Purification and kinetic properties of ATP: citrate oxaloacetate lyase from rat brain.

Abstract— A method for a partial purification of ATP:citrate oxaloacetate lyase from rat brain is described. The Lineweaver–Burk plots of velocity vs citrate concentration are biphasic in the presence of fixed concentrations of MgCl₂. Therefore two values of K_m, corresponding to low and high concentrations of citrate, can be determined. When MgCl₂ is added in equimolar concentrations with citrate, a monophasic plot with one K_m of 0.13 mm is obtained. The K_m value for MgATP^2- was independent of citrate concentration, being equal to 0.40–0.43 mm. The K_m for CoA was 0.0007 mm. ADP and P_i are competitive inhibitors with respect to ATP. K_i for MgADP⁻ is equal to 0.13 mm. dl -isocitrate and cis-aconitate are partially competitive inhibitors with respect to citrate with K_i values of 5.8 and 4.8 mm, respectively. α-Ketoglutarate and pyruvate are noncompetitive inhibitors with respect to ATP and citrate, with K_i values equal to 9 and 45 mm, respectively. The physiological significance of these effectors for the regulation of citrate lyase activity in brain is discussed.     

Apparent Inhibition of ATP Citrate Lyase by l-Glutamate In Vitro Is Due to the Presence of Glutamine Synthetase

Preincubation in assay mixture for 30 min at 37 degrees C of ATP citrate lyase from rat brain and liver results in 65-70% inhibition in the presence of 10 mM L-glutamate. This inhibition is specific since none of the known brain metabolites of glutamate shows this effect. ATP and ammonium sulphate-suspended, commercially purified malate dehydrogenase are both important in the generation of inhibition; citrate and NADH are not. The ATP citrate lyase activity in desalted crude extracts and 11% polyethylene glycol-precipitated fractions is inhibited but the enzyme purified by dye affinity chromatography is unaffected. Such purification reveals the presence of a factor responsible for the generation of the inhibition shown to be of Mr 380,000. These lines of evidence implicate endogenous glutamine synthetase, and the involvement of this enzyme is established by the use of its inhibitor L-methionine sulphoximine and by the addition of purified glutamine synthetase to restore the glutamate inhibition of purified ATP citrate lyase. The phenomenon probably arises from the production by glutamine synthetase of ADP, a known product inhibitor of ATP citrate lyase. Therefore contrary to previous reports elsewhere, L-glutamate has no role in the regulation of brain ATP citrate lyase and thus the supply of cytoplasmic acetyl groups for biosynthesis.     

Two mechanisms for spreading depression in the chicken retina

Two mechanisms have been proposed to explain spreading depression (SD): one based on a release of glutamate (Van Harreveld, 1959), and the other on a release of potassium (Grafstein, 1956) from neuronal elements. Both glutamate and KCl cause transparency changes in the retina, comparable to those occurring in this tissue during SD. The glutamate effect is inhibited by MgCl₂ (10 mM), in contrast to the transparency change due to KCl which is not affected by Mg⁺⁺. Also SD is usually inhibited by MgCl₂ which suggests that such SDs are based on a glutamate release. Impairment of the tissue metabolism promotes SDs which are insensitive to MgCl₂. The resulting failure of the mechanisms that transport K⁺ and glutamate which leak out of the intracellular compartment back into the cells and fibers, seems to be involved in the generation of Mg⁺⁺ insensitive SDs. This may facilitate either K-based SDs or glutamate-based SDs since the inhibitory effect of Mg⁺⁺ is counteracted by an enhanced glutamate concentration. Both proposed mechanisms for SD seem to be possible under special circumstances.     

Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase

The (Na⁺ + K⁺)-dependent ATPase exhibits substrate sites with both high affinity (K _m near 1 µM) and low affinity (K _m near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl₂, the nonhydrolyzable - imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theK _D for AMP-PNP was 4.2 µM in the absence of MgCl₂. Adding 50 µM MgCl₂, however, decreased theK _D to 2.2 µM; by contrast, higher concentrations of MgCl₂ increased theK _D until, with 2 mM MgCl₂, theK _D was 6 µM. The half-maximal effect of MgCl₂ on increasing theK _D occurred at approximately 1 mM. This biphasic effect of MgCl₂ is interpreted as Mg²⁺ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl₂ concentrations would represent Mg²⁺ acting through the low-affinity substrate sites. NaCl in the absence of MgCl₂ increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl₂, however, NaCl increased theK _D for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl₂, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K⁺-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.     

Association of ATP citrate lyase with mitochondria

(1) The association of ATP citrate lyase with mitochondria was studied with isolated rat hepatocytes and mitochondria. (2) When hepatocytes were treated with digitonin, about 25% of the lyase activity was released like a mitochondrial enzyme. (3) The effect of temperature on release of lyase from hepatocytes was different from that on the release of other cytosolic or mitochondrial enzymes. (4) The fraction of total hepatic lyase in mitochondrial preparations made with exogenous MgCl₂ was 30 times greater than that for a cytosolic marker enzyme, phosphoglycerate kinase. (5) Lyase substrates enhanced the release of the enzyme both from hepatocytes and from isolated mitochondria. (6) The metabolic significance of association of ATP citrate lyase with mitochondria is discussed. (7) Data obtained in the course of these experiments indicate that less than 3% of adenylate kinase is cytosolic.     

Citrate inhibition of rat-kidney cortex phosphofructokinase

The regulatory properties of citrate on the activity of phosphofructokinase (PFK) purified from rat-kidney cortex has been studied. Citrate produces increases in the K_0.5 for Fru-6-P and in the Hill coefficient as well as a decrease in the V_max of the reaction without affecting the kinetic parameters for ATP as substrate. ATP potentiates synergistically the effects of citrate as an inhibitor of the enzyme. Fru-2,6-P₂ and AMP at concentrations equal to K_a were not able to completely prevent citrate inhibition of the enzyme. Physiological concentrations of ATP and citrate produce a strong inhibition of renal PFK suggesting that may participate in the control of glycolysisin vivo.Abbreviations PFK 6-Phosphofructo-1-kinase (EC 2.7.1.11) - Fru-6-P Fructose 6-phosphate - Fru-2,6-P₂ Fructose 2,6-bisphosphate     

Acetate oxidation to CO2 via a citric acid cycle involving an ATP-citrate lyase: a mechanism for the synthesis of ATP via substrate level phosphorylation in Desulfobacter postgatei growing on acetate and sulfate

Desulfobacter postgatei is an acetate-oxidizing, sulfate-reducing bacterium that metabolizes acetate via the citric acid cycle. The organism has been reported to contain a si-citrate synthase (EC 4.1.3.7) which is activated by AMP and inorganic phosphate. It is show now, that the enzyme mediating citrate formation is an ATP-citrate lyase (EC 4.1.3.8) rather than a citrate synthase. Cell extracts (160,000xg supernatant) catalyzed the conversion of oxaloacetate (apparent K _m=0.2 mM), acetyl-CoA (app. K _m=0.1 mM), ADP (app. K _m=0.06 mM) and phosphate (app. K _m=0.7 mM) to citrate, CoA and ATP with a specific activity of 0.3 mol·min^-1·mg^-1 protein. Per mol citrate formed 1 mol of ATP was generated. Cleavage of citrate (app. K _m=0.05 mM; V _max=1.2 mol · min^-1 · mg^-1 protein) was dependent on ATP (app. K _m=0.4 mM) and CoA (app. K _m=0.05 mM) and yielded oxaloacetate, acetyl-CoA, ADP, and phosphate as products in a stoichiometry of citrate:CoA:oxaloacetate:ADP=1:1:1:1. The use of an ATP-citrate lyase in the citric acid cycle enables D. postgatei to couple the oxidation of acetate to 2 CO₂ with the net synthesis of ATP via substrate level phosphorylation.     

Initial steps of sophoroselipid biosynthesis by Candida bombicola ATCC 22214 grown on glucose

 Candida bombicola ATCC 22214 produces the glycolipid sophoroselipid when cultivated on a medium with glucose as the sole carbon source. Under phosphate-limiting conditions the product yield rises from 0.033 to 0.143 and the specific product formation rate rises from 0.004 h^-1 to 0.007 h^-1. Enhanced sophoroselipid synthesis is initiated by the decline of the specific activities of NAD- and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42) to 2% and 0% of the initial activities respectively. Constantly high specific activity of citrate synthase (EC 4.1.3.7) causes an accumulation of isocitrate and citrate in the mitochondria. Both acids are transported into the cytosol where citrate is cleaved by ATP: citrate lyase (EC 4.1.3.8) giving rise to acetyl-CoA, the precursor of fatty acid synthesis. The ATP: citrate lyase is unaffected by different energy charges; the apparent K _m values for coenzyme A, ATP and citrate are 23 μM, 250 μM and 256 μM respectively. NADPH for fatty acid synthesis might be generated by further metabolism of oxaloacetate, the other product of the citrate-cleaving reaction, by oxidation of the isocitrate by the cytosolic NADP-dependent isocitrate dehydrogenase or via the hexose monophosphate shunt. A possible explanation for sophoroselipid formation during exponential growth is given. Received: 7 November 1995/Received revision: 19 March 1996/Accepted: 25 March 1996     

INHIBITION OF ALANINE AND ASPARTATE AMINOTRANSFERASES BY α-OXODERIVATIVES OF THE BRANCHED-CHAIN AMINO ACIDS

Abstract—

• 1 L-Alanine: α-oxoglutarate aminotransferase was partly purified from rat brain and liver. The enzyme from the brain has about 10 times less activity than that from the liver.
• 2 Both enzymes have identical apparent K_m values for L-alanine, L-glutamate, α-oxoglutarate and pyruvate. Moreover they are competitively inhibited by L-leucine. α-oxoisocaproate and α-oxotsovalerate. Obtained K, values are very similar and do not depend on the course of reaction.
• 3 α-Oxoisocaproate inhibits the activity of crystalline L-aspartate: α-oxoglutarate aminotransferase; Kj is about 4–7 mM.
• 4 The pyridoxamine form of L-alanine: α-oxoglutarate aminotransferase seems to be more sensitive to the inhibitory effect of the compounds investigated.
• 5 The effect of branched-chain amino acids and their α-oxoanalogues on the metabolism of amino groups in maple syrup urine disease is discussed.

     

Catabolism of d-fructose and d-ribose by Pseudomonas doudoroffii

1. The 1-P-fructokinase (1-PFK) and 6-P-fructokinase (6-PFK) from Pseudomonas doudoroffii were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. The pH optima of these enzymes were 9.0 and 8.5, respectively.
2. When the concentrations of the substrates of the 1-PFK reaction were varied, Michaelis-Menten kinetics were observed. The Kms for d-fructose-1-P (F-1-P) and ATP were 3.03×10^-4 M and 3.39×10^-4 M, respectively. Variation of MgCl₂ at fixed concentrations of F-1-P and ATP resulted in sigmoidal kinetics; about 10 mM MgCl₂ was necessary for maximal activity. Activity of 1-PFK was inhibited when the ratio of ATP: Mg⁺⁺ was higher than 0.5, suggesting that ATP: 2Mg⁺⁺ was the substrate and that free ATP was inhibitory. Although an absolute requirement for K⁺ or NH ₊ ⁴ could not be demonstrated, these cations stimulated the rate of the reaction. Activity of 1-PFK was not significantly affected by 3 mM AMP, cyclic-AMP, P_i, d-fructose-6-P (F-6-P), ADP, P-enolpyruvate (PEP), pyruvate, citrate, or l-glutamate.
3. Sigmoidal kinetics were observed for 6-PFK when the concentration of F-6-P was increased and the level of ATP was kept constant. Activity of 6-PFK was increased by ADP, inhibited by PEP, and unaffected by 3 mM AMP, cyclic-AMP, P_i, F-1-P, pyruvate, or citrate.

     

Purification and characterization of benzoyl-CoA ligase from a syntrophic,benzoate-degrading,anaerobic mixed culture

Summary The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optimum was 37–40° C, the optimum pH around 8.0 and optimal activity required 50–100 mM TRIS-HCI buffer, pH 8.0 and 3–7 mM MgCl₂; MgCl₂ in excess of 10 mM was inhibitory. The activation energy for benzoate was 11.3 kcal/mol. Although growth occured only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3- and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The K _m values for benzoate, 2-, 3- and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 mM, the V _max values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a K _m of 0.17 mM and a V _max of 1.05 U/mg and for ATP a K _m of 0.16 mM and a V _max of 1.08 U/mg was determined. Benzoate activation was inhibited by more than 6 mM ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (K _i) for pyrophosphate was 5.7 mM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found. Correspondence to: J. Winter     

Spinach pyruvate kinase isoforms : partial purification and regulatory properties

Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide K_m values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The K_i for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a K_a of 0.05 millimolar, and glutamate is an inhibitor with a K_i of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, we suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation.     

Modification of glutamate- effects on neuroblastoma cells in culture by heavy metals

Sodium L-glutamate inhibited the growth (due to inhibition of cell division and cell death) of mouse neuroblastoma (NB) cells in culture in a dose dependent fashion. The sensitivity of adrenergic (NBA₂₍₁₎) and cholinergic (NBE^?) clones to L-glutamate was similar. Sodium D-glutamate, L-aspartate, α-ketoglutarate, glutamine, γ-aminobutyric acid and carbachol did not inhibit the growth of NB cells. Methylmercuric chloride (CH₃HgCl), inorganic mercury (HgCl₂), manganese chloride (MnCl₂) and lead tri-butyl acetate, by themselves inhibited the growth of NB cells in culture to a varying degree ranging from 41% to 49%. However, the combination of glutamate with CH₃HgCl, HgCl₂ and MnCl₂, produced a synergestic effect on growth inhibition of NB cells in culture. The combination of glutamate with lead tri-butyl acetate produced only an additive effect. Sodium kainate neither inhibited the growth nor potentiated the growth inhibitory effect of L-glutamate on NB cells. Neuroblastoma cells contained high levels of receptors for glutamate but not for kainate. These results show that neuroblastoma culture may be a useful model to study the mechanisms of glutamate effects and their modification by various agents.     

Ammonia assimilation and oxygen evolution by a reconstituted chloroplast system in the presence of 2-oxoglutarate and glutamate

(Ammonia plus 2-oxoglutarate)-dependent O₂ evolution by intact chloroplasts was enhanced three- to five fold by 2 mM L- and D-malate, attaining rates of 9–15 μmol mg^-1 Chl h^-1. Succinate and fumarate also promoted activity but D-aspartate and, in the presence of aminooxyacetate, L-aspartate inhibited the malate-promoted rate. A reconstituted chloroplast system supported (ammonia plus 2-oxoglutarate)-dependent O₂ evolution at rates of 6-11 μmol mg^-1 Chl h^-1 in the presence of MgCl₂, NADP(H), ADP plus Pi (or ATP), ferredoxin and L-glutamate. The concentrations of L-glutamate and ATP required to support 0.5 V _max were 5 mM and 0.25 mM, respectively. When the reaction was initiated with NH₄Cl, O₂ evolution was preceded by a lag phase before attaining a constant rate. The lag phase was shortened by addition of low concentrations of L-glutamine or by preincubating in the dark in the presence of glutamate, ATP and NH₄Cl. Oxygen evolution was inhibited by 2 mM azaserine and, provided it was added initially, 2 mM methionine sulphoximine. The (ammonia plus 2-oxoglutarate)-dependent O₂ evolution was attributed to the synthesis of glutamine from NH₄Cl and glutamate which reacted with 2-oxoglutarate in a reaction catalysed by ferredoxin-specific glutamate synthase using H₂O as the ultimate electron donor. The lag phase was attributed to the establishment of a steady-state pool of glutamine. L-Malate did not affect the activity of the reconstituted system.     

L-Glutamate Supplementation Improves Small Intestinal Architecture and Enhances the Expressions of Jejunal Mucosa Amino Acid Receptors and Transporters in Weaning Piglets

L-Glutamate is a major oxidative fuel for the small intestine. However, few studies have demonstrated the effect of L-glutamate on the intestinal architecture and signaling of amino acids in the small intestine. The aim of this study was to investigate the effects of dietary L-glutamate supplementation on the intestinal architecture and expressions of jejunal mucosa amino acid receptors and transporters in weaning piglets. A total of 120 weaning piglets aged 35±1 days with an average body weight at 8.91±0.45 kg were randomly allocated to two treatments with six replicates of ten piglets each, fed with diets containing 1.21% alanine, or 2% L-glutamate. L-Glutamate supplementation increased the activity of glutamate oxaloacetate transaminase (GOT) in the jejunal mucosa. Also, the mRNA expression level of jejunal mucosa glutamine synthetase (GS) was increased by L-glutamate supplementation. The height of villi in duodenal and jejunal segments, and the relative mRNA expression of occludin and zonula occludens protein-1 (ZO-1) in jejunal mucosa were increased by dietary L-glutamate supplementation. L-Glutamate supplementation increased plasma concentrations of glutamate, arginine, histidine, isoleucine, leucine, methionine, phenylalanine and threonine. L-Glutamate supplementation also increased the relative mRNA expression of the jejunal mucosa Ca²⁺-sensing receptor (CaR), metabotropic glutamate receptor 1 (mGluR1) and metabotropic glutamate receptor 4 (mGluR4), and neutral amino acid transporter B⁰-like (SLC1A5) in the jejunal mucosa. These findings suggest that dietary addition of 2% L-glutamate improves the intestinal integrity and influences the expression of amino acid receptors and transporters in the jejunum of weaning, which is beneficial for the improvement of jejunal nutrients for digestion and absorption.     

Mechanisms Underlying Regulation of a Barium -sensitive K<Superscript>+ </Superscript>Conductance by ATP in Single Proximal Tubule Cells Isolated from Frog Kidney

K⁺ channels play an important role in pump-leak coupling and volume regulation in the renal proximal tubule. Previous experiments have identified a barium-sensitive K⁺ conductance (G_Ba) in proximal tubule cells isolated from frog kidneys. In this paper we examine the regulation of G_Ba by ATP. G_Ba was measured in single cells isolated from frog kidney using the whole-cell patch-clamp technique. G_Ba was activated by 2 mM intracellular ATP. This activation was enhanced by inhibition of protein kinase C and attenuated by inhibition of protein kinase A, indicating reciprocal regulation by these kinases. Activation by ATP was reduced in the presence of a hypertonic bath solution, suggesting that cell swelling was required. However, after activation to steady-state, G_Ba was not sensitive to cell-volume changes. Hypotonic shock-induced volume regulation was inhibited by barium and quinidine, inhibitors of G_Ba. The effect of maximal inhibitory concentrations of barium and quinidine on volume regulation was similar and addition of both blockers together did not augment the inhibitory response. G_Ba was also activated by ADP, via a mechanism dependent on the presence of Mg²⁺. However, the responses to ADP and ATP were not additive, suggesting that these nucleotides may share a common mechanism of activation. The regulation of G_Ba by ATP was biphasic, with a half-maximal activating concentration of 0.89 mM and a half maximal inhibitory concentration of 6.71 mM. The sensitivity to nucleotides suggests that G_Ba may be regulated by the metabolic state of the cell. Furthermore, the sensitivity to solution osmolality, coupled with the blocker profile of inhibition of volume regulation, suggests that G_Ba could play a role in volume regulation.     

Effects of Mg(2+) on activation of the (Na(+) + K(+)-dependent ATPase by Na(+1).

The effects of MgCl₂ on Na activation of three different enzymatic reactions catalyzed by a rat brain (Na + K)-dependent ATPase (adenosine 5′-triphosphatase) were studied. For the Na⁺-dependent ATPase reaction measured with 6 μm ATP, the K_0.5 for Na increased from 0.4 to 1.7 mm as the MgCl₂ concentration was raised from 50 to 2000 μm; the half-maximal effect occurred at a free Mg²⁺ concentration near 0.8 mm. By contrast, with 3 mm ATP and 3 mm MgCl₂ the K_0.5 for Na was again 0.4 mm, but further addition of 2 mm MgCl₂ then had little effect on the K_0.5 for Na. For the Na-dependent phosphorylation of the enzyme, measured with 6 μm ATP, the K_0.5 for Na increased similarly, from 0.2 to 0.8 mM, as the MgCl₂ concentration was raised from 50 to 2000 μm, but for the (Na + K)-dependent ATPase reaction the K_0.5 for Na was 13 mm and increased by only one-third as the MgCl₂ concentration was raised. The K_0.5 for K was also little affected by changes in MgCl₂ concentration. Finally, with 3 mm ATP and 3 mm MgCl₂ the K_0.5 for Na in the (Na + K)-dependent ATPase reaction decreased to 5 mm. These observations are considered in terms of an enzyme having high-affinity and low-affinity substrate sites, with occupancy of the low-affinity sites modifying Na activation differently, depending both on the specific reaction catalyzed and on whether occupancy is by free Mg²⁺ or by Mg-ATP.     

Studies on glutamine synthetase. Purification of the enzyme from mung bean (Phaseolus aureus) seedlings and modulation of the enzyme-antibody reaction by the substrates

Glutamine synthetase (L-glutamate : ammonia ligase, EC 6.3.1.2) fromPhaseolus aureus (mung bean) seedlings was purified to homogeneity by ammonium sulphate fractionation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and affinity chromatography on histidine-Sepharose. The enzyme had a molecular weight of 775,000 ± 25,000. The enzyme consisted of identical subunits with an approximate subunit molecular weight of 50,000. Hyperbolic saturation curves were obtained with the substrates, glutamate, ATP and hydroxylamine. Antibody, raised in the rabbit, against mung bean glutamine synthetase, completely inhibited the activity of the enzyme. Preincubation of the enzyme with glutamate and ATP, prior to the addition of the antibody, partially protected the enzyme against inhibition. TheK _m values of this enzyme-antibody complex and the native enzyme were identical (glutamate, 2.5mM; ATP, 1 mM; hydroxylamine, 0.5 mM). The Km values of the partially inhibited enzyme (the enzyme pretreated with antibody prior to the addition of substrates) were 2-fold higher than those of the native enzyme. These results suggested that the substrate-induced conformational changes in the enzyme were responsible for the protection against inhibition of the enzyme activity by the antibody.     

Binding of L-[3H]Glutamate to Fresh or Frozen Synaptic Membrane and Postsynaptic Density Fractions Isolated from Cerebral Cortex and Cerebellum of Fresh or Frozen Canine Brain

Abstract: Synaptic membrane (SPM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brain, either fresh or frozen and isolated from either fresh or frozen tissue, were found to contain L-[³H]glutamate binding sites. It was found that there was a concentration of L-glutamate binding sites in CTX-PSD and CL-PSD over the respective membrane fractions, and the B_max value of CL-PSD (92.0 pmol/mg protein) was about three times that of CTX-PSD (28.9 pmol/mg). The results, together with those of others, suggest that the thin CL-PSD are probably derived from the excitatory synapses in the molecular layer. The ion dependency of L-glutamate binding to canine CTX-SPM fraction was found to be similar to that reported for a rat brain SPM fraction: (a) Cl^? increased the number of L-glutamate binding sites and the effect was enhanced by Ca²⁺; Ca²⁺ alone had no significant effect; (b) the Cl^?/Ca²⁺ -sensitive binding sites were abolished by 2-amino-4-phosphonobutyrate (APB) or freezing and thawing: (c) the effect of Na⁺ ion was biphasic: low concentration of Na⁺ (< 5 mM) decreased Cl^?7Ca²⁺ -de-pendent L-glutamate binding sites, whereas at higher concentrations of Na⁺ the binding of glutamate was found to increase either in the presence or absence of Ca²⁺ and Cl^?. In addition, the K⁺ ion (50 mM) was found to decrease the Na⁺-independent and Cl^?/Ca^2--independent binding of L-glutamate to fresh CTX-SPM by 18%, but it decreased the Na^?-dependent and Cl^?/Ca²⁺-independent L-glutamate binding by 93%; in the presence of Cl, ^?/Ca²⁺, the K⁺ ion decreased the Na⁺-dependent binding by 78%. Freezing and thawing of CTX-SPM resulted in a 50% loss of the Na⁺-dependent L-glutamate binding sites assayed in the absence of Ca²⁺ and Cl^?. The CL-SPM fraction showed similar ion dependency of L-glutamate binding except for the absence of Na^?-dependent glutamate binding sites. The CTX-PSD fraction contained neither Na⁺-dependent nor APB (or Cl^?/Ca²⁺)-sensitive L-glutamate binding sites and its L-glutamate binding was unaffected by freezing and thawing, in agreement with the reported findings using rat brain PSD preparation. L-Glutamate binding to CTX-SPM or CTX-PSD fraction was not affected by pretreatment with 10 mM L-glutamate, nor by simultaneous incubations with calmodulin. Also, phosphorylation of CTX-SPM or CTX-PSD fraction, whether incubated simultaneously or after removal of the phosphorylating reagents, had no effect on binding of L-glutamate. Furthermore, binding of L-glutamate to CTX-SPM or CTX-PSD was found to have no significant effect on subsequent phosphorylation of the fractions. Treatment of the CTX-PSD fraction with 0.5% deoxycholate, 1.0% N-lauroyl sarcosinate, 4 M guanidine-HCl, pH 7.0, 0.5 M KCl, and 1.0 M KCl removed the L-glutamate receptors from the PSD by 25%, 44%, 40%, 8%, and 11%. respectively. The respective percentages of total protein solubilized by these reagents were similar, indicating no preferential dissociation of the receptors, and suggesting that the L-glutamate receptor is an intrinsic PSD component. The present findings, together with the earlier ones showing the presence of γ-aminobutyric acid and flunitrazepam binding sites, of the Ca²⁺-dependent K⁺ channel, and of the voltage-dependent Ca²⁺ channel proteins in the isolated PSD fraction, suggest that many, if not all, neurotransmitter receptor proteins and ion channel proteins are anchored in the PSD at the synapse, and thus the PSD may play an important role in neurotransmission at the postsynaptic site.     

Effects of Cations on (Ca2++ Mg2+)-Activated ATPase from Rat Brain

Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K_0.5 for activation by Ca²⁺ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl₂, 100 m_M-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca²⁺, but marked inhibition occurred at higher concentrations. Free Mg²⁺ increased ATPase activity, with an estimated K_0.5, in the presence of 100 μm -CaCl₂, of 2.5 mm ; raising the MgCl₂ concentration diminished the inhibition due to millimolar concentrations of CaCl₂, but antagonized activation by submicromolar concentrations of Ca²⁺. Dimethylsulfoxide (10%, v/v) had no effect on the K_0.5 for activation by Ca²⁺, but decreased activation by free Mg²⁺ and increased the inhibition by millimolar CaCl₂. The monovalent cations K⁺, Na⁺, and TI⁺ stimulated ATPase activity; for K⁺ the K_0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca²⁺ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-³²P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s^?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s^?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s^?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K⁺ stimulation of both dephosphorylation and inhibition by vanadate.