Affordable Access

Glutathione disulfide-stimulated Mg2+-ATPase of human erythrocyte membranes.

Authors
  • Kondo, T
  • Kawakami, Y
  • Taniguchi, N
  • Beutler, E
Type
Published Article
Journal
Proceedings of the National Academy of Sciences
Publisher
Proceedings of the National Academy of Sciences
Publication Date
Nov 01, 1987
Volume
84
Issue
21
Pages
7373–7377
Identifiers
PMID: 2959960
Source
Medline
License
Unknown

Abstract

Inside-out erythrocyte membranes attached to polycationic beads manifested glutathione disulfide (GSSG)-stimulated ATPase activity. A Lineweaver-Burk plot of the ATPase activity as a function of GSSG concentration was biphasic and gave apparent Km values of 0.13 mM and 2.0 mM. These kinetics are similar to those reported for the ATP-requiring GSSG-transport systems in human erythrocytes and for the GSSG-stimulated ATPase activity in the plasma membranes of rat hepatocytes. Erythrocyte membranes that were depleted of extrinsic proteins were solubilized in 0.5% Triton X-100. Affinity chromatography on S-hexylglutathione-Sepharose 6B, with elution by a linear gradient of S-hexyl-glutathione, resulted in the resolution of two peaks of enzyme activity. One enzyme, which was eluted at approximately 0.5 mM S-hexylglutathione, had a high affinity for GSSG (apparent Km of 150 microM) and for ATP (80 microM). The other enzyme, which was eluted at approximately 1 mM S-hexylglutathione, had a low affinity for GSSG (apparent Km of 2.0 mM) and ATP (140 microM). GSSG-independent Mg2+-ATPase, Ca2+-dependent Mg2+-ATPase and Na+, K+-dependent Mg2+-ATPase were undetectable in the fractions. Addition of Ca2+, ouabain, or vanadate neither activated nor inhibited the activities, further indicating that the enzymes are distinguishable from ion-pumping ATPases. The enzymes required GSSG for activation; reduced glutathione (GSH) was ineffective. The ATPase activity of the high-Km enzyme was inhibited by addition of p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide and was activated by treatment with dithiothreitol, whereas the ATPase activity of the low-Km enzyme was not modified by these thiol reagents. The properties of the enzymes are similar to those of ATP-dependent GSSG-transport systems in human erythrocytes, suggesting that these ATPases may function in the active transport of GSSG.

Report this publication

Statistics

Seen <100 times