THE EFFECTS OF MGADP ON CROSS-BRIDGE KINETICS - A LASER FLASH-PHOTOLYSIS STUDY OF GUINEA-PIG SMOOTH-MUSCLE

1. The effects of MgADP on cross-bridge kinetics were investigated using laser flash photolysis of caged ATP (P3-1(2-nitrophenyl) ethyladenosine 5'-triphosphate) in guinea-pig portal vein smooth muscle permeabilized with Staphylococcus aureus alpha-toxin. Isometric tension and in-phase stiffness transitions from rigor state were monitored upon photolysis of caged ATP. The estimated concentration of ATP released from caged ATP by high-pressure liquid chromatography (HPLC) was 1.3 mm. 2. The time course of relaxation initiated by photolysis. of caged ATP in the absence of Ca2+ was well fitted during the initial 200 ms by two exponential functions with time constants of, respectively, tau1 = 34 ms and tau2 = 1.2 s and relative amplitudes of 0.14 and 0-86. Multiple exponential functions were needed to fit longer intervals; the half-time of the overall relaxation was 0.8 s. The second order rate constant for cross-bridge detachment by ATP, estimated from the rate of initial relaxation, was 0.4-2.3 x 104 M-1 s-1. 3. MgADP dose dependently reduced both the relative amplitude of the first component and the rate constant of the second component of relaxation. Conversely, treatment of muscles with apyrase, to deplete endogenous ADP, increased the relative amplitude of the first component. In the presence of MgADP, in-phase stiffness decreased during force maintenance, suggesting that the force per crossbridge increased. The apparent dissociation constant (K(d)) of MgADP for the crossbridge binding site, estimated from its concentration-dependent effect on the relative amplitude of the first component, was 1.3 mum. This affinity is much higher than the previously reported values (50-300 mum for smooth muscle; 18-400 mum for skeletal muscle; 7-10 mum for cardiac muscle). It is possible that the high affinity reflects the properties of a state generated during the co-operative reattachment cycle, rather than that of the rigor bridge. 4. The rate constant of MgADP release from cross-bridges, estimated from its concentration-dependent effect on the rate constant of the second (tau2) component. was 0.35-7.7 s-1. To the extent that reattachment of cross-bridges could slow relaxation even during the initial 200 ms, this rate constant may be an underestimate. 5. Inorganic phosphate (P(i), 30 mm) did not affect the rate of relaxation during the initial approximately 50 ms, but accelerated the slower phase of relaxation, consistent with a cyclic cross-bridge model in which P(i) increases the proportion of cross-bridges in detached ('weakly bound') states. In the presence of 100 mum MgADP, P(i) (10-50 mm) predominantly accelerated the final slow phase of relaxation, rather than the initial (tau1 and tau2) components, and shortened the duration of the plateau phase that followed the initial rapid relaxation. 6. Muscles, in which the myosin light chains were thiophosphorylated, relaxed during the initial 40-50 ms after photolysis of caged ATP at the same rate as muscles with non-thiophosphorylated light chains both in the absence and presence of MgADP. This result suggests that myosin light chain phosphorylation does not affect the rate of detachment of nucleotide-free cross-bridges. MgADP also reduced the amplitude of the first component of relaxation in thiophosphorylated muscles, with a K(d) of 4.4 mum in the absence of Ca2+ and of 4.0 mum in the presence of 30 mum Ca2+, suggesting that ADP affinity is not a major site of regulation by myosin light chain phosphorylation and is not directly modulated by Ca2+. 7. The high affinity of MgADP for cross-bridges raises the possibility that micromolar accumulation of ADP, in conjunction with co-operative reattachment of non-phosphorylated cross-bridges, contributes to tension maintenance at low levels of myosin light chain phosphorylation and low energy cost ('latch'), and to the slower rates of relaxation than myosin dephosphorylation.