ABSTRACT
In the search of Na+,K(+)-ATPase modulators, we have reported the isolation by gel filtration and HPLC of a brain fraction, termed endobain E, which highly inhibits Na+,K(+)-ATPase activity. In the present study we compared some properties of endobain E with those of ascorbic acid. Kinetic experiments assaying synaptosomal membrane K(+)-p-nitrophenylphosphatase (K(+)-p-NPPase) activity in the presence of endobain E or ascorbic acid showed that in neither case did enzyme inhibition prove competitive in nature versus K+ or p-NPP concentration. At pH 5.0, endobain E and ascorbic acid maximal UV absorbance was 266 and 258 nm, respectively; alkalinization to pH 14.0 led to absorption drop and shift for endobain E but to absorbance disappearance for ascorbic acid. After cysteine treatment, endobain E absorbance decreased, whereas that of ascorbic acid remained unaltered; iodine treatment led to absorbance drop and shift for endobain E but to absorbance disappearance for ascorbic acid. HPLC analysis of endobain E disclosed the presence of two components: one eluting with retention time and UV spectrum indistinguishable from those of ascorbic acid and a second, as yet unidentified, both exerting Na+,K(+)-ATPase inhibition.
Subject(s)
Ascorbic Acid/pharmacology , Brain/enzymology , Ouabain/analogs & derivatives , Ouabain/pharmacology , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , 4-Nitrophenylphosphatase/metabolism , Animals , Antioxidants/pharmacology , Brain/drug effects , Cysteine/pharmacology , Iodine/pharmacology , Kinetics , Male , Rats , Rats, Wistar , Spectrophotometry, UltravioletABSTRACT
Previous work from this laboratory led to the isolation by gel filtration and anionic exchange HPLC of a rat brain fraction named II-E, which highly inhibits synaptosomal membrane Na+, K+-ATPase activity. In this study we evaluated the kinetics of such inhibition and found that inhibitory potency was independent of Na+ (1.56-200 mM), K+ (1.25-40 mM), or ATP (1-8 mM) concentration. Hanes-Woolf plots indicated that II-E decreases Vmax but does not alter KM value, and suggested uncompetitive inhibition for Na+, K+ or ATP. However, II-E became a stimulator at 0.5 mM ATP concentration. It is postulated that this brain factor may modulate ionic transport at synapses, thus participating in central neurotransmission.
Subject(s)
Brain Chemistry , Enzyme Inhibitors/pharmacology , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Animals , Enzyme Inhibitors/isolation & purification , Kinetics , Male , Potassium/metabolism , Rats , Rats, Wistar , Sodium/metabolism , Synaptic Membranes/enzymologyABSTRACT
Na+,K+-ATPase activity of rat brain synaptosomal membranes was evaluated in the presence of an inhibitory fraction II-E (termed endobain E), isolated by gel filtration and anionic exchange HPLC of a rat brain soluble fraction. We studied endobain E aging, analyzed its inhibitory potency in the absence or presence of ouabain as well as its ability to block high affinity [3H]ouabain binding to cerebral cortex membranes. Similar loss of endobain E activity was observed when samples were stored either dried or in solution. Endobain E fraction inhibited synaptosomal membrane Na+,K+-ATPase activity in a concentration-dependent manner and the slope of the corresponding curve strongly resembled that of ouabain. Assays performed in the presence of endobain E and ouabain indicated that the inhibitory effect was additive or less than additive, depending on their respective concentrations during preincubation and/or incubation. High affinity [3H]ouabain binding to cerebral cortex membranes proved concentration-dependent from 0.10 to 0.50 mg protein per ml; binding inhibition by endobain E was independent of protein concentration within the above range. [3H]ouabain binding inhibition by endobain E was concentration-dependent over a 10-fold range, an effect similar to that found for Na+,K+-ATPase inhibition. The extent of endobain E effect on Na+,K+-ATPase inhibition was much higher (90-100%) than that on [3H]ouabain binding blockade (50%). Findings suggest some type of interaction between endobain E and ouabain inhibitory mechanisms and favour the view that the former behaves as an endogenous ouabain.
Subject(s)
Cerebral Cortex/metabolism , Enzyme Inhibitors/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Cerebral Cortex/enzymology , Male , Ouabain/metabolism , Protein Binding , Rats , Rats, Wistar , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Synaptosomes/enzymologySubject(s)
Biological Factors/isolation & purification , Cerebral Cortex/metabolism , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Synaptosomes/enzymology , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Animals , Biological Factors/pharmacology , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/pharmacology , Intracellular Membranes/enzymology , Kinetics , Neurons/enzymology , Ouabain/pharmacology , Potassium/metabolism , Rats , Rats, WistarABSTRACT
Since a brain soluble fraction (peak II) is known to be able to inhibit synaptosomal membrane Na+, K(+)-ATPase activity, here we attempted to compare its effect on cellular and subcellular brain components such as synaptosomal and astrocytic membranes, as well as mitochondrial preparations. The difference between total and Mg(2+)-ATPase activity was noteworthy in synaptosomal membranes but proved unremarkable in astrocytic and mitochondrial preparations. Peak II highly inhibited total ATPase in synaptosomal membranes but failed to modify enzyme activity in astrocytic and mitochondrial preparations. Findings suggest cellular and subcellular specificity of peak II on brain ATPase activity.
Subject(s)
Cerebral Cortex/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Subcellular Fractions/enzymology , Synaptosomes/enzymology , Animals , Astrocytes/enzymology , In Vitro Techniques , Male , Mitochondria/enzymology , Ouabain/metabolism , Rats , Rats, Wistar , Subcellular Fractions/pathologyABSTRACT
Since a brain soluble fraction (peak II) is known to be able to inhibit synaptosomal membrane Na+, K(+)-ATPase activity, here we attempted to compare its effect on cellular and subcellular brain components such as synaptosomal and astrocytic membranes, as well as mitochondrial preparations. The difference between total and Mg(2+)-ATPase activity was noteworthy in synaptosomal membranes but proved unremarkable in astrocytic and mitochondrial preparations. Peak II highly inhibited total ATPase in synaptosomal membranes but failed to modify enzyme activity in astrocytic and mitochondrial preparations. Findings suggest cellular and subcellular specificity of peak II on brain ATPase activity.