ACTIVATION OF MYOSIN LIGHT-CHAIN KINASE AND NITRIC-OXIDE SYNTHASE ACTIVITIES BY CALMODULIN FRAGMENTS

We have investigated the abilities of calmodulin (CaM) tryptic fragmen ts 1-75 (TRCI) or 78-148 (TRCII) to activate gizzard smooth muscle myo sin light chain kinase (gMLCK), rabbit skeletal muscle myosin light ch ain kinase (skMLCK), and neural nitric oxide synthase (nNOS) activitie s. Our results indicate for all three enzymes that binding of CaM foll ows an ordered mechanism wherein the C-terminal lobe, represented by T RCII, binds specifically to a site we designated as A, followed by bin ding of the N-terminal lobe, represented by TRCI, to a site designated as B. With TRCII and TRCI bound to their respective sites, skMLCK and gMLCK activities are both activated to about 80% of their maximum lev els. Occupancy of both sites in the MLCK enzymes by TRCI results in on ly low levels of enzyme activation; occupancy of both sites by TRCII a lso results in low levels of gMLCK activity, but activates skMLCK acti vity to 65% of the maximum level. With TRCI bound at site B and either TRCII or TRCI bound at site A, nNOS activity is 50% of the maximum le vel. Apparent dissociation constants for TRCII binding to site A and T RCI binding to site B are, respectively; 0.3 and 3 mu M (skMLCK); 1.2 and 0.8 mu M (gMLCK); 10 nM and 150 mu M (nNOS). Our results demonstra te that the CaM lobes can make distinct contributions to binding and/o r activation of different CaM-dependent enzymes and that the tethering function of the central helix can be mimicked by sufficiently high co ncentrations of the CaM fragments. We have modeled tethering as if it stabilizes the CaM-enzyme complex by creating a high effective concent ration of the N-terminal lobe. Calculated values for this concentratio n term indicate essentially identical contributions by the central hel ix to the observed nanomolar dissociation constants of the three CaM-e nzyme complexes examined.