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Functional activity of oligosaccharide-deficient (Na,K)ATPase expressed in Xenopus oocytes
K Takeda1, S Noguchi, A Sugino
1Department of Biology, University of Occupational and Environmental Health, Kitakyushu, Japan.
Abstract:
(Na,K)ATPase from Torpedo californica was expressed in Xenopus laevis oocytes in the presence of tunicamycin by injecting mRNAs for the alpha- and beta-subunits derived from the cloned cDNAs into the oocytes. The oligosaccharide-deficient ATPase thus synthesized was transported to the oocyte plasma membrane, where it exhibited virtually the same ATPase activity, ouabain-binding capacity and 86Rb+ transport activity as the fully glycosylated enzyme. We conclude that the oligosaccharide chains on the beta-subunit has no effect on the catalytic activities of (Na,K)ATPase.
Insights
Oligosaccharide chains on the beta-subunit of Torpedo californica (Na,K)ATPase do not affect its catalytic functions. This enzyme, expressed in Xenopus oocytes, maintained activity even without glycosylation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The sodium-potassium pump ((Na,K)ATPase) is crucial for maintaining cellular ion gradients.
- Glycosylation is a common post-translational modification, but its role in (Na,K)ATPase function is not fully understood.
Purpose of the Study:
- To investigate the functional significance of oligosaccharide chains on the beta-subunit of (Na,K)ATPase.
- To determine if glycosylation impacts the catalytic activity and membrane transport function of the enzyme.
Main Methods:
- Messenger RNAs (mRNAs) encoding the alpha- and beta-subunits of Torpedo californica (Na,K)ATPase were injected into Xenopus laevis oocytes.
- Oocyte expression was performed in the presence of tunicamycin, an inhibitor of N-linked glycosylation, to produce oligosaccharide-deficient enzyme.
- Enzyme activity was assessed by measuring ATPase activity, ouabain-binding capacity, and 86Rb+ (rubidium-86) transport.
Main Results:
- The oligosaccharide-deficient (Na,K)ATPase was successfully synthesized and transported to the plasma membrane of Xenopus oocytes.
- The non-glycosylated enzyme exhibited comparable ATPase activity to the fully glycosylated form.
- Ouabain-binding capacity and 86Rb+ transport activity were also similar between the glycosylated and non-glycosylated (Na,K)ATPase.
Conclusions:
- Olighosaccharide chains on the beta-subunit of (Na,K)ATPase are not essential for its catalytic functions.
- Glycosylation does not appear to play a significant role in the membrane transport activity or ligand binding of this enzyme.