Kinetics of interaction of the myristoylated alanine-rich C kinase substrate, membranes, and calmodulin

被引:75
作者
Arbuzova, A
Wang, JY
Murray, D
Jacob, J
Cafiso, DS
McLaughlin, S
机构
[1] SUNY STONY BROOK,DEPT PHYSIOL & BIOPHYS,HLTH SCI CTR,STONY BROOK,NY 11794
[2] UNIV VIRGINIA,DEPT CHEM,CHARLOTTESVILLE,VA 22901
[3] UNIV VIRGINIA,BIOPHYS PROGRAM,CHARLOTTESVILLE,VA 22901
关键词
D O I
10.1074/jbc.272.43.27167
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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) = 10(11) M-1 s(-1) 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 similar to 10 s(-1) (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 mu M 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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页码:27167 / 27177
页数:11
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