Abstract
Human cerebrospinal fluid (CSF) inhibits the Na+/K+ pump in human red cells and the activity of purified Na+/K+-ATPase (Halperin, J. A., Shaeffer, R., Galvez, L., and Malavé, S. (1985) Proc. Natl. Acad. Sci. U.S. A. 80, 6102-6104, 1983; Halperin, J. A., Martin, A. M., and Malavé, S. (1985) Life Sci. 37, 561-566. We describe here some properties of the CSF inhibitor of the Na+/K+ pump. Active material was extracted from human CSF with 50% methanol and then concentrated and desalted by ultrafiltration. This extract inhibited, in a dose-dependent manner, the ouabain-sensitive influx of K+ into human red cells and the activity of purified Na+/K+-ATPase. Partial separation of the inhibitory activity was achieved by gel filtration and reverse-phase high performance liquid chromatography. Inhibition of both pump and enzyme was specific in that other red cell membrane transport systems or enzymes examined were not influenced by CSF extracts. Dialysis and ultrafiltration experiments indicate that the molecular weight of the inhibitor is approximately equal to 600. The inhibitory activity is sensitive to proteolytic enzymes indicating that the inhibitor might be a small peptide. In the presence of CSF extract the K0.5 for external K+ to stimulate the Na+/K+ pump increased from 1.4 to 3.1 mM, suggesting that the CSF inhibitor competes with external K+ for stimulation of the pump. We estimate that the concentration of the inhibitor in CSF might be approximately equal to 50 pg/ml, a value close to the concentration of other active peptides found in human CSF.
Highlights
From the $Departmentsof Physiology and Biophysics and TBiological Chemistry, Haruard MedicalSchool and the
We describe here some properties of the cerebrospinal fluid (CSF) inhibitor of the Na+/K+pump
Active material was extracted from human CSFwith 50%methanol and concentrated and desalted by ultrafiltration
Summary
An aliquot of the methanol extract was injected onto a Superose 12 column(PharmaciaLKB Biotechnology Inc.) connectedto a Waters Associates high performance liquid chromatography (HPLC) system. The column was equilibrated and eluted witbhuffer containing: 130 mM NaCl, 2.8 mM KCl, 1mM MgCl,, 10 mM glucose, and 10 mM MOPS adjusted to pH7.4 with Tris base. The flow rate was 0.6 outward and two K+ ions inward across the cell membrane ml/min. The absorbance was monitored at 214 nm. Fractions of 1.2 [2]. Since this enzyme contains high affinityreceptors for ml were collected and assayed for biological activity. Digitalis glycosides [3], it has been postulated that there are endogenous ligands for these receptors that might regulate
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