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 (MLC(20)) 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) = similar to 200 ms) saturated at 40 mu M. Photolysis of caged ATP following preequilibration with identical [Ca(4)CaM] shortened t(d) to 41 ms. The rate of phosphorylation was very fast (3.5 s(-1) at 22 degrees C in the presence of 5 mu M 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 mu M CaM and a rate of MLC(20) 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 mu M) 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 mu M) to shorten the t(d). We estimate the endogenous CaM available under steady state conditions in vivo to be approximately 0.25 mu M and probably less during a rapid Ca2+ transient. We conclude that the [CaM] dependence of the kinetics of MLC(20) phosphorylation and force development (t1/2 and t(d)) initiated by Ca-2+ 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 Ca(4)CaM myosin light chain kinase complex (Torok, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820). The relatively short delay (108 ms in the presence of 40 mu M 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 Ca(2)CaM myosin light chain kinase complex.