Analysis of thiol-topography in Na,K-ATPase using labelling with different maleimide nitroxide derivatives

Mikael Esmann, K. Hideg, Derek Marsh

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Spin-label EPR spectroscopy of shark rectal gland Na,K-ATPase modified at cysteine residues with a variety of maleimide-nitroxide derivatives is used to characterize the different classes of sulphydryl groups. The spin-labelled derivatives vary with respect to charge and lipophilicity, and the chemical reactivity towards modification and inactivation of the Na,K-ATPase is dependent on these properties. Ascorbate is used to reduce the spin-labels in situ, and the kinetics of reduction of the protein-bound spin-labels are found also to depend on the nature of the maleimide-nitroxide derivative. The Na,K-ATPase is labelled either at Class I groups (with retention of enzymatic activity) or at Class II groups (where the enzymatic activity is lost). Although Class I groups are labelled more readily than are Class II groups they are only slightly more susceptible to reduction by ascorbate than the Class II groups, indicating no major difference in environment. The spectral difference observed between immobilized and mobile spin-labels with both Class I and Class II groups labelling is not reflected in widely different reduction kinetics for these two spectral components. Solubilization of the enzyme in an active form does not change the protein structure in terms of increased accessibility of the SH-groups to reduction by ascorbate. The results are discussed in terms of the location of the different SH-groups and the origins of the differences in mobility evident in the EPR spectra of the spin-labelled SH-groups.

Original languageEnglish
Pages (from-to)215-225
Number of pages11
JournalBBA - Biomembranes
Volume1112
Issue number2
DOIs
Publication statusPublished - Dec 9 1992

Fingerprint

Spin Labels
Sulfhydryl Compounds
Labeling
Topography
Adenosine Triphosphatases
Derivatives
Paramagnetic resonance
Salt Gland
Sharks
Chemical reactivity
Kinetics
Cysteine
Spectrum Analysis
Proteins
Spectroscopy
nitroxide maleimide
sodium-translocating ATPase
Enzymes

Keywords

  • ATPase, Na/K-
  • Enzyme structure
  • EPR
  • Spin label

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology

Cite this

Analysis of thiol-topography in Na,K-ATPase using labelling with different maleimide nitroxide derivatives. / Esmann, Mikael; Hideg, K.; Marsh, Derek.

In: BBA - Biomembranes, Vol. 1112, No. 2, 09.12.1992, p. 215-225.

Research output: Contribution to journalArticle

@article{9caff4ce128540b3abc2d567bd7e7f4a,
title = "Analysis of thiol-topography in Na,K-ATPase using labelling with different maleimide nitroxide derivatives",
abstract = "Spin-label EPR spectroscopy of shark rectal gland Na,K-ATPase modified at cysteine residues with a variety of maleimide-nitroxide derivatives is used to characterize the different classes of sulphydryl groups. The spin-labelled derivatives vary with respect to charge and lipophilicity, and the chemical reactivity towards modification and inactivation of the Na,K-ATPase is dependent on these properties. Ascorbate is used to reduce the spin-labels in situ, and the kinetics of reduction of the protein-bound spin-labels are found also to depend on the nature of the maleimide-nitroxide derivative. The Na,K-ATPase is labelled either at Class I groups (with retention of enzymatic activity) or at Class II groups (where the enzymatic activity is lost). Although Class I groups are labelled more readily than are Class II groups they are only slightly more susceptible to reduction by ascorbate than the Class II groups, indicating no major difference in environment. The spectral difference observed between immobilized and mobile spin-labels with both Class I and Class II groups labelling is not reflected in widely different reduction kinetics for these two spectral components. Solubilization of the enzyme in an active form does not change the protein structure in terms of increased accessibility of the SH-groups to reduction by ascorbate. The results are discussed in terms of the location of the different SH-groups and the origins of the differences in mobility evident in the EPR spectra of the spin-labelled SH-groups.",
keywords = "ATPase, Na/K-, Enzyme structure, EPR, Spin label",
author = "Mikael Esmann and K. Hideg and Derek Marsh",
year = "1992",
month = "12",
day = "9",
doi = "10.1016/0005-2736(92)90394-2",
language = "English",
volume = "1112",
pages = "215--225",
journal = "Biochimica et Biophysica Acta - Biomembranes",
issn = "0005-2736",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Analysis of thiol-topography in Na,K-ATPase using labelling with different maleimide nitroxide derivatives

AU - Esmann, Mikael

AU - Hideg, K.

AU - Marsh, Derek

PY - 1992/12/9

Y1 - 1992/12/9

N2 - Spin-label EPR spectroscopy of shark rectal gland Na,K-ATPase modified at cysteine residues with a variety of maleimide-nitroxide derivatives is used to characterize the different classes of sulphydryl groups. The spin-labelled derivatives vary with respect to charge and lipophilicity, and the chemical reactivity towards modification and inactivation of the Na,K-ATPase is dependent on these properties. Ascorbate is used to reduce the spin-labels in situ, and the kinetics of reduction of the protein-bound spin-labels are found also to depend on the nature of the maleimide-nitroxide derivative. The Na,K-ATPase is labelled either at Class I groups (with retention of enzymatic activity) or at Class II groups (where the enzymatic activity is lost). Although Class I groups are labelled more readily than are Class II groups they are only slightly more susceptible to reduction by ascorbate than the Class II groups, indicating no major difference in environment. The spectral difference observed between immobilized and mobile spin-labels with both Class I and Class II groups labelling is not reflected in widely different reduction kinetics for these two spectral components. Solubilization of the enzyme in an active form does not change the protein structure in terms of increased accessibility of the SH-groups to reduction by ascorbate. The results are discussed in terms of the location of the different SH-groups and the origins of the differences in mobility evident in the EPR spectra of the spin-labelled SH-groups.

AB - Spin-label EPR spectroscopy of shark rectal gland Na,K-ATPase modified at cysteine residues with a variety of maleimide-nitroxide derivatives is used to characterize the different classes of sulphydryl groups. The spin-labelled derivatives vary with respect to charge and lipophilicity, and the chemical reactivity towards modification and inactivation of the Na,K-ATPase is dependent on these properties. Ascorbate is used to reduce the spin-labels in situ, and the kinetics of reduction of the protein-bound spin-labels are found also to depend on the nature of the maleimide-nitroxide derivative. The Na,K-ATPase is labelled either at Class I groups (with retention of enzymatic activity) or at Class II groups (where the enzymatic activity is lost). Although Class I groups are labelled more readily than are Class II groups they are only slightly more susceptible to reduction by ascorbate than the Class II groups, indicating no major difference in environment. The spectral difference observed between immobilized and mobile spin-labels with both Class I and Class II groups labelling is not reflected in widely different reduction kinetics for these two spectral components. Solubilization of the enzyme in an active form does not change the protein structure in terms of increased accessibility of the SH-groups to reduction by ascorbate. The results are discussed in terms of the location of the different SH-groups and the origins of the differences in mobility evident in the EPR spectra of the spin-labelled SH-groups.

KW - ATPase, Na/K-

KW - Enzyme structure

KW - EPR

KW - Spin label

UR - http://www.scopus.com/inward/record.url?scp=0026446386&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026446386&partnerID=8YFLogxK

U2 - 10.1016/0005-2736(92)90394-2

DO - 10.1016/0005-2736(92)90394-2

M3 - Article

C2 - 1333803

AN - SCOPUS:0026446386

VL - 1112

SP - 215

EP - 225

JO - Biochimica et Biophysica Acta - Biomembranes

JF - Biochimica et Biophysica Acta - Biomembranes

SN - 0005-2736

IS - 2

ER -