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 (td) 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 td (470 ms at 0 added CaM) that followed Ca2+ release, but its effect (td = approximately 200 ms) saturated at 40 microM. Photolysis of caged ATP following preequilibration with identical [Ca4CaM] shortened td to 41 ms. The rate of phosphorylation was very fast (3.5 s-1 at 22 degrees C in the presence of 5 microM 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 td of 194 ms at 5 microM 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 microM) of protal vein smooth muscles td 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 microM) to shorten the td. We estimate the endogenous CaM available under steady state conditions in vivo to be approximately 0.25 microM and probably less during a rapid Ca2+ transient. We conclude that the [CaM] dependence of the kinetics of MLC20 phosphorylation and force development (t1/2 and td) initiated by Ca2+ reflects the recruitment of a slowly diffusible component of total CaM. The relatively long duration of td (197 ms) at saturating [CaM] suggests the contribution to td 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 microM 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.
Zimmermann
B
B
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908, USA.
Somlyo
A V
AV
Ellis-Davies
G C
GC
Kaplan
J H
JH
Somlyo
A P
AP
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008642:Mesenteric Veins / Q000378:metabolism; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation; D010782:Photolysis; D011817:Rabbits; D013997:Time Factors
D002147:Calmodulin; D002614:Chelating Agents; D018994:Myosin Light Chains; C085112:2-nitrophenyl-EGTA; D004533:Egtazic Acid; C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D002118:Calcium