Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

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Abstract
To characterize the molecular properties conveyed by the isoforms of the α 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 α isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the α1 isoform, whereas the intestinal enzyme exhibits both the α1 and the α2 isoforms. After 32 hours of development, Na,K-ATPase activity [in μmol 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+, 16.6±2.2mm vs. 8.29±1.5mm for K+ (Pvs. 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 α subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.