Isoforms of Na,K-ATPase in Artemia salina: II. Tissue distribution and kinetic characterization.
To characterize the molecular properties conveyed by the isoforms of the alpha 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 alpha isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the alpha 1 isoform, whereas the intestinal enzyme exhibits both the alpha 1 and the alpha 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mumol Pi/mg protein/hr (1 mu)] 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 (K1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7 +/- 0.6 mM vs. 23.5 +/- 4 mM (P less than 0.01) for Na+, 16.6 +/- 2.2 mM vs. 8.29 +/- 1.5 mM for K+ (P less than 0.01), and 0.87 +/- 0.8 mM vs. 0.79 +/- 1.1 mM for ATP (NS). The apparent Ki's for ouabain inhibition were 1.1 x 10(-4) M vs. 2 x 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Cortas
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Department of Biochemistry & Molecular Biophysics, College of Physicians & Surgeons, Columbia University, New York, New York 10032.
Arnaout
M
M
Salon
J
J
Edelman
I S
IS
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001156:Artemia / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D006651:Histocytochemistry; D007422:Intestines / Q000166:cytology / Q000201:enzymology; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics*; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D012491:Salt Gland / Q000166:cytology / Q000201:enzymology; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics*; D013861:Thiocyanates / Q000378:metabolism; D014018:Tissue Distribution
D005452:Fluoresceins; D007527:Isoenzymes; D013861:Thiocyanates; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D011188:Potassium