Abstract
Myosin light chain kinase is activated by Ca2+/calmodulin. Insights into the kinetic mechanism of this activation by Ca2+/calmodulin have now been obtained using extrinsically labeled fluorescent calmodulin, a fluorescent peptide substrate, and a stopped-flow spectrophotofluorimeter. We employed spinach calmodulin labeled with the sulfhydryl-selective probe, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid, to measure changes in the fluorescence intensity of the 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin upon binding to rabbit skeletal muscle myosin light chain kinase. The fluorescent peptide substrate KKRAARAC(sulfobenzo-furazan)SNVFS-amide was used to measure kinase activity. Our results showed that the binding interaction could be modeled as a two-step process: a bimolecular reaction with an association rate of 4.6 x 10(7) M-1 s-1 followed by an isomerization with a rate of 2.2 s-1. Phosphorylation of the peptide during stopped-flow experiments could be modeled by a two-step process with a catalytic association rate of 6.5 x 10(6) M-1 s-1 and a turnover rate of 10-20 s-1. Our results also indicated that kinase activity occurred too rapidly for the slower isomerization rate of 2.2 s-1 to be linked specifically to the activation process.
Highlights
Residue located at position 27 in thefirst Ca'+-binding domain (Toda et al, 1985)
Design and Analysis of Stopped-flow Experiments-The data obtained from a typical stopped-flowexperiment involving the interaction of MIANS-calmodulin and myosin light chain kinase was generated from the average of the signal from eight separate time courses
The results of several experiments in which both fluorescencechanges concentrations of myosin light chain kinase used in this experiment and 32Pincorporation into SBF-labeled peptide were measured are were 0.8 (A), 1.3 ( B ),1.9 ( C ),and 2.5 (D) pM
Summary
Residue located at position 27 in thefirst Ca'+-binding domain (Toda et al, 1985). A variety of sulfhydryl-specific probes attached at this position are sensitive to thebinding of target peptides andproteins (Mills et al, 1988; Yuan and Haug, 1988; Zot et al, 1990). For several environmentally sensitive fluorescent probes, a pronounced change in fluorescence intensity occurs when Ca2+/calmodulinbinds to a Myosin light chain kinase is a Ca'+/calmodulin-activated enzyme which catalyzes the phosphorylation of the regulatory light chain of myosin. Others have observed that the target protein, but littloer no change occurs upon the binding ofCa'+ or certain peptides to Ca2+/calmodulin(Mills et al, 1988; Yuan and Haug, 1988) These types of observations, in addition to results obtained from steady-state measurements of the activation of skeletal muscle myosinlight chain kinase by fluorescently labeled wheat calmodulin, haveled to the proposal that thechanges in fluorescent intensity correspond to activation of a Ca'+/calmodulin/enzyme complex (Zot et al, 1990). MLCK, the Ca'+-calmodulin-skeletal musclemyosin light chain kinase complex; SBF-peptide, a cysteine containing peptide labeled with 4-chloro-7-sulfobenzofurazan
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