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PubMed1999

Significance of repeatedly normal aminotransferase activities in HCV-infected patients.

The significance of repeatedly normal serum aminotransferase activities in antihepatitis C virus (anti-HCV)-positive patients is not clear. To address this issue, the authors analyzed clinical, virologic, histopathologic, and biological characteristics of such subjects. Among their active file of 1,200 anti-HCV-positive immunocompetent patients, they identified 36 subjects (3%) with repeatedly normal aminotransferase activities, as defined by at least four normal values of aminotransferase over a minimum period of 6 months without any abnormal value (mean of this period, 31 +/- 21 months). The 36 patients included 11 men and 25 women with a mean age of 45 +/- 15 years. Twenty-three of these 36 subjects (64%) had detectable HCV viremia by polymerase chain reaction. Their genotype distribution was as follows: genotype 1a or 1b, 57%; genotype 2, 26%; and genotype 3, 17%. Of the HCV ribonucleic acid (RNA)-positive and HCV RNA-negative subjects, 17 and 5 had a liver biopsy respectively. In the former, the mean Knodell score was 5.6 +/- 3.5 (range, 1 to 14), and was < 5 in 9 patients (53%) and > or = 5 in 8 (47%), including extensive fibrosis (n = 2) or cirrhosis (n = 2). In the HCV RNA-negative subjects, one patient had a Knodell score > or = 5. Comparing the 23 immunocompetent viremic subjects with repeatedly normal serum aminotransferase activities with our group (n = 564) of immunocompetent viremic patients with abnormal aminotransferase activities, there was a significant predominance of women (70% versus 44%, p < 0.05) and of genotype 2 in the former (26% versus 7%, p < 0.05), but no differences according to quantitative viremia, alcohol consumption, or distribution of risk factor were observed. Most of viremic HCV-infected patients with long-term and repeatedly normal aminotransferase values have indeed chronic active hepatitis, including extensive fibrosis or cirrhosis in as many as 20% of patients. This emphasizes the need for serum HCV RNA determination in anti-HCV-positive patients with normal aminotransferase activities. In these patients liver biopsy may be necessary and should be discussed.

Adolescent↗

PubMed1993

Expression of the soybean (Glycine max) glutamate 1-semialdehyde aminotransferase gene in symbiotic root nodules.

Extracts of soybean (Glycine max) root nodules and greening etiolated leaves catalyzed radiolabeled delta-aminolevulinic acid (ALA) formation from 3,4-[3H]glutamate but not from 1-[14C]glutamate. Nevertheless, those tissue extracts expressed the activity of glutamate 1-semialdehyde (GSA) aminotransferase, the C5 pathway enzyme that catalyzes ALA synthesis from GSA for tetrapyrrole formation. A soybean nodule cDNA clone that conferred ALA prototrophy, GSA aminotransferase activity, and glutamate-dependent ALA formation activity on an Escherichia coli GSA aminotransferase mutant was isolated. The deduced product of the nodule cDNA shared 79% identity with the GSA aminotransferase expressed in barley leaves, providing, along with the complementation data, strong evidence that the cDNA encodes GSA aminotransferase. GSA aminotransferase mRNA and enzyme activity were expressed in nodules but not in uninfected roots, indicating that the Gsa gene is induced in the symbiotic tissue. The Gsa gene was strongly expressed in leaves of etiolated plantlets independently of light treatment and, to a much lesser extent, in leaves of mature plants. We conclude that GSA aminotransferase, and possibly the C5 pathway, is expressed in a nonphotosynthetic plant organ for nodule heme synthesis and that Gsa is a regulated gene in soybean.

Amino Acid Sequence↗

PubMed2004

Viral clearance in HCV viraemic patients with normal alanine aminotransferase after combination therapy: a controlled, open-labelled study.

BACKGROUND: In patients with chronic hepatitis C virus infection and persistently normal alanine aminotransferase levels, liver fibrosis has been reported in 0-22% of cases and advanced liver disease in 5-10% of cases. AIM: To determine whether patients with persistently normal alanine aminotransferase levels clear infection after anti-viral therapy at equal or different rates from infected patients with raised alanine aminotransferase levels. METHODS: Thirty-five hepatitis C virus RNA-positive patients with fibrosis at liver histology (Group 1) were matched for genotype, sex, age and histology with patients with raised alanine aminotransferase levels (Group 2). Both groups were treated with 3 MU interferon-alpha2b plus ribavirin (1000-1200 mg) for 12 months. RESULTS: End-of-therapy response was achieved in 71.4%[95% confidence interval (CI), 56.4-86.3] of patients in Group 1 and in 52.3% (95% CI, 42.8-61.9) of those in Group 2 (P = 0.04). At week 72, 22 patients (62.8%; 95% CI, 46.8-78.1) in Group 1 and 50 patients (47.5%; 95% CI, 38.0-57.1) in Group 2 showed a sustained virological response (P = 0.11). Non-1 genotype was the only independent predictor of sustained response (P = 0.002), with an odds ratio of 3.45 (95% CI, 1.58-7.50). At month 3 of therapy, the positive predictive values for non-response were 100% and 96% in Groups 1 and 2, respectively. CONCLUSIONS: Interferon and ribavirin induce comparable sustained virological response in patients with persistently normal or raised alanine aminotransferase levels. Stage 1 fibrosis, rather than alanine aminotransferase levels, may be the criterion on which to decide whether or not to treat patients with persistently normal alanine aminotransferase levels.

Adult↗

PubMed1981

Studies on the posttranscriptional site of cAMP action in the regulation of the synthesis of tyrosine aminotransferase.

Synthesis of L-tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5) can be induced by N6,O2'-dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP) in Reuber H35 cell cultures. Actinomycin D fails to block this induction which indicates a target for Bt2cAMP at a posttranscriptional level. We have determined the influence of Bt2cAMP on several translational events during the tyrosine aminotransferase synthesis with the following results. (1) The number of nascent tyrosine aminotransferase chains increased, whereas no effect was measured on the number of nascent total protein chains. (2) The rate of elongation along the tyrosine aminotransferase mRNA and total mRNA is not enhanced by Bt2cAMP. (3) The induced synthesis of tyrosine aminotransferase is more sensitive to the inhibition of elongation. We conclude from our results that Bt2cAMP induces the synthesis of tyrosine aminotransferase by an increase in the rate of initiation on the tyrosine aminotransferase mRNA.

Animals↗

PubMed1998

The apo-enzyme content of aminotransferases in healthy and diseased domestic animals.

We investigated the apo-enzyme content of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in clinically normal and ill canine, feline, equine and bovine patients. Aminotransferase activity was measured with and without the addition of exogenous pyridoxal-5'-phosphate (P5P). The amount of apo-enzyme was expressed as the percentage change in aminotransferase activity with the inclusion of P5P. The results of aminotransferase assays without P5P (holo-enzyme activity) were highly correlated to the results obtained with P5P (total enzyme activity) in all four species (Spearman rank correlations > 0.980). The median apo-aminotransferase percentage in clinically normal patients was: 11% ALT and 0% AST in 115 dogs, 7% ALT and -5% AST in 50 cats, 6% AST in 46 horses and 9% AST in 50 cattle. The amount of apo-enzyme did not increase as holo-enzyme activity increased in any of the species. The apo-aminotransferase content was not significantly different in canine, feline and equine patients with diseases affecting the liver, kidney or musculature compared to clinically normal animals. The apo-enzyme content did not differ significantly between specific liver diseases in canine and feline patients.

Journal Article↗

PubMed1989

Reactions catalyzed by purified L-glutamine: keto-scyllo-inositol aminotransferase, an enzyme required for biosynthesis of aminocyclitol antibiotics.

Dialyzed extracts of the gentamicin producer Micromonospora purpurea catalyze reactions which represent transaminations proposed for 2-deoxystreptamine biosynthesis. To determine whether these transaminations were catalyzed by a single aminotransferase or by multiple enzymes, we purified and characterized an L-glutamine:keto-scyllo-inositol aminotransferase from M. purpurea. This enzyme was purified 130- to 150-fold from late-log-phase mycelia of both wild-type M. purpurea and a 2-deoxystreptamine-less idiotroph. The cofactor pyridoxal phosphate was found to be tightly bound to the enzyme, and spectral analysis demonstrated its participation in the transamination reactions of this enzyme. The major physiological amino donor for the enzyme appears to be L-glutamine; the keto acid product derived from glutamine was characterized as 2-ketoglutaramate, indicating that the alpha amino group of glutamine participates in the transamination. We found that crude extracts contained omega-amidase activity, which may render transaminations with glutamine irreversible in vivo. The substrate specificity of the aminotransferase was shown to be restricted to deoxycyclitols, monoaminocyclitols, and diaminocyclitols, glutamine, and 2-ketoglutaramate, which contrasts with the broader substrate specificity of mammalian glutamine aminotransferase. The appearance of the enzyme in late-log phase, coupled with its narrow substrate specificity, indicates that it participates predominantly in 2-deoxystreptamine biosynthesis rather than in general metabolism. The enzyme catalyzes reactions which represent both transamination steps of 2-deoxystreptamine biosynthesis. Although copurification of two aminotransferases cannot be ruled out, our data are consistent with the participation of a single aminotransferase in the formation of both amino groups of 2-deoxystreptamine during biosynthesis by M. purpurea. We propose that this aminotransferase participates in a key initial step in the biosynthesis of most aminocyclitol antibiotics.

Amidohydrolases↗

PubMed1981

Purification and properties of aromatic amino acid aminotransferase from Klebsiella aerogenes.

We describe the complete purification of aromatic aminotransferase I, the enzyme responsible for the ability of Klebsiella aerogenes to use tryptophan and phenylalanine as sole sources of nitrogen, as well as the partial purification of aromatic aminotransferase IV. An examination of the properties of these enzymes revealed that aminotransferase I had much greater affinity for the aromatic amino acids than aminotransferase IV, explaining the essential role of aminotransferase I in the utilization of exogenously supplied aromatic amino acids. The properties of aminotransferase IV suggest that this enzyme is actually an aspartate aminotransferase (EC 2.6.1.1), corresponding to the product of the aspC gene of Escherichia coli.

Amino Acids↗

PubMed2004

Normal serum aminotransferase concentration and risk of mortality from liver diseases: prospective cohort study.

OBJECTIVE: To examine the relation between the normal range of serum aminotransferase concentration and mortality from liver disease. DESIGN: Prospective cohort study. SETTING: Korea Medical Insurance Corporation study with eight years' follow up. PARTICIPANTS: 94,533 men and 47,522 women aged 35-59 years. MAIN OUTCOME MEASURE: Mortality from liver diseases according to death certificate. RESULTS: There was a positive association between the aminotransferase concentration, even within normal range (35-40 IU/l), and mortality from liver disease. Compared with the concentration < 20 IU/l, the adjusted relative risks for an aspartate aminotransferase concentration of 20-29 IU/l and 30-39 IU/l were 2.5 (95% confidence interval 2.0 to 3.0) and 8.0 (6.6 to 9.8) in men and 3.3 (1.7 to 6.4) and 18.2 (8.1 to 40.4) in women, respectively, The corresponding risks for alanine aminotransferase were 2.9 (2.4 to 3.5) and 9.5 (7.9 to 11.5) in men and 3.8 (1.9 to 7.7) and 6.6 (1.5 to 25.6) in women, respectively. According to receiver operating characteristic curves the best cut-off values for the prediction of liver disease in men were 31 IU/l for aspartate aminotransferase and 30 IU/l for alanine aminotransferase. CONCLUSION: People with slightly increased aminotransferase activity, but still within the normal range, should be closely observed and further investigated for liver diseases.

Adult↗

PubMed1983

[Aspartate And Alanine Aminotransferase In Fasciola Hepatica]

The activity and distribution of aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2) in adult Fasciola hepatica have been studied. Fasciola hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of GOT and GPT was measured by the method of Reitman and Frankel. Isozyme patterns of those enzyme were also examined by DEAE-cellulose column chromatography. The results obtained were as follows: 1. The activity of aspartate and alanine aminotransferase was about 0.55 unit and 0.92 unit per 1 g of Fasciola hepatica, respectively. 2. The activity of those enzymes was relatively low compared with those in mammalian tissues. 3. The distribution of aspartate aminotransferase in the subcellular organelles showed that 71 % of the activity was in cytosolic, 24 % in mitochondrial and 5 % was in nuclear fraction. 4. About 22 % of the total alanine aminotransferase activity was found in the mitochondrial fraction, about 66 percent in the cytosolic fraction. 5. Aspartate aminotransferase from cytosolic fraction was separated into two types of isozymes, whereas alanine aminotransferase from cytosolic fraction gave only one active peak on DEAE-cellulose column chromatography.

Journal Article↗

PubMed1976

Immunochemical studies of serine dehydratase and ornithine aminotransferase regulation in rat liver in vivo.

Previous studies of serine dehydratase (EC 4.2.1.13) and ornithine aminotransferase (EC 2.6.1.13) adaptation in rat liver showed that in rats on a high protein diet, glucocorticoid administration increased serine dehydratase activity while simultaneously reducing the activity of ornithine aminotransferase. The present study examines the role of enzyme synthesis in the expression of these and other dissimilar adaptive characteristics of the two enzymes. Both enzymes were purified to crystallinity and used to prepare specific antibodies. Changes in the rate of synthesis of each enzyme during adaptation were then measured immunochemically. In rats fed ad libitum, the synthetic rates for both enzymes exhibited circadian rhythm, although enzyme levels remained relatively constant. The circadian cycle for ornithine aminotransferase synthesis was in phase with the cycles for body weight and relative liver weight (maxima at 9 a.m., minima at 9 p.m.) but was approximately 12 hours out of phase with the cycle for serine dehydratase synthesis. 9alpha-Fluoro-11beta, 21-dihydroxy-16alpha, 17alpha-isopted at 9 a.m., increased serine dehydratase synthesis and simultaneously decreased the synthesis of ornithine aminotransferase. When triamcinolone was injected at 9 p.m., however, serine dehydratase synthesis was not stimulated, although the reduction of ornithine aminotransferase synthesis was still produced. These results suggest that: (a) circadian cycling of synthesis may be a general phenomenon in enzyme regulation even though for enzymes with relatively long half-lives, such cycling may not be reflected as fluctuations in enzyme levels; (b) such circadian rhythmicity may also involve cyclic changes in the responsiveness of the enzyme-forming system to regulatory stimuli; (c) whereas the adaptive behavior of serine dehydratase typifies that of amino acid-catabolizing enzymes in general, the responses of ornithine aminotransferase denote a functional association of this enzyme with anabolic processes. On this basis, the possibility that ornithine aminotransferase plays a pivotal role in the regulation of urea cycle activity and nitrogen balance is discussed.

Animals↗

PubMedDate not supplied

Alanine aminotransferase (ALT) levels in a normal population and interferon therapy in chronic hepatitis C patients with normal ALT.

BACKGROUND/AIMS: The aims of this study were to determine the distribution of serum alanine aminotransferase levels in a normal population and to clarify whether interferon treatment is justified in HCV-infected patients with persistently normal alanine aminotransferase levels. METHODOLOGY: The distribution of alanine aminotransferase levels was examined among 949 normal subjects who were negative for hepatitis viruses, denied regular alcohol use. Nineteen patients with chronic hepatitis C and persistently normal alanine aminotransferase levels were treated with alpha interferon (six or ten million units thrice weekly for six months). RESULTS: Peaks of alanine aminotransferase distribution among the normal subjects were seen at 16-20 IU/L and 11-15 IU/L in males and females, respectively. Fourteen of the 19 patients who received interferon treatment had favorable factors of response to interferon (eight with low pretreatment virus load, four with HCV genotype 2 and two with both). A sustained virological response was achieved in eight (57%) of 14, and alanine aminotransferase levels decreased significantly to within the normal range after interferon treatment in six of eight. CONCLUSIONS: Patients with chronic hepatitis C and persistently normal alanine aminotransferase levels should be treated with high doses of interferon if they have favorable factors of response to interferon treatment.

Adult↗

PubMed1985

Serum aminotransferases and gamma-glutamyl transferase in protein energy malnutrition.

Forty six children suffering from Protein Energy Malnutrition (PEM) were classified according to the Wellcome classification. Their aspartate aminotransferase, alanine aminotransferase and gamma-glutamyl transferase were measured. Aspartate aminotransferase was raised in 20 patients (43.5%) and alanine aminotransferase was raised in 12 patients (26%). Y-glutamyl transferase was raised in only one patient suffering from marasmic kwashiorkor, who, in contrast to the rest of the patients had a marked rise in aminotransferases. The aminotransferase elevation correlated positively with a Severity Index calculated from height and weight retardation and serum albumin levels. It is suggested that the moderate rise in aminotransferases found in PEM is not due to damage to the liver. However, marked enzyme elevations can occur in a small minority of patients, suggestive of liver injury, probably caused by hepatotoxins.

Child↗

PubMed1983

Direct evidence that the protein kinase catalytic subunit mediates the effects of cAMP on tyrosine aminotransferase synthesis.

The effect of purified beef heart cAMP-dependent protein kinase catalytic subunit on tyrosine aminotransferase activity in intact cultured rat H35 hepatoma cells was directly tested by micro-injection using human red blood cell ghosts as vehicles. Although the micro-injection procedure itself produced temporary fluctuations in protein synthesis and in tyrosine aminotransferase activity in H35 cells, after a recovery period of 8-12 h, these parameters returned to normal in parallel with restoration of full inducibility of the aminotransferase by both 8-Br-cAMP and dexamethasone. Eight to sixteen hours after fusion of H35 cells with unloaded ghosts, ghosts loaded with bovine serum albumin or mock-loaded with the partially purified protein kinase catalytic subunit, no significant change in the activity of the aminotransferase was detected. In contrast, fusion with ghosts loaded with the catalytic subunit at concentrations between 0.1-2 mg/ml caused reproducible 2-3-fold increases in enzyme activity. Homogeneous preparations of the catalytic subunit exhibited even greater potency as an inducer. The effect was both time- and concentration-dependent and was abolished by inactivation of the catalytic subunit with N-ethylmaleimide prior to loading. The partially purified inhibitor of protein kinase from beef heart, while not affecting basal tyrosine aminotransferase activity, selectively inhibited the ability of 8-Br-cAMP but not that of dexamethasone to stimulate the activity of this enzyme. In addition, micro-injection of the pure regulatory subunit of the kinase blocked the response of the aminotransferase to low concentrations of 8-Br-cAMP. These results provide strong support for the proposition that the catalytic subunit of protein kinase mediates the effects of cAMP on the synthesis of tyrosine aminotransferase.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1984

Transcriptional control of ornithine aminotransferase synthesis in rat kidney by estrogen and thyroid hormone.

Ornithine aminotransferase, a mitochondrial matrix enzyme, is expressed constitutively in many mammalian tissues and is subject to complex hormonal and dietary regulation in rat liver and kidney. The kidney enzyme is induced by estrogen and thyroid hormone (3,3',5-triiodo-L-thyronine, T3). We demonstrated previously that the hormonal induction in kidney is the result of an increase in ornithine aminotransferase mRNA levels (Mueckler, M. M., and Pitot, H. C. (1983) J. Biol. Chem. 258, 1781-1784). In order to define further the level at which the enzyme is induced in the kidney, the transcriptional activity of the ornithine aminotransferase gene was measured after hormonal induction. This was accomplished by incubating purified kidney nuclei in an in vitro transcription reaction in the presence of [32P] UTP and hybridizing the nascent, 32P-labeled, nuclear RNA to cloned ornithine aminotransferase cDNA immobilized on nitrocellulose filters. Administration of estrogen resulted in an increase in the rate of ornithine aminotransferase gene transcription by 1 h, and the rate remained elevated until at least 20 h after administration. The activity of the gene was elevated 12 h after T3 administration. A lag time of at least 4 h was observed for the effect of T3 on ornithine aminotransferase gene transcription. When administered in combination, estrogen and T3 exerted a synergistic effect on the activity of the gene. The administration of estrogen or both estrogen and T3 also resulted in the increased steady state level of two putative ornithine aminotransferase nuclear precursor RNAs, 3190 and 4260 nucleotides in length.

Animals↗

PubMed1990

The significance of serum mitochondrial aspartate aminotransferase activity in obstructive jaundice: experimental and clinical studies.

The serum level of mitochondrial aspartate aminotransferase was determined in experimental and clinical obstructive jaundice, using an immunoabsorbance method which allowed the differential determination of cytosolic and mitochondrial isozymes in the serum. In experimental obstructive jaundice using dogs, the serum mitochondrial aspartate aminotransferase value rapidly decreased to normal after biliary decompression following a period of biliary obstruction of within 3 weeks. On the other hand, when the period of jaundice was prolonged for 5 weeks, the activity of the enzyme after biliary drainage still continued to show high values, being 14.2 +/- 1.8 Karmen units at 4 weeks following biliary decompression. Determination of aspartate aminotransferase activity in tissue from such organs as the liver, heart, kidney, skeletal muscle and brain, as well as serum samples withdrawn from local veins, confirmed that high serum values of the enzyme in experimental obstructive jaundice were mostly attributable to hepatic impairment induced by biliary obstruction not by secondarily damaged tissues of other organs. Mitochondrial aspartate aminotransferase proved to be a more useful marker than other routine tests in icteric dogs. In 13 clinical patients with obstructive jaundice, decreasing rates of serum mitochondrial aspartate aminotransferase on the 7th and 14th postoperative days could be applied to evaluate the viability of the icteric liver. The decreasing rates were more advantageous than the preoperative activity itself in predicting the postoperative function of the liver. Thus, mitochondrial aspartate aminotransferase appears to serve as a useful marker for assessing the liver function in obstructive jaundice.

Adolescent↗

PubMed1986

Source of increased serum aspartate and alanine aminotransferase: cycloheximide effect on carbon tetrachloride hepatotoxicity.

Repeated doses of the protein synthesis inhibitor cycloheximide prevented the increases in rat liver mitochondrial and cytosolic aspartate aminotransferase, in alanine aminotransferase and in protein content observed 24 h after a single carbon tetrachloride injection. Serum aminotransferase activity increases induced by carbon tetrachloride were also decreased as much as 75.7% with cycloheximide. Increased synthesis is, therefore, suggested as an important and sometimes major source of increased serum aminotransferases in hepatocellular injury. This effect of cycloheximide lends support to the hypothesis that the liver enzyme increases after CCl4 are probably due to increased synthesis, in addition to the classically held mechanisms of leakage from necrotic or damaged hepatocytes. This explanation of the mechanisms of release of aminotransferases in rat liver injury would clarify many clinical observations if the same phenomenon were to occur in humans in response to hepatic injury. These data suggest that increased serum aminotransferase activities represent a healing, in addition to a degenerative, process.

Alanine Transaminase↗

PubMed1987

In vivo inhibition of aspartate aminotransferase in mice by L-hydrazinosuccinate.

L-Hydrazinosuccinate, which has been shown to be a slow-, tight-binding inhibitor of aspartate aminotransferase (EC 2.6.1.1) in vitro, was tested as an inhibitor in vivo of the enzyme as well as other pyridoxal enzymes. Intraperitoneal administration to mice at a dose of 0.6 mmol/kg rapidly decreased aspartate aminotransferase activities in liver and kidney cytosols to a minimal level lower than 10% of the original, and no appreciable reversal of the inhibition was observed after 24 h; at lower doses the activities were significantly recovered during the same period following an initial marked decrease. Of the other pyridoxal enzymes tested, alanine aminotransferase in liver was the most sensitive to the inhibitor. It was initially inhibited as severely as aspartate aminotransferase, but the inhibition was reversed considerably faster. Aspartate aminotransferase activities in brain and heart were less severely affected than those in liver and kidney; they were less markedly lowered initially and were substantially recovered after 24 h. Consistent with the observed organ specificity, heated extracts from brain and heart in the mice administered with the inhibitor showed relatively weak inhibitory activities in vitro to aspartate aminotransferase purified from pig heart, while the extracts from liver and kidney were strongly inhibitory.

Animals↗

PubMed1989

Intracellular predominance of the pyridoxal 5'-phosphate form of aspartate aminotransferase in Escherichia coli B and reversible transformation of this form by extracellular substances.

The intracellular proportion of the pyridoxal 5'-phosphate form of aspartate aminotransferase to the total enzyme in E. coli B cells was determined by a newly devised method, dependent on selective inactivation of the intracellular pyridoxal 5'-phosphate form of the enzyme by extracellularly added sodium borohydride. A large portion (80-99%) of the intracellular aspartate aminotransferase was in pyridoxal 5'-phosphate form in both natural and synthetic medium-grown bacterial cells. The intracellular predominancy of pyridoxal 5'-phosphate did not vary during the growth of bacteria and during incubation of bacterial cells in various kinds of buffers with different pH values. In contrast, the saturation levels generally used to describe in vivo the proportions of the apo and holo vitamin B6-dependent enzymes did not reflect the intracellular amount of the pyridoxal 5'-phosphate (holo) form of aspartate aminotransferase probably because the intracellular pyridoxal 5'-phosphate form was changed to an apo form by the disruption of bacterial cells for preparing crude extract. Various extracellularly-added vitamin B6 antagonists decreased the intracellular amount of pyridoxal 5'-phosphate without decrease in the total intracellular activity of the enzyme. The modified forms were stable in E. coli B cells and reversed into pyridoxal 5'-phosphate form by incubation of the antagonist-treated cells in the buffer containing pyridoxal. The present results showed that the sodium borohydride reduction method can be used for further analysis of the in vivo interaction of pyridoxal 5'-phosphate and apoaspartate aminotransferase. The fact that about 50% of the intracellular pyridoxal 5'-phosphate form was changed to a modified form without impairment of cell growth in the presence of 4-deoxypyridoxine, and that about 50% of intracellular modified aspartate aminotransferase was reversed to pyridoxal 5'-phosphate by the removal of antagonist followed by incubation suggested that there exists characteristically 2 different fractions of pyridoxal 5'-phosphate forms of aspartate aminotransferase in E. coli cells.

Aspartate Aminotransferases↗