Spectrofluorometric analysis of length-dependent conformational changes in cardiac troponin C.
Length modulation of cardiac muscle is manifested in the Frank-Starling relation of the heart. Recently, it has been shown that length-dependent changes in SH reactivity of cardiac troponin C (cTnC) occurred in association with cross-bridge attachment and Ca2+. However, the presence of two SH groups (Cys-35 and Cys-84) in the regulatory region of cTnC complicates efforts to detect conformational changes. In this study skinned porcine cardiac fibers were reacted with 7-diethylamino-3-[4'maleimidylphenyl]-4-methylcoumarin (CPM). Alkaline urea gel electrophoresis, along with protein elution, was used to isolate filament bound cTnC. Analysis of fluorescence measurement showed that there is a Ca(2+)-increased fluorescence for CPM-labeled cTnC in long fibers (sarcomere length = 2.2 approximately 2.5 microm) but not in short fibers (sarcomere length = 1.6 approximately 1.8 microm). In addition, the labeled cTnC was measured for the fluorescence decrease over time by adding a non-fluorescence energy acceptor, 4-dimethylaminophenylazophenyl-4'maleimide (DABMI), in the presence and absence of Ca2+. Fluorescence quenching by DABMI is not affected by Ca2+ in long fibers but it is significantly increased in short fibers. However, the fibers maintained in the relaxed state with 5 mM MgATP and 1 mM Vanadate showed no length effect on the CPM-labeled cTnC in terms of the Ca(2+)-mediated changes in fluorescence spectrum and in fluorescence quenching by DABMI. All together, our results suggest that the relative reactivities of Cys-35 and Cys-84 vary with sarcomere length.
Liou
Y M
YM
Department of Zoology, College of Life Science, National Chung-Hshing University, Taichung 402, Taiwan. ymlion@dragon.nchu.edu.tw
Tseng
Y C
YC
Cheng
J C
JC
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D003374:Coumarins; D003545:Cysteine / Q000378:metabolism; D005456:Fluorescent Dyes; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D012518:Sarcomeres / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D034421:Sus scrofa; D019209:Troponin C / Q000187:drug effects / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives* / Q000494:pharmacology
D003374:Coumarins; D005456:Fluorescent Dyes; D019209:Troponin C; D014638:Vanadates; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; C049748:4-dimethylaminophenylazophenyl-4'-maleimide; D000255:Adenosine Triphosphate; D004124:p-Dimethylaminoazobenzene; D003545:Cysteine; D002118:Calcium