The FibroScan-aspartate aminotransferase score can stratify the disease severity in a Japanese cohort with fatty liver diseases.

This study aimed to prove that the FibroScan-aspartate aminotransferase (FAST) scores can be used to stratify disease severity in a Japanese cohort with fatty liver diseases [metabolic dysfunction-associated fatty liver disease (MAFLD) and nonalcoholic fatty liver disease (NAFLD)]. All the participants (n = 2254) underwent liver stiffness measurements and controlled attenuation parameter assessments. We compared the clinical characteristics of the patients with MAFLD and NAFLD using the FAST scores and explored the independent determinants of FAST scores ≥ 0.35, which indicated possible progressive disease. Overall, MAFLD was diagnosed in 789 patients (35.0%), while NAFLD was diagnosed in 618 (27.4%). The proportion of patients that had a condition that suggested progressive liver disease was higher in those with MAFLD than in those with NAFLD [68 (8.6%) vs 48 (7.7%)]. The area under the receiver-operating characteristic curve of the FAST score for diagnosing advanced fibrosis was 0.969 in MAFLD and 0.965 in NAFLD. Multivariate analyses determined that diabetes mellitus, alanine aminotransferase (ALT) levels, fatty liver index, and Fibrosis-4 index independently predict FAST scores ≥ 0.35 in patients with MAFLD. ALT levels had the strongest correlation with the FAST scores (p = 0.7817). The FAST score could stratify the disease severity in the Japanese cohort with fatty liver diseases.

A quantitative electrochemical assay for liver injury.

Liver diseases represent a vastly underestimated and historically neglected public health problem, disproportionately affecting those in low- and middle- income countries (LMICs). Patients on hepatotoxic medications, such as HIV and TB medications, need consistent monitoring of liver function as part of their standard of care. In high resource settings, this is often the case, but in LMICs traditional methods fail due to high cost and lack of proper equipment, supplies and trained personnel. To address this gap in technology and patient care, we have developed a quantitative, electrochemical assay capable of quantifying levels of alanine aminotransferase (ALT), a primary biomarker associated with liver function. We can quantify ALT with increased sensitivity (1.53 nA/(U/L*mm2) and over a wide, linear concentration range (40-1990 U/L). The assay demonstrated in this study can be used to overcome several pressing challenges associated with effective, timely treatment of liver disease in LMICs.

A novel C-S lyase from the latex-producing plant Taraxacum brevicorniculatum displays alanine aminotransferase and l-cystine lyase activity.

We isolated a novel pyridoxal-5-phosphate-dependent l-cystine lyase from the dandelion Taraxacum brevicorniculatum. Real time qPCR analysis showed that C-S lyase from Taraxacum brevicorniculatum (TbCSL) mRNA is expressed in all plant tissues, although at relatively low levels in the latex and pedicel. The 1251 bp TbCSL cDNA encodes a protein with a calculated molecular mass of 46,127 kDa. It is homologous to tyrosine and alanine aminotransferases (AlaATs) as well as to an Arabidopsis thaliana carbon-sulfur lyase (C-S lyase) (SUR1), which has a role in glucosinolate metabolism. TbCSL displayed in vitrol-cystine lyase and AlaAT activities of 4 and 19nkatmg(-1) protein, respectively. However, we detected no in vitro tyrosine aminotransferase (TyrAT) activity and RNAi knockdown of the enzyme had no effect on phenotype, showing that TbCSL substrates might be channeled into redundant pathways. TbCSL is in vivo localized in the cytosol and functions as a C-S lyase or an aminotransferase in planta, but the purified enzyme converts at least two substrates specifically, and can thus be utilized for further in vitro applications.

Biochemical characterization and kinetic properties of alanine aminotransferase homologues partially purified from wheat (Triticum aestivum L.).

Four homologues of alanine aminotransferase have been isolated from shoots of wheat seedlings and purified by saline precipitation, gel filtration, preparative electrophoresis and anion exchange chromatography on Protein-Pak Q 8HR column attached to HPLC. Alanine aminotransferase 1 (AlaAT1) and 2 (AlaAT2) were purified 303- and 452-fold, respectively, whereas l-glutamate: glyoxylate aminotransferase 1 (GGAT1) and 2 (GGAT2) were purified 485- and 440-fold, respectively. Consistent inhibition of AlaAT (EC 2.6.1.2) and GGAT (EC 2.6.1.4) activities by p-hydroxymercuribenzoate points on participation of cysteine residues in the enzyme activity. The molecular weight of AlaAT1 and AlaAT2 was estimated to be 65kDa and both of them are monomers in native state. Nonsignificant differences between K(m) using alanine as substrate and catalytic efficiency (k(cat)/K(m)) for l-alanine in reaction with 2-oxoglutarate indicate comparable kinetic constants for AlaAT1 and AlaAT2. Similar kinetic constants for l-alanine in reaction with 2-oxoglutarate and for l-glutamate in reaction with pyruvate for all four homologues suggest equally efficient reaction in both forward and reverse directions. GGAT1 and GGAT2 were able to catalyze transamination between l-glutamate and glyoxylate, l-alanine and glyoxylate and reverse reactions between glycine and 2-oxoglutarate or pyruvate. Both GGATs also consisted of a single subunit with molecular weight of about 50kDa. The estimated K(m) for GGAT1 (3.22M) and GGAT2 (1.27M) using l-glutamate as substrate was lower in transamination with glyoxylate than with pyruvate (9.52 and 9.09mM, respectively). Moreover, distinctively higher values of catalytic efficiency for l-glutamate in reaction with glyoxylate than for l-glutamate in reaction with pyruvate confirm involvement of these homologues into photorespiratory metabolism.

The enzymology of alanine aminotransferase (AlaAT) isoforms from Hordeum vulgare and other organisms, and the HvAlaAT crystal structure.

In this paper we describe the expression, purification, kinetics and biophysical characterization of alanine aminotransferase (AlaAT) from the barley plant (Hordeum vulgare). This dimeric PLP-dependent enzyme is a pivotal element of several key metabolic pathways from nitrogen assimilation to carbon metabolism, and its introduction into transgenic plants results in increased yield. The enzyme exhibits a bi-bi ping-pong reaction mechanism with a K(m) for alanine, 2-oxoglutarate, glutamate and pyruvate of 3.8, 0.3, 0.8 and 0.2 mM, respectively. Barley AlaAT catalyzes the forward (alanine-forming) reaction with a k(cat) of 25.6 s(-1), the reverse (glutamate-forming) reaction with k(cat) of 12.1 s(-1) and an equilibrium constant of ~0.5. The enzyme is also able to utilize aspartate and oxaloacetate with ~10% efficiency as compared to the native substrates, which makes it much more specific than related bacterial/archaeal enzymes (that also have lower K(m) values). We have crystallized barley AlaAT in complex with PLP and l-cycloserine and solved the structure of this complex at 2.7 Å resolution. This is the first example of a plant AlaAT structure, and it reveals a canonical aminotransferase fold similar to structures of the Thermotoga maritima, Pyrococcus furiosus, and human enzymes. This structure bridges our structural understanding of AlaAT mechanism between three kingdoms of life and allows us to shed some light on the specifics of the catalysis performed by these proteins.

A blue native polyacrylamide gel electrophoretic technology to probe the functional proteomics mediating nitrogen homeostasis in Pseudomonas fluorescens.

As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This electrophoretic technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.

[Expression of recombinant human alanine aminotransferase (ALT) isoenzyme 2 and preparation of anti-ALT2 monoclonal antibody].

AIM: To clone and express human alanine aminotransferase 2 (ALT2) in E.coli Rosetta (DE3), and to prepare monoclonal antibodies(mAb) against ALT2 for diagnostic purpose. METHODS: The gene encoding alanine aminotransferase 2 (ALT2) was cloned from hepatoma carcinoma cell by RT-PCR, and then inserted into pET28a vector. Recombination plasmids (pET28a-ALT2) were transformed into E.coli BL21. Human ALT2 was expressed as His-tagged fusion proteins and purified by immobilized Ni(2+);-affinity chromatography. The purified fusion ALT2 protein was used as an antigen to prepare mAb against it. RESULTS: The fusion ALT2 protein was expressed in recombinant E.coli Rosetta (DE3). The enzymatic activity of purified His-tag ALT2 is over 10 000 U/L. Mice were immunized with the purified fusion ALT2 protein, and 5 mAbs against ALT2 were generated. CONCLUSION: Two mAbs with high specificity for ALT2 were selected for further quantitative diagnostic reagent development.

Purification of three aminotransferases from Hydrogenobacter thermophilus TK-6--novel types of alanine or glycine aminotransferase: enzymes and catalysis.

Aminotransferases catalyse synthetic and degradative reactions of amino acids, and serve as a key linkage between central carbon and nitrogen metabolism in most organisms. In this study, three aminotransferases (AT1, AT2 and AT3) were purified and characterized from Hydrogenobacter thermophilus, a hydrogen-oxidizing chemolithoautotrophic bacterium, which has been reported to possess unique features in its carbon and nitrogen anabolism. AT1, AT2 and AT3 exhibited glutamate:oxaloacetate aminotransferase, glutamate:pyruvate aminotransferase and alanine:glyoxylate aminotransferase activities, respectively. In addition, both AT1 and AT2 catalysed a glutamate:glyoxylate aminotransferase reaction. Interestingly, phylogenetic analysis showed that AT2 belongs to aminotransferase family IV, whereas known glutamate:pyruvate aminotransferases and glutamate:glyoxylate aminotransferases are members of family Igamma. In contrast, AT3 was classified into family I, distant from eukaryotic alanine:glyoxylate aminotransferases which belong to family IV. Although Thermococcus litoralis alanine:glyoxylate aminotransferase is the sole known example of family I alanine:glyoxylate aminotransferases, it is indicated that this alanine:glyoxylate aminotransferase and AT3 are derived from distinct lineages within family I, because neither high sequence similarity nor putative substrate-binding residues are shared by these two enzymes. To our knowledge, this study is the first report of the primary structure of bacterial glutamate:glyoxylate aminotransferase and alanine:glyoxylate aminotransferase, and demonstrates the presence of novel types of aminotransferase phylogenetically distinct from known eukaryotic and archaeal isozymes.

Seroprevalence of hepatitis delta virus infection among subjects with underlying hepatic diseases in Chennai, southern India.

Four hundred million people are carriers of hepatitis B virus (HBV) worldwide and approximately 5% of these are reportedly positive for hepatitis delta virus (HDV). Several reports indicate a declining trend in the occurrence of HDV infection in the north of tropical India. To our knowledge, no study has been conducted to evaluate whether a similar epidemiological change is occurring in southern India. Therefore we evaluated the seroprevalence of HDV among 153 individuals with HBV-related liver diseases in Chennai, and assessed any change in epidemiological pattern by comparing the results with seroprevalence figures reported previously. Of the 153 patients screened, nine (5.9%) were reactive to anti-delta antibodies, six (3.9%) presented an evidence of past infection (IgG anti-delta positive) and three (2.0%) showed anti-HDV IgM, suggestive of recent HDV infection. Alanine transaminase elevation was not significant in HDV-associated infection compared with HBV alone-infected acute viral hepatitis (AVH) (P=0.82) and chronic liver disease (P=0.77) patients. The anti-HDV positivity in AVH was considerably low (6.6%), compared with previous Indian reports varying from 10.7% to >30%. HDV infection was relatively low and seems to play a minor determining factor of liver diseases in the tropical south Indian population.

Expression, purification, and initial characterization of human alanine aminotransferase (ALT) isoenzyme 1 and 2 in High-five insect cells.

Alanine aminotransferase (ALT) is a key enzyme for gluconeogenesis as well as a widely used serum marker for liver injury. We have identified two ALT isoenzymes, ALT1 and ALT2, which are encoded by separate genes. In this study, we described the expression, purification and initial characterization of human ALT1 and ALT2 proteins in High-five insect cells. Human ALT1 and ALT2 were expressed as His-tagged fusion proteins by recombinant baculovirus in insect cells and purified into homogeneity in one step by using immobilized Ni2+-affinity chromatography. Tag-free ALT1 and ALT2 were obtained by cleavage of enterokinase digestion and used for initial characterization of the enzymes. The specific ALT activity of purified fusion or His-tag-removed ALT1 was about 15-fold higher than that of ALT2 and their enzymatic activities decreased quickly at 37 degrees C and -20 degrees C, but were well preserved at -80 degrees C. Nevertheless, the ALT1 and ALT2 activities remained stable in a buffer containing 25% glycerol. The pH profile was similar between hALT1 and hALT2 in that both enzymes remained fully active between pH 6.5 and 8.0. The purified ALT recombinant proteins can not only be used as a reference protein standard for the ALT assay in clinical chemistry, but also will be useful for understanding the biochemical and biological significance of the isoenzymes and for developing ALT isoform-specific assays for clinical or preclinical diagnostic use.

A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast.

In humans, beta-alanine (BAL) and the neurotransmitter gamma-aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, and SkPYD4 encodes an enzyme involved in both BAL and GABA transamination. SkPYD4 and SkUGA1 as well as S. cerevisiae UGA1 and Schizosaccharomyces pombe UGA1 were subcloned, over-expressed and purified. One discontinuous and two continuous coupled assays were used to characterize the substrate specificity and kinetic parameters of the four enzymes. It was found that the cofactor pyridoxal 5'-phosphate is needed for enzymatic activity and alpha-ketoglutarate, and not pyruvate, as the amino group acceptor. SkPyd4p preferentially uses BAL as the amino group donor (V(max)/K(m)=0.78 U x mg(-1) x mm(-1)), but can also use GABA (V(max)/K(m)=0.42 U x mg(-1) x mm(-1)), while SkUga1p only uses GABA (V(max)/K(m)=4.01 U x mg(-1) x mm(-1)). SpUga1p and ScUga1p transaminate only GABA and not BAL. While mammals degrade BAL and GABA with only one enzyme, but in different tissues, S. kluyveri and related yeasts have two different genes/enzymes to apparently 'distinguish' between the two reactions in a single cell. It is likely that upon duplication approximately 200 million years ago, a specialized Uga1p evolved into a 'novel' transaminase enzyme with broader substrate specificity.

cDNA cloning, expression, purification, distribution, and characterization of biologically active canine alanine aminotransferase-1.

Alanine aminotransferase (ALT) is a pyridoxal enzyme found mainly in the liver and kidney, but also in small amounts in the heart, muscle, fat, and brain. Serum aminotransferase activities have been used broadly as surrogate markers for tissue injury and disease in human and veterinary clinical settings and in safety assessment of chemicals and pharmaceuticals. Because of its relative abundance in liver, increased serum ALT activity is generally considered indicative of liver damage. Two ALT isoenzymes, ALT1 and ALT2, are known and have been cloned and sequenced from human, rat, and mouse. In this study, we have cloned the complementary DNA encoding the canine orthologue of ALT1 (cALT1). The complete cDNA sequence comprised 1852 bases and contained a 1485-base open reading frame, which encodes a polypeptide of 494 amino acid residues. Canine ALT1 shares 87.7, 87.2, and 87.0% amino acid identity to its human, mouse, and rat orthologues, respectively. The cDNA was expressed in Escherichia coli, with a N-terminal His (6x) tag, and the recombinant enzyme was purified using immobilized metal-affinity chromatography. The final yield of the purified recombinant cALT1 was greater than 5mg/L culture. The alanine transaminase activity of purified cALT1 was 229.81U/mg protein, which is approximately 38-fold higher than that of total soluble recombinant E. coli cell lysate, confirming that the enzyme is a functional ALT. Evaluation of various canine tissues by RT-PCR revealed that the level of ALT1 expression is in the order of: heart>liver>fat approximately brain approximately gastrocnemius>kidney. The purified cALT1 will be helpful to develop isoenzyme-specific anti-bodies, which could further improve the diagnostic resolution of current ALT assays in drug safety studies.

A novel low molecular weight alanine aminotransferase from fasted rat liver.

Alanine is the most effective precursor for gluconeogenesis among amino acids, and the initial reaction is catalyzed by alanine aminotransferase (AlaAT). Although the enzyme activity increases during fasting, this effect has not been studied extensively. The present study describes the purification and characterization of an isoform of AlaAT from rat liver under fasting. The molecular mass of the enzyme is 17.7 kD with an isoelectric point of 4.2; glutamine is the N-terminal residue. The enzyme showed narrow substrate specificity for L-alanine with Km values for alanine of 0.51 mM and for 2-oxoglutarate of 0.12 mM. The enzyme is a glycoprotein. Spectroscopic and inhibition studies showed that pyridoxal phosphate (PLP) and free -SH groups are involved in the enzymatic catalysis. PLP activated the enzyme with a Km of 0.057 mM.

Avaliação hematológica e dosagem bioquímica de ALT, AST e creatinina em elefante-marinho-do-sul, Mirounga leonina (linnaeus, 1758), encontrado no litoral de Salvador, Bahia / Haematological analysis and clinical chemistry of ALT, AST and creatinine of a southern elephant seal, Mirounga leonina (linnaeus, 1758), found on the Salvador coast, Bahia

O Centro de Resgate de Mamíferos Aquáticos (CRMA) tem trabalhado desde 1999 com o resgate e a reabilitação de cetáceos e pinípedes em situação de encalhe no litoral do Estado da Bahia. O presente trabalho objetiva apresentar e avaliar os resultados de hemogramas e dosagens séricas de alanina aminotranferase (ALT), aspartato aminotransferase (AST) e creatinina realizados em um exemplar de elefante-marinho-do-sul, Mirounga leonina (LINNAEUS, 1758), encontrado no dia 11 de fevereiro de 2002 na praia da Barra, litoral de Salvador, BA. Tratava-se de um filhote macho, com 137cm de comprimento e peso aproximado de 49kg, com estado nutricional debilitado, um ferimento no lado direito na altura da escápula. O exemplar permaneceu em acompanhamento clínico por 56 dias, sendo vermifugado com Febendazole, o ferimento suturado e tratado com álcool iodado, Nitrofurazona solução e Tartarato de Ketanserina, simultaneamente com Enrofloxacina 10%, Potenay®, Complexo B e Benerva®.No dia 16 seguinte apresentou um quadro de conjuntivite unilateral direita, sendo tratado até o final de sua estadia com pomada oftálmica à base de Cloridrato de Cloranfenicol. Durante este período colheu-se um total de seis amostras de sangue, sendo três para a realização de hemograma e as demais para dosagem de ALT, AST e creatinina. De acordo com os resultados dos eritrogramas o filhote desenvolveu um quadro de anemia, classificada como microcítica e normocrômica. Com relação aos leucogramas, destacou-se linfopenia, eosinopenia e monocitopenia, possivelmente influenciado pelas condições de estresse e o manejo. Constatou-se nos exames bioquimicos valores diminuídos para a AST e creatinina, porém não representando a existência de um quadro patológico de significância clínica.

Since 1999 the Aquatic Mammals Rescue Center - AMRC has been working in the rescue and rehabilitation of stranded cetaceans and pinnipeds on the coast of Bahia, Brazil. This paper presents and analyses the blood cells count and clinical chernistry of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine of a southern elephant seal, Mirounga leonina (LINNAEUS, 1758), found on February the 11 th at Barra Beach, Salvador, BA. The specimen was an orphan male calf, with 137cm of length and estimated weight of 49kg. It presemed bad nutritional conditions and a shark bite on the right shoulder area. Clinical management was performed for 56 days, anthelrnintic Febendazole was utilized, and the bite was treated with iodined alcohol, Nitrofurazone solution and Kethanserin, simultaneously with Enrofloxacin 10%, Potenay®, Vitamin B Complex and Benerva®. On the 16th the animal presented a right unilateral conjuntivitis, treated with Cloranphenicol oftalmic pomade until the end of its stay in the captive. During this period a total of six blood samples were collected, three for total blood counts and the others for the biochernistry determination of ALT, AST and creatinine. According to the haematological analysis the seal developed an anaemia which was classified as microcytic and normochromic. Lymphopenia, eosinopenia and monocytopenia were also observed, possibly due to its handling and stress conditions. The clinical chemistry presented low values for AST and creatinine, although this did not represent the existence of any pathologic context or disease with clinical significance.

Strenuous physical labor is important as a cause of elevated alanine aminotransferase levels in Japanese patients with chronic hepatitis C viremia.

To clarify the influence of lifestyle habits on the elevated alanine aminotransferase (ALT) levels deterioration of Japanese patients with chronic hepatitis C virus (HCV) viremia, we investigated the effects of smoking, drinking, and physical labor on the disease course of the residents living in a rural area of Kyushu, Japan. The data of patients with chronic HCV viremia and control subjects without HCV infection were analyzed retrospectively from 1986 to 1992 and prospectively from 1993 to 2000. In 2000, a questionnaire was given to 268 HCV-infected patients and 275 control subjects to survey for the lifestyle habits. The data of serial ALT level testing during the observation period was used as a measure of liver damage: 183 HCV patients (68.3%) and 10 control subjects (3.6%) had abnormal ALT levels greater than 35 IU/1 for more than half of their observation period. The percentage of HCV patients with elevated ALT levels significantly increased with the daily consumption of alcohol (p < 0.0001), the length of time spent in strenuous physical labor per day (p = 0.0056), and the number of cigarettes smoked per day (p = 0.0003). A stepwise logistic regression analysis showed male sex (p = 0.003), platelet counts (p < 0.001), strenuous physical labor (p = 0.002), and drinking history (p = 0.007) to be significantly associated with the elevated ALT levels of HCV patients. When strenuous physical labor was done for over 2 h, the probability of elevated ALT levels was increased compared with patients engaging in strenuous physical labor under 2 h (estimated odds ratio = 1.82 [under 2 h], 20.60 [over 2 h]). Interestingly, strenuous physical labor was extracted before alcohol consumption as a significant factor in the elevated ALT levels. Among the control subjects, only the amount of alcohol consumed per day (p = 0.0001) was significantly associated with the elevated levels. These data suggests that strenuous physical labor over a long period of time might be related to elevated ALT levels in patients with chronic HCV viremia as well as drinking.

Purification and properties of cytosolic alanine aminotransferase from the liver of two freshwater fish, Clarias batrachus and Labeo rohita.

Cytosolic alanine aminotransferase (c-AAT) was purified up to 203- and 120-fold, from the liver of two freshwater teleosts Clarias batrachus (air-breathing, carnivorous) and Labeo rohita (water-breathing, herbivorous), respectively. The enzyme from both fish showed similar elution profiles on a DEAE-Sephacel ion exchange column. SDS-PAGE of purified enzymes revealed two subunits of 54 and 56 kDa, in both fish. The apparent Km values for l-alanine were 18.5+/-0.48 and 23.55+/-0.60 mM, whereas for 2-oxoglutarate the Km values were observed to be 0.29+/-0.023 and 0.33+/-0.028 mM for the enzyme from C. batrachus and L. rohita, respectively. With l-alanine as substrate, aminooxyacetic acid was found to act as a competitive inhibitor with KI values of 6.4 x 10(-4) and 3.4 x 10(-4) mM with c-AAT of C. batrachus and L. rohita, respectively. However, when 2-oxoglutarate was used as substrate, aminooxyacetic acid showed uncompetitive inhibition with similar KI values for purified c-AAT from both fish. Temperature and pH profiles of the enzyme did not show any marked differences between the two fish examined. These results suggest that liver c-AAT, isolated from these two fish species adapted to different modes of life, remain unaltered structurally. However, at the kinetic level, liver c-AAT from C. batrachus exhibits significantly higher affinity for the substrate l-alanine and decreased affinity for its metabolic inhibitor, in comparison to that of the enzyme purified from L. rohita. Such functional changes seem to be of physiological significance and also provide preliminary evidence for subtle changes in the enzyme as a mark of metabolic adaptation in the fish to different physiological demands.

Isolation and characterization of cytosolic alanine aminotransferase isoforms from starved rat liver.

Alanine is the most effective precursor for gluconeogenesis among amino acids and the initial reaction is catalyzed by alanine aminotransferases (AlaATs). It is a less extensively studied enzyme under starvation and known to that the enzyme activity increases in liver under starvation. The present study describes the purification and characterization of two isoforms of alanine aminotransferases from starved male rat liver under starvation. The molecular mass of isoforms was found to be 17.7 and 112.2 kDa with isoelectric points of 4.2 and 5.3 respectively for AlaAT I and AlaAT II. Both the enzymes showed narrow substrate specificity for L-alanine with different Km for alanine and 2-oxoglutarate. Both the enzymes were glycoprotein in nature. Inhibition, modification and spectroscopic studies showed that both PLP and free-SH groups are directly involved in the enzymatic catalysis. PLP activated both the enzymes with a Km 0.057 mM and 0.2 mM for AlaAT I and II respectively.

3-Hydroxykynurenine transaminase identity with alanine glyoxylate transaminase. A probable detoxification protein in Aedes aegypti.

This study describes the functional characterization of a specific mosquito transaminase responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). The enzyme was purified from Aedes aegypti larvae by ammonium sulfate fractionation, heat treatment, and various chromatographic techniques, plus non-denaturing electrophoresis. The purified transaminase has a relative molecular mass of 42,500 by SDS-PAGE. N-terminal and internal sequencing of the purified protein and its tryptic fragments resolved a partial N-terminal sequence of 19 amino acid residues and 3 partial internal peptide sequences with 7, 10, and 7 amino acid residues. Using degenerate primers based on the partial internal sequences for PCR amplification and cDNA library screening, a full-length cDNA clone with a 1,167-bp open reading frame was isolated. Its deduced amino acid sequence consists of 389 amino acid residues with a predicted molecular mass of 43,239 and shares 45-46% sequence identity with mammalian alanine glyoxylate transaminases. Northern analysis shows the active transcription of the enzyme in larvae and developing eggs. Substrate specificity analysis of this mosquito transaminase demonstrates that the enzyme is active with 3-HK, kynurenine, or alanine substrates. The enzyme has greater affinity and catalytic efficiency for 3-HK than for kynurenine and alanine. The biochemical characteristics of the enzyme in conjunction with the profiles of 3-HK transaminase activity and XA accumulation during mosquito development clearly point out its physiological function in the 3-HK to XA pathway. Our data suggest that the mosquito transaminase was evolved in a manner precisely reflecting the physiological requirement of detoxifying 3-HK produced in the tryptophan oxidation pathway in the mosquito.

Purification and characterization of the alanine aminotransferase from the hyperthermophilic Archaeon pyrococcus furiosus and its role in alanine production.

Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6. 5 to 7.8 and at a temperature of over 95 degrees C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in the P. furiosus genome database. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. The k(cat)/K(m) values for alanine and pyruvate formation were 41 and 33 s(-1) mM(-1), respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aat gene. In addition, both the aat and gdh (encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdh genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.

Alanine aminotransferase and glycine aminotransferase from maize (Zea mays L.) leaves.

Alanine aminotransferase (AlaAT, EC 2.6.1.2) and glycine aminotransferase (GlyAT, EC 2.6.1.4), two different enzymes catalyzing transamination reactions with L-alanine as the amino-acid substrate, were examined in maize in which alanine participates substantially in nitrogen transport. Preparative PAGE of a partially purified preparation of aminotransferases from maize leaves gave 6 fractions differing in electrophoretic mobility. The fastest migrating fraction I represents AlaAT specific for L-alanine as amino donor and 2-oxoglutarate as amino acceptor. The remaining fractions showed three aminotransferase activities: L-alanine-2-oxoglutarate, L-alanine-glyoxylate and L-glutamate-glyoxylate. By means of molecular sieving on Zorbax SE-250 two groups of enzymes were distinguished in the PAGE fractions: of about 100 kDa and 50 kDa. Molecular mass of 104 kDa was ascribed to AlaAT in fraction I, while the molecular mass of the three enzymatic activities in 3 fractions of the low electrophoretic mobility was about 50 kDa. The response of these fractions to: aminooxyacetate, 3-chloro-L-alanine and competing amino acids prompted us to suggest that five out of the six preparative PAGE fractions represented GlyAT isoforms, differing from each other by the L-glutamate-glyoxylate:L-alanine-glyoxylate:L-alanine-2-oxoglutarate activity ratio.