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pubmed:abstractText |
It has recently been suggested that activation of smooth muscle myosin light chain kinase (MLCK) can be modulated by formation of supramolecular structures (Sobieszek, A. 1991. Regulation of smooth muscle myosin light chain kinase. Allosteric effects and co-operative activation by CaM. J. Mol. Biol. 220:947-957). The present light scattering data demonstrate that the inactive (calmodulin-free) MLCK apoenzyme exists in solution as a mixture of oligomeric (2% by weight), dimeric (53%), and monomeric (45%) species at physiological ionic strength (160 mM salt). These long-living assemblies, the lifetime of which was measured by minutes, were in equilibrium with each other. The most likely form of the oligomer was a spiral-like hexamer, the dimensions of which fit very well the helical structure of self-assembled myosin filaments (Sobieszek, A. 1972. Cross-bridges on self-assembled smooth muscle myosin filaments. J. Mol. Biol. 70:741-744). After activation of the kinase by calmodulin (CaM) we could not detect any appreciable changes in the distribution of the kinase species either when the kinase was saturated with CaM or when its molar concentration exceeded that of CaM. Our fluorescent measurements suggest that the earlier observed inhibition of kinase at substoichiometric amounts of CaM (Sobieszek, A., A. Strobl, B. Ortner, and E. Babiychuk. 1993. Ca2+-calmodulin-dependent modification of smooth-muscle myosin light chain kinase leading to its co-operative activation by calmodulin. Biochem. J. 295:405-411) is associated with slow conformational change(s) of the activated (CaM-bound) kinase molecules. Such conformational rearrangements also took place with equimolar kinase to CaM; however, in this case there was no decrease in MLCK activity. The nature of these conformational changes, which are accompanied by reduction of the kinase for CaM affinity, is discussed.
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