Transients in orientation of a fluorescent cross-bridge probe following photolysis of caged nucleotides in skeletal muscle fibres.
In muscle fibres labelled with iodoacetamidotetramethylrhodamine at Cys707 of the myosin heavy chain, the probes have been reported to change orientation when the fibre is activated, relaxed or put into rigor. In order to test whether these motions are indications of the cross-bridge power stroke, we monitored tension and linear dichroism of the probes in single glycerol-extracted fibres of rabbit psoas muscle during mechanical transients initiated by laser pulse photolysis of caged ATP and caged ADP. In rigor dichroism is negative, indicating average probe absorption dipole moments oriented more than 54.7 degrees away from the fibre axis. During activation from rigor induced by photoliberation of ATP from caged ATP in the presence of calcium, the dichroism reversed sign promptly (half-time 12.5 ms for 500 microM-ATP) upon release of ATP, but then changed only slightly during tension development 20 to 100 milliseconds later. During the onset of rigor following transfer of the fibre from an ATP-containing relaxing solution to a rigor medium lacking ATP, force generation preceded the change in dichroism. The dichroism change occurred slowly (half-time 47 s), because binding of ADP to sites within the muscle fibre limited its rate of diffusion out of the fibre. When ADP was introduced or removed, the dichroism transient was similar in time course and magnitude to that obtained after the introduction or removal of ATP. Neither adding nor removing ADP produced substantial changes in force. These results demonstrate that orientation of the rhodamine probes on the myosin head reflects mainly structural changes linked to nucleotide binding and release, rather than rotation of the cross-bridge during force generation.
Tanner
J W
JW
Department of Physiology, University of Pennsylvania, Philadelphia 19104.
Thomas
D D
DD
Goldman
Y E
YE
D000205:Actomyosin / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D005454:Fluorescence Polarization; D066298:In Vitro Techniques; D007700:Kinetics; D009038:Motion; D009119:Muscle Contraction; D009218:Myosins / Q000737:chemistry*; D010782:Photolysis; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins