Fluorescence polarization of skeletal muscle fibers labeled with rhodamine isomers on the myosin heavy chain.
Fluorescence polarization was used to examine orientational changes of Rhodamine probes in single, skinned muscle fibers from rabbit psoas muscle following either photolysis of caged nucleotides or rapid length changes. Fibers were extensively and predominantly labeled at SH1 (Cys-707) of the myosin heavy chain with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine. Results from spectroscopic experiments utilizing the two Rhodamine isomers were quite similar. Following photolysis of either caged ATP or caged ADP, probes promptly reoriented toward the muscle fiber axis. Changes in the fluorescence polarization signals with transients elicited by the photolysis of caged ATP in the presence of saturating Ca2+ greatly preceded active force generation. Photolysis of caged ADP caused only a small, rapid decrease in force but elicited changes in the fluorescence polarization signals with time course and amplitude similar to those following photolysis of caged ATP. Fluorescence polarization signals were virtually unchanged by rapid length steps in both rigor and active muscle fibers. These results indicate that structural changes monitored by Rhodamine probes at SH1 are not associated directly with the force-generating event of muscle contraction. However, the fluorescence polarization transients were slightly faster than the estimated rate of cross-bridge detachment following photolysis of caged ATP, suggesting that the observed structural changes at SH1 may be involved in the communication pathway between the nucleotide- and actin-binding sites of myosin.
Berger
C L
CL
Department of Physiology, University of Pennsylvania, Philadelphia, USA. berger@salus.med.uvm.edu
Craik
J S
JS
Trentham
D R
DR
Corrie
J E
JE
Goldman
Y E
YE
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D066298:In Vitro Techniques; D007700:Kinetics; D009119:Muscle Contraction; D018485:Muscle Fibers, Skeletal / Q000502:physiology* / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000502:physiology* / Q000648:ultrastructure; D009210:Myofibrils / Q000502:physiology / Q000648:ultrastructure; D018995:Myosin Heavy Chains / Q000502:physiology* / Q000648:ultrastructure; D009218:Myosins / Q000378:metabolism; D009578:Nitrobenzenes / Q000378:metabolism; D010782:Photolysis; D011817:Rabbits; D012235:Rhodamines; D013050:Spectrometry, Fluorescence
D009578:Nitrobenzenes; C101520:P(2)-(1-(2-nitrophenyl)ethyl) ADP; D012235:Rhodamines; D000244:Adenosine Diphosphate; C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D018995:Myosin Heavy Chains; D009218:Myosins