Studies on sodium transport in rat brain nerve-ending particles

(1) Nerve-ending particles isolated from crude mitochondrial preparations from rat brain by discontinuous Ficoll density gradient ultracentrifugation were shown to possess a Mg2+ and energy-dependent transport system for Na+. (2) Ouabain or iodoacetate plus cyanide exerted an inhibitory effect on the outflux but not the influx of Na+. (3) When K+ was added to a medium containing particles loaded with Na+ (22Na), an immediate release of Na+ from these particles was observed; this suggests the existence of a Na+-K+ exchange transport system. (4) The K+ effect was inhibited by 10(-4) M-ouabain only at low (about 3.3 mM), but not at high (20 mM), K+ concentrations. (5) The uptake and release of Na+ by the nerve-ending particles were found to be temperature-dependent. (6) Only nerve-ending particles with intact synaptosomal membranes were found to transport Na+ actively.     

Alkaloid neurotoxins-dependent sodium transport in insect synaptic nerve-ending particles

1. Sodium uptake associated with the activation of voltage-sensitive sodium channels by alkaloid activators, batrachotoxin, veratridine, and aconitine in presynaptic nerve terminals isolated from the central nervous system of cockroach (Periplaneta americana) was investigated. 2. Batrachotoxin (K0.5, 0.2 microM) was full agonist as for most effective activator of Na+ uptake; veratridine (K0.5, 2.5 microM) and aconitine (K0.5, 7.6 microM) produced a maximal stimulation of 22Na+ uptake that were 71% and 43% respectively of that produced by batrachotoxin. 3. Veratridine-dependent 22Na+ uptake was completely inhibited by tetrodotoxin (I0.5, 11 nM), a specific inhibitor of the nerve membrane sodium channels. 4. The present study describes appropriate conditions for measuring neurotoxins--stimulated sodium transport in insect central nervous system synaptosomes. The data show that voltage-sensitive sodium channels as defined by specific activation by the alkaloid neurotoxins are qualitatively distinct in insect synaptosomes than those previously described for vertebrate brain synaptosomes, cultured neuronal cell, nerve membrane vesicles and neuroblastoma cells.     

The adenosine-triphosphatase activity of dissociated acto-heavy-meromyosin

1. At low ionic strength, when turbidity and viscosity measurements indicated dissociation of acto-heavy-meromyosin, its adenosine triphosphatase was strongly activated by Mg(2+) and Ca(2+). 2. The characteristics of the adenosine triphosphatase of dissociated acto-heavy-meromyosin in the presence of Mg(2+) were similar to those reported for myofibrils and actomyosin. 3. In the presence of Ca(2+) the adenosine-triphosphatase activity was much less sensitive to ionic strength than was the case with Mg(2+). 4. At low ionic strength Mg(2+) was more effective in maintaining the dissociation of acto-heavy-meromyosin in the presence of ATP than was Ca(2+). This difference was not apparent when ATP was replaced by ITP. 5. Although the recovery of viscosity was complete on reassociation of acto-heavy-meromyosin the turbidity did not return to the original value. 6. The general implications of Mg(2+) activation of acto-heavy-meromyosin when classical interpretation indicates dissociation of the complex are discussed.     

The adenosine-triphosphatase activity of desensitized actomysin

1. A simple procedure involving repeated washings of actomyosin, extracted as the complex from myofibrils (natural actomyosin) at ionic strength less than 0.002, is described for the preparation of a desensitized actomyosin. 2. The Mg(2+)-activated adenosine triphosphatase of natural actomyosin was markedly inhibited by ethylenedioxybis(ethyleneamino)tetra-acetic acid, whereas that of the desensitized actomyosin was unaffected. 3. The activity of the Ca(2+)-activated adenosine triphosphatase of natural actomyosin was generally lower than that of the Mg(2+)-activated adenosine triphosphatase, whereas in the desensitized actomyosin the difference between the activities was considerably less. In both natural and desensitized actomyosin the adenosine-triphosphatase activities in the presence of Mg(2+) were similar. 4. The conversion of the natural into the desensitized actomyosin was accompanied by the removal of a protein fraction containing the factors responsible for the sensitivity to ethylenedioxybis(ethyleneamino)tetra-acetic acid and for modifying the Ca(2+)-activated adenosine triphosphatase. When added to a desensitized actomyosin this fraction effected a reversal to the natural form. The recombination was facilitated by increasing the ionic strength of the medium. The two factors showed different stabilities to heat and tryptic digestion.     

The adenosine-triphosphatase activity of adrenal chromaffin granules

1. The preparation of a fraction containing highly purified chromaffin granules from the bovine adrenal medulla is described. 2. The fraction contains an adenosine-triphosphatase activity that is stimulated by Mg²⁺ and that cannot be explained by contamination with mitochondria or microsomes. 3. It is suggested that the adenosine-triphosphatase activity is related to the uptake of cate-cholamines by the chromaffin granules.     

The mode of inhibition by calcium of cell-membrane adenosine-triphosphatase activity

1. The mechanism of the inhibition of Na(+)-plus-K(+)-activated adenosine triphosphatase by calcium was investigated with an enzyme preparation from rabbit kidney cortex and with membranes of human erythrocytes. 2. CaATP, rather than ionic Ca(2+), acts as a competitive inhibitor, competing with MgATP in the Na(+)-plus-K(+)-activated adenosine-triphosphatase reaction. 3. There appears to be no competition between calcium and Na(+) for the activation of adenosine triphosphatase. 4. The inhibition of Na(+)-plus-K(+)-activated adenosine triphosphatase of cell membranes by low concentrations of CaATP and the consequent need of intact cells to keep the cytoplasmic concentration of calcium low relative to that of magnesium suggests a raison d'être for the mitochondrial calcium pump.     

Phosphate binding by cerebral microsomes in relation to adenosine-triphosphatase activity

1. Microsomes prepared from guinea-pig and ox brain were incubated for periods of a few seconds with low concentrations of Mg-[(32)P]ATP, the reaction was stopped with trichloroacetic acid and determinations were made of the phosphate bound to the acid-washed, and in some cases solvent-extracted, residue. 2. At 20 mum-ATP, at 37 degrees and in the presence of Na(+) ions, 30-50 mumumoles of phosphate/mg. of microsomal protein were bound by the preparation within 1 sec. of starting the reaction; little further change in level occurred until hydrolysis of ATP exceeded 50%, when the bound phosphate began to decline fairly rapidly to the zero-time value. 3. At 20mum-ATP without Na(+) ions present or in the presence of K(+) ions, the level of bound phosphate increased gradually and did not decline as ATP hydrolysis approached completion. 4. Potassium ions either inhibited the formation of Na(+)-dependent bound phosphate or, when added during the course of the reaction, rapidly reduced its level. 5. At 200 mum-ATP the bound phosphate formed in the presence of Na(+) ions appeared to consist of a mixture of the unstable Na(+)-dependent type and the stable type requiring only Mg(2+) ions for its formation. 6. Non-radioactive ATP added during the course of the reaction at 20 mum-ATP with Na(+)ions present rapidly discharged virtually all the bound (32)P counts; at 200 mum-ATP only a proportion of the label was similarly discharged. The Na(+)-dependent bound phosphate is therefore turning over, in contrast with that formed in the absence of Na(+)ions, which proved more stable. 7. The Na(+)-dependent bound phosphate was not in the form of ATP; experiments with [(14)C]ATP instead of [(32)P]ATP showed a small and invariable binding of ATP by the preparation unaffected by Na(+) ions or time of incubation. 8. Under the usual conditions employed in this work ouabain stimulated formation of Na(+)-dependent bound phosphate when Na(+) ions were suboptimum and inhibited it when optimum Na(+) ions were present. 9. The Na(+)-dependent binding reaction under present conditions did not involve incorporation into phosphorylserine groups. 10. The relation of the findings to the (Na(+),K(+))-ATPase of the preparation, and to observations in brain slices appearing to implicate phosphorylserine groups in cation transport, is discussed.     

Properties of phosphate bound to cerebral microsomes during adenosine-triphosphatase activity

1. Ox-brain microsomes were incubated with [gamma-(32)P]ATP under various conditions. After the reaction, which was stopped with trichloroacetic acid, a small amount of phosphate remained bound to the washed precipitate. 2. Properties of the bound phosphate were studied by treatment with buffers and solvents. 3. The Na(+)-dependent increment in bound phosphate, predominant at low ATP concentration and features of which suggest involvement in the concomitant adenosine-triphosphatase activity, was rapidly released in both circumstances. 4. In aqueous media the labile phosphate was released entirely as inorganic phosphate at faster rates with increasing alkalinity. 5. In acidified chloroform-alcohol mixtures the released phosphate appeared both as inorganic phosphate and different single (32)P-labelled organic phosphates, which were tentatively identified as the relevant mono-alkyl phosphates, presumably derived by acid-catalysed alcoholysis of a labelled microsomal component, or components. 6. The labile phosphate corresponded to the P exchangeable with non-radioactive ATP added during the enzyme reaction. 7. The possible molecular nature of the labile fraction of the bound phosphate is discussed.     

Effects of lower alcohols on potassium transport and microsomal adenosine-triphosphatase activity of rat cerebral cortex

1. Slices of rat cerebral cortex, incubated anaerobically at 37 degrees , lost K(+) from an initial concentration of 102m-equiv./kg. to a concentration of 57m-equiv./kg. after 10min. On subsequent aerobic incubation they regained K(+) rapidly at a rate that varied with the K(+) concentration of the medium. 2. Lower aliphatic alcohols, present at equal thermodynamic activity, produced approximately equal degrees of inhibition of K(+) uptake during the aerobic incubation. This inhibition was reduced by an increase in K(+) content of the medium. Ethanol did not affect the rate of K(+) loss during anaerobic incubation. 3. Li(+), in concentrations of 1-10mm, also inhibited K(+) uptake by brain-cortex slices, the degree of inhibition varying with the Li(+) concentration. Ouabain also inhibited K(+) uptake. 4. The same series of alcohols, at equal thermodynamic activity, produced comparable degrees of inhibition of Na(+),K(+),Mg(2+)-stimulated adenosine-triphosphatase activity in brain microsomes. 5. It is suggested that inhibition of cation transport is an important, but not a primary, mechanism in the production of central nervous depression by alcohols and other substances.     

Evidence for an electrogenic adenosine-triphosphatase in Hevea tonoplast vesicles

The function of the Mg-dependent ATPase of Hevea tonoplast in active proton transport was investigated by using a purified tonoplast fraction containing tightly sealed vesicles. In the used experimental conditions, the uptake of [¹⁴C]triphenylmethyl-phosphonium ion ([¹⁴C]TPMP⁺) and [³H]tetraphenyl-phosphonium ion ([³H]TPP⁺) by the vesicles indicated a transmembrane potential difference, negative inside. In parallel, the uptake of [¹⁴C]methylamine into the vesicles monitored a transmembrane pH gradient, interior acid. The addition of 5 mM Mg-ATP markedly depolarized the membrane and increased the magnitude of trnasmembrane pH gradient. These ATP-driven events were substrate specific for Mg-ATP. They were strongly inhibited by ATPase inhibitors such as N, N′-dicyclohexylcar-bodiimide. They were completely eliminated by proton conductors such as carbonylcyanide-p-trifluoromethoxy-phenylhydrazone and 5-chloro-3-tert-butyl-2′-chloro-4-nitro-salicylanilide. They depended on the pH of the medium, the maximum being reached at about pH 7.0. These data provide in vitro evidence that the Mg-ATPase localized at tonoplast level is an electrogenic pump. They are consistent with the hypothesis that an electrogenic H⁺ pump is catalyzed by the tonoplast ATPase of higher plants.     

Specific template activity of ribonucleoprotein particles isolated from germinating Triticum aestivum embryo

A ribonucleoprotein (RNP) complex formed in wheat embryo at early germination stage was shown to be composed of 40-S RNP particle monomers and oligomers. RNA purified from this complex stimulated incorporation in vitro of various amino acids with efficiences dependent upon the frequencies of the corresponding codons calculated for this RNA from its base composition. Methionine was incorporated over the expected rate when the crude RNP complex, instead of the purified RNA, was used as the template. It is believed that the RNP complex represents a crude informosome fraction. The informosomes seem to contain a protein component that promotes the initiation of translation but does not involve the subsequent production of protein.