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Interaction of ouabain with sarcolemma-enriched membrane preparations from canine ventricle :...

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Interaction of ouabain with sarcolemma-enriched membrane preparations from canine ventricle : evidence for two forms of Na[superscript +],K[superscript +] -ATPase

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Title	Interaction of ouabain with sarcolemma-enriched membrane preparations from canine ventricle : evidence for two forms of Na[superscript +],K[superscript +] -ATPase
Author	Wellsmith, Nancy Verne
Date	1981
Affiliation/College	College of Graduate Studies
Department	Pharmacology
Extent	viii, 108 leaves : ill.; 29 cm
Subject.MeSH	Sodium-Potassium-Exchanging ATPase Ouabain--pharmacology Academic Dissertations
Collection	MUSC Dissertations and Theses
Contributing Institution	Waring Historical Library (MUSC)
Rights	Copyright is held by the author.
Language	English
Type	Text
Format	application/pdf
Media Type	Manuscripts
Resource Identifier	mtd1981_wellsmith_nv
Abstract	Ouabain binding to sarcolemma enriched preparations from canine ventricle was studied under ligand conditions known to promote binding to the Na[superscript +],K[superscript +] -ATPase. This sarcolemma preparation was enriched 19-27 fold over homogenate in membrane markers, and electron microscopy showed that the major configuration was vesicular. A fraction of these vesicles was judged intact and impermeable, but the majority were freely permeable. Profiles for binding and dissociation were characterized by two kinetic components: (1) fast association, slow dissociation and lower limit of K[subscript eq] = 1.21 X 10[superscript 9] M[superscript -1]; (2) slow association, fast dissociation and lower limit of K[subscript eq] = 5.15 X 10[superscript 6] M[superscript -1] (estimates for K[subscript eq] from profiles in presence of magnesium, sodium plus ATP). Ouabain binding and dissociation in the absence of supporting ligands were characterized by reaction to a single species of receptors with slow association, fast dissociation, lower limit of K[subscript eq] = 5.25 X 10[superscript 6] M[superscript -1] and minimal effect on Na[superscript +],K[superscript +] -ATPase activity. Binding in the presence of magnesium plus inorganic phosphate caused 86% inhibition of Na[superscript +],K[superscript +] -ATPase activity. The two binding components were lost at different rates upon heating at 55°C. It was concluded that the membrane preparation possesses two receptors for ouabain, one of which is the Na[superscript +],K[superscript +] -ATPase and the second of which is a "new" receptor. A likely possibility is that "new" receptor is the Na[superscript +],K[superscript +] -ATPase in a physiochemical state characterized by low catalytic activity, low affinity for ouabain (i.e., two to three orders of magnitude less than that for the active Na[superscript +],K[superscript +] –ATPase) and no requirement of specific ligands for ouabain binding. This conclusion is based on the following observations: (a) The ratios between the two forms were affected by ligands which promote ouabain binding to the Na[superscript +],K[superscript +] -ATPase; (b) binding to both receptors was inhibited by potassium; (c) pretreatment with sodium dodecyl sulfate may have converted the "new" receptor into the active form of the Na[superscript +],K[superscript +] -ATPase; and (d) prior binding to the "new" receptor in the isolated membrane preparation reduced the rate of binding to the active form of the Na[superscript +],K[superscript +] -ATPase. These results could not eliminate, however, the possibilities, that the "new" receptor was chemically different from the Na[superscript +],K[superscript +] -ATPase, or reflected Na[superscript +],K[superscript +] -ATPase to which access was limited. Further studies were carried out in a more highly enriched sarcolemma preparation, consisting of approximately 76% intact right-side out vesicles, and 24% freely permeable vesicles. The presence of a second binding site for ouabain in sarcolemma was confirmed. Ouabain binding in buffer occurred largely to two components, with compartment sizes, R1=61.3±4.0 pmoles/mg and R2=22.8±lO.7 pmoles/mg, and pseudo-first order rate constants k[subscript 1] =0.19±0.02 min[superscript -1] and k[subscript 2]=0.020±O.009 min[superscript -1] (32°C, l0[superscript -6]M ouabain). Intravesicular magnesium increased the rate of binding to R[subscript 2] to k'[subscript 2] =5.2±2.1 min[superscript -1]. Thus, this component manifests characteristics of Na[superscript +],K[superscript +] -ATPase: slow binding in buffer, which is increased by intravesicular magnesium. Freeze-thaw cycles increased Na[superscript +],K[superscript +] -ATPase activity 4-5 fold, and ouabain binding in buffer was then characterized by a single component, 84.8±12.4 pmoles/mg, with a rate constant, k = 0.019±0.004 min[superscript -1] (32°C, 10[superscript -6] M ouabain). Prior exposure of sarcolemma preparation to ouabain in buffer decreased Na[superscript +],K[superscript +] -ATPase activation to 22.7±3.5 μmole/mg/hr vs 60.4±8.3 μmole/mg/hr activation in control vesicles. Thus, R[subscript 1] is a form of Na[superscript +],K[superscript +] -ATPase, but does not reflect limited access of site to ouabain since k[subscript l] > k[subscript 2]. It was concluded that R[subscript l] is probably an alternate form of Na[superscript +],K[superscript +] -ATPase which may be converted to active Na[superscript +],K[superscript +] -ATPase by a physical perturbation (freeze-thaw cycles or detergent treatment) of the membrane.

Interaction of ouabain with sarcolemma-enriched membrane preparations from canine ventricle : evidence for two forms of Na[superscript +],K[superscript +] -ATPase

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