Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization |
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Authors: | Nadim Cortas May Arnaout John Salon I S Edelman |
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Institution: | (1) Department of Biochemistry & Molecular Biophysics, College of Physicians & Surgeons, Columbia University, 10032 New York, New York;(2) Present address: Vollum Institute for Advanced Biomedical Research, 3181 S.W. Sam Jackson Park Road, 97201 Portland, OR |
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Abstract: | Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity in mol Pi/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K
1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm
vs. 23.5±4mm (P<0.01) for Na+, 16.6±2.2mm
vs. 8.29±1.5mm for K+ (P<0.01), and 0.87±0.8mm
vs. 0.79±1.1mm for ATP (NS). The apparentK
i's for ouabain inhibition were 1.1×10–4
m
vs. 2×10–5
m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK
1/2 for Na+ and K+ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship. |
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Keywords: | Na K-ATPase isoforms Na K-ATPase kinetics Artemia salina brine shrimp fluorescein-isothiocyanate salt glands intestines ouabain inhibition |
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