1. The interaction between MgADP and rigor cross-bridges in glycerol-extracted single fibres from rabbit psoas muscle has been investigated using laser pulse photolysis of caged ATP (P3-1(2-nitrophenyl)ethyladenosine 5'-triphosphate) in the presence of MgADP and following small length changes applied to the rigor fibre. 2. Addition to 465-mu-M-MgADP to a rigor fibre caused rigor tension to decrease by 15.3 +/- 0.7% (S.E.M., n = 24 trials in thirteen fibres). The half-saturation value for this tension reduction was 18 +/- 4-mu-M (n = 23, thirteen fibres). 3. Relaxation from rigor by photolysis of caged ATP in the absence of CA2+ was markedly slowed by inclusion of 20-mu-M-2 mM-MgADP in the photolysis medium. 4. Four phases of tension relaxation occurred with MgADP in the medium: a(t), a quick partial relaxation (in pre-stretch fibres); b(t), a slowing of relaxation or a rise in tension for 50-100 ms; c(t), a sudden acceleration of relaxation; and d(t), a final, nearly exponential relaxation. 5. Experiments at varied MgATP and MgADP concentrations suggested that phase, a(t) is due to MgATP binding to nucleotide-free cross-bridges. 6. Phase b(t) was abbreviated by including 1-20 mM-orthophosphate (P(i)) in the photolysis medium, or by applying quick stretches before photolysis or during phase b(t). These results suggest that phases b(t) and c(t) are complex processes involving ADP dissociation, cross-bridge reattachment and co-operative detachment involving filament sliding and the Ca2+-regulatory system. 7. Stretching relaxed muscle fibres to 3.2-3.4-mu-m striation spacing followed by ATP removal and release of the rigor fibre until tension fell below the relaxed level allowed investigation of the strain dependence of relaxation in the regions of negative cross-bridge strain. In the presence of 50-mu-M-2 mM-MgADP and either 10 mM-P(i) or 20 mM-2,3-butanedione monoxime, relaxation following photolysis of caged ATP was 6- to 8-fold faster for negatively strained cross-bridges than for positively strained ones. This marked strain dependence of cross-bridge detachment is predicted from the model of A. F. Huxley (1957). 8. In the presence of Ca2+, activation of contraction following photolysis of caged ATP was slowed by inclusion of 20-500-mu-M-MgADP in the medium. An initial decrease in tension related to cross-bridge detachment by MgATP was markedly suppressed in the presence of MgADP. 9. Ten millimolar P(i) partly suppressed active tension generation in the presence of MgADP. The tension transients obtained when MgADP, Ca2+ and P(i) were included in the photolysis medium showed an unexpected initial tension rise following photolysis of caged ATP and then a decrease to the steady tension level. 10. Computer simulations of the cross-bridge reactions involving ADP release, MgATP binding, detachment, and reattachment into force-generating intermediates were fitted to the transients recorded following photolysis of caged ATP. In the absence of ADP the time course of the transients could be simulated using a simple model without strain-dependent rate constants and assuming that attached cross-bridge states in rapid equilibrium with detached states [GRAPHICS] and [GRAPHICS] exerted zero force. However, in the presence of MgADP the transients simulated with these assumptions showed a deeper tension dip following photolysis of caged ATP than the experimental records. 11. Two explanations of this discrepancy are considered. In the first hypothesis, rigor cross-bridges are assumed to be distributed over a wide range of forces, including negative forces, and ADP dissociates more rapidly from negatively strained cross-bridges than from positively strained ones. In the presence of MgADP, rapid detachment of the negatively strained cross-bridges limits the magnitude of tension dip following ATP release. 12. In the second explanation, in the presence of MgADP, attached cross-bridge states that are in rapid equilibrium with detached states [GRAPHICS] and [GRAPHICS] are considered to bear significant force. Force in these states limits the magnitude of the initial tension dip following release of ATP. Since [GRAPHICS] and [GRAPHICS] cannot bind MgADP, the assignment of tension to these states with MgADP present implies that in this model, interactions between cross-bridges or between individual heads of a myosin molecule occur in the presence of MgADP. Kinetic simulations on the basis of both models approximated the experimental records in the absence and presence of MgADP. However, the first model better accommodated data obtained in the presence of Ca2+, MgADP and P(i).
