Cerebral Alanine Transport and Alanine Aminotransferase Reaction: Alanine as a Source of Neuronal Glutamate

Abstract: Alanine transport and the role of alanine amino-transferase in the synthesis and consumption of glutamate were investigated in the preparation of rat brain synaptosomes. Alanine was accumulated rapidly via both the high-and low-affinity uptake systems. The high-affinity transport was dependent on the sodium concentration gradient and membrane electrical potential, which suggests a cotransport with Na⁺. Rapid accumulation of the Na⁺-alanine complex by synaptosomes stimulated activity of the Na⁺/K⁺ pump and increased energy utilization; this, in turn, activated the ATP-producing pathways, glycolysis and oxidative phosphorylation. Accumulation of Na⁺ also caused a small depolarization of the plasma membrane, a rise in [Ca²⁺]₁, and a release of glutamate. Intra-synaptosomal metabolism of alanine via alanine aminotransferase, as estimated from measurements of N fluxes from labeled precursors, was much slower than the rate of alanine uptake, even in the presence of added oxoacids. The velocity of [¹⁵N]alanine formation from [¹⁵N]glutamine was seven to eight times higher than the rate of [¹⁵N]glutamate generation from [¹⁵N]alanine. It is concluded that (a) overloading of nerve endings with alanine could be deleterious to neuronal function because it increases release of glutamate; (b) the activity of synaptosomal alanine aminotransferase is much slower than that of glutaminase and hence unlikely to play a major role in maintaining [glutamate] during neuronal activity; and (c) alanine aminotransferase might serve as a source of glutamate during recovery from ischemia/hypoxia when the alanine concentration rises and that of glutamate falls.     

Alanine Aminotransferase in Bovine Brain: Purification and Properties

Abstract: Mitochondrial and cytosolic alanine aminotransferases (EC 2.6.1.2) were partially purified (140- and 180-fold, respectively) from bovine brain cortex by means of (NH₄)₂SO₄ precipitation, gel filtration on Sephadex G-150, and ion-exchange chromatography on DEAE A-50 and characterized. The enzymes exhibited identical molecular weights (110,000 ± 10,000) and pH optima (7.8), but were eluted from CM Sephadex C-50 at different ionic strengths. Isoelectric focusing of the enzymes indicated a pi value of 5.2 for the cytosolic enzyme and 7.2 for the mitochondrial enzyme. The K _m values of the mitochondrial enzyme were 5.1 m M , 6.6 m M , 0.7 m M , and 0.4 m M and of the cytosolic isozyme were 30.3 m M , 4.3 m M , 0.7 m M , and 0.5 m M for alanine, glutamate, 2-oxoglutarate, and pyruvate, respectively. The results indicated that two forms of alanine aminotransferase exist in nerve tissue, which suggests that they may play different roles in the cellular metabolism of nerve tissue.     

TAT-Mediated Delivery of Human Alanine:Glyoxylate Aminotransferase in a Cellular Model of Primary Hyperoxaluria Type I

Defects in liver peroxisomal alanine:glyoxylate aminotransferase (AGT), as a consequence of inherited mutations on the AGXT gene, lead to primary hyperoxaluria type I (PH1), a rare metabolic disorder characterized by the formation of calcium oxalate stones at first in the urinary tract and then in the whole body. The curative treatments currently available for PH1 are pyridoxine therapy, effective in only 10–30 % of the patients, and liver transplantation, an invasive procedure with potentially serious complications. A valid therapeutic option for PH1 patients would be the development of an enzyme administration therapy. However, the exogenous administration of the missing AGT would require the crossing of the plasma membrane to deliver the protein to liver peroxisomes. In this study, we constructed, purified and characterized the fusion protein of AGT with the membrane-penetrating Tat peptide (Tat-AGT). Although Tat-AGT shows subtle active site conformational changes as compared with untagged AGT, it retains a significant transaminase activity. Western-blot analyses, enzymatic assays and immunofluorescence studies show that active Tat-AGT can be successfully delivered to a mammalian cellular model of PH1 consisting of chinese hamster ovary cells expressing glycolate oxidase (CHO-GO), whereas untagged AGT cannot. Moreover, the intracellular transduced Tat-AGT makes CHO-GO cells able to detoxify endogenously produced glyoxylate to an extent similar to that of CHO-GO cells stably expressing AGT. Altogether, these results show that the Tat peptide is capable of delivering a functional AGT to mammalian cells, thus paving the way for the possibility to use Tat-AGT as an enzyme replacement therapy to counteract PH1.     

Allele-specific Characterization of Alanine: Glyoxylate Aminotransferase Variants Associated with Primary Hyperoxaluria

Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive kidney stone disease caused by deficiency of the peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT), which is involved in glyoxylate detoxification. Over 75 different missense mutations in AGT have been found associated with PH1. While some of the mutations have been found to affect enzyme activity, stability, and/or localization, approximately half of these mutations are completely uncharacterized. In this study, we sought to systematically characterize AGT missense mutations associated with PH1. To facilitate analysis, we used two high-throughput yeast-based assays: one that assesses AGT specific activity, and one that assesses protein stability. Approximately 30% of PH1-associated missense mutations are found in conjunction with a minor allele polymorphic variant, which can interact to elicit complex effects on protein stability and trafficking. To better understand this allele interaction, we functionally characterized each of 34 mutants on both the major (wild-type) and minor allele backgrounds, identifying mutations that synergize with the minor allele. We classify these mutants into four distinct categories depending on activity/stability results in the different alleles. Twelve mutants were found to display reduced activity in combination with the minor allele, compared with the major allele background. When mapped on the AGT dimer structure, these mutants reveal localized regions of the protein that appear particularly sensitive to interactions with the minor allele variant. While the majority of the deleterious effects on activity in the minor allele can be attributed to synergistic interaction affecting protein stability, we identify one mutation, E274D, that appears to specifically affect activity when in combination with the minor allele.     

Alanine Aminotransferase and Risk of the Metabolic Syndrome: A Linear Dose-Response Relationship

Background

Elevated baseline circulating alanine aminotransferase (ALT) level has been demonstrated to be associated with an increased risk of the metabolic syndrome (MetS), but the nature of the dose-response relationship is uncertain.

Methods

We performed a systematic review and meta-analysis of published prospective cohort studies to characterize in detail the nature of the dose-response relationship between baseline ALT level and risk of incident MetS in the general population. Relevant studies were identified in a literature search of MEDLINE, EMBASE, and Web of Science up to December 2013. Prospective studies in which investigators reported relative risks (RRs) of MetS for 3 or more categories of ALT levels were eligible. A potential nonlinear relationship between ALT levels and MetS was examined using restricted cubic splines.

Results

Of the 489 studies reviewed, relevant data were available on 29,815 non-overlapping participants comprising 2,125 incident MetS events from five prospective cohort studies. There was evidence of a linear association (P for nonlinearity = 0.38) between ALT level and risk of MetS, characterised by a graded increase in MetS risk at ALT levels 6–40 U/L. The risk of MetS increased by 14% for every 5 U/L increment in circulating ALT level (95% CI: 12–17%). Evidence was lacking of heterogeneity and publication bias among the contributing studies.

Conclusions

Baseline ALT level is associated with risk of the MetS in a linear dose-response manner. Studies are needed to determine whether the association represents a causal relationship.     

Aminotransferase and the Production of Alanine During Hyperosmotic Stress in Paramecium calkinsi

When Paramecium calkinsi encounter hyperosmotic stress, intracellular free alanine increases. In vivo assays indicate that the reaction catalyzed by alanine aminotransferase contributes to the build up of alanine in response to hyperosmotic shock. ¹⁴C-pyruvate is converted to ¹⁴C-alanine in cells grown axenically at 200 mosm. When shifted to 600 mosm, the rate of conversion of pyruvate to alanine increases, and conversion at either 200 or 600 mosm is blocked by 1 mM aminooxyacetic acid (AOA), an inhibitor of aminotransferase. Intracellular free alanine increase is partially inhibited by AOA, and AOA prevents cells living in fresh water from acclimating to higher salinities, an indication that the increase in intracellular alanine is physiologically significant.     

家蚕丙氨酸转氨酶理化性质的研究

丙氨酸转氨酶(ALT)是家蚕丝心蛋白合成中的关键酶,为了阐明丝心蛋白的合成机理及利用ALT活力调控丝物质的形成,有必要深入了解ALT的分子性质。作者在分离纯化家蚕后部丝腺ALT的基础上,应用聚丙烯酰胺凝胶电泳等生化方法进一步研究了该酶的若干理化性质。结果表明,家蚕ALT由两种不同亚基组成,分子量分别是54kd和21kd;最适底物是丙酮酸;最适温度为50℃;最适DH为8．5;钾、钠、钙、镁等离子对酶有激活作用,而锰、铜、锌等离子对酶有抑制作用。研究还表明,该酶是一种依赖于金属离子的酶类,活性中心含有巯基或咪唑基。     

Enzyme Activities in Urtica dioica: Effects of Daylength and Leaf Age on Glutamate Dehydrogenase, Aspartate Aminotransferase and Alanine Aminotransferase

Enzymes, important to protein synthesis, were investigated in young and old leaves of Urtica dioica. The plants, divided into two groups, were exposed to either 18-hour or 12-hour photo-periods. One group of plants from each photoperiodic regime was subjected to an irradiance of 28 W × m^-2, and the other group of plants to 42 W × m^-2. The enzymes investigated were glutamate dehydrogenase (GDH), aspartate aminotransferase (glutamate-oxaloacetate transaminase, GOT), and alanine aminotransferase (glutamate-pyruvate transaminase, GPT), GDH and GOT were determined by means of electrophoretic separation on polyacrylamide and spectrophotometric measurements. GPT was determined only by the latter method. Plants exposed to 18-hour photoperiods showed much higher GDH activity than did those exposed to 12-hour photoperiods. The activity of GDH also increased with leaf age. Besides one uniform NAD⁺-dependent GDH, two other NAD⁺-independent enzymes, showing GDH activity, were identified on polyacryl-amide gel electrophoresis. The distribution of NADH and NAD⁺-dependent GDH activity between young and old leaves was similar under different growth conditions. The activity of GOT was insensitive to environmental changes. The results regarding GPT indicate that this enzyme responded to different photoperiods in the same way as GDH. A correlation coefficient of 0.928 was obtained for the relationship between GDH and GPT activity.     

丙氨酸转氨酶修饰对大肠杆菌L-色氨酸合成的影响

探究大肠杆菌细胞内负责L-丙氨酸合成的转氨酶对菌株代谢及L-色氨酸合成的影响。运用Red重组技术分别对编码L-丙氨酸转氨酶的基因alaA、alaC和avtA进行敲除。通过摇瓶和50 L罐中探究其对L-色氨酸积累、L-丙氨酸代谢及菌体生长变化情况。结果显示,除3种L-丙氨酸转氨酶全部缺失的工程菌L-丙氨酸合成受阻、菌体生长受到较强抑制外,其它各任意一种或两种丙氨酸转氨酶缺失菌株的生长并未有较大差异,但色氨酸的合成变化显著。其中alaA和alaC双基因缺失的E.coli FS-T4工程菌,摇瓶发酵L-色氨酸产量达6.08 g/L,L-丙氨酸含量仅0.16 g/L,较出发菌株分别提高了26.7%和降低了91.0%。在50 L罐中E.coli FS-T4工程菌L-色氨酸产量最高可达41.9 g/L,糖酸转化率达20.5%,分别较出发菌株提高了13.8%和5.1%。转氨酶AlaA和AlaC的同时缺失,既可以满足细胞整体氨基酸池的需要,而且有利于减少杂酸的积累,使得更多的碳源流向L-色氨酸的合成。     

Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism.     

Elevated Alanine Aminotransferase Is Strongly Associated with Incident Metabolic Syndrome: A Meta-Analysis of Prospective Studies

Background

The incidence of metabolic syndrome (MetS) is rapidly increasing worldwide and associated with alanine aminotransferase (ALT) activity. However, the impact of ALT activity on MetS incidence is inconsistent in published literature. We therefore estimated the association between elevated ALT activity and incident MetS through a meta-analysis of prospective cohort studies.

Methods/Principal Findings

All published prospective cohort studies on the association between elevated ALT activity and incident MetS were retrieved from Pubmed, Embase, and the Institute for Scientific Information (ISI). In all, seven prospective cohort studies, with 31545 participants and 2873 cases of incident MetS were recruited. If there was insignificant heterogeneity (P-value>0.05 and I²<50%), the fixed-effect model was used to calculate the pooled relative risks (RRs) of incident MetS induced by raised ALT. Otherwise, the random-effect model was used. The calculated RR was 1.81 (95% confidence interval [CI]: 1.49–2.14) when the incidence of MetS was compared between the highest versus the lowest classification of ALT activities. The pooled RR was 1.13 (95% CI: 1.11–1.16) in dose-response analysis with 5 units per liter (U/l) of ALT increment. Subgroup analysis suggested that gender disparity might be the main origin of heterogeneity in overall analysis (P = 0.007 between RRs of gender-specific subgroups evaluated with 5 U/l increments of ALT). Women had a higher dose-response risk of MetS incidence (1.38, 95% CI: 1.20–1.55) than men. Furthermore, sensitivity analysis confirmed the stability of results. No publication bias was found in our meta-analysis.

Conclusions/Significance

Current evidence from prospective studies supports the association between ALT elevation and increasing MetS incidence. This association is closer and more consistent in female population. Further studies are needed to confirm this association and to investigate the potential mechanism of ALT activity on MetS occurrence.     

Diurnal Rhythm in Rat Liver Serine: Pyruvate Aminotransferase Activity

Abstract

In male rats, hepatic serine: pyruvate aminotransferase shows a diurnal rhythm of activity. The periodicity differs from those of other rate‐limiting enzymes in the amino acid catabolism by a biphasic pattern. Fasting did not prevent the diurnal increase in enzyme activity.     

Prokaryotic-Type Aspartate Aminotransferase in Sorghum Leaves: Localization,Distribution and Potential Role

Russian Journal of Plant Physiology - The first part of this work consists in the identification of a prokaryotic-type bifunctional enzyme (aspartate aminotransferase, PT-AAT) in photosynthetic...     

Subcellular Localization of Asparaginase and Asparagine Aminotransferase in Pisum sativum Leaves

Protoplasts isolated from young and mature pea leaves (Pisum sativum L.) were broken and their contents fractionated by differential centrifugation or on sucrose-density gradients. Asparaginase was found only in the cytosol of young leaves. Asparagine aminotransferase was found in both young and mature leaves and was localized exclusively in the peroxisome. This corroborates the observation that asparagine transamination is catalyzed by the serine:glyoxylate aminotransferase.     

Diurnal Changes in Asparaginase Activity in Pea Leaves: I. THE REQUIREMENT FOR LIGHT FOR INCREASED ACTIVITY

During the growth of leaves of Pisum sativum L., levels of asparaginase(E.C. 3.5.1.1 [EC] ) showed a diurnal variation during a 3 d periodof leaf expansion, increasing in the light and decreasing inthe dark period; the greatest diurnal variation being foundin half-expanded leaves. Asparaginase activity in half-expandedleaves reached a maximum after 4 h exposure to light and thisactivity was maintained over the rest of the light period. Changesin asparaginase activity were not influenced by diurnal temperaturechanges. The increase in asparaginase activity during the lightperiod was directly proportional to the photon flux densityover the range 0–285 µmol m^-2 s^-1 PAR. The increaseof asparaginase activity during illumination of detached leaveswas inhibited by the photosynthetic electron transport inhibitors3-(3', 4'-dichlorophenyl)-1, 1-dimethylurea (DCMU) and atrazine.These observations indicate that the increase in asparaginaseactivity in half-expanded leaves is dependent upon non-cyclicelectron transport. Key words: Pisum sativum, asparaginase, photosynthetic electron transport