METABOLIC PARAMETERS OF ONTOGENESIS OF ELECTRICAL ACTIVITY IN THE BRAIN. SODIUM-POTASSIUM ACTIVATED ADENOSINE TRIPHOSPHATASE IN DEVELOPING CHICK EMBRYO BRAIN

—(1) The activity of the Na-K ATPase in the particulate fraction of the chick embryo brain has been assayed at different stages of development with the objective of finding whether or not changes in the activity of this enzyme bear any relation to the maturation of spontaneous and evoked electrical activity of the growing chick brain. (2) The specific activity of the enzyme is low on day 6 and it rises rapidly between days 10 and 12, at which time it attains a plateau and remains essentially unchanged from day 12 until day 20. Experimental evidence rules out the possible presence of an inhibitor of the enzyme in 8-day-old brain homogenates, suggesting that these developmental changes in the activity of the enzyme may represent new synthesis of enzyme rather than its activation. The period between days 10 and 12 does not represent a unique stage of general protein synthesis. (3) The chick brain particulate enzyme has an optimum activity at pH 7·4 and at 37°. It is optimally activated by a Na⁺ concentration of 100mm and K⁺ concentration of 20 mm . The enzyme is inhibited by ouabain and Ca²⁺. (4) The results have been discussed.     

THE SUBCELLULAR DISTRIBUTION AND NUCLEOTIDE SPECIFICITIES OF Na+, K+-STIMULATED ADENOSINE TRIPHOSPHATASE AND [14C]ADENOSINE DIPHOSPHATE-ADENOSINE TRIPHOSPHATE EXCHANGE REACTIONS IN RAT BRAIN

(1) The subcellular distributions of Na-K ATPase and [¹⁴C]ADP-ATP exchange activities were studied in rat brain. The data presented are not consistent with a discrete localization of these enzymes in any given fraction, but nerve endings and microsomes had similar specific activities. The supernatant fraction had the highest exchange and the lowest Na-K ATPase activities, measured at a concentration of 3 mm -MgCl₂. (2) Nucleotide specificity of the Na-K ATPase was determined in all fractions, and this enzyme system showed an absolute requirement for ATP. The [¹⁴C]ADP-ATP exchange, measured at 3mm -MgCl₂, possessed broader specificity and also was active toward ITP, UTP and GTP; this serves to differentiate it from the Na-K ATPase. (3) Treatment of nerve ending fractions with NaI medium removed the bulk of the [¹⁴C]ADP-ATP exchange activity without loss in Na-K ATPase activity. (4) The exchange activity in NaI-insoluble fractions was insensitive to NaCl in the presence of 3 mm -MgCl₂, but it was stimulated 502-820 percent at low MgCl₂ concentrations, a finding which may be consistent with the postulated role of this exchange reaction in the Na-K ATPase system.     

Na-K]ATPase activity in proximal and distal tubules of the rat kidney: modification and application of a quantitative cytochemical technique

A modified cytochemical assay for [Na-K]ATPase in cryostat sections of kidney was further characterized and used to quantify activity in seven functionally distinct sites along the rat nephron. The activity of [Na-K]ATPase was defined as the difference in ATPase activity in specifically identified tubules contained in serial sections incubated with and without ouabain. Preincubation of sections with ouabain was required for maximal inhibition of [Na-K]ATPase activity in several distal sites. The concentration of ouabain necessary for maximal inhibition of activity was 3.0 mM and half-maximal inhibition was obtained in all regions with 30-100 microM ouabain. In distal sites, [Na-K]ATPase formed a higher proportion of total ATPase activity (60-80 per cent) than in proximal sites (20-40 per cent). Enzyme activity was quantified using two different methods. The first measured activity over the basal region of tubules and gave an index of the concentration of [Na-K]ATPase over the basal lateral infoldings of cells composing the tubule. The second read activity over the entire cross section of tubules and provided an estimate of [Na-K]ATPase per length of tubule. The highest activities over the basal basal region were obtained from tubules of the distal nephron including the inner (MALin) and outer (MALout) medullary ascending limb, distal convoluted tubule (DCT) and connecting segment (CS). Lower activities were obtained in proximal convoluted (PCT) tubules, proximal straight (PS) tubules and the papillary collecting duct (PD). Distal convoluted tubules contained the highest activity per length of tubule. Other sites contained lower levels of activity in the following order: MALin greater than MALout greater than PCT greater than PD greater than PS. The modifications introduced increase the sensitivity and precision of this assay and permit the application of this technique to studies of [Na-K]ATPase activity in the major functional regions of the rat nephron.     

USE OF ARRHENIUS PLOTS OF Na-K ATPase and ACETYLCHOLINESTERASE AS A TOOL FOR STUDYING CHANGES IN LIPID-PROTEIN INTERACTIONS IN NEURONAL MEMBRANES DURING BRAIN DEVELOPMENT

Abstract— The activities of Na-K ATPase and acetylcholinesterase in the rat brain cortex were measured at different postnatal ages as a function of temperature. It was found that compared to acetylcholinesterase, Na-K ATPase is more strongly affected by the rise in temperature and that this response is further enhanced with age. Arrhenius plots of the data were prepared and the apparent energies of activation were computed for each plot. It was observed that all plots were biphasic except that for Na-K ATPase of the immature (5-day-old) brain which showed no transition temperature, with an apparent energy of activation of 15.5 kcal/mol. The enzyme from the mature brain (25-day-old) showed an average transition temperature of 22.6°C, with average apparent energies of activation of 15.3 and 27.2 kcal/mol above and below the transition temperature respectively. The cortex of 1-day-old rat showed no Na-K ATPase activity. Arrhenius plots of acetylcholinesterase studied at ages 1, 5 and 25 days postnatally all showed transition temperatures which increased from an average of 16.1°C for 1-day-old to 17 and 21.5°C for 5- and 25-day-old animals respectively. The average apparent energies of activation for acetylcholinesterase below the transition temperature changed from 8.3 kcal/mole at day 1 to 8.7 and 7.2 kcal/mol at days 5 and 25, while above the transition temperature they were 4.3, 5.2 and 4.1 at days 1, 5 and 25 respectively. The results are discussed in terms of the differences and changes in the interactions of Na-K ATPase and acetylcholinesterase with membrane lipids during the postnatal phase of brain development.     

Effect of Na-K adenosinetriphosphatase activity on relaxation of canine tracheal smooth muscle

The effect of Na-K adenosinetriphosphatase (ATPase) on relaxation induced by isoproterenol, prostaglandin E2, sodium nitroprusside, and forskolin, a specific stimulant of adenylate cyclase, was investigated in canine tracheal smooth muscle strips. Relaxation in response to isoproterenol, prostaglandin E2, and forskolin was significantly decreased after inhibition of the Na-K ATPase by ouabain or a potassium-free medium, but relaxation to sodium nitroprusside was not affected. Relaxation to isoproterenol was greater in muscles contracted by 5-hydroxytryptamine than in those contracted by acetylcholine. The stimulation of Na-K ATPase activity with potassium also caused differences in relaxation between tissues contracted with 5-hydroxytryptamine or acetylcholine. Relaxation caused by isoproterenol by activation of the Na-K-ATPase was also decreased by the Ca2+-channel antagonists, verapamil and diltiazem. The results suggest 1) Na-K ATPase activity modulates relaxation caused by isoproterenol, prostaglandin E2, and forskolin in canine tracheal smooth muscle, 2) isoproterenol or activation of the Na-K ATPase may cause relaxation partly by reducing Ca2+ influx through potential-dependent Ca2+ channels, and 3) the differences in the inhibitory effects of isoproterenol and Na-K ATPase activity on muscles contracted by acetylcholine and 5-hydroxytryptamine could be due to differences between these contractile agents in their dependence on extracellular Ca2+ for activation.     

Renal sodium handling and stimulation of medullary Na-K-ATPase during blockade of prostaglandin synthesis

The effect of suppression of prostaglandin synthesis on renal sodium handling and microsomal Na-K ATPase was studied in control and indomethacin treated intact rats maintained on a normal sodium diet (series A) and chronically salt loaded (series B). Indomethacin administration resulted in a decreased GFR and a significantly depressed urinary excretion and an increased fractional reabsorption of sodium in animals fed the normal sodium diet or chronically salt loaded. In rats maintained on a normal Na diet, the activity of the renal medullary Na-K ATPase after indomethacin was 206.3 +/- 6.4 ug Pi/mg protein, i.e. significantly higher as compared with the enzyme activity in the medullary renal fraction from control animals in which it averaged 148 +/- 7.79 ug Pi/mg protein (p less than 0.001). While after chronic salt load a similar increment in the activity of renal medullary Na-K ATPase was observed, no additional stimulation was elicited by subsequent indomethacin administration. The addition of exogenous PGE2, 0.1 mM to microsomal fractions obtained from kidneys of normal rats, was associated with a moderate suppression of the medullary Na-K-ATPase activity, from a basal level of 170 +/- 16 to 151.3 +/- 13 umol Pi/mg protein/hr (p less than 0.005). In isolated segments of medullary thick ascending limb of Henle's loop (MTAL) addition of PGE2 to the incubation medium resulted in a significant inhibition of Na-K ATPase from 37.2 +/- 2 to 21.25 +/- 1.17 x 10(-11) mol/mm/min (p less than 0.0001). These findings suggest that the increased renal Na reabsorption after inhibition of PG synthesis might be related, at least partly, to stimulation of medullary Na-K ATPase. In parallel, the reported natriuretic effect of prostaglandins might imply a direct inhibitory effect of these mediators on renal Na-K ATPase.     

Determination of Fatty Acid in Surfactin and Elucidation of the Total Structure of Surfactin

Several properties of ATPase bound to the inner membrane of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 were examined. The membrane-bound ATPase had two optimal peaks of the activity at pH 5.8 and 7.3. The ATPase activity was strongly inhibited by N,N’- dicyclohexylcarbodiimide (DCCD) and NaN₃ at pH 5.8 and 8.0, and stimulated by MgCl₂ and CaCl₂ at pH 8.0. At pH 8.0, the enzyme hydrolyzed GTP and ITP as well as ATP but not AMP or p-nitrophenylphosphate. CTP, UTP, and ADP were poor substrates. These characteristics indicate that there is a F₀F₁-type ATPase in the inner membrane of this bacterium. In addition, the ATPase activity was also significantly inhibited by Na₃ Vo₄, suggesting the coexistence of a P-type ATPase as a minor constituent. The membrane-bound ATPase activity was maximum at 50°C, but the strong DCCD-sensitivity observed at 20°C was greatly reduced at this temperature.     

THE EFFECTS OF LITHIUM ON ATPase ACTIVITY IN SUBCELLULAR FRACTIONS FROM RAT BRAIN

Abstract— The effects of lithium chloride in vitro and in vivo were investigated on Na-K ATPase and Mg ATPase activities in synaptic plasma membrane, mitochondrial and synaptic vesicle fractions prepared from rat brain. In vitro , lithium chloride (10⁻³-10⁻⁸ m ) had no effect on ATPase activity in any of the fractions studied. Lithium chloride given chronically by i.p. injection (30 mg/rat/day) for 9 days had little effect on synaptic plasma membrane ATPases. Dietary administration of lithium chloride (60 mmol/kg food) produced a small but significant increase in synaptic plasma membrane Mg ATPase activity after 3 weeks administration and mitochondrial Mg ATPase activity after 1 week. There was no effect on synaptic plasma membrane Na-K ATPase activity. Salt supplementation reduced the toxic effects of lithium administration and it is suggested that toxicity may account for some of the previously reported changes in synaptic membrane ATPases produced by lithium.     

Synthetic Analogues of the Termite Trail-following Substance

The effect of tripropyltin chloride (TPT) on some functional reactions in E. coli was investigated. The inhibition on respiration and protein, DNA and RNA syntheses were examined in vivo. Oxygen consumption by E. coli cells was scarcely inhibited even at the concentration of 30 µg/ml TPT. The incorporations of ¹⁴C-labeled amino acids into protein fraction were inhibited. Especially, in the case of l-leucine, it was inhibited 60% even at 10 µg/ml TPT. Both incorporations of ¹⁴C-adenine into DNA and RNA fraction were inhibited 50–60% at 20 µg/ml TPT. RNA polymerase was prepared from E. coli cells and the effect of organotin compounds on the enzyme activity was examined. Organotin compounds inhibited the enzyme activity only at high concentrations (5-10mm). and dialkyltin chlorides which possess no antimicrobial action showed the inhibition more intensely than trialkyltin chlorides. The effect on membrane-bound ATPase was also examined in vitro. We found that TPT has high inhibitory action on membrane-bound ATPase. However, it slightly inhibited the activity of ATPase separated from membrane.     

Norepinephrine induced alpha-adrenoceptor mediated increase in rat brain Na-K ATPase activity is dependent on calcium ion

It has been reported that norepinephrine increases Na-K ATPase activity by acting on -1 adrenoceptors. The mechanism of such an increase was investigated. The norepinephrine induced increase in synaptosomal Na-K ATPase activity was prevented by pretreating the rat brain homogenate with either EDTA, a divalent cation chelator or prazosin, an -1 adrenoceptor blocker. The norepinephrine and EGTA increased the Na-K ATPase activity in the synaptosome prepared from rat brain homogenate untreated with EDTA. The EGTA was ineffective in stimulating the enzyme activity if the synaptosome was prepared from homogenate treated with norepinephrine. However, the EGTA was effective in increasing the enzyme activity if the synaptosome was prepared from the homogenate treated with norepinephrine in the presence of prazosin.

Thus, norepinephrine did not increase the Na-K ATPase activity in the presence of EDTA or -1 adrenoceptor blocker. Similarly, the Ca⁺⁺ chelator, EGTA, could not increase the enzyme activity if the homogenate was pretreated with norepinephrine alone. However, if norpeinephrine action was blocked by -1 antagonist prazosin, EGTA increased the enzyme activity possibly by chelation of Ca⁺⁺. Further, chlorotetracycline fluorescence study showed that norepinephrine removes membrane bound Ca⁺⁺. Thus, it is likely that norepinephrine acts on adrenoceptors and removes membrane bound Ca⁺⁺ and thereby increases the Na-K ATPase activity in the synaptosome.     

(Na-K)ATPase activity along the nephrons in normal and adrenalectomized rats measured by quantitative cytochemistry

A cytochemical method was used to measure total, ouabain insensitive and specific (Na-K)ATPase activities along the rat nephron. Enzyme activity was expressed as per cent of mean integrated extinction with reference to a calibrated filter. The lowest mean values of total, ouabain-insensitive, and (Na-K)ATPase activities were found in the proximal convoluted tubule (PCT). In the distal convoluted tubule (DCT), total and ouabain-insensitive activities (77.8 per cent and 45.8 per cent, respectively) were significantly higher than in the medullary thick ascending limb (MAL) (66.0 per cent and 24.6 per cent, respectively). Mean values of (Na-K)ATPase activity were significantly lower in DCT than in MAL (32.0 per cent and 41.3 per cent, respectively). Using Lineweaver-Burk plots, the KM ATP value for total ATPase activity was found to be 2.33, 1.79, and 3.63 mM in DCT, MAL, and PCT respectively. Maximal velocity was lower in PCT than in MAL and DCT. For (Na-K)ATPase, the smallest KM value was found in MAL (0.95 mM) and was 2.73 and 5.71 mM in DCT and PCT respectively. Maximal velocity was the highest in MAL (49.3 per cent), lower in DCT (36.1 per cent) and least in PCT (22.5 per cent). ATPase was measured in the MAL and DCT from rats fed a normal (N-Na+) or a high (Hi-Na+) sodium diet, and from Hi-Na+ rats one week after adrenalectomy (ADX). In the MAL, (Na-K)ATPase tended to be higher in Hi-Na+ than in rats, but was significantly lower in ADX than in Hi-Na+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basolateral membrane lipid dynamics alter Na-K ATPase activity in rabbit small intestine.

The role of basolateral membrane fluidity in regulating Na-K ATPase activity along the crypt-villus axis in rabbit distal small intestine was assessed. Basolateral membranes were prepared from isolated villus and crypt enterocytes at 24- to 28-fold enhancement. Villus basolateral membranes were significantly (p < 0.001) more fluid than crypt basolateral membranes as measured by 1,6-diphenyl-1,3,5-hexatriene. No difference was seen between the two groups as measured by either 2-(9-anthroyloxy)-stearic fatty acid or 16-(9-anthroyloxy)-palmitic acid. Fluidity alterations were accompanied by an increased phospholipid content in villus membranes, which resulted in a decreased cholesterol:phospholipid ratio and an increased lipid:protein molar ratio. Na-K ATPase activity was significantly (p < 0.01) greater in villus basolateral membranes than in crypt membranes, and demonstrated a greater sensitivity to ouabain inhibition. Ouabain inhibition curves calculated from villus data fit well (p < 0.001) with a two binding site model, with a high affinity (Ki 16 nM) and a low affinity (Ki 4.2 microM) ouabain binding site. In crypt basolateral membranes, only a low affinity site was apparent (Ki 3.0 microM). Fluidizing crypt basolateral membranes in vitro with benzyl alcohol to levels seen in villus basolateral membranes resulted in the appearance of a high affinity ouabain binding site (Ki 110 nM) and an increased sensitivity of Na-K ATPase to ouabain inhibition. The fluidization of villus basolateral membranes eliminated the binding associated with the high affinity site. Treatment with methanol, as a control, did not alter Na-K ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na-K-dependent ATPase in red cells and thyroid status

The Relationship between ouabain-sensitive ATPase (Na-K ATPase) activity in erythrocytes and the thyroid status was studied in 36 patients with Graves' disease and 58 patients receiving L-thyroxine (T4) replacement therapy. Forty normal children served as control. Total ATPase activity in 4 untreated hypothyroid patients was significantly reduced (11.0 +/- 4.6 vs 17.3 +/- 4.1 micrograms-P/h/mg-protein, P less than 0.01), and Na-K ATPase was undetectable, both of which were normalized after 4 weeks of L-T4 therapy. Na-K ATPase in hyperthyroid patients was also decreased (0.9 +/- 0.8 vs. 4.0 +/- 2.7, P less than 0.01), but was gradually normalized after 3 months of euthyroid state. Clinically euthyroid children treated with L-T4 were divided into 2 groups with regard to Na-K ATPase activity, normal and low. Analysis of the possible factors producing this difference revealed that, in primary hypothyroidism, the factor appeared to be the endogenous T4 level, while in patients with dwarfism, the secretory capacity of TSH or TSH-releasing hormone (TRH) was contributory. Thus Na-K ATPase activity in red cells remains within the normal range after L-T4 replacement in the presence of a severe degree of primary hypothyroidism or in association with secondary or tertiary hypothyroidism. Other factors such as the L-T4 dose, duration of the therapy, serum T4 and T3 concentrations, were not significantly different in the two groups. These results indicate that (1) Na-K ATPase in red cells is decreased in hyper- or hypothyroid state, (2) restoration of normal activity requires 1-3 months of euthyroid period, and (3) it is a sensitive index of peripheral thyroid status over the preceding few months.     

The effect of phospholipase C on the activity of adenosine triphosphatase and acetylcholinesterase in synaptic membranes isolated from the cerebral cortex of squirrel monkey

A synaptic-membrane fraction rich in junctional components and Na-K ATPase and AChE activity was isolated from the cerebral cortex of the squirrel monkey. Incubation of membrane preparations with phospholipase C decreased the activity of Na-K ATPase by 50 per cent but had no effect on the activity of AChE. Analysis of the membrane fraction showed that phospholipase C cleaved both choline phosphoglyceride and the diacyl type of ethanolamine phosphoglyceride from membrane lipids. Addition of egg lecithin at low concentrations partially restored the activity of Na-K ATPase. Kinetic studies revealed that treatment with phospholipase C may produce a non-competitive type of inhibition as a result of the cleavage of a charged phosphorylated nitrogen base from membrane lipids.     

Solubilization of a phospholipid-stimulated adenosine triphosphatase complex from membranes of Escherichia coli.

A phospholipid-stimulated adenosine triphosphatase (ATPase) complex was solubilized and partially purified from membrane particles of Escherichia coli ML308-225. The complex was of large molecular size and contained 16 polypeptides, five of which were subunits of the F₁-type ATPase of E. coli. Components of the respiratory chain were absent. Enzyme activity was stimulated by lysophosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, and cardiolipin but not by phosphatidylethanolamine. The ATPase activity of the complex was inhibited by N,N′-dicyclohexylcarbodiimide and by Dio-9 at lower inhibitor:protein ratios than required for inhibition of the F₁-type ATPase of E. coli. However, the ATPase complex was less sensitive than the membrane-bound enzyme to inhibition by these compounds.     

Characterization and solubilization of the membrane-bound ATPase of Mycoplasma gallisepticum.

The membrane-bound ATPase of Mycoplasma gallisepticum selectively hydrolyzed purine nucleoside triphosphates and dATP. ADP, although not a substrate, inhibited ATP hydrolysis. The enzyme exhibited a pH optimum of 7.0 to 7.5 and an obligatory requirement for divalent cations. Dicyclohexylcarbodiimide at a concentration of 1 mM inhibited 95% of the ATPase activity at 37 degrees C, with 50% inhibition occurring at 22 microM dicyclohexylcarbodiimide. Sodium or potassium (or both) failed to stimulate activity by greater than 37%. Azide (2.6 mM), diethylstilbestrol (100 micrograms/ml), p-chloromercuribenzoate (1 mM), and vanadate (50 microM) inhibited 50, 91, 89, and 60%, respectively. The ATPase activity could not be removed from the membrane without detergent solubilization. Although most detergents inactivated the enzyme, the dipolar ionic detergent N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (0.1%) solubilized approximately 70% of the enzyme with only a minor loss in activity. The extraction led to a twofold increase in specific activity and retention of inhibition by dicyclohexylcarbodiimide and ADP. Glycerol greatly increased the stability of the solubilized enzyme. The properties of the membrane-bound ATPase are not consistent with any known ATPase. We postulate that the ATPase functions as an electrogenic proton pump.     

MEMBRANE-BOUND NEURAMINIDASE IN THE BRAIN OF DIFFERENT ANIMALS: BEHAVIOUR OF THE ENZYME ON ENDOGENOUS SIALO DERIVATIVES AND RATIONALE FOR ITS ASSAY

—The activity of brain membrane-bound neuraminidase on endogenous and exogenous substrates was comparatively studied in various animals (rat, chicken, rabbit, pig, calf and human). The maximum rate of hydrolysis of endogenous substrates by membrane-bound neuraminidase (using a crude preparation of the enzyme) was different in the various animals (from 0·05 to 0·73 units, referred to 1 mg protein) and was obtained under similar but not identical optimum conditions (pH from 4·1 to 5·1; requirement or not of Triton X-100). The maximum degree of hydrolysis of endogenous substates was also different (from 15 to 27 nmol released NeuNAc/mg protein) and was obtained within different incubation periods (from 2 to 18 h). It corresponded (in rabbit, calf, human brain only), or not, to the actual exhaustion of the endogenous substrates. The endogenous substrates were recognized as both gangliosides and sialoglycoproteins. The extent of hydrolysis of sialoglycoproteins varied from 1·5% in rabbit to 15·6% in chicken brain; the hydrolysis of gangliosides (ranging from 14·1% in pig to 53·7% in rabbit brain) reached only in some animals (rabbit, calf, human) the complete transformation of major oligosialogangliosides into the neuraminidase resistant monosialoganglioside GMI. Upon addition of exogenous substrates (sialyl-lactose, ganglioside GD1a, brain sialopeptides, ovine submaxillary mucin) the actual rate of liberation of total NeuNAc (from both endogenous and exogenous substrates) considerably exceeded, although at a different extent (depending on the animal and on the added substrate used) the rate of hydrolysis of sole endogenous substrates. The possibility of an accurate assay of brain membrane-bound neuraminidase in a crude enzyme preparation is evaluated and guidelines for the assay procedure suggested.     

FORMATION OF ASPARAGINE FROM ASPARTIC ACID IN RAT BRAIN

The synthesis of asparagine in rat brain was studied both in vitro and in vivo. A conversion in vitro of about 2 per cent of the added l -[¹⁴C]aspartic acid into asparagine was found after a 2 h incubation with the 100,000 g supernatant fraction from brain. This corresponded to a formation of 4·8 nmol of asparagine/mg of protein/h. The reaction required ATP and glutamine, and was linear with time during the 2 h incubation. When the crude mitochondrial fraction was added to the incubation mixture the reaction was inhibited, probably because of the presence of ATPase activity in the mitochondrial preparation. Inhibition by the reaction product seemed unlikely since removal of endogenous asparagine did not stimulate the reaction; only when asparagine was added at levels of 0·5 or 1·0 mm was significant inhibition found. Ammonium chloride was less effective than glutamine as an amide donor. Endogenous asparaginase (EC 3.5.1.1.) activity was low in the in vitro preparation and did not significantly affect the conversion. Synthesis of asparagine from aspartic acid did not occur in slices of brain nor was there a significant conversion of aspartic acid or glucose to asparagine after their intracerebral administration in vivo.     

Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis

The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 microM hop compounds. The extent of activation depended on the concentration of hop compounds and was maximal at the highest concentration tested. The ATPase activity was strongly inhibited by N,N'-dicyclohexylcarbodiimide, a known inhibitor of FoF1-ATPase. Western blots of membrane proteins of L. brevis with antisera raised against the alpha- and beta-subunits of FoF1-ATPase from Enterococcus hirae showed that there was increased expression of the ATPase after hop adaptation. The expression levels, as well as the ATPase activity, decreased to the initial nonadapted levels when the hop-adapted cells were cultured further without hop compounds. These observations strongly indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance of L. brevis to hop compounds.