Effects of the binding of myosin light chain kinase on the reactivities of calmodulin lysines

A. E. Jackson, Kermit L Carraway, D. Puett, K. Brew

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

The effects of the binding of smooth muscle myosin light chain (MLC) kinase on the microenvironments of different regions of calmodulin (CaM) were investigated by comparing the acylation rate constants of the seven lysine amino groups of free CaM with those of CaM complexed with MLC kinase. Equimolar amounts of CaM and CaM-MLC kinase complex were trace labeled with [3H]acetic anhydride in the presence of phenylalanine as a standard nucleophile. After completion of the reaction, equal amounts of a trace 14C-acetylated CaM sample, together with [14C]acetylphenylalanine, were added to each reaction mixture. The 3H/14C-labeled CaM and acetylphenylalanine were then isolated from each solution. After complete reaction with nonradioactive acetylating reagent, 3H/14C ratios (r) were determined for each ε-N-acetyllysine in the two CaM samples. These values were obtained either from isolated peptide fragments containing one lysine or from ε-N-acetyl phenylthiohydantoin lysine obtained by Edman degradation of peptide fragments containing two lysines. From the ratios, protection factors (= r(free)/r(complex)) were determined as a measure of the perturbation produced by MLC kinase binding. These protection factors were corrected, using the isotope ratios of the internal standard, for differences in the degree of competition for labeling reagent between the two mixtures. In two separate labeling experiments employing different levels of trace labeling, very little change was observed in the reactivities of four lysines on MLC kinase binding (lysines 13, 30, 77, and 94). Small but reproducible decreases (about 2-fold) were observed in the reactivities of lysines 21 and 148, while lysine 75 underwent a major (more than 7-fold) decrease in labeling. In conjunction with previously published data, these results are interpreted as suggesting that the major perturbation in lysine 75 is a direct effect of MLC kinase contact with CaM-binding site for MLC kinase. The smaller changes in reactivities at lysines 21 and 148 may reflect a conformational change that occurs in CaM as a result of binding to MLC kinase.

Original languageEnglish (US)
Pages (from-to)12226-12232
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number26
StatePublished - 1986
Externally publishedYes

Fingerprint

Myosin-Light-Chain Kinase
Calmodulin
Lysine
Labeling
Peptide Fragments
Phenylthiohydantoin
Smooth Muscle Myosins
Acylation
Nucleophiles
Phenylalanine
Isotopes
Rate constants
Binding Sites

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effects of the binding of myosin light chain kinase on the reactivities of calmodulin lysines. / Jackson, A. E.; Carraway, Kermit L; Puett, D.; Brew, K.

In: Journal of Biological Chemistry, Vol. 261, No. 26, 1986, p. 12226-12232.

Research output: Contribution to journalArticle

@article{b23b0780aa724b1dbacf44a9dfa3f237,
title = "Effects of the binding of myosin light chain kinase on the reactivities of calmodulin lysines",
abstract = "The effects of the binding of smooth muscle myosin light chain (MLC) kinase on the microenvironments of different regions of calmodulin (CaM) were investigated by comparing the acylation rate constants of the seven lysine amino groups of free CaM with those of CaM complexed with MLC kinase. Equimolar amounts of CaM and CaM-MLC kinase complex were trace labeled with [3H]acetic anhydride in the presence of phenylalanine as a standard nucleophile. After completion of the reaction, equal amounts of a trace 14C-acetylated CaM sample, together with [14C]acetylphenylalanine, were added to each reaction mixture. The 3H/14C-labeled CaM and acetylphenylalanine were then isolated from each solution. After complete reaction with nonradioactive acetylating reagent, 3H/14C ratios (r) were determined for each ε-N-acetyllysine in the two CaM samples. These values were obtained either from isolated peptide fragments containing one lysine or from ε-N-acetyl phenylthiohydantoin lysine obtained by Edman degradation of peptide fragments containing two lysines. From the ratios, protection factors (= r(free)/r(complex)) were determined as a measure of the perturbation produced by MLC kinase binding. These protection factors were corrected, using the isotope ratios of the internal standard, for differences in the degree of competition for labeling reagent between the two mixtures. In two separate labeling experiments employing different levels of trace labeling, very little change was observed in the reactivities of four lysines on MLC kinase binding (lysines 13, 30, 77, and 94). Small but reproducible decreases (about 2-fold) were observed in the reactivities of lysines 21 and 148, while lysine 75 underwent a major (more than 7-fold) decrease in labeling. In conjunction with previously published data, these results are interpreted as suggesting that the major perturbation in lysine 75 is a direct effect of MLC kinase contact with CaM-binding site for MLC kinase. The smaller changes in reactivities at lysines 21 and 148 may reflect a conformational change that occurs in CaM as a result of binding to MLC kinase.",
author = "Jackson, {A. E.} and Carraway, {Kermit L} and D. Puett and K. Brew",
year = "1986",
language = "English (US)",
volume = "261",
pages = "12226--12232",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "26",

}

TY - JOUR

T1 - Effects of the binding of myosin light chain kinase on the reactivities of calmodulin lysines

AU - Jackson, A. E.

AU - Carraway, Kermit L

AU - Puett, D.

AU - Brew, K.

PY - 1986

Y1 - 1986

N2 - The effects of the binding of smooth muscle myosin light chain (MLC) kinase on the microenvironments of different regions of calmodulin (CaM) were investigated by comparing the acylation rate constants of the seven lysine amino groups of free CaM with those of CaM complexed with MLC kinase. Equimolar amounts of CaM and CaM-MLC kinase complex were trace labeled with [3H]acetic anhydride in the presence of phenylalanine as a standard nucleophile. After completion of the reaction, equal amounts of a trace 14C-acetylated CaM sample, together with [14C]acetylphenylalanine, were added to each reaction mixture. The 3H/14C-labeled CaM and acetylphenylalanine were then isolated from each solution. After complete reaction with nonradioactive acetylating reagent, 3H/14C ratios (r) were determined for each ε-N-acetyllysine in the two CaM samples. These values were obtained either from isolated peptide fragments containing one lysine or from ε-N-acetyl phenylthiohydantoin lysine obtained by Edman degradation of peptide fragments containing two lysines. From the ratios, protection factors (= r(free)/r(complex)) were determined as a measure of the perturbation produced by MLC kinase binding. These protection factors were corrected, using the isotope ratios of the internal standard, for differences in the degree of competition for labeling reagent between the two mixtures. In two separate labeling experiments employing different levels of trace labeling, very little change was observed in the reactivities of four lysines on MLC kinase binding (lysines 13, 30, 77, and 94). Small but reproducible decreases (about 2-fold) were observed in the reactivities of lysines 21 and 148, while lysine 75 underwent a major (more than 7-fold) decrease in labeling. In conjunction with previously published data, these results are interpreted as suggesting that the major perturbation in lysine 75 is a direct effect of MLC kinase contact with CaM-binding site for MLC kinase. The smaller changes in reactivities at lysines 21 and 148 may reflect a conformational change that occurs in CaM as a result of binding to MLC kinase.

AB - The effects of the binding of smooth muscle myosin light chain (MLC) kinase on the microenvironments of different regions of calmodulin (CaM) were investigated by comparing the acylation rate constants of the seven lysine amino groups of free CaM with those of CaM complexed with MLC kinase. Equimolar amounts of CaM and CaM-MLC kinase complex were trace labeled with [3H]acetic anhydride in the presence of phenylalanine as a standard nucleophile. After completion of the reaction, equal amounts of a trace 14C-acetylated CaM sample, together with [14C]acetylphenylalanine, were added to each reaction mixture. The 3H/14C-labeled CaM and acetylphenylalanine were then isolated from each solution. After complete reaction with nonradioactive acetylating reagent, 3H/14C ratios (r) were determined for each ε-N-acetyllysine in the two CaM samples. These values were obtained either from isolated peptide fragments containing one lysine or from ε-N-acetyl phenylthiohydantoin lysine obtained by Edman degradation of peptide fragments containing two lysines. From the ratios, protection factors (= r(free)/r(complex)) were determined as a measure of the perturbation produced by MLC kinase binding. These protection factors were corrected, using the isotope ratios of the internal standard, for differences in the degree of competition for labeling reagent between the two mixtures. In two separate labeling experiments employing different levels of trace labeling, very little change was observed in the reactivities of four lysines on MLC kinase binding (lysines 13, 30, 77, and 94). Small but reproducible decreases (about 2-fold) were observed in the reactivities of lysines 21 and 148, while lysine 75 underwent a major (more than 7-fold) decrease in labeling. In conjunction with previously published data, these results are interpreted as suggesting that the major perturbation in lysine 75 is a direct effect of MLC kinase contact with CaM-binding site for MLC kinase. The smaller changes in reactivities at lysines 21 and 148 may reflect a conformational change that occurs in CaM as a result of binding to MLC kinase.

UR - http://www.scopus.com/inward/record.url?scp=0023036889&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023036889&partnerID=8YFLogxK

M3 - Article

VL - 261

SP - 12226

EP - 12232

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 26

ER -