence, force and rate constants - pCa relations...................74
2.2.3.4 Determination of rate constants from monophasic force transients and
monophasic and biphasic fluorescence transients.......................................74
3. Results.......................................................................................................76
3.1 Kinetic mechanism of Ca2+-induced structural changes of sTnl in psoas
myofibrils.......................................................................................................76
3.1.1 Labelling of sTn with IAF and IANBD...................................................76
3.1.2 Fluorescence emission of isolated sTnIAF and sTnIANBD.......................76
3.1.3 Ca2+-dissociation kinetics from isolated sTn determined with Quin-2 .. 77
3.1.4 Sarcomeric localization of the labelled sTn in the exchanged myofibrils
......................................................................................................................79
3.1.5 Testing fluorescence emission from incorporated sTnIAF and sTnIANBD 80
3.1.6 Testing fluorescence emission from Tn complexes selectively labelled
either on sTnl or on sTnC.............................................................................81
3.1.7 sTnIAF switch-on kinetics in the absence of ATP..................................83
3.1.8 Ca2+-dependence of the switch-on of sTnIAF........................................83
3.1.9 Ca2+-inactivation: switch-off of sTnIAF in comparison with force relaxation
......................................................................................................................89
3.2 Effect of strong-binding force-generating cross-bridges on sTn switch off
kinetics..........................................................................................................91
3.2.1 Exchange of endogenous sTn in the mounted skinned fiber...............91
3.2.2 Ca2+-dependent thin filament activation and steady-state force
development of a single skinned fiber during isometric contraction..............91
3.2.3. Attempts to measure thin filament inactivation simultaneously with force
relaxation......................................................................................................97
4. Discussion...............................................................................................102
4.1 Kinetic mechanism of Ca2+-induced structural changes of sTnl in psoas
myofibrils.....................................................................................................102
4.1.1 Sumary of the most important results................................................102
4.1.2 Origin and interpretation of the fluorescence signals.........................103
4.1.3 Effects of sarcomeric incorporation on sTn switch kinetics................106
4.1.4 Relation of switch-on and switch-off kinetics to kinetics of force
contraction and relaxation...........................................................................107
4.1.5 Conclusion.........................................................................................111
4.2 Effect of strong-binding force-generating cross-bridges on sTn switch-off
kinetics........................................................................................................112
4.2.1 Summary...........................................................................................112
4.2.2 Ca2+-induced thin filament activation and resulting force development of
a single skinned fiber in steady-state isometric contraction........................112
4.2.2.1 Steady state fluorescence of TnIAF-exchanged fibers.....................112
4.2.2.2 Steady state fluorescence of sTnIANBD-exchanged fibers................115
4.2.3 Measurements of the Ca2+-induced sTn conformational change in
parallel with force relaxation of a single skinned fiber.................................116
4.2.3.1 Experiments using non-exchanged muscle fibers...........................116
4.2.3.2 Limited suitability of current models of regulation of muscle
contraction to explain the diazo-2 induced force relaxation........................117
4.2.3.3 Thin filament inactivation and force relaxation of sTnIAF-exchanged
fibers...........................................................................................................119
4.2.3.4 Thin filament inactivation and force relaxation of sTnIANBD-exchanged
fibers...........................................................................................................121
4.2.3.5 Experiments recorded before eliminating the artefact in the
fluorescence transients...............................................................................123
4.2.3.6 Additional experiments which could be recorded using the established
setting.........................................................................................................124
4.2.3.6.1 Diazo-2 experiments using labelled sTn in presence of S1 and ATP-
yS and in rigor conditions............................................................................124
4.2.3.6.2 Diazo-2 experiments using rabbit psoas fibers to study sarcomere
dynamics during force relaxation................................................................124
4.2.3.6.3 Application of the acquired experience and developed techniques to
cardiac muscle basics and clinics...............................................................125
4.2.4 Conclusions.......................................................................................126
5. References..............................................................................................127
6. List of abbreviations.................................................................................134
7. Aknowledgements...................................................................................141
8. Erklarung (entsprechend §4 Abs. 1 Nr 9 der Promotionsordnung der
Mathematisch-Naturwissenschaftlichen Fakultatder Universitat zu Kbln)......143
9. Publications.............................................................................................145
10. Curriculum vitae....................................................................................147
|
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| spelling | López Dávila, Alfredo Jesus 1976- Verfasser (DE-588)1020422483 aut Kinetic mechanism of calcium-induced structural changes of skeletal troponin vorgelegt von Alfredo Jesus López Dávila 2012 XIII, 147 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache Köln, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=024883119&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | López Dávila, Alfredo Jesus 1976- Kinetic mechanism of calcium-induced structural changes of skeletal troponin |
| subject_GND | (DE-588)4113937-9 |
| title | Kinetic mechanism of calcium-induced structural changes of skeletal troponin |
| title_auth | Kinetic mechanism of calcium-induced structural changes of skeletal troponin |
| title_exact_search | Kinetic mechanism of calcium-induced structural changes of skeletal troponin |
| title_full | Kinetic mechanism of calcium-induced structural changes of skeletal troponin vorgelegt von Alfredo Jesus López Dávila |
| title_fullStr | Kinetic mechanism of calcium-induced structural changes of skeletal troponin vorgelegt von Alfredo Jesus López Dávila |
| title_full_unstemmed | Kinetic mechanism of calcium-induced structural changes of skeletal troponin vorgelegt von Alfredo Jesus López Dávila |
| title_short | Kinetic mechanism of calcium-induced structural changes of skeletal troponin |
| title_sort | kinetic mechanism of calcium induced structural changes of skeletal troponin |
| topic_facet | Hochschulschrift |
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