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 m uscle following either photolysis of caged nucleotides or rapid length changes. Fibers were extensively and predominantly labeled at SH1 (Cy s-707) of the myosin heavy chain with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine. Results from spectroscopic experim ents utilizing the two Rhodamine isomers were quite similar. Following photolysis of either caged ATP or caged ADP, probes promptly reorient ed toward the muscle fiber axis. Changes in the fluorescence polarizat ion transients elicited by the photolysis of caged ATP in the presence of saturating Ca2+ greatly preceded active force generation. Photolys is of caged ADP caused only a small, rapid decrease in force but elici ted changes in the fluorescence polarization signals with time course and amplitude similar to those following photolysis of caged ATP. Fluo rescence 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 a ssociated directly with the force-generating event of muscle contracti on. However, the fluorescence polarization transients were slightly fa ster than the estimated rate of cross-bridge detachment following phot olysis of caged ATP, suggesting that the observed structural changes a t SH1 may be involved in the communication pathway between the nucleot ide- and actin-binding sites of myosin.