Competition among oxidizable substrates in brains of young and adult rats. Dissociated cells.

The rates of conversion of D-(-)-3-hydroxy[3-14C]butyrate, [3-14C]acetoacetate, [6-14C]glucose and [U-14C]glutamine into 14CO2 were measured in the presence and absence of alternative oxidizable substrates in intact dissociated cells from the brains of young and adult rats. When unlabelled glutamine was added to [6-14C]glucose or unlabelled glucose was added to [U-14C]glutamine, the rate of 14CO2 production was decreased in both young and adult rats. The rate of oxidation of 3-hydroxy[3-14C]butyrate was also decreased by the addition of unlabelled glutamine in both age groups, but in the reverse situation, i.e. unlabelled 3-hydroxybutyrate added to [U-14C]glutamine, only the brain cells from young rats were affected. No significant effects were seen when glutamine and acetoacetate were combined. The addition of either of the two ketone bodies to [6-14C]glucose markedly lowered the rate of 14CO2 production in young rats, but in the adult only 3-hydroxybutyrate was effective and the magnitude of decrease in the rate of [6-14C]glucose oxidation was much lower than in young animals. Unlabelled glucose decreased the rate of [3-14C]acetoacetate oxidation to a minor extent in brain cells from both age groups; when added to 3-hydroxy[3-14C]butyrate, glucose had no effect in young rats and greatly enhanced 14CO2 production in adult brain cells. Many of these patterns of substrate interaction in dissociated brain cells differ from those in whole homogenates; they may be a function of the plasma membranes and the role of a carrier-mediated transport system or a reflection of a difference in the population of cell types or subcellular organelles in these two preparations.     

Competition of glycerol with other oxidizable substrates in rat brain.

The rate of conversion of [1,3-14C]glycerol into 14CO2 was measured in the presence and absence of unlabelled alternative substrates in whole homogenates from the brains of young (4-6 and 18-20 days old) and adult rats. Unlabelled glucose decreased 14CO2 production from [1,3-14C]glycerol by about 40% at all ages studied. Unlabelled 3-hydroxybutyrate significantly decreased the 14CO2 production from both low (0.2 mM) and high (2.0 mM) concentrations of glycerol in 4-6- and 18-20-day-old rat pups. However, the addition of 3-hydroxybutyrate had no effect on the rate of 14CO2 production from 2.0 mM-glycerol in adult rats, suggesting that the interaction of 3-hydroxybutyrate with glycerol in adult rat brain is complex and may be related to the biphasic kinetics previously reported for glycerol oxidation. Unlabelled glutamine decreased the production of 14CO2 by brain homogenates from 18-20-day-old and adult rats, but not in 4-6-day-old rat pups. In the converse situation, the addition of unlabelled glycerol to whole brain homogenates had little effect on the rate of 14CO2 production from [6-14C]glucose, 3-hydroxy[3-14C]butyrate and [U-14C]glutamine, although some significant differences were noted. Collectively these results suggest that glycerol and these other substrates may be metabolized in separate subcellular compartments in brain such that the products of glucose, 3-hydroxybutyrate and glutamine metabolism can dilute the oxidation of glycerol, but the converse cannot occur. The data also demonstrate that there are complex age-related changes in the interaction of glycerol with 3-hydroxybutyrate and glutamine. The fact that glycerol oxidation was only partially suppressed by the addition of 1-5 mM-glucose, -3-hydroxybutyrate or -glutamine could also suggest that glycerol may be selectively utilized as an energy substrate in some discrete brain region.     

Regulation of glutamine and pyruvate oxidation in cultured adrenocortical cells by cortisol,antioxidants, and oxygen: Effects on cell proliferation

The regulation of CO₂ production from [U-¹⁴C]glutamine and C₂ of [2-¹⁴C]pyruvate was investigated in cultured bovine adrenocortical cells, and the effect of alterations in the relative rates of oxidation of these substrates on cell proliferation, particularly in the presence of an inhibitor of transamination reactions, was examined. ¹⁴CO₂ production from 2 mM [U-¹⁴C]glutamine and 2 mM [2-¹⁴C]pyruvate was measured in the presence of 100 μM 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. Treatment of primary cultures for 24 h with 50 μM cortisol increased the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate, an effect dependent on prior low cell density. Cortisol treatment also resulted in a prolonged delay in the onset of proliferation from low density, and completely inhibited growth in the presence of 2 mM aminooxyacetate, which reduces mitochondrial utilization of glutamine. The effects on glutamine and pyruvate metabolism and on cell growth, with or without aminooxyacetate, were prevented by simultaneous treatment with the antioxidants dimethyl sulfoxide (10 mM) and butylated hydroxyanisole (100 μM), suggesting the involvement of lipid peroxidation in the action of cortisol, as previously demonstrated for its action on 11β-hydroxylase. During continued proliferation of adrenocortical cells in the absence of cortisol there was also a slower increase in the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate as a function of population doubling level. The rate of this increase was slowed by growth of cells in 2% O₂ rather than the standard 19% O₂, and accelerated by continued growth of cells in the presence of cortisol. The rate of increase in the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate under these three conditions correlated with inhibition of cell growth by aminooxyacetate. In contrast to the complete inhibition of growth in aminooxyacetate demonstrated by cortisol-treated cells, control cells (19% O₂) did proliferate, although growth was limited, whereas cells at 2% O₂ proliferated to a much greater extent. In the absence of aminooxyacetate the rate of growth in primary adrenocortical cell cultures under these three conditions was similar. Lipid peroxidation appears to make cultured adrenocortical cells dependent on glutamine for mitochondrial function and proliferation by inhibiting the utilization of the normal substrate, pyruvate.     

Characterization of sheep liver and lung microsomal ethylmorphine N-demethylase

Ethylmorphine N-demethylase activity of the sheep liver and lung microsomes was reconstituted in the presence of solubilized microsomal cytochrome P-450, NADPH-cytochrome c reductase and synthetic lipid, phosphatidylcholine dilauroyl. The Km of the lung microsomal ethylmorphine N-demethylase was calculated to be 4.84 mM ethylmorphine from its Lineweaver-Burk graph and lung enzyme was inhibited by its substrate, ethylmorphine, when its concn was 25 mM and above, reaching to 67% inhibition at 50 mM concn. The Lineweaver-Burk and Eadie-Hofstee plots of the liver enzyme were found to be curvilinear. From these graphs, two different Km values were calculated for the liver enzyme as 4.17 mM and 0.40 mM ethylmorphine. Ethylmorphine N-demethylase activities of both liver and lung microsomes were inhibited by NiCl2, CdCl2 and ZnSO4. Ethylalcohol inhibited N-demethylation of ethylmorphine in lung and liver microsomes. Acetone (5%) slightly enhanced the N-demethylase activity of the liver enzyme, whereas 5% acetone completely inhibited the lung enzyme. Phenylmethylsulfonyl fluoride at 0.10 mM and 0.25 mM concn had no effect on liver enzyme activity, while at these concns, it inhibited the activity of the lung enzyme by about 35%.     

Differential substrate oxidation by dissociated brain cells and homogenates during development.

The rates of oxidation of 3-hydroxy[3-14C]butyrate, [3-14C]acetoacetate and [6-14C]glucose were compared by using two different preparations of brain from the same animals (i.e. whole homogenates and dissociated brain cells) at various ages during development. In homogenates the rates of oxidation of 3-hydroxy[3-14C]butyrate and [3-14C]acetoacetate were high in young rats and low in adults, and were significantly higher at most ages during development than those obtained for intact cells. In contrast, rates of [6-14C]glucose oxidation by homogenates and intact cells were essentially the same at early ages; however, the rate by homogenates did not change throughout development, whereas that by intact cells increased severalfold by adulthood. In adult animals the initial glucose concentration affected the rate of glucose oxidation in homogenates, but not in intact cells. These data suggest a role for the intact cell membrane in the regulation of alternative substrate utilization by brain cells and that this process changes during development. However, the data may reflect selective differences in the cellular and subcellular components in these two preparations.     

Induction of sugar transport in chick embryo fibroblasts by hexose starvation. Evidence for transcriptional regulation of transport.

Incubation of chick embryo fibroblasts in glucose-free medium resulted in a dramatic increase in the rate of 2-deoxy-D-glucose transport. The greatest increase in rate occurred during the first 20 hours of incubation in glucose-free medium and was blocked by actinomycin D, dordycepin, or cycloheximide. The conditions of 2-deoxy-D-glucose concentration and time of incubation with the sugar were determined where transport rather than phosphorylation was rate-limiting in sugar uptake. These studies demonstrated that the transport of 2-deoxy-D-glucose was rate-limiting for only 1 or 2 min when the concentration of sugar in the medium was near the Km for transport, i.e. 2mM. No difference was found in the level of hexokinase activity in homogenates prepared from cells incubated glucose-free medium or standard medium when either 2-deoxy-D-[14C]glucose or D-glucose was used as substrate. A kinetic analysis of the initial rates of 2-deoxy-D-glucose transport by Lineweaver-Burk plots showed that the Vmax for sugar transport increased from 18 to 95 nmol per mg of protein per min when fibroblasts were incubated in glucose-free medium for 40 hours. The Km remained constant at 2 mM. Analysis of the initial rates of 3-omicron-methyl-D-glucose transport by Lineweaver-Burk plots further substantiated that the increase in sugar transport was due to an increase in the Vmax for transport with the Km remaining constant. The activation energy for the transport reaction calculated from an Arrhenius plot was 17.4 Cal per mol for cells cultured in the standard medium and 17.2 Cal per mol for cells cultured in the glucose-free medium. These results are consistent with the interpretation that the Vmax increase observed in hexose-starved cells is due to an increase in the number of transport sites.     

The effect of inhibitors on the oxygen kinetics of terminal oxidases of Acanthamoeba castellanii.

1. Respiration of growing cultures of Acanthamoeba castellanii is inhibited less than 60% by azide (35 mM); the respiration of early-exponential-phase cultures differs from that of late-exponential-phase cultures in being stimulated by up to 120% by low concentrations (less than 1 mM) of this inhibitor. Azide (0.5 mM) plus 1 mM-salicylhydroxamic acid gives 80% inhibition of respiration in early- or late-exponential-phase cultures. 2. Lineweaver-Burk plots of 1/v against 1/[O2] for growing and stationary-phase cultures give values of less than 1 muM for the apparent Km for oxygen. 3. These values are not significantly altered when determined in the presence of 1 mM-salicylhydroxamic acid. 4. Higher values (greater than 7 muM) for apparent Km values for oxygen were obtained in the presence of azide, which gives non-linear Lineweaver-Burk plots. 5. Competitive inhibition of respiration by CO occurs with Ki 2.4 muM. 6. The results are discussed in terms of the presence of three terminal oxidases in this organism, namely two oxidases with high affinities for oxygen (cytochrome c oxidase of the main phosphorylating electron-transport chain and the salicylhydroxamic acid-sensitive oxidase) and a third oxidase with a low affinity for oxygen, sensitive to inhibition by cyanide but not by azide or salicylhydroxamic acid. The relative contributions to oxygen utilization by these oxidases change during the growth of a batch culture.     

Formation of N-nitrosamines from seconday animes and nitrite by resting cells of Escherichia coli B.

In the presence of resting cells of Escherichia coli B, the formation of N-nitrosamines from nitrite and secondary amines, such as dimethylamine and piperidine, was proportional to the incubation time and to the cell concentration. Optimum pH was 8.0. Boiled cells were incapable of nitrosating secondary amines. Although these experiments were carried out by using intact cells of E. coli B, the reaction followed Michaelis-Menten kinetics, and the apparent Km values calculated from Lineweaver-Burk plots were 0.12 +/- 0.03 M for dimethylamine and 0.07 +/- 0.02 M for nitrite. The apparent Km for piperidine was 0.15 +/- 0.05 M. The nitrosation was inhibited by high substrate concentrations. These results suggested that the formation of n-nitrosamines by resting cells of E. coli B apparently depends on their enzyme activities.     

Comparative studies on glutamate metabolism in synpatic and non-synaptic rat brain mitochondria.

1. The apparent Michaelis constants of the glutamate dehydrogenase (EC 1.4.1.3), the glutamate-oxaloacetate transaminase (EC 2.6.1.1) and the glutaminase (EC 3.5.1.2) of rat brain mitochondria derived from non-synaptic (M) and synaptic (SM2) sources were studied. 2. The kinetics of oxygen uptake of both populations of mitochondria in the presence of a fixed concentration of malate and various concentrations of glutamate or glutamine were investigated. 3. In both mitochondrial populations, glutamate-supported respiration in the presence of 2.5 mM-malate appears to be biphasic, one system (B) having an apparent Km for glutamate of 0.25 +/- 0.04 mM (n=7) and the other (A) of 1.64 +/- 0.5 mM (n=7) [when corrected for low-Km process, Km=2.4 +/- 0.75 mM (n=7)]. Aspartate production in these experiments followed kinetics of a single process with an apparent Km for glutamate of 1.8-2 mM, approximating to the high-Km process. 4. Oxygen-uptake measurement with both mitochondrial populations in the presence of malate and various glutamate concentrations in which amino-oxyacetate was present showed kinetics approximating only to the low-Km process (apparent Km for glutamate approximately 0.2 mM). Similar experiments in the presence of glutamate alone showed kinetics approximating only to the high-Km process (apparent Km for glutamate approximately 1-1.3 mM). 5. Oxygen uptake supported by glutamine (0-3 mM) and malate (2.5 mM) by the free (M) mitochondrial population, however, showed single-phase kinetics with an apparent Km for glutamine of 0.28 mM. 6. Aspartate and 2-oxoglutarate accumulation was measured in 'free' nonsynaptic (M) brain mitochondria oxidizing various concentrations of glutamate at a fixed malate concentration. Over a 30-fold increase in glutamate concentration, the flux through the glutamate-oxaloacetate transaminase increased 7--8-fold, whereas the flux through 2-oxoglutarate dehydrogenase increased about 2.5-fold. 7. The biphasic kinetics of glutamate-supported respiration by brain mitochondria in the presence of malate are interpreted as reflecting this change in the relative fluxes through transamination and 2-oxoglutarate metabolism.     

Formation of N-nitrosamines from seconday animes and nitrite by resting cells of Escherichia coli B.

In the presence of resting cells of Escherichia coli B, the formation of N-nitrosamines from nitrite and secondary amines, such as dimethylamine and piperidine, was proportional to the incubation time and to the cell concentration. Optimum pH was 8.0. Boiled cells were incapable of nitrosating secondary amines. Although these experiments were carried out by using intact cells of E. coli B, the reaction followed Michaelis-Menten kinetics, and the apparent Km values calculated from Lineweaver-Burk plots were 0.12 +/- 0.03 M for dimethylamine and 0.07 +/- 0.02 M for nitrite. The apparent Km for piperidine was 0.15 +/- 0.05 M. The nitrosation was inhibited by high substrate concentrations. These results suggested that the formation of n-nitrosamines by resting cells of E. coli B apparently depends on their enzyme activities.     

Mechanisms related to reduction of radical in mouse lung using an L-band ESR spectrometer

Reduction of radicals in mouse lung was characterized in whole animals using an L-band ESR technique and nitroxide radicals as probes. An aqueous solution of nitroxide radical was immediately instilled intratracheally to mouse after euthanasia. Nitroxide radicals without charged groups were reduced significantly in the lung, while radicals with charged groups were only slightly reduced. Permeation rates across lung plasma membrane were not rate limiting of the stage of reduction of the noncharged nitroxides. Michaelis parameters, apparent Km and apparent Vmax, were obtained from the Lineweaver-Burk plots of the reduction. Among noncharged nitroxides with constant apparent Vmax, radicals with a larger n-octanol/water partition coefficient showed a lower apparent Km, thereby suggesting that the concentration of these nitroxides in the membrane contributes to apparent Km. The reduction rate of noncharged nitroxide, hydroxy-TEMPO, was influenced by noncharged SH reagents instilled together with the nitroxide; dithiothreitol stimulated the reduction, while the oxidized reagent inhibited it. The Lineweaver-Burk plots of the nitroxide reduction in the presence of various concentrations of dithiothreitol suggest the possibility that the reduction system for hydroxy-TEMPO is based on a kind of ping pong bi-reactant mechanism, and that the reduction system utilizes SH as an electron donor. Endogenous glutathione contributed partially to the reduction.     

Alanine reverses the inhibitory effect of phenylalanine on acetylcholinesterase activity

The aim of this work was to evaluate, in vitro, the effect of L-alanine (Ala) on suckling rat brain acetylcholinesterase (AChE) and on eel Electrophorus electricus pure AChE inhibited by L-phenylalanine (Phe) as well as to investigate whether Phe or Ala is a competitive inhibitor or an effector of the enzyme. AChE activity was determined in brain homogenates and in the pure enzyme after 1 h preincubation with 1.2 mM of Phe or Ala as well as with Phe plus Ala. The activity of the pure AChE was also determined using as a substrate different amounts of acetylthiocholine. Ala reversed completely the inhibited AChE by Phe (18-20% in 500-600 microM substrate, p<0.01). Lineweaver-Burk plots showed that Vmax remained unchanged. However, Km was found increased with Phe (150%, p<0.001), decreased with Ala alone (50%, p<0.001) and unaltered with Phe plus Ala. It is suggested that: a) Phe presents a competitive inhibitory action with the substrate whereas Ala a competitive activation; b) Ala competition with Phe might unbind the latter from AChE molecule inducing the enzyme stimulation; c) Ala might reverse the inhibitory effect of Phe on brain AChE in phenylketonuric patients, if these results are extended into the in vivo reality.     

Kinetic studies of four enzyme forms of beta-N-acetylhexosaminidase from embryonic chicken brain

The separation of four different hexosaminidase forms from embryonic chicken brain (16-day-old) has been performed by ion-exchange chromatography. Two different DEAE-cellulose columns have been used: a first one at pH 7.2 and a second one at pH 6.0. Km and Vmax values were estimated from the Lineweaver-Burk or Dixon plots and ki from the Dixon plots, using N-acetyl-D-glucosamine or N-acetyl-D-galactosamine as inhibitors. In both cases we found a kind of competitive inhibition in which Lineweaver-Burk and Dixon plots curve downwards.     

Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes

The activity of an ethanolamine and serine base exchange enzyme of rat brain microsomes was copurified to near homogeneity. The purification sequence involved detergent solubilization, Sepharose 4B column chromatography, phenyl-Sepharose 4B column chromatography, glycerol gradient sedimentation, and agarose-polyacrylamide gel electrophoresis under non-denaturing conditions. The ratio of the ethanolamine and serine base exchange activities remained almost constant during purification, and both enzyme activities were enriched 25-fold over the initial microsomal suspension. The final enzyme preparation which contained both enzyme activities showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel, having an apparent molecular mass of about 100 kDa. Serine inhibited the ethanolamine incorporation by this preparation and ethanolamine inhibited the serine incorporation. The competitive nature of this inhibition was apparent from Lineweaver-Burk plots, suggesting that the enzyme catalyzes the incorporation of both ethanolamine and serine into their corresponding phospholipids. The Km and Ki values for ethanolamine were quite similar, being 0.02 and 0.025 mM, respectively. The Km and Ki values for serine were also quite similar being 0.11 and 0.12 mM, respectively. The pH optimum was the same at 7.0 with both substrates. The optimum Ca2+ concentration was 8 mM for serine incorporation.     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

Ornithine-σ-Aminotransferase Exhibits Different Kinetic Properties in Astrocytes, Cerebral Cortex Interneurons, and Cerebellar Granule Cells in Primary Culture

To evaluate a possible role of ornithine-delta-aminotransferase (EC 2.6.1.13; Orn-T) as a rate-limiting enzyme for the synthesis of transmitter glutamate and gamma-aminobutyric acid (GABA), respectively, its activity and kinetic properties were analyzed in cultured astrocytes as well as in neuronal cultures consisting mainly of glutamatergic neurons (cerebellar granule cells) or GABAergic neurons (cerebral cortex interneurons). For comparison the activity and kinetics of Orn-T were also assayed in mouse brain homogenates. The highest activity of Orn-T was found in astrocytes and in cerebral cortical neurons (5.3 +/- 0.5 and 5.3 +/- 0.4 nmol X mg-1 X min-1, respectively) whereas the activities of Orn-T in cerebellar granule cell cultures and in mouse brain were found to be about half of these values (3.1 +/- 0.3 and 2.8 +/- 0.1 nmol X min-1 X mg-1, respectively). From a kinetic study of Orn-T in the different preparations only a relatively low affinity for the enzyme with respect to ornithine was found in cerebellar granule cells, astrocytes, and whole brain [apparent Km values (at 0.5 mM alpha-ketoglutarate): 4.7 +/- 0.9, 4.3 +/- 2.2, and 6.8 +/- 2.2 mM, respectively] whereas the corresponding Km value for Orn-T in cerebral cortex interneurons was found to be significantly lower (apparent Km: 0.8 +/- 0.3 mM). The enzyme was not found to be inhibited by GABA (range 0.1 - 10 mM) in any of the preparations.     

Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts

Human diploid fibroblasts utilize both glucose and glutamine as energy sources. The utilization of glutamine by fibroblasts is regulated by glucose, and vice versa. This conclusion is supported by the following observations: (1) essentially identical growth rates were observed in Eagle's minimum essential medium (MEM)3 in which the glucose concentration was either 5.5 mM or was maintained between 25 and 40 micrometer, (2) the total glutamine utilization by fibroblasts increase at least 30% in medium with 25 micrometer to 70 micrometer glucose compared to medium with 5.5 mM glucose, while the rate of glutamine-1 or 5-14C oxidation to CO2 increased 5-fold as the glucose concentration was decreased to zero, (3) 2 mM glutamine inhibited glucose-6-14C oxidation by 88% and stimulated glucose-1-14C by 77% in log phase cells and (4) glutamine oxidation in normal medium contributed approximately 30% of the energy requirement of human diploid fibroblasts.     

Thiamine Triphosphatase in the Membranes of the Main Electric Organ of Electrophorus electricus: Substrate-Enzyme Interactions

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate (TTP). Membrane fractions prepared from this tissue contain a thiamine triphosphatase that is strongly activated by anions and irreversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion transport inhibitor. Kinetic parameters of the enzyme are markedly affected by the conditions of enzyme preparation: In crude membranes, the apparent Km is 1.8 mM and the pH optimum is 6.8, but trypsin treatment of these membranes or their purification on a sucrose gradient decreases both the apparent Km (to 0.2 mM) and the pH optimum (to 5.0). Anions such as NO3- (250 mM) have the opposite effect, i.e., even in purified membranes, the pH optimum is now 7.8 and the Km is 1.1 mM; at pH 7.8, NO3- increases the Vmax 24-fold. TTP protects against inhibition by DIDS, and the KD for TTP could be estimated to be 0.25 mM, a value close to the apparent Km measured in the same purified membrane preparation. Thiamine pyrophosphate (0.1 mM) did not protect against DIDS inhibition. At lower (10(-5)-10(-6) M) substrate concentrations, Lineweaver-Burk plots of thiamine triphosphatase activity markedly deviate from linearity, with the curve being concave downward. This suggests either anticooperative binding or the existence of binding sites with different affinities for TTP. The latter possibility is supported by binding data obtained using [gamma-32P]TTP. Our data suggest the existence of a high-affinity binding site (KD of approximately 0.5 microM) for the Mg-TTP complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen Hormones Reduce Lipid Peroxidation in Cells and Tissues of the Central Nervous System

Effects of estrogen hormones on lipid peroxidation (LPO) were examined in rat brain homogenates (RBHs), hippocampal HT 22 cells, rat primary neocortical cultures, and human brain homogenates (HBHs). Dose-response curves indicated half-maximal effective concentrations (EC50) of 5.5 and 5.6 mM for iron-induced LPO in RBHs and HT 22 homogenates. Incubation of living rat primary neocortical cultures with iron resulted in an EC50 of 0.5 mM, whereas culture homogenates showed an EC50 of 1.2 mM. Estrogen hormones reduced LPO in all systems: In RBHs, estrone inhibited iron-induced LPO to 74.1 +/- 5.8% of control levels (17beta-estradiol: 71.3 +/- 0.1%) at a concentration of 10 microM. In hippocampal HT 22 cell homogenates, levels of LPO were reduced to 74.8 +/- 5.5% by estrone and to 47.8 +/- 6.2% by 17beta-estradiol. In living neocortical cultures, 17beta-estradiol decreased iron-induced LPO to 79.2 +/- 4.8% and increased the survival of cultured neuronal cells. Of the other steroid compounds tested (corticosterone, progesterone, testosterone), only progesterone decreased LPO in HT 22 cell homogenates. In HBHs, LPO was dose-dependently increased by iron concentrations from 2.7 to 6.0 mM. Incubation with estrogens resulted in a dose-dependent inhibition of LPO to 53.8 +/- 8.6% with 10 microM 17beta-estradiol, whereas estrone failed to affect iron-induced LPO to a significant extent. Nonestrogenic steroids, including hydrocortisol, did not show significant effects on LPO in HBHs.     

Transglutaminase Activity in Rat Brain: Characterization, Distribution, and Changes with Age

The activity of transglutaminase was characterized in the rat brain. In adults, comparable levels of transglutaminase activity are present in all brain regions examined. The activity is present in all subcellular fractions, as studied by differential centrifugation, but the soluble fraction contains the highest specific activity. The endogenous activity (enzyme activity assayed in the absence of the exogenous substrate casein) is very low in all subcellular fractions, except in the synaptosomal fraction where its highest levels are about 40-60% of the activity assayed in the presence of casein. Furthermore, enzyme activity is present on the external surface of synaptosomes. In the soluble fraction, maximal activity can be detected between pH values of 9 and 10 when assayed in the presence of 5 mM CaCl2 (with half-maximal activity requiring 0.75 mM CaCl2) and 0.4 mM putrescine (with an apparent Km for putrescine of 0.1 mM). The activity can be partially inhibited by ZnCl2 (with an IC50 of 4.5 mM) and by AlCl3 (with an IC50 of 5.1 mM). In the cerebellum, where the full span of neuronal development can be studied after birth, the highest specific activity is observed just after birth, thereafter the activity starts to decline and by 14 days, after a reduction of about 65%, it reaches levels observed throughout life.