Kinetics of prephosphorylation reactions and myosin light chain phosphorylation in smooth muscle: Flash photolysis studies with caged calcium and caged ATP

The pre-myosin light chain (MLC₂₀) phosphorylation components of the lag phase (t_d) of contractile activation were determined in permeabilized smooth muscles activated by photolytic release of ATP from caged ATP and/or Ca²⁺ from 4-(2-nitrophenyl)-EGTA (NP-EGTA). Calmodulin (CaM) shortened the t_d (470 ms at 0 added CaM) that followed Ca²⁺ release, but its effect (t_d = ∼200 ms) saturated at 40 μM. Photolysis of caged ATP following preequilibration with identical [Ca₄CaM] shortened t_d to 41 ms. The rate of phosphorylation was very fast (3.5 s^-1 at 22 °C in the presence of 5 μM exogenous CaM) following photolysis of caged ATP, and, following Ca²⁺ release, phosphorylation was accelerated by CaM. Simultaneous photolysis of caged ATP and NP-EGTA was followed by a t_d of 194 ms at 5 μM CaM and a rate of MLC₂₀ phosphorylation intermediate between these parameters following photolysis of, respectively, NP-EGTA and caged ATP. In the presence of the normal, total endogenous CaM content (37 ±4 μM) of portal vein smooth muscles t_d was 565 ms. Steady state maximum force at pCa 5.5 was increased by much lower (100 nM) exogenous [CaM] than was required (>2.5 μM) to shorten the t_d. We estimate the endogenous CaM available under steady state conditions in vivo to be approximately 0.25 μM and probably less during a rapid Ca²⁺ transient. We conclude that the [CaM] dependence of the kinetics Of MLC₂₀ phosphorylation and force development (t_1/2, and t_d) initiated by Ca²⁺ reflects the recruitment of a slowly diffusible component of total CaM. The relatively long duration of t_d (197 ms) at saturating [CaM] suggests the contribution to t_d of an additional component, possibly a prephosphorylation activation/isomerization of the Ca₄CaM myosin light chain kinase complex (Török, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820). The relatively short delay (108 ms in the presence of 40 μM CaM) following simultaneous photolysis of NP-EGTA and caged ATP suggests that preincubation with ATP (prior to photolysis of NP-EGTA) may inhibit the formation of a preactive Ca₂CaM myosin light chain kinase complex.