A new simple preparation method for NaK-ATPase-rich membrane fragments

Role of sorbitol accumulation and myo-inositol depletion in paranodal swelling of large myelinated nerve fibers in the insulin-deficient spontaneously diabetic bio-breeding rat. Reversal by insulin replacement, an aldose reductase inhibitor, and myo-inositol.

“…The baseline diabetics were immediately sacrificed for baseline morphometric studies, while the remaining groups were entered into a 3-wk study period, during which the insulin-deficient diabetic rats were maintained hyperglycemic on small daily doses of insulin, the "insulinreplaced" diabetics were treated intensively with insulin to normalize blood glucose, and the "ARI-treated" and "MI-treated diabetics were main- At the completion of the 3-wk study period, nondiabetic controls and BB diabetic rats treated as described in Methods and in Table I were anesthetized. Mid-thigh segments of the sciatic nerves were surgically removed, and either deproteinized and processed for gas-liquid chromatographic determination of MI, or homogenized in sucrose containing buffer for determination of composite and non-ouabain-inhibitable ATPase activity, using the enzymatic adenine-nucleotide-linked kinetic spectrophotometric method of Yoda and Yoda (22). Ouabain-inhibitable ATPase activity, a measure of (Na,K)-ATPase activity in rat nerve (7,11,13,17), was defined as the arithmetic difference between total and non-ouabain-inhibitable ATPase activity, and was expressed per mg wet wt of nerve.…”

“…Briefly, nerve segments were minced and homogenized at 4VC in 2 ml of0.2 M sucrose-0.02 M Tris-HCI pH 7.5. 5-20 ,g ofhomogenate was assayed spectrophotometrically in I ml of 100 mM NaCi, 10 mM KCa, 2.5 mM MgCI2, I mM Tris-ATP, I mM tri(cyclohexylammonium) phosphoenolpyruvate, 30 mM imidazole-HCI buffer (pH 7.3), 0.15 mM NADH, 50 ug of lactate dehydrogenase and 30 sg of pyruvate kinase (22). ATPase activity in the presence of 0.10 mM ouabain was defined as the ouabain-uninhibitable ATPase fraction, since preliminary experiments had indicated that maximum ouabain inhibition is obtained at this concentration (11).…”

Action of Sorbinil in Diabetic Peripheral Nerve: Relationship of Polyol (Sorbitol) Pathway Inhibition to a myo-Inositol-mediated Defect in Sodium-Potassium ATPase Activity

“…Five to 20 |xl of homogenate were assayed enzymatically for ATPase activity in 1 ml of reaction mixture containing 100 mM NaCI, 10 mM KCI, 2.5 mM MgCI 2 , 1 mM Tris-ATP, 1 mM tri(cyclohexylammonium) phosphoenolypyruvate, 30 mM imidazole-HCI buffer (pH 7.3), 0.15 mM NADH, 50 |xg of lactate dehydrogenase, and 30 |xg of pyruvate kinase. 16 After initial stabilization, composite ATPase activity was monitored spectrophotometrically as the oxidation of NADH. Sodium-plus-potassium-stimulated ATPase activity was computed by subtracting the reaction rate in an identical cuvette from which NaCI and KCI were omitted.…”

Impaired rat sciatic nerve sodium-potassium adenosine triphosphatase in acute streptozocin diabetes and its correction by dietary myo-inositol supplementation.

“…Fresh sciatic nerve segments were minced and homogenized at 40°C in 2 ml of 0.2 M sucrose-0.02 M Tris-HCI buffer solution pH 7.5, using a Polytron homogenizer (model , Brinkman Instruments, Inc, Westbury, NY), for at least three periods, not exceeding 15 s each (16) and centrifuged at 100 g for 2 min at 4°C. 5-20 Ml of supernatant was added to 1.0 ml X 1.0 cm disposable cuvettes (Fisher Scientific, Pittsburgh, PA) containing (final concentration) 100 mM NaCl, 10 mM KCI, 2.5 mM MgCl2, 1 mM Tris-ATP, 1 mM tri(cyclohexylammonium) phosphoenolpyruvate, 30 mM imidazole-HCl buffer (pH 7.3), 0.15 mM NADH, and 50 Mg of lactate dehydrogenase and 30 Mg of pyruvate kinase (22).…”