The effects of MgADP on cross‐bridge kinetics: a laser flash photolysis study of guinea‐pig smooth muscle.

Abstract

SUMMARY1. The effects of AMgADP 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 atoxin. 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 1P3 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 tine constants of. respectively, T1 = 34 ms and T2 = 1P2 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 0A4-23 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 (Kd) of MgADP for the crossbridge binding site, estimated from its concentration-dependent effect on the relative amplitude of the first component. was 1P3/M. This affinity is much higher than the previously reported values (50-300 JtM for smooth muscle; 18-400 /tM for skeletal muscle: 7-10 /um 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 (T2) component, was 0'35-7'7 s-'. To the extent that reattachment of cross-bridges could slow relaxation even during the initial 200 ms. this rate constant may be an underestimate. § To whom all correspondence should be addressed.Ms 1080 2E. XISHIY-E AND OTHERS 5. Inorganic phosphate (Pi, 30 mM) did not affect the rate of relaxation during the initial -50 ms, but accelerated the slower phase of relaxation, consistent with a cyclic cross-bridge model in which Pi increases the proportion of cross-bridges in detached ('weakly bound') states. In the presence of 100 /tM MgADP, Pi (10-50 mM) predominantly accelerated the final slow phase of relaxation, rather than the initial (T1 and T2) 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-thiophos...

Smooth muscle myosin: regulation and properties

“…For example, solution studies (Gollub et al 1996;Cremo & Geeves 1998) show high affinity ðK d ¼ 5 mMÞ of MgADP for the non-phosphorylated complex of chicken gizzard S1 and actin. These studies also report that the association constant of ATP for actomyosin is close to values obtained in skinned smooth muscle preparations (Nishiye et al 1993;Khromov et al 1996). Such comparative data and those reported elsewhere in this review give confidence in interpreting our in situ data in terms of their physiological significance.…”

“…Gollub et al 1996Gollub et al , 1999) and on rigor force (e.g. Nishiye et al 1993;Dantzig et al 1999;Khromov et al 2001) have been used to estimate ADP binding. Here, we describe studies with fluorescent nucleotide probes.…”

Mechanical study of rat soleus muscle using caged ATP and X‐ray diffraction: high ADP affinity of slow cross‐bridges

“…6) indicated that soleus fibres are more sensitive to ADP than psoas fibres. The situation in both cardiac muscle (Martin & Barsotti, 1994) and smooth muscle (Nishiye, Somlyo, Torok & Somlyo, 1993) is similar to that described above for soleus fibres, in that their responses to ATP are complex and become simpler when apyrase is used. The complex results with the soleus and these muscles in the nominal absence of ADP are probably caused by small amounts of ADP being left in the rigor fibres.…”

Phosphorylation of Smooth Muscle Myosin Heads Regulates the Head-induced Movement of Tropomyosin

“…In contrast, the movement of tropomyosin by skeletal muscle myosin heads appears to be the same regardless of the presence of ADP (23). This difference could be related to the finding that ADP binds more strongly to smooth muscle myosin than to the skeletal muscle isoform (47,48), resulting in a large ADP-induced structural change in smooth muscle myosin but not in skeletal myosin (49,50).…”