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Volume 272, Number 43, Issue of October 24, 1997 pp. 27167-27177
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Kinetics of Interaction of the Myristoylated Alanine-rich C Kinase Substrate, Membranes, and Calmodulin

(Received for publication, July 1, 1997, and in revised form, August 12, 1997)

Anna Arbuzova , Jiyao Wang , Diana Murray , Jaison Jacob ^¶ , David S. Cafiso ^¶ and Stuart McLaughlin

From the  Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8661 and the ^¶ Department of Chemistry and Biophysics Program, University of Virginia, Charlottesville, Virginia 22901

Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic "effector" region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151-175), showed that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. Here we describe the kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151-175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example, k_on = 10¹¹ M¹ s¹ for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically, k_on is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (k_off) is ~10 s¹ (lifetime = 0.1 s) for vesicles with 10% acidic lipid in 100 mM KCl. Ca²⁺-calmodulin (Ca²⁺·CaM) decreases markedly the lifetime of the peptide on vesicles, e.g. from 0.1 to 0.01 s in the presence of 5 µM Ca²⁺·CaM. Our results suggest that Ca²⁺·CaM collides with the membrane-bound MARCKS-(151-175) peptide and pulls the peptide off rapidly. We discuss the biological implications of this switch mechanism, speculating that an increase in the level of Ca²⁺-calmodulin could rapidly release phosphatidylinositol 4,5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151-175).

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