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1.
4-Aminobutyrate aminotransferase. Conformational changes induced by reduction of pyridoxal 5-phosphate 总被引:1,自引:0,他引:1
Conformational changes induced in 4-aminobutyrate aminotransferase (4-aminobutyrate:2-oxoglutarate aminotransferase, EC 2.6.1.19) by conversion of pyridoxal-5-P to pyridoxyl-5-P were examined by two independent methods. The reactivity of the SH groups of the reduced enzyme is increased by chemical modification of the cofactor. 1.8 SH per dimer of modified enzyme react with DTNB, whereas 1.2 SH per dimer of the native enzyme react with the attacking reagent under identical experimental conditions. The modified and native forms of the enzyme bind the fluorescent probe ANS, but the number of binding sites for ANS is increased as result of conversion of P-pyridoxal to P-pyridoxyl. After the conformational changes onset by reduction of the cofactor, the modified enzyme binds one molecule of pyridoxal-5-P with a Kd of 0.1 microM to become catalytically competent. The catalytic site of the reduce enzyme was probed with P-pyridoxal analogs. Like resolved 4-aminobutyrate aminotransferase, the reduced species recognize the phosphorothioate analog and regain 40% of the total enzymatic activity. Since the catalytic parameters of reduced and native 4-aminobutyrate aminotransferase are indistinguishable, it is concluded that the additional catalytic site of the reduced enzyme is functionally identical to that of the native enzyme. 相似文献
2.
Induction of cytosolic aspartate aminotransferase by glucagon in primary cultured rat hepatocytes 总被引:2,自引:0,他引:2
Y Horio H Fukui M Taketoshi T Tanaka H Wada 《Biochemical and biophysical research communications》1988,153(1):410-416
The activity and the mRNA content of cytosolic aspartate aminotransferase (EC 2.6.1.1) were examined in cultured rat hepatocytes. Addition of glucagon (1 x 10(-7) M) in the presence of dexamethasone (1 x 10(-7) M) caused about 2-fold increase in the activity and mRNA content. Dibutyryl cAMP (1 x 10(-4) M) could replace glucagon for this effect. Maximal induction of cytosolic aspartate aminotransferase mRNA was observed 8 h after their additions. Insulin (1 x 10(-7) M) did not inhibit the enzyme induction by glucagon or dibutyryl cAMP. These results suggest that the cytosolic aspartate aminotransferase gene is regulated by cAMP, and not by insulin. 相似文献
3.
Influence of alloxan diabetes and insulin treatment on the activity of alanine aminotransferase in rat brain regions, liver and heart 总被引:1,自引:0,他引:1
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. 相似文献
4.
Alanine aminotransferase (EC 2.6.1.2) was obtained from the fungus Leptosphaeria michotii (West) Sacc, and enriched 714-fold by a 5-step purification procedure as a dimer of Mr 110000, associated with a polypeptide of Mr 25000. Its isoelectric point was 5.25. The enzyme was active from pH 3.5 to 9.5 with a maximum at pH 7.5. Its specific activity was 6000 nkat (mg protein)−1 ; the Km was 6.85 m M for L-alanine and 0.2 m M for 2-oxoglutarate. The enzyme did not show any detectable activity in the presence of L-aspartate, cysteine sulfinate, α-aminobutyrate or cyclic amino acids as substrates. It did not express alanine:glyoxylate aminotransferase activity. Alanine aminotransferase in L. michotii has previously been shown to have an activity rhythm in constant temperature and darkness. The enzyme level was quantified along the activity rhythm by enzyme-linked immunosorbent assay (ELISA), using a monospecific polyclonal antibody against the purified enzyme. The cyclic variations of alanine aminotransferase activity were correlated with cyclic variations in the enzyme level. 相似文献
5.
A mutant of Hordeum vulgare L. (LaPr 85/84) deficient in serine: glyoxylate aminotransferase (EC 2.6.1.45) activity has been isolated. The plant also lacks serine: pyruvate aminotransferase and asparagine: glyoxylate aminotransferase activities. Genetic analysis of the mutation strongly indicates that these three activities are all carried on the same enzyme protein. The mutant is incapable of normal rates of photosynthesis in air but can be maintained at 0.7% CO2. The rate of photosynthesis cannot be restored by supplying hydroxypyruvate, glycerate, glutamate or ammonium sulphate through the xylem stream. This photorespiratory mutant demonstrates convincingly that photorespiration still occurs under conditions in which photosynthesis becomes insensitive to oxygen levels. Two major peaks and one minor peak of serine: glyoxylate aminotransferase activity can be separated in extracts of leaves of wild-type barley by diethylaminoethyl-sephacel chromatography. All three peaks are missing from the mutant, LaPr 85/84. The mutant showed the expected rate (50%) of ammonia release during photorespiration but produced CO2 at twice the wild-type rate when it was fed [14C]glyoxylate. The large accumulation of serine detected in the mutant under photorespiratory conditions shows the importance of the enzyme activity in vivo. The effect of the mutation on transient changes in chlorophyll a fluorescence initiated by changing the atmospheric CO2 concentration are presented and the role of the enzyme activity under nonphotorespiratory conditions is discussed.Abbreviations DEAE
diethylaminoethyl
- PFR
photon fluence rate
- SGAT
serine:glyoxylate aminotransferase 相似文献
6.
K Wagner M D Roper B H Leichtling J Wimalasena W D Wicks 《The Journal of biological chemistry》1975,250(1):231-239
A variety of 6- and 8-substituted analogs of cAMP (cyclic adenosine 3:5-monophosphate) have been tested for their ability to increase activity of tyrosine aminotransferase (EC 2.6.1.5) in cultured Reuber H35 hepatoma cells. Some analogs, particularly the 8-thio-substituted ones, produced effects approximately equivalent to those generated by N-6, O2'-dibutyryl cAMP. In contrast, cAMP and its O-2-monobutyryl derivative were relatively ineffective even at very high concentrations, whereas three other analogs actually depressed the activity of the aminotransferase. Changes in enzyme activity generated by the various analogs were paralleled closely by changes in the relative rate of aminotransferase synthesis. An excellent correlation was found to exist between the ability of any given analog to influence the activity of tyrosine aminotransferase and that of phosphoenolpyruvate carboxykinase (EC 4.1.1.32). A similar correlation was found to exist between the ability of various analogs to evelate the activity of these enzymes and to inhibit reversibly the growth of H35 cells. Only one of five inhibitors of cAMP phosphodiesterase activity tested produce any increase in aminotransferase activity when added alone. All of the 6- and 8-substituted analogs tested, including noniducers, stimulated f1 histone phosphorylation in crude rat liver extracts with approximately equal potencies. On the other hand, dibutyryl cAMP was only a weak activator of protein kinase in vitro, even though it is a potent enzyme inducer. A possible resolution of this apparent discrepancy has been provided by preliminary analyses of site-specific f1 histone phosphorylation in whole cells. Only compounds active as aminotransferase inducers are capable of stimulating phosphorylation of the serine-37 residue of endogenous f1 histone (3- to 10-fold). 相似文献
7.
S Ueno A Sano T Hineno K Kondoh T Mizuno H Morino Y Kakimoto 《Biochimica et biophysica acta》1990,1035(2):128-131
D-3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40, D-BAIB aminotransferase) participates in the metabolism of thymine. Recently we purified this enzyme from rat liver. We have studied D-BAIB aminotransferase further to clarify its physiological function. Among our findings were the following. (1) The enzyme activity was widely distributed in the organs of guinea pigs and rats. The kidney, liver, and lung showed high specific activities. (2) Using the livers of six vertebrates, differences between species were studied. Activity was detected in all species, the human liver showing the lowest activity among them. (3) Developmental study using rat liver showed that the activity was low at birth, increased sharply thereafter for 10 days, and then subsequently declined to the adult level. (4) Intraperitoneal injection of BAIB and beta-alanine in rats was performed to determine whether they induce activity of this aminotransferase. Only BAIB increased the activity of the aminotransferase in the liver significantly. (5) Subcellular distribution study of this aminotransferase in rat liver revealed that it is a mitochondrial enzyme. 相似文献
8.
Rat cytosolic aspartate aminotransferase: regulation of its mRNA and contribution to gluconeogenesis
Induction of cytosolic aspartate aminotransferase (cAspAT) was observed in rat liver on administration of a high-protein diet, or glucagon and during fasting. The enzyme activity in the liver of rats given 80% protein diet or glucagon injection during starvation increased to 2- to 2.4-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of hybridizable cAspAT mRNA, measured by blot analysis using the cloned rat cAspAT cDNA as a probe. No increase in the enzyme was detected in kidney, heart, brain, or skeletal muscle. The activity of mitochondrial aspartate aminotransferase (mAspAT) did not increase. Induction of cAspAT was observed when glucose metabolism tended toward gluconeogenesis. The physiological function of the induction of cAspAT is considered to be to increase the supply of oxaloacetate as a substrate for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) [EC 4.1.1.32] for gluconeogenesis. 相似文献
9.
A five-step procedure is described for preparing highly purified aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC.2.6.1.1) from cell-freee enzyme extracts of Pediococcus cerevisiae. An overall purification of 130-fold was achieved. Some of P. cerevisiae aspartate aminotransferase properties were studied, i.s. pH optimum (7.8--8.0), optimum of temperature (37 degrees), Michaelis constans for 4 enzyme substrates and substrate specificity of enzyme. The enzyme is very thermolabile. During purification the enzyme was stabilizated by 2-oxoglutarate. The highly purified preparation was stored in the solution containing ammonium sulphate. The obtained aspartate aminotransferase preparation was free of alanine and aromatic amino acids aminotransferase activites and did not reveal malate dehydrogenase activity. 相似文献
10.
Y Horio Y Nishida R Sakakibara S Inagaki Y Kamisaki H Wada 《Biochemistry international》1988,16(3):579-586
Induction of cytosolic aspartate aminotransferase (glutamic oxaloacetic transaminase) was observed in rat liver on administration of a high-protein diet. The enzyme activity in the liver of rats given 60% and 80% protein diet increased to 1.8- and 1.9-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of functional sGOT mRNA, measured by in vitro translation. Whereas the activity of mitochondrial aspartate aminotransferase did not increase. Induction of cytosolic aspartate aminotransferase was also detected immunocytochemically. 相似文献
11.
12.
《Biochimica et Biophysica Acta (BBA) - Enzymology》1978,522(1):96-103
The reversible heat activation and cold inactivation of tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) of chick liver were investigated. When the enzyme obtained by gel filtration was preincubated at 37°C for 10 min with 50 μM pyridoxal 5′-phosphate (pryidoxal-5′-P), a 7-fold increase in enzyme activitiy was detected. When the preincubated enzyme was cooled to 0°C, it lost its activity. Furthermore, the dramatic cyclical changes in enzyme activity occurred by sequential heating at 37°C and cooling to 0°C of the enzyme, in the presence of pyridoxal-5′-P, over shorter periods of time without loss of enzyme activity. However, when α-ketoglutarate was added to the enzyme during cold exposure, no further decrease in activity was observed. This protective effect was seen at a concentration of 5 μM. 相似文献
13.
The development of tyrosine aminotransferase (TAT) activity in Xenopus laevis embryos was studied. Undivided eggs can transaminate tyrosine to some extent. The enzyme activity increases after hatching on the third day of development. In the early stages of development, the transamination of tyrosine is due to aspartate aminotransferase (ASAT, EC 2.6.1.1), both isoenzymes of which are present in the undivided egg. No specific TAT (EC 2.6.1.5) can be detected until the age of about 1 day, at which time neurulation is complete and the rapid development of the foregut and visceral pouches and arches has begun. The appearance of the enzyme is immediately preceded by a steep increase in the concentration of free tyrosine. Tyrosine aminotransferase is known to be induced by its substrate in the adult liver, and a similar effect may operate in the embryo. 相似文献
14.
Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase. 总被引:5,自引:1,他引:4
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The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes. 相似文献
15.
Possible involvement of cGMP in the control of tyrosine aminotransferase degradation in rat hepatocytes 总被引:1,自引:0,他引:1
Addition of theophylline to primary cultures of rat hepatocytes in which tyrosine aminotransferase had been preinduced with dexamethasone caused a further increase in specific activity of the enzyme. This increase was due in part to a reduction in the rate of tyrosine aminotransferase degradation that began about 2 hr after theophylline was added. The level of cGMP also increased with a similar time lag following the addition of theophylline. The concentration of theophylline which produced the above effects (1 mM) did not alter the rate of general protein degradation in hepatocytes. Addition of 8-bromo-cGMP (0.5 mM) resulted in an immediate reduction in the rate of tyrosine aminotransferase degradation and in an increase in the activity of the enzyme. Treating hepatocytes with MnCl2 (0.9 mM) caused an elevation of cGMP and a concomitant slowing of tyrosine aminotransferase degradation without changing the level of cAMP significantly. These results suggest an inverse relationship between the level of cGMP and the rate of tyrosine aminotransferase degradation in hepatocytes. 相似文献
16.
The subcellular distribution of asparagine:oxo-acid aminotransferase (EC 2.6.1.14) in rat liver was examined by centrifugation in a sucrose density gradient. About 30% of the homogenate activity after the removal of the nuclear fraction was recovered in the peroxisomes, about 56% in the mitochondria, and the remainder in the soluble fraction from broken peroxisomes. The mitochondrial asparagine aminotransferase had identical immunological properties with the peroxisomal one. Glucagon injection to rats resulted in the increase of its activity in the mitochondria but not in the peroxisomes. Immunological evidence was obtained that the enzyme was identical with alanine:glyoxylate aminotransferase 1 (EC 2.6.1.44) which had been reported to be identical with serine:pyruvate aminotransferase (EC 2.6.1.51) (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The same results as described above were obtained with mouse liver. All of alanine:glyoxylate aminotransferase 1 in livers of mammals other than rodents, which cross-react with the antibody against rat liver alanine:glyoxylate aminotransferase 1, had no asparagine aminotransferase activity. 相似文献
17.
Hosono A Mizuguchi H Hayashi H Goto M Miyahara I Hirotsu K Kagamiyama H 《Journal of biochemistry》2003,134(6):843-851
A subfamily I aminotransferase gene homologue containing an open reading frame encoding 381 amino acid residues (Mr=42,271) has been identified in the process of the genome project of an extremely thermophilic bacterium, Thermus thermophilus HB8. Alignment of the predicted amino acid sequence using FASTA shows that this protein is a member of aminotransferase subfamily Igamma. The protein shows around 40% identity with both T. thermophilus aspartate aminotransferase [EC 2.6.1.1] and mammalian glutamine:phenylpyruvate aminotransferase [EC 2.6.1.64]. The recombinant protein expressed in Escherichia coli is a homodimer with a subunit molecular weight of 42,000, has one pyridoxal 5'-phosphate per subunit, and is highly active toward glutamine, methionine, aromatic amino acids, and corresponding keto acids, but has no preference for alanine and dicarboxylic amino acids. These substrate specificities are similar to those described for mammalian glutamine: phenylpyruvate aminotransferase. This is the first enzyme reported so far that has the glutamine aminotransferase activity in non-eukaryotic cells. As the presence of aromatic amino acid:2-oxoglutarate aminotransferase [EC 2.6.1.57] has not been reported in T. thermophilus, this enzyme is expected to catalyze the last transamination step of phenylalanine and tyrosine biosynthesis. It may also be involved in the methionine regeneration pathway associated with polyamine biosynthesis. The enzyme shows a strikingly high pKa value (9.3) of the coenzyme Schiff base in comparison with other subfamily I aminotransferases. The origin of this unique pKa value and the substrate specificity is discussed based on the previous crystallographic data of T. thermophilus and E. coli aspartate aminotransferases. 相似文献
18.
1. Butan-1-ol solubilizes that portion of rat liver mitochondrial aspartate aminotransferase (EC 2.6.1.1) that cannot be solubilized by ultrasonics and other treatments. 2. A difference in electrophoretic mobilities, chromatographic behaviour and solubility characteristics between the enzymes solubilized by ultrasonic treatment and by butan-1-ol was observed, suggesting the occurrence of two forms of this enzyme in rat liver mitochondria. 3. Half the aspartate aminotransferase activity of rat kidney homogenate was present in a high-speed supernatant fraction, the remainder being in the mitochondria. 4. A considerable increase in aspartate aminotransferase activity was observed when kidney mitochondrial suspensions were treated with ultrasonics or detergents. 5. All the activity after maximum activation was recoverable in the supernatant after centrifugation at 105000g for 1hr. 6. The electrophoretic mobility of the kidney mitochondrial enzyme was cathodic and that of the supernatant enzyme anodic. 7. Cortisone administration increased the activities of both mitochondrial and supernatant aspartate aminotransferases of liver, but only that of the supernatant enzyme of kidney. 相似文献
19.
Genevieve Bourdel Anik Girard-Globa Marguerite Forestier Betty Gouhot-Nubel 《Biochimica et Biophysica Acta (BBA)/General Subjects》1975,399(2):339-344
Hepatic tyrosine aminotransferase (EC 2.6.1.5) was induced in rats by intubation of amino acid mixtures (complete or tryptophan-free). Enzyme activity was increased 4-fold by the complete mixture and 8-fold by the tryptophan-free mixture. The enzyme was analyzed by chromatography on CM-Sephadex. Chromatographic patterns were characteristic of the type of inducer rather than of the chronology of the induction cycle: after induction by the complete amino acid mixture the three forms of the enzyme were equally increased whereas after induction by the tryptophan-free mixture Form I was preferentially increased. 相似文献
20.
Anaerobic induction in conifers: Expression of endogenous and chimeric anaerobically-induced genes 总被引:1,自引:0,他引:1
A. G. Good F. Bekkaoui G. Pilate D. I. Dunstan W. L. Crosby 《Physiologia plantarum》1990,78(3):441-446
Alcohol dehydrogenase (ADH; EC 1.1.1.1) activity was measured in Picea glauca (Moench) Voss cell suspensions under differing conditions of hypoxia. ADH activity increased 4.5 fold after 48 h of induction. When cells were induced under different levels of hypoxia (2, 5 and 20% O2 ) changes in ADH activity were found to increase with lower levels of oxygen. Alanine aminotransferase (AlaAT; EC 2.6.1.2) activity increased under hypoxia in a pattern similar to ADH, however lactate dehydrogenase (LDH; EC 1.1.1.27) activity did not increase under hypoxic conditions. The ability of white spruce cells to accurately regulate heterologous anaerobic promotors was tested by electroporating chimeric ADH reporter genes into protoplasts. While protoplasts were capable of anaerobically regulating a maize ADH reporter construct, constructs with dicotyledonous promoters (pea and Arabidopsis ) were not expressed. 相似文献