The influence of short-term <Emphasis Type="SmallCaps">l</Emphasis>-arginine supplementation on rats’ muscular and hepatic cells in ischemia–reperfusion syndrome

Due to the complex mechanisms of l-arginine activity, it is difficult to determine the clinical significance of supplementation with this amino acid. The objective of this study was to determine the influence of short-term supplementation with l-arginine in stress conditions, induced by ischemia–reperfusion syndrome, by assessing the damage to muscular and hepatic cells on the basis of creatine kinase (CK), alanine aminotransferase (ALAT) and aspartic aminotransferase (AspAT) activity in blood and the level of oxygen free radicals in analyzed tissues of rats. We observed that induced ischemia of hind limb caused an increase in CK, ALAT and AspAT activity and an increase in the level of free radicals in liver, but not in skeletal muscle. Supplementation with l-arginine led to a reduction in serum activity of CK and AspAT and reduction of the level of free radicals in analysed tissues. Simultaneous supplementation with l-arginine AND l-NAME resulted in a reversal of changes induced by l-arginine supplementation in the case of AspAT and free radicals in skeletal muscle. The results indicate that under conditions of ischemia–reperfusion, short-term administration of l-arginine has a protective effect on skeletal muscle manifesting itself by reduction of CK in the serum and reduction of free radicals level in THIS tissue.     

Effect of LED light spectra on starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus

The present study aimed to test starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus illuminated by light-emitting diodes (LEDs): red (peak at 630nm), green (peak at 530nm), and blue (peak at 450nm) within a visible light. We investigated the oxidative stress induced by starvation for 12days during illumination with 3 LED light spectra through measuring antioxidant enzyme (superoxide dismutase [SOD] and catalase [CAT]) mRNA expression and activity; CAT western blotting; and measuring lipid peroxidation [LPO]), plasma H(2)O(2), lysozyme, glucose, alanine aminotransferase (AlaAT), aspartate aminotransferase (AspAT), and melatonin levels. In green and blue lights, expression and activity of antioxidant enzyme mRNA were significantly lower than those of other light spectra, results that are in agreement with CAT protein expression level by western blot analysis. Also, in green and blue lights, plasma H(2)O(2), lysozyme, glucose, AlaAT, AspAT, and melatonin levels were significantly lower than those in other light spectra. These results indicate that green and blue LEDs inhibit oxidative stress and enhance immune function in starved cinnamon clownfish.     

The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments

Here, a complete study is described of all the genes and isoenzymes for aspartate aminotransferase (AspAT) present in Arabidopsis thaliana . Four classes of cDNAs representing four distinct AspAT genes ( ASP1—ASP4 ) have been cloned from Arabidopsis . Sequence analysis of the cDNAs suggests that the encoded proteins are targeted to different subcellular compartments. ASP1 encodes a mitochondrial form of AspAT, ASP3 encodes a chloroplastic/plastidic form of AspAT, whereas ASP2 and ASP4 each encode cytosolic forms of AspAT. Three distinct AspAT holoenzymes (AAT1—AAT3) were resolved by activity gel analysis. Organelle isolation reveals that AAT1 is mitochondrial-localized, AAT3 is plastid-localized, and AAT2 is cytosolic. Gene-specific Northern analysis reveals that each Asp mRNA accumulates differentially with respect to organ-type. However, the individual Asp mRNAs show no dramatic fluctuations in response to environmental stimuli such as light. Southern analysis reveals that four distinct nuclear genes probably represent the entire AspAT gene family in Arabidopsis . These molecular studies shed light on the subcellular synthesis of aspartate in Arabidopsis and suggest that some of the AspAT isoenzymes may play overlapping roles in plant nitrogen metabolism.     

Characterization of amino acid aminotransferases of Methanococcus aeolicus.

Four aminotransferases were identified and characterized from Methanococcus aeolicus. Branched-chain aminotransferase (BcAT, EC 2.6.1.42), aspartate aminotransferase (AspAT, EC 2.6.1.1), and two aromatic aminotransferases (EC 2.6.1.57) were partially purified 175-, 84-, 600-, and 30-fold, respectively. The apparent molecular weight, substrate specificity, and kinetic properties of the BcAT were similar to those of other microbial BcATs. The AspAT had an apparent molecular weight of 162,000, which was unusually high. It had also a broad substrate specificity, which included activity towards alanine, a property which resembled the enzyme from Sulfolobus solfataricus. An additional alanine aminotransferase was not found in M. aeolicus, and this activity of AspAT could be physiologically significant. The apparent molecular weights of the aromatic aminotransferases (ArAT-I and ArAT-II) were 150,000 and 90,000, respectively. The methanococcal ArATs also had different pIs and kinetic constants. ArAT-I may be the major ArAT in methanococci. High concentrations of 2-ketoglutarate strongly inhibited valine, isoleucine, and alanine transaminations but were less inhibitory for leucine and aspartate transaminations. Aromatic amino acid transaminations were not inhibited by 2-ketoglutarate. 2-Ketoglutarate may play an important role in the regulation of amino acid biosynthesis in methanococci.     

Chromosomal localization of human aspartate aminotransferase genes by in situ hybridization

Summary The localization of the human genes for cytosolic and mitochondrial aspartate aminotransferase (AspAT) has been determined by chromosomal in situ hybridization with specific human cDNA probes previously characterized in our laboratory. The cytosolic AspAT gene is localized on chromosome 10 at the interface of bands q241–q251. Mitochondrial AspAT is characterized by a multigene family located on chromosomes 12 (p131–p132), 16 (q21), and 1 (p32–p33 and q25–q31). Genomic DNA from ten blood donors was digested by ten restriction enzymes, and Southern blots were hybridized with the two specific probes. Restriction fragment length polymorphism was revealed in only one case for cytosolic AspAT, with PvuII, while no polymorphism for mitochondrial AspAT was found.     

A relationship between heat load and plasma enzyme concentration

1. 1. The sensitivity of serum enzyme levels as indicators of tissue damage is less well established in the prodromal period of heatstroke, especially for sub-lethal stress conditions.

2. 2. Anaesthetized rats were exposed to two different sets of thermal conditions.

3. 3. Plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LD) were assayed in each group upon termination of stress, 6 h post-stress and 24 h post-stress.

4. 4. The tissue “damage” sustained was mild to moderate and completely reversible.

5. 5. The rate of rise in body temperature may constitute an important factor in the ultimate pathology.

6. 6. CK proved to be the most sensitive parameter of tissue “damage”.

Enzymatic synthesis of L-tert-leucine with branched chain aminotransferase

In this study, we demonstrated the asymmetric synthesis of L-tert-leucine from trimethylpyruvate using branchedchain aminotransferase (BCAT) from Escherichia coli in the presence of L-glutamate as an amino donor. Since BCAT was severely inhibited by 2-ketoglutarate, in order to overcome this here, we developed a BCAT/aspartate aminotransferase (AspAT) and BCAT/AspAT/pyruvate decarboxylase (PDC) coupling reaction. In the BCAT/ AspAT/PDC coupling reaction, 89.2 mM L-tert-leucine (ee >99%) was asymmetrically synthesized from 100 mM trimethylpyruvate.     

Activity of antioxidant enzymes and physiological responses in ark shell,Scapharca broughtonii,exposed to thermal and osmotic stress: Effects on hemolymph and biochemical parameters

Changes in water temperature and salinity are responsible for a variety of physiological stress responses in aquatic organisms. Stress induced by these factors was recently associated with enhanced reactive oxygen species (ROS) generation, which caused oxidative damage. In the present study, we investigated the time-related effects of changes in water temperature and salinity on mRNA expression and the activities of antioxidant enzymes (SOD and CAT) and lipid peroxidation (LPO) in the gills and digestive glands of the ark shell, Scapharca broughtonii. To investigate physiological responses, hydrogen peroxide (H₂O₂), lysozyme activity, aspartate aminotransferase (AspAT), and alanine aminotransferase (AlaAT) were measured in the hemolymph. Water temperature and salinity changes significantly increased antioxidant enzyme mRNA expression and activity in the digestive glands and gills in a time-dependent manner. H₂O₂ concentrations increased significantly in the high-temperature and hyposalinity treatments. LPO, AspAT and AlaAT levels also increased significantly in a time-dependent manner, while lysozyme activity decreased. These results suggest that antioxidant enzymes play important roles in reducing oxidative stress in ark shells exposed to changes in water temperature and salinity.     

Diurnal and Seasonal Variations of Serum Enzyme Activity in Cattle and Sheep

In order to test the variation of enzyme activity in serum of cattle and sheep during the day, blood samples were taken at three hrs. interval from 6 a.m. to 9 p.m. The following enzymes were assayed: Aspartate aminotransferase (AspAT = GOT), alanine aminotransferase (AlAT = GPT), total lactate dehydrogenase (LDH), and a-hydroxybutyrate dehydrogenase (HBD). The variation between animals contributed by far to the greatest part of the total variation in clinical healthy animals. The time-of-day-dependant variation was less than 3 %, except for alanine aminotransferase. During the first two weeks of spring pasture serum aspartate and alanine aminotransferase levels were significantly raised in both cows and ewes, compared with serum levels of the same animals on indoor feeding. No such increase occurred in total lactate dehydrogenase.     

Nickel-induced depression of nitrogen assimilation in wheat roots

The influence of 50 and 100 μM Ni on the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) was studied in the wheat roots. Root fresh weight, tissue Ni, nitrate, ammonium, glutamate and protein concentrations were also determined. Exposure to Ni resulted in a marked reduction in fresh weight of the roots accompanied by a rapid accumulation of Ni in these organs. Both nitrate and ammonium contents in the root tissue were considerably enhanced by Ni stress. While protein content was not significantly influenced by Ni application, glutamate concentration was slightly reduced on the first day after treatment with the higher Ni dose. Treatment of the wheat seedlings with 100 μM Ni led to a decrease in NR activity; however, it did not alter the activation state of this enzyme. Decline in NiR activity observed after application of 100 μM Ni was more pronounced than that in NR. The activities of GS and NADH-GOGAT also showed substantial decreases in response to Ni stress with the latter being more susceptible to this metal. Starting from the fourth day, both aminating and deaminating GDH activities in the roots of the seedlings supplemented with Ni were lower in comparison to the control. While the activity of AspAT remained unaltered after Ni application that of AlaAT showed a considerable enhancement. The results indicate that exposure of the wheat seedlings to Ni resulted in a general depression of nitrogen assimilation in the roots. Increase in the glutamate-producing activity of AlaAT may suggest its involvement in supplying the wheat roots with this amino acid under Ni stress.     

AAT1, a gene encoding a mitochondrial aspartate aminotransferase in Saccharomyces cerevisiae.

We have isolated a gene, AAT1, encoding an aspartate aminotransferase (AspAT) from a Saccharomyces cerevisiae genomic library. AAT1 encodes a 451 amino acid protein with a predicted molecular weight of 51,687, which is likely to be the yeast mitochondrial AspAT. Sequence comparison of this yeast AspAT with AspATs from other organisms shows a high degree of homology in regions previously shown to be important for catalysis. However, the yeast mitochondrial AspAT contains four obvious insertions with respect to all other known AspATs, suggesting that the AAT1-encoded protein represents a distinct AspAT.     

Tyr225 in aspartate aminotransferase: contribution of the hydrogen bond between Tyr225 and coenzyme to the catalytic reaction

Tyr225 in the active site of Escherichia coli aspartate aminotransferase (AspAT) was replaced by phenylalanine or arginine by site-directed mutagenesis. X-ray crystallographic analysis of Y225F AspAT showed that the benzene ring of Phe225 was situated at the same position as the phenol ring of Tyr225 in wild-type AspAT. The mutations resulted in a great decrease in the rate of the transamination reaction, suggesting that Tyr225 is important for efficient catalysis. The kinetic analysis of half-transamination reactions of Y225F AspAT with four substrates (aspartate, glutamate, oxalacetate, and 2-oxoglutarate) and some analogues (2-methylaspartate, succinate, and glutarate) revealed a considerable increase in the affinities for all these compounds. In contrast, affinity for the amino acid substrates was decreased by mutation to arginine, but affinities for the keto acid substrates and the two dicarboxylates (succinate and glutarate) were increased. The electrostatic interaction between O(3') of the coenzyme [pyridoxal 5'-phosphate (PLP)] and the residue at position 225 affected the pKa value of the Schiff base, which is formed between the epsilon-amino group of Lys258 and the aldehyde group of PLP; based on the spectrophotometric titration the pKa values were determined to be 6.8 for wild-type AspAT, 8.5 for Y225F AspAT, and 6.1 for Y225R AspAT in the absence of substrate. The absorption spectra of the three AspATs were almost identical in the acidic pH region, but the spectrum of Y225F AspAT differed from that of wild-type or Y225R AspAT in the alkaline pH region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Physical Stress on Serum Enzymes and Other Blood Constituents in Sheep

In connection with transfer of sheep from the lowland near Oslo to mountain pastures at an altitude of 1,200 m above sea level, investigations were carried out in 37 animals to study the effect of physical stress on serum enzymes and other blood constituents. The sheep were adult ewes and lambs. About half of the animals had been accustomed to outdoor life on pasture for more than one month, while the others were moved directly from indoor feeding. Blood was collected before departure, after six hrs. of long-distance transportation by lorry, and after three hrs. of subsequent continuous herding on foot. The following blood components were determined: Aspartate aminotransferase (AspAT = GOT), alanine aminotransferase (A1AT = GPT), α-hydroxybutyrate dehydrogenase (HBD), total lactate dehydrogenase (LDH), LDH isoenzymes, alkaline phosphatase, calcium, inorganic phosphorus, magnesium, blood sugar, total serum proteins, and haemoglobin. In summary, it may be said that the lambs reacted with greater changes of the blood components than adult animals, and that untrained, indoor fed lambs were distinctly more sensitive than those taken from pasture. The “indoor” lambs showed a statistical significant increase from the initial values in AspAT, HBD, total LDH, the isoenzymes LDH3 and LDH4, and blood sugar. Significantly decreased values were recorded in Ga, P, Mg, and total serum protein. Some of these changes, as in Mg and P, were most pronounced after transportation, while elevations of serum enzyme levels continued to increase during the subsequent herding. Based upon the shift in LDH isoenzyme distribution towards a more cathodically dominated pattern it is supposed that the main origin of increased serum enzyme activity was skeletal muscle.     

Hepatic accumulation and effects of microcystin-LR on juvenile goldfish Carassius auratus L

After intraperitoneal injection of microcystin-LR (MC-LR) (125 microg kg(-1) body wt.), the concentration of MC-LR in the liver of juvenile goldfish Carassius auratus (30 g body wt.) was assayed by a modified protein phosphatase inhibition method. A temporary accumulation occurred from 3 to 48 h post-injection, followed by a significant decrease between 48 and 96 h. Under our experimental conditions, contamination by MC-LR did not change ionic homeostasis, as attested by blood osmolality values and gill Na(+)/K(+) ATPase activity. Light microscopy observations revealed lesions and cellular necrosis progression, which was concomitant with an increase in enzyme activity of plasma aspartate aminotransferase (AspAT), alanine aminotransferase (AlaAT) and L-lactate dehydrogenase (LDH) and with a decrease of hepatic glutathione-S-transferase (GST) activity. Structural alterations and enzymatic activity modifications became significant within 24 h post-injection. Recovery of hepatocytes on day 21 after MC-LR injection was evident, together with a decrease in the MC-LR equivalent content of the liver.     

Effect of pyridoxine-deficiency on the turnover of aspartate aminotransferase isozymes in rat liver

DL-[14C]Leucine or L-[3H]leucine was injected intraperitoneally into pyridoxine-deficient and control rats, and the subsequent incorporation of the radioactivities into aspartate aminotransferase (AspAT) isozymes and the total soluble protein in the liver was measured. AspAT in the cytosol (AspATc) was separated into 3 subfractions with different characteristics on chromatofocusing. The results showed that in the liver of pyridoxine-deficient rats, the syntheses of all 3 subfractions of AspATc and degradation of AspATc (total) were increased, but that the syntheses and degradation of the total soluble protein and mitochondrial AspAT (AspATm) were not much different from those in control rats. The half-lives of soluble protein and AspATm were calculated to be 3.26-3.72 and 5.02-6.67 days, respectively, in both groups, and that of AspATc in control liver was found to be 4.78 days. The rate of degradation of AspATc in pyridoxine-deficient rat liver could not be calculated, because its kinetics were very complicated; there were apparently at least 2 components with different rates of degradation. Thus pyridoxal 5'-phosphate (PLP) apparently affects both the synthesis and degradation of AspATc, but does not affect the turnover of AspATm in rat liver.     

Characterization of histidinol phosphate aminotransferase from Escherichia coli

Histidinol phosphate aminotransferase (HPAT) is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase classified into Subgroup I aminotransferase, in which aspartate aminotransferase (AspAT) is the prototype. In order to expand our knowledge on the reaction mechanism of Subgroup I aminotransferases, HPAT is an enzyme suitable for detailed mechanistic studies because of having low sequence identity with AspAT and a unique substrate recognition mode. Here we investigated the spectroscopic properties of HPAT and the effect of the C4-C4' strain of the PLP-Lys(214) Schiff base on regulating the Schiff base pK(a) in HPAT. Similar to AspAT, the PLP-form HPAT showed pH-dependent absorption spectral change with maxima at 340 nm at high pH and 420 nm at low pH, having a low pK(a) of 6.6. The pK(a) value of the methylamine-reconstituted K214A mutant enzyme was increased from 6.6 to 10.6. Mutation of Asn(157) to Ala increased the pK(a) to 9.2. Replacement of Arg(335) by Leu increased the pK(a) to 8.6. On the other hand, the pK(a) value of the N157A/R335L double mutant enzyme was 10.6. These data indicate that the strain of the Schiff base is the principal factor to decrease the pK(a) in HPAT and is crucial for the subsequent increase in the Schiff base pK(a) during catalysis, although the electrostatic effect of the arginine residue that binds the negatively charged group of the substrate is larger in HPAT than that in AspAT. Our findings also support the idea that the strain mechanism is common to Subgroup I aminotransferases.     

Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliaceum, an NAD-malic-enzyme-type C4 plant and screened the library for AspAT isozymes. A full-length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondrial AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAT are synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28-amino-acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P. miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.     

The effect of long-term enrichment of diet with selenium, vitamin E and B15 on the activity of certain enzymes in rat brain

The aim of this study was tracing of changes in the activity of glutathione peroxidase (GSHPx), glutathione transferase (GSH S-Tr), aspartate aminotransferase (AspAT) and alanine aminotransferase (A1AT) in the brain as a result of diet enrichment with antioxidants: selenium (Se), vitamin E and vitamin B15 (pangamic acid). The experiment was carried out on Wistar rats with initial body weight 150 g. Following prolonged enrichment of diet with Se (0.1 ppm of sodium selenite), vitamin E (6 mg/100 g of diet) and vitamin B15 (2.5 mg/100 g of diet) the following results were obtained. The activity of GSHPx in brain microsomes was not changed after one year of vitamin E administration when it was measured against hydrogen hydroxide and against cumene hydrochloride; vitamin E administration increased the activity of GSH S-Tr in the cytoplasmic fraction of brain cells. Diet enrichment with selenium increased after 12 and 18 months the activity of GSHPx measured against both substrates, and GSH S-Tr activity increased considerably. Presence of vitamin B15 in diet reduced GSHPx activity after one-year or longer administration, after 18 months the activity of GSH S-Tr was reduced also. No changes were noted in the activity of AspAT and A1AT.     

Molecular analysis of the role of two aromatic aminotransferases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism of Pyrococcus furiosus

The genes encoding aromatic aminotransferase II (AroAT II) and aspartate aminotransferase (AspAT) from Pyrococcus furiosus have been identified, expressed in Escherichia coli and the recombinant proteins characterized. The AroAT II enzyme was specific for the transamination reaction of the aromatic amino acids, and uses a-ketoglutarate as the amino acceptor. Like the previously characterized AroAT I, AroAT II has highest efficiency for phenylalanine (k(cat)/Km = 923 s(-1) mM(-1)). Northern blot analyses revealed that AroAT I was mainly expressed when tryptone was the primary carbon and energy source. Although the expression was significantly lower, a similar trend was observed for AroAT II. These observations suggest that both AroATs are involved in amino acid degradation. Although AspAT exhibited highest activity with aspartate and alpha-ketoglutarate (k(cat) approximately 105 s(-1)), it also showed significant activity with alanine, glutamate and the aromatic amino acids. With aspartate as the amino donor, AspAT catalyzed the amination of alpha-ketoglutarate, pyruvate and phenyl-pyruvate. No activity was detected with either branched-chain amino acids or alpha-keto acids. The AspAT gene (aspC) was expressed as a polycistronic message as part of the aro operon, with expression observed only when the aromatic amino acids were absent from the growth medium, indicating a role in the biosynthesis of the aromatic amino acids.