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PubMed2003

CodY-regulated aminotransferases AraT and BcaT play a major role in the growth of Lactococcus lactis in milk by regulating the intracellular pool of amino acids.

Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.

Amino Acids, Aromatic↗

PubMed1981

Properties of crystalline L-ornithine: alpha-ketoglutarate delta-aminotransferase from Bacillus sphaericus.

The distribution of bacterial L-ornithine: alpha-ketoglutarate delta-aminotransferase (L-ornithine:2-oxo-acid aminotransferase [EC 2.6.1.13]) was investigated, and Bacillus sphaericus (IFO 3525) was found to have the highest activity of the enzyme, which was inducibly formed by addition of L-ornithine or L-arginine to the medium. L-Ornithine:alpha-ketoglutarate delta-aminotransferase, purified to homogeneity and crystallized from B. sphaericus, had a molecular weight of about 80,000 and consisted of two subunits identical in molecular weight (41,000) and in amino-terminal residue (threonine). The enzyme exhibited absorption maxima at 278,343, and 425 nm and contained 1 mol of pyridoxal 5'-phosphate per mol of enzyme. The formyl group of pyridoxal 5'-phosphate was bound through an aldimine linkage to the epsilon-amino group of a lysine residue of the protein. The enzyme-bound pyridoxal 5'-phosphate, absorbing at 425 nm, was released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive enzyme, which was reactivated by addition of pyridoxal 5'-phosphate, still had a 343-nm peak and contained 1 mol of a vitamin B6 compound. The holoenzyme showed positive circular dichroic bands at 340 and 425 nm, whereas the inactive form had no band at 425 nm. The enzyme was highly specific for L-ornithine and alpha-ketoglutarate and catalyzed delta-transamination between them to produce L-glutamate and L-glutamate-gamma-semialdehyde, which as spontaneously converted to delta 1-pyrroline-5-carboxylate. The enzyme activity was significantly affected by nonsubstrate amino acids, amines, and carbonyl reagents.

Amino Acids↗

PubMed1997

Identification and analysis of a gene encoding L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase involved in the 1,3-diaminopropane production pathway in Acinetobacter baumannii.

The ca. 2.2-kbp region upstream of the ddc gene encoding L-2,4-diaminobutyrate decarboxylase in Acinetobacter baumannii was sequenced and found to contain another open reading frame of 1,338 nucleotides encoding a protein with a deduced molecular mass of 47,423 Da. Analysis of the homologies observed from the deduced amino acid sequence indicated that the gene product is an enzyme belonging to subgroup II of the aminotransferases. This was first verified when examination of the crude extracts from Escherichia coli transformants led to detection of a novel aminotransferase activity catalyzing the following reversible reactions: L-2,4-diaminobutyric acid + 2-ketoglutaric acid<-->L-glutamic acid + L-aspartic beta-semialdehyde. Further confirmation was obtained when the gene was overexpressed in E. coli and the corresponding protein was purified to homogeneity. It catalyzed the same reactions and its N-terminal amino acid sequence was consistent with that deduced from the nucleotide sequence. Therefore, the gene and its product were named dat and L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase (DABA AT), respectively. Feeding experiments of A. baumannii with L-[U-14C]aspartic acid resulted in the incorporation of the label into 1,3-diaminopropane. Apparent homologs of dat and DABA AT were detected in other Acinetobacter species by PCR amplification and Western blotting. These results indicate that the dat gene (as well as the ddc gene) participates in the synthesis of 1,3-diaminopropane, the only diamine found in this genus. However, the biological role, if one exists, of 1,3-diaminopropane synthesis is unknown.

Acinetobacter↗

PubMed1993

The distal enhancer implicated in the developmental regulation of the tyrosine aminotransferase gene is bound by liver-specific and ubiquitous factors.

Tyrosine aminotransferase gene expression is confined to parenchymal cells of the liver, is inducible by glucocorticoids and glucagon, and is repressed by insulin. Three enhancers control this tissue-specific and hormone-dependent activity, one of which, located at -11 kb, is implicated in establishing an active expression domain. We have studied in detail this important regulatory element and have identified a 221-bp fragment containing critical enhancer sequences which stimulated the heterologous thymidine kinase promoter more than 100-fold in hepatoma cells. Within this region, we have characterized two essential liver-specific enhancer domains, one of which was bound by proteins of the hepatocyte nuclear factor 3 (HNF3) family. Analyses with the dedifferentiated hepatoma cell line HTC suggested that HNF3 alpha and/or -gamma, but not HNF3 beta, are involved in activating the tyrosine aminotransferase gene via the -11-kb enhancer. Genomic footprinting and in vitro protein-DNA binding studies documented cell-type-specific binding of ubiquitous factors to the second essential enhancer domain, which by itself stimulated the thymidine kinase promoter preferentially in hepatoma cells. These results will allow further characterization of the role of these enhancer sequences in developmental activation of the tyrosine aminotransferase gene.

Animals↗

PubMed1982

Effect of excess leucine on tryptophan oxygenase, 3-hydroxyanthranilate oxygenase and leucine aminotransferase in livers of young rats.

Hepatic tryptophan oxygenase (EC 1.13.1.12) 3-hydroxyanthranilate oxygenase (EC 1.13.1.6) and leucine aminotransferase (EC 2.6.1.6) activities were determined in livers of rat pups of various ages whose mothers were fed on diets with or without excess leucine. Tryptophan oxygenase activity was detectable on the 20th day in both the groups and thereafter increased with age. Low activity of 3-hydroxyanthranilate oxygenase was observed at birth, the levels increased on the 10th day and thereafter remained unaltered on both diets. Leucine aminotransferase activity was highest at birth and thereafter decreased with age. Tryptophan oxygenase and leucine aminotransferase activities were significantly higher at all ages in livers of pups born to mothers given excess leucine in their diet.

Age Factors↗

PubMed1985

Influence of alloxan diabetes and insulin treatment on the activity of alanine aminotransferase in rat brain regions, liver and heart.

Studies with brain alanine aminotransferase showed higher activity of the enzyme in the soluble fraction of cerebellum. Among the tissues, the liver soluble fraction was the richest source of the enzyme. Alloxan-induced diabetes caused both regional and time-dependent variations in the activity of brain alanine aminotransferase. Significant among these changes were the decrease in both soluble and particulate enzyme from cerebral hemispheres and an increase in the soluble enzyme activity from cerebellum at early stages of diabetes. Brain stem did not show any marked change in enzyme activity. Liver and heart enzyme, however, increased significantly after 1-2 weeks of diabetes. Insulin treatment to diabetic animals caused an 'over-shoot' in soluble alanine aminotransferase activity, particularly in cerebellum and liver.

Alanine Transaminase↗

PubMed1982

Development of tyrosine aminotransferase and para-hydroxyphenylpyruvate dioxygenase activities in fetal and neonatal human liver.

In livers of fetuses of 220--340 g body wt, total cytosolic tyrosine aminotransferase activity was 1.0 nmol of product/mg of protein per min, and the corresponding values for autopsy livers of newborns of 740--1,475 g and 2,600--3,650 g were 1.5 and 5.7, respectively, as compared with the adult value of 12.7. On the other hand, para-hydroxyphenylpyruvate dioxygenase activity is at adult level already in fetuses less than 340 g body wt. The Km value for tyrosine of tyrosine aminotransferase (1 mM) was considerably higher than the corresponding value for para-hydroxyphenylpyruvate of para-hydroxyphenylpyruvate dioxygenase (50 micro M). These results suggest that tyrosine aminotransferase is the rate limiting enzyme in the catabolism of tyrosine in premature infants.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed1977

Relation of triiodothyronine and reverse triiodothyronine administration in rats to hepatic L-triiodothyronine aminotransferase activity.

The effects of administration of 3,5,3'-triiodothyronine (T3) to normal and to hypothyroid male rats upon the hepatic activity of L-triiodothyronine aminotransferase were determined using 3,5-dinitro-L-tyrosine as substrate in the assay. Initial studies in normal rats demonstrated that basal enzyme activity was highest in liver and kidney of the organs tested, and that virtually no activity was detectable in skeletal muscle, serum, thyroid or pituitary gland. Hepatic enzyme activity increased from birth to a peak at 80-120 days and declined thereafter. Daily administration of T3 to normal rats in doses of 5 mug/100g BW for 8 days significantly elevated hepatic enzyme activity above normal. In daily doses of 2.5mug/100g BW, T3 restored the depressed enzyme activities in hypothyroid rats to normal. Daily administration of 3,3',5'-triiodothyronine (reverse T3) to normal rats in doses of 17.5 mug/100g BW and greater for 3 days increased L-T3 aminotransferase activity more than 30% above normal levels. Reverse T3 appeared to be approximately as active as T3 in increasing the hepatic activity of L-T3 aminotransferase.

Aging↗

PubMed1994

Evidence for diverse roles of protein kinase-C in the inhibition of gene expression by insulin: the tyrosine aminotransferase, albumin, and phosphoenolpyruvate carboxykinase genes.

We have previously shown that insulin is less effective in inducing expression of several genes in H4 hepatoma cells with reduced functional protein kinase-C (PKC) activity. However, other reports suggest that insulin regulation of gene transcription is not PKC dependent. Insulin and phorbol 12-myristate 13-acetate (PMA) rapidly inhibit transcription of the tyrosine aminotransferase and albumin genes. Prolonged PMA pretreatment, to desensitize cells to PMA, resulted in a loss of insulin ability to inhibit albumin transcription. Insulin was still able to inhibit tyrosine aminotransferase transcription, but less than in non-PMA-pretreated cells, and there was also a slight decrease in the ability of insulin to inhibit phosphoenolpyruvate carboxykinase transcription. We previously demonstrated decreased responsiveness of PMA-induced gene expression in insulin-desensitized cells. In the present work, using insulin-desensitized H4 cells (insulin pretreatment for 24 h), subsequent treatment with PMA did not alter phosphoenolpyruvate carboxykinase transcription rates, whereas PMA did inhibit tyrosine aminotransferase transcription rates to an extent similar to observed in nonpretreated cells. Unexpectedly, there was a significant increase in albumin transcription after PMA addition to insulin-pretreated cells. These findings support our hypothesis that the role of PKC in the regulation of gene expression by insulin varies for different insulin-regulated genes.

Animals↗

PubMed1982

Conversion of chloramphenicol degradation products by tyrosine aminotransferase from Flavobacteria.

The tyrosine aminotransferase of Flavobacterium strain CB 60, strain CB 6 and F. devorans r - a partially purified enzyme was used - is able to deaminate oxidatively p-aminophenylalanine and the intermediate products of chloramphenicol degradation p-nitrophenylserine and p-aminophenylserine. The aminotransferases of the strains CB 6 and CB 60 also convert p-aminophenylserinol. p-Nitrophenylserinol only reacts with the enzyme from strain CB 6. Determination of substrate specificity from strain CB 6 shows that an alcoholic group in C3 position (ring proximal) and to a lower degree an alcoholic group in C1 position (ring distal) decrease the turnover rate. Based on its broad substrate specificity the tyrosine aminotransferase has the ability not only to metabolize physiological compounds but also degradation products of chloramphenicol.

Chloramphenicol↗

PubMed1977

Pyridoxal 5'-phosphate as an activator of the apoenzyme of alanine aminotransferase in human serum.

The relationship between reaction conditions and the stimulation of alanine aminotransferase by pyridoxal 5'-phosphate was investigated. Reaction conditions given for optimized alanine aminotransferase measurements are also optimal for measurements in the presence of pyridoxal 5'-phosphate taking into consideration the inhibitory effect of phosphate buffer. Addition of 150 mumol/1 pyridoxal 5'-phosphate to the reaction mixture guarantees a maximal stimulation after a pre-incubation period of 10 min. Factors influencing the magnitude of stimulation rates of both aminotransferases in different patient groups by pyridoxal 5'-phosphate are discussed.

Alanine Transaminase↗

PubMedDate not supplied

On the mechanism of inactivation and ATP-dependent reactivation of rat liver tyrosine aminotransferase.

The mechanism of in vitro inactivation and ATP-dependent rapid reactivation of rat liver tyrosine aminotransferase by a membrane-bound system from rat liver and kidney cortex and the nucleotide specificity of this process was investigated using partially purified tyrosine amino-transferase as a substrate. Adenosine 5'-triphosphate (ATP) could be replaced by guanosine 5'-triphosphate (GTP), Whereas inosine 5'-triphosphate (ITP) was less effective. During reactivation [gamma-32P]ATP was incorporated into the enzyme and not excorporated by incubation of the labeled enzyme with excess non-radioative ATP. Inactivation of labeled tyrosine aminotransferase by a particulate fraction led to a decrease protein-bound radioactivity concomitant with an increase of [32P]orthophosphate. This points to a phosphorylation and dephosphorylation mechanism in the regulation of tyrosine aminotransferase activity.

Adenosine Triphosphate↗

PubMedDate not supplied

Purification, characterization and N-terminal sequence of phosphoserine aminotransferase from the green alga Scenedesmus obliquus, mutant C-2 A'.

Phosphoserine aminotransferase (EC 2.6.1.52), an enzyme of the "phosphorylated pathway" leading to the formation of serine, was purified from Scenedesmus obliquus, mutant C-2 A'. Purification started from the soluble supernatant of a crude cell homogenate and included different affinity and DEAE chromatographic techniques, as well as gel filtration. The purified phosphoserine aminotransferase was enriched 1537-fold and identified to be a homodimer with subunit molecular masses of 40 kDa, each. The absorption spectrum is consistent with the presence of pyridoxal-5-phosphate as cofactor. From the purified enzyme 18 amino acids of the N-terminus could be determined, showing at least 67% homology with the serC gene encoding phosphoserine aminotransferases from bacterial organisms.

Amino Acid Sequence↗

PubMed1978

Branched-chain amino acid aminotransferase in mouse testicular tissue.

Branched-chain amino acid aminotransferase (L-leucine:2-oxoglutarate aminotransferase, EC 2.6.1.6) activity was determined in several tissues of the mouse. Testis homogenates presented a specific activity very close to that of heart extracts which were the most active. Enzyme activity was detectable in testes from 5-day-old mice and increased steadily during development to reach a maximum at the 20th day of life. The transaminase was present in the cytosol of testicular homogenates and also associated, probably in the matrix, with a special type of mitochondria present in spermatozoa and gametogenic cells. The enzyme from testis is active against the three branched-chain amino acids and catalyses the reaction in both directions. Highest activity and lowest Km were obtained with L-leucine. Activity with L-valine was the lowest. The enzyme from the mitochondrial fraction showed identical properties to that from the soluble phase. The possible participation of this aminotransferase in a shuttle system transferring reducing equivalents from cytoplasm to mitochondria is postulated.

Animals↗

PubMed1984

Glutamine fructose-6-phosphate aminotransferase and glycoprotein synthesis in the developing chick eye.

Glutamine fructose-6-phosphate aminotransferase is an essential enzyme in the synthesis of glycoproteins and other complex carbohydrates. The specific activity of this enzyme was measured in posterior ocular tissues of the chick embryo from day 6 of development until hatching. The enzyme is present at high concentrations in the early embryo, then decreases 15-fold by day 9 in both retina and retinal pigment epithelium (PE). Thus, aminotransferase activity is highest during the period when retinal cells are migrating and beginning to differentiate; these maturational events may require cell-surface glycoproteins. On day 17-18 of chick embryo development, the neural retina suddenly adheres firmly and irreversibly to the underlying PE. Aminotransferase activity and glucosamine incorporation into retinal macromolecules were examined near this critical time, as were the effects of glycoprotein-synthesis inhibitors upon retinal adhesion. No evidence was found for involvement of complex carbohydrates (on cell surfaces or in the interphotoreceptor matrix, IPM) in formation of retinal adhesion in the chick. Histological studies show an absence of glycosaminoglycans in the IPM of embryonic chicks (at all stages) and of adult chickens, although these compounds are present in mammalian matrix.

Animals↗

PubMed1983

A new sensitive and convenient assay of ornithine aminotransferase.

A new radioisotopic assay for ornithine aminotransferase with [U-14C]ornithine as a substrate was developed. The dihydroquinazolinium compound formed was extracted from the reaction mixture with n-butanol and an addition of high concentration of sodium sulfate. This assay is sensitive and convenient and more than 50 assays can be done in one day without any special apparatus. The activities of ornithine aminotransferase in rat liver and human lymphoblastoid cells could be measured at nanomole levels by this method. Thus, the assay should be useful for measurement of OAT activity in cultured cells and for studies on the metabolic bases of the different types of ornithine aminotransferase deficiency: vitamin B6-responsive and -nonresponsive types.

Animals↗

PubMed1989

Competitive inhibition of mouse brain gamma-aminobutyrate aminotransferase by ATP.

The nucleotide ATP was shown to be a reversible inhibitor of partially purified gamma-aminobutyrate aminotransferase isolated from mouse brain. This inhibition was of the competitive type with respect to the substrate, gamma-aminobutyric acid (Ki = 3.7 +/- 0.6 mM), but was noncompetitive with respect to both the second substrate alpha-ketoglutarate and the cofactor pyridoxal 5'-phosphate. Two analogues of ATP, ADP and GTP, also gave rise to an inhibition gamma-aminobutyrate aminotransferase that was similar to that produced by ATP. These results are consistent with the view that mouse brain gamma-aminobutyric acid aminotransferase could be under regulatory control by ATP and certain other nucleotides within the mitochondria.

4-Aminobutyrate Transaminase↗

PubMed1976

Heterogeneity of hepatic tyrosine aminotransferase. Separation of the multiple forms from rat and frog liver by isoelectric focussing and hydroxylapatite column chromatography and their partial characterization.

L-Tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5; TAT) and other enzymes that transaminate tyrosine in rat liver cytosol have been separated into four fractions by isoelectric focussing. One of the forms is probably identical to mitochondrial L-aspartate:2-oxoglutarate aminotransferase (EC 2.6.1.1.; mASAT). The other three forms have pI's of 4.72, 4.98 and 5.30 and Km values of 1.3 and 0.3 mM for tyrosine and alpha-ketoglutarate. These heat stable forms have little or no ASAT activity. Rat liver TAT is also separated into three peaks by hydroxylapatite. Each fraction gives only one peak of activity when electrofocussed separately. In the frog, three groups of peaks of TAT activity have been separated by hydroxylapatite column chromatography. The first group is connected with ASAT activity. These peaks (pI's 6.35, 6.50 and 6.90) are heat stable and have a Km value for tyrosine of 4 mM. These fractions probably represent cytoplasmic ASAT (sASAT). The second group of peaks has at least two subforms (pI's 9.0 and 9.4, Km for tyrosine 15 mM). These forms probably represent mASAT. The third group consists of three forms that resemble the major forms of rat liver TAT. These results indicate that heterogeneity is common to many aminotransferases and independent of regulation by glucocorticoids.

Animals↗