Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant... The stimulation by Mg2+, Na+, K+, NH4 +, and ATP of (Na+, K+)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na+, K+)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg2+, Na+, and K+ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V M = 115.0 ± 2.3 U mg−1, K 0.5 = 0.10 ± 0.01 mmol L−1. Stimulation by Na+ (V M = 110.0 ± 3.3 U mg−1, K 0.5 = 1.30 ± 0.03 mmol L−1), Mg2+ (V M = 115.0 ± 4.6 U mg−1, K 0.5 = 0.96 ± 0.03 mmol L−1), NH4 + (V M = 141.0 ± 5.6 U mg−1, K 0.5 = 1.90 ± 0.04 mmol L−1), and K+ (V M = 120.0 ± 2.4 U mg−1, K M = 2.74 ± 0.08 mmol L−1) followed single saturation curves and, except for K+, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K I = 12.4 ± 1.3 mol L−1. Complementary inhibition studies suggest the presence of F0F1–, Na+-, or K+-ATPases, but not V(H+)- or Ca2+-ATPases, in the gill microsomal preparation. K+ and NH4 + synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4 +, and K+ of the gill enzyme. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

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Publisher
Springer New York
Copyright
Copyright © 2013 by Springer Science+Business Media New York
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-013-9565-4
Publisher site
See Article on Publisher Site

Abstract

The stimulation by Mg2+, Na+, K+, NH4 +, and ATP of (Na+, K+)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na+, K+)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg2+, Na+, and K+ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V M = 115.0 ± 2.3 U mg−1, K 0.5 = 0.10 ± 0.01 mmol L−1. Stimulation by Na+ (V M = 110.0 ± 3.3 U mg−1, K 0.5 = 1.30 ± 0.03 mmol L−1), Mg2+ (V M = 115.0 ± 4.6 U mg−1, K 0.5 = 0.96 ± 0.03 mmol L−1), NH4 + (V M = 141.0 ± 5.6 U mg−1, K 0.5 = 1.90 ± 0.04 mmol L−1), and K+ (V M = 120.0 ± 2.4 U mg−1, K M = 2.74 ± 0.08 mmol L−1) followed single saturation curves and, except for K+, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K I = 12.4 ± 1.3 mol L−1. Complementary inhibition studies suggest the presence of F0F1–, Na+-, or K+-ATPases, but not V(H+)- or Ca2+-ATPases, in the gill microsomal preparation. K+ and NH4 + synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4 +, and K+ of the gill enzyme.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Jun 20, 2013

References

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