Fluorescence analysis of calmodulin mutants containing tryptophan: conformational changes induced by calmodulin-binding peptides from myosin light chain kinase and protein kinase II

Biochemistry ◽  
1991 ◽  
Vol 30 (30) ◽  
pp. 7615-7630 ◽  
Author(s):  
Marie Chabbert ◽  
Thomas J. Lukas ◽  
D. Martin Watterson ◽  
Paul H. Axelsen ◽  
Franklyn G. Prendergast
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Fluorescence Analysis ◽  
Calmodulin Binding ◽  
Protein Kinase Ii ◽  
Binding Peptides

scholarly journals Inhibition by melittin of phospholipid-sensitive and calmodulin-sensitive Ca2+ -dependent protein kinases

1982 ◽  
Vol 202 (1) ◽  
pp. 217-224 ◽  
Author(s):  
N Katoh ◽  
R L Raynor ◽  
B C Wise ◽  
R C Schatzman ◽  
R S Turner ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Dependent Protein Kinase ◽  
Calmodulin Binding ◽  
Substrate Protein ◽  
Dependent Protein ◽  
A Site

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.

scholarly journals Isolation of the native form of chicken gizzard myosin light-chain kinase

1984 ◽  
Vol 218 (3) ◽  
pp. 863-870 ◽  
Author(s):  
P K Ngai ◽  
C A Carruthers ◽  
M P Walsh
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Binding Protein ◽  
Cross Reactivity ◽  
Native Form ◽  
Chicken Gizzard ◽  
Calmodulin Binding ◽  
Chymotryptic Peptide ◽  
Dependent Protein

A simple and rapid procedure for the purification of the native form of chicken gizzard myosin light-chain kinase (Mr 136000) is described which eliminates problems of proteolysis previously encountered. During this procedure, a calmodulin-binding protein of Mr 141000, which previously co-purified with the myosin light-chain kinase, is removed and shown to be a distinct protein on the basis of lack of kinase activity, different chymotryptic peptide maps, lack of cross-reactivity with a monoclonal antibody to turkey gizzard myosin light-chain kinase, and lack of phosphorylation by the purified catalytic subunit of cyclic AMP-dependent protein kinase. This Mr-141000 calmodulin-binding protein is identified as caldesmon on the basis of Ca2+-dependent interaction with calmodulin, subunit Mr, Ca2+-independent interaction with skeletal-muscle F-actin, Ca2+-dependent competition between calmodulin and F-actin for caldesmon, and tissue content.

Sign in / Sign up

Export Citation Format

Share Document