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Comparison of Digitalis Sensitivities of Na+/K+-ATPases from Human and Pig Kidneys

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      Abstract

      Digitalis drugs are selective inhibitors of the plasma membrane Na+/K+-ATPase. There are many studies on molecular mechanisms of digitalis interaction with purified pig kidney enzyme, with the tacit assumption that it is a good model of human kidney enzyme. However, previous studies on crude or recombinant human kidney enzymes are limited, and have not resulted in consistent findings on their digitalis sensitivities. Hence, we prepared comparably purified enzymes from human and pig kidneys and determined inhibitory constants of digoxin, ouabain, ouabagenin, bufalin, and marinobufagenin (MBG) on enzyme activity under optimal turnover conditions. We found that each compound had the same potency against the two enzymes, indicating that (i) the pig enzyme is an appropriate model of the human enzyme, and (ii) prior discrepant findings on human kidney enzymes were either due to structural differences between the natural and recombinant enzymes or because potencies were determined using binding constants of digitalis for enzymes under nonphysiological conditions. In conjunction with previous findings, our newly determined inhibitory constants of digitalis compounds for human kidney enzymes indicate that (i) of the compounds that have long been advocated to be endogenous hormones, only bufalin and MBG may act as such at kidney tubules, and (ii) beneficial effects of digoxin, the only digitalis with extensive clinical use, does not involve its inhibitory effect on renal tubular Na+/K+-ATPase.

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      Most cited references 41

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      An improved assay for nanomole amounts of inorganic phosphate.

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        Biochemistry of Na,K-ATPase.

         Jack Kaplan (2002)
        The Na,K-ATPase or sodium pump carries out the coupled extrusion and uptake of Na and K ions across the plasma membranes of cells of most higher eukaryotes. It is a member of the P-type ATPase superfamily. This heterodimeric integral membrane protein is composed of a 100-kDa alpha-subunit with ten transmembrane segments and a heavily glycosylated beta subunit of about 55 kDa, which is a type II membrane protein. Current ideas on how the protein achieves active transport are based on a fusion of results of transport physiology, protein chemistry, and heterologous expression of mutant proteins. Recently acquired high resolution structural information provides an important new avenue for a more complete understanding of this protein. In this review, the current status of knowledge of Na,K-ATPase is discussed, and areas where there is still considerable uncertainty are highlighted.
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          Crystal structure of the sodium-potassium pump.

          The Na+,K+-ATPase generates electrochemical gradients for sodium and potassium that are vital to animal cells, exchanging three sodium ions for two potassium ions across the plasma membrane during each cycle of ATP hydrolysis. Here we present the X-ray crystal structure at 3.5 A resolution of the pig renal Na+,K+-ATPase with two rubidium ions bound (as potassium congeners) in an occluded state in the transmembrane part of the alpha-subunit. Several of the residues forming the cavity for rubidium/potassium occlusion in the Na+,K+-ATPase are homologous to those binding calcium in the Ca2+-ATPase of sarco(endo)plasmic reticulum. The beta- and gamma-subunits specific to the Na+,K+-ATPase are associated with transmembrane helices alphaM7/alphaM10 and alphaM9, respectively. The gamma-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the alpha-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.
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            Author and article information

            Affiliations
            []Department Biochemistry & Cancer Biology, College of Medicine & Life Sciences, University of Toledo , 3000 Arlington Avenue, MS 1010, Toledo, Ohio 43614, United States
            []Laboratory of Cardiovascular Science, National Institute of Aging, National Institutes of Health , Baltimore, Maryland 21224, United States
            [§ ]Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences , St. Petersburg 194223, Russia
            Author notes
            [* ]E-mail: Amir.Askari@ 123456utoledo.edu . Phone: 419-383-3982.
            Journal
            ACS Omega
            ACS Omega
            ao
            acsodf
            ACS Omega
            American Chemical Society
            2470-1343
            13 July 2017
            31 July 2017
            : 2
            : 7
            : 3610-3615
            5537699
            10.1021/acsomega.7b00591
            Copyright © 2017 American Chemical Society

            This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

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            ao-2017-00591e

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