Excess generation of endogenous heme inhibits L-alanine:4,5-dioxovalerate transaminase in rat liver mitochondria

In the present study, we examined the possibility that the excess heme generation within mitochondria may provide a local concentration, sufficient to inhibit the activity of L-alanine:4,5-dioxovalerate transaminase, the enzyme proposed for an alternate route of delta-aminolevulinic acid biosynthesis in mammalian system. This was accomplished by assaying together L-alanine:4,5-dioxovalerate transaminase and heme synthetase activities in intact mitochondria isolated from rat liver. Endogenous heme in intact mitochondria has been generated in excess, by increasing the concentration of the substrate of heme synthetase. Our studies showed that the activity of L-alanine:4,5-dioxovalerate transaminase decreased as the rate of heme formation increased. In intact mitochondria, almost 50% inhibition of alanine:4,5-dioxovalerate transaminase was obtained with 4.0 mumole of heme generation. We conclude that end product inhibition of L-alanine:4,5-dioxovalerate transaminase by hemin, which was proposed in earlier report by us (FEBS Letter (1985), 189, 129), is an important physiological mechanism for the regulation of hepatic heme biosynthesis.     

Purification and characterization of rat kidney L-alanine: 4,5-dioxovalerate transaminase and inhibition by hemin

Rat kidney L-alanine:4,5-dioxovalerate transaminase (EC 2.6.1.43), which may be involved in the formation of aminolevulinic acid in mammalian cells, was purified 82-fold to apparent homogeneity with a 19% yield. Molecular weight of the enzyme, as estimated by gel filtration, was found to be 225 000. In polyacrylamide gel electrophoresis under denaturing conditions, the enzyme moved as a single band corresponding to an Mr of 37 000, suggesting that the enzyme is composed of six identical subunits. The Km values of L-alanine and 4,5-dioxovalerate are 2.9 and 0.25 mM, respectively. The enzyme had an optimum activity at pH 6.6 and was most active at 65 degrees C. Among some amino acids tested, L-alanine proved to be the most efficient amino donor, and the enzyme was also stereospecific for the L-isomer. The effect of intermediate metabolites of heme biosynthesis, for example, delta-aminolevulinic acid, protoporphyrin, hemin and bilirubin has been studied on purified L-alanine:4,5-dioxovalerate transaminase. Amongst these metabolites, hemin and protoporphyrin were found to be effective inhibitors.     

Localization of L-alanine: 4,5-dioxovalerate transaminase in the matrix of the rat kidney mitochondria

The bulk of the enzyme L-alanine: 4,5-dioxovalerate transaminase, which catalyses the transamination reaction between L-alanine and 4,5-dioxovalerate to synthesize delta-aminolevulinic acid was predominantly recovered in the mitochondrial matrix. Sub-fractionation procedure of the mitochondria involved the use of digitonin and lubrol followed by differential centrifugation to separate soluble and particulate enzymes. Lubrol did not inhibit this enzyme. Presence of this enzyme in the mitochondrial matrix was further confirmed by western blot analysis. The results support the conclusion that L-alanine: 4,5-dioxovalerate transaminase is localized and functions in the mitochondrial matrix.     

Evidence of hemin as an end product inhibitor of L-alanine: 4,5-dioxovalerate transaminase in rat liver mitochondria

This study describes the in vitro and in vivo effect of hemin on L-alanine:4,5-dioxovalerate transaminase activity. Hemin was shown to be an inhibitor of the purified enzyme and this inhibition was proportional to the concentration of hemin. The examined kinetic data with hemin showed uncompetitive inhibition for both alanine and 4,5-dioxovalerate. An apparent Ki of 30 and 42 microM for hemin were obtained with both alanine and 4,5-dioxovalerate, respectively. Moreover, the enzyme activity in liver was considerably decreased after the intravenous hemin administration and such an inhibition is dose and time dependent. Furthermore, maximum inhibition of the enzyme was observed 30 min after hemin injection and 60% enzyme inhibition was achieved with a dose of 1.2 mg/kg body wt of rat. Thus is suggests the important role of this enzyme on heme biosynthesis.     

Role of L-alanine: 4,5-dioxovalerate transaminase in chlorophyll synthesis in Bajra (Pennisetum typhoideum) seedlings

L-alanine:4,5-dioxovalerate transaminase activity and chlorophyll levels were estimated in lead and mercury treated Bajra seedlings. The enzyme activity increased with age upto 2nd day of germination and decreased on consequent days, where as the chlorophyll content increased with age upto 4th day and remained constant on day 5. Both the metals have no effect on L-alanine: 4,5-dioxovalerate transaminase activity but reduced chlorophyll levels. In vitro incubation of the enzyme with metal solutions showed that the enzyme activity was inhibited by mercury, while lead had no effect. Studies on sub-cellular localization of the L-alanine:4,5-dioxovalerate transaminase showed that it is present in all fractions. The non-correlation between L-alanine: 4,5-dioxovalerate transaminase activity and chlorophyll synthesis is evident from different activity profiles with age and response to heavy metal treatment in the seedlings. Hence, our results suggest the non-involvement of L-alanine:4,5-dioxovalerate transaminase in chlorophyll synthesis in bajra seedlings.     

Affinity purification and properties of rat liver mitochondrial L-alanine:4,5-dioxovalerate transaminase and its inhibition by hemin

The present study describes a new rapid procedure for purification of L-alanine:4,5-dioxovalerate transaminase from rat liver mitochondria which was purified 243-fold with a 32% yield to apparent homogeneity. The purification procedure involved protamine sulfate treatment, followed by phenyl-Sepharose CL-4B column chromatography and alanine-Sepharose 4B affinity chromatography. The Km values for L-alanine and 4,5-dioxovalerate were 3.3 and 0.28 mM, respectively. The enzyme-bound pyridoxal phosphate content was estimated to be two molecules per enzyme molecule. The purified enzyme was inhibited by the reaction product pyruvic acid, substrate analog, methylglyoxal, and sulfhydryl inhibitors. Excess concentrations of 4,5-dioxovalerate was also found to inhibit the enzyme and our experiments failed to demonstrate reversibility of the reaction. Only hemin among the intermediate compounds of heme metabolism tested was shown to be an inhibitor of purified alanine:4,5-dioxovalerate transaminase. Hemin was further shown as an uncompetitive inhibitor of both alanine and dioxovalerate.     

L-alanine: 4,5-dioxovalerate aminotransferase from Pseudomonas riboflavina: purification and inactivation by methylglyoxal

L-Alanine:4,5-dioxovalerate aminotransferase, which catalyzes transamination between L-alanine and 4,5-dioxovalerate to yield delta-aminolevulinate and pyruvate, has been purified from Pseudomonas riboflavina IFO 3140. The enzyme had a molecular weight of 190,000 and consisted of four identical subunits. It was crystallized as pale yellow needles. The enzyme used L-alanine (relative activity 100), beta-alanine (39), and L-ornithine (14) as amino donors. gamma-aminobutyrate (55) and epsilon-aminocaproate (34) were also effective as amino donors. The reaction proceeded according to a ping-pong mechanism and the Km values for L-alanine and 4,5-dioxovalerate were 1.7 and 0.75 mM, respectively. The activity of the enzyme is strongly inhibited by pyruvate, hemin, and methylglyoxal. Methylglyoxal interacted with the enzyme and brought about a complete inactivation.     

Purification and properties of L-alanine:4,5-dioxovalerate aminotransferase from Chlorella regularis

The enzyme L-alanine:4,5-dioxovalerate aminotransferase (EC 2.6.1.43), which catalyzes the synthesis of 5-aminolevulinic acid, was purified 161-fold from Chlorella regularis. The enzyme also showed L-alanine:glyoxylate aminotransferase activity (EC 2.6.1.44). The activity of glyoxylate aminotransferase was 56-fold greater than that of 4,5-dioxovalerate aminotransferase. The ratio of the two activities remained nearly constant during purification, and when the enzyme was subjected to a variety of treatments. 4,5-Dioxovalerate aminotransferase activity was competitively inhibited by glyoxylate, with a Ki value of 0.5 mM. Double-reciprocal plots of velocity versus 4,5-dioxovalerate with varying L-alanine concentrations indicate a ping-pong reaction mechanism. The apparent Km values for 4,5-dioxovalerate and L-alanine were 0.12 and 3.5 mM, respectively. The enzyme is an acidic protein having an isoelectric point of 4.8. The molecular weight of the enzyme was estimated to be 126,000, with two identical subunits. These results suggest that, in Chlorella, as in bovine liver mitochondria and Euglena, both 4,5-dioxovalerate and glyoxylate aminotransferase activities are associated with the same protein. From the activity ratio of transamination and catalytic properties, it is concluded that this enzyme does not function primarily as a part of the 5-carbon pathway to 5-aminolevulinic acid synthesis.     

Purification and some properties of L-alanine:4,5-dioxovaleric acid transaminase from rat liver mitochondria

L-alanine:4,5-dioxovalerate transaminase (EC 2.6.1.44) has been purified to homogeneity from rat liver mitochondria. Molecular weight of the native enzyme is estimated to be 230,000 +/- 3000 by gel filtration. Under denaturing condition, the dissociated enzyme has a subunit of approximately 41,000 +/- 2000, indicating the enzyme apparently is composed of six identical subunits. The enzyme is heat stable and has optimal activity at pH 6.9. Km values for L-alanine and 4,5-dioxovalerate are 3.3 X 10(-3) M and 2.8 X 10(-4) M respectively. Excess dioxovalerate inhibits the enzyme activity. Pyridoxal phosphate and dithiothreitol also inhibit the enzyme activity.     

A Rapid, High-Yield Purification of L-Alanine:4,5-Dioxovalerate Transaminase from Rat Kidney Mitochondria Using an Improved Enzyme Assay Method

The present report documents an improved enzyme assay method for the mammalian L-alanine:4,5-dioxovalerate transaminase which is of significant utility in work with crude tissue homogenates, cell cultures, or purified enzyme preparations. We also describe a new and rapid purification procedure for this enzyme from rat kidney mitochondria. The three-step procedure involves the use of digitonin and lubrol for mitochondrial matrix preparation and L-alanine-Sepharose 4B column chromatography followed by gel filtration on Sepharose 6B. By this procedure it is possible to obtain a highly purified enzyme preparation in a relatively short time with a 37.5% yield.     

Response to light of a specific aminotransferase for delta-aminolevulinate formation in higher plants

It is thought that the C-5 pathway is the major, possibly the sole, route for the formation of delta-aminolevulinic acid for the biosynthesis of tetrapyrroles, including chlorophylls, in higher plants; a route involving 4,5-dioxovalerate as an intermediate followed by transamination to delta-aminolevulinic acid has been supported as one of the C-5 pathways (Granick, S., and Beale, S. I. (1978) Adv. Enzymol. Relat. Areas Mol. Biol. 46, 33-203). A specific aminotransferase for L-alanine and 4,5-dioxovalerate was found in the cucumber seeds. In dark-grown cucumber seedlings, alanine:4,5-dioxovalerate aminotransferase activity in the transitional region between shoot and root was remarkably high compared with that in the cotyledons. The exposure of the dark-grown seedlings to illumination resulted in a rapid and dramatic increase in the activity only in this transitional region. In contrast, the enzyme in the cotyledons, stem, and roots did not respond to illumination. After a 27-h illumination, the enzyme activity in the transitional region was 100-fold higher than that in the cotyledons. Other aminotransferases assayed in the transitional region did not respond to illumination. Alanine:4,5-dioxovalerate aminotransferase in the transitional region was also specific for L-alanine and 4,5-dioxovalerate.     

L-Alanine: 4,5-dioxovalerate transaminase does not regulate heme synthesis in rat liver

L-Alanine: 4,5-dioxovalerate transaminase (ADT) was determined in liver homogenates of rats treated by either inducers of porphyrin synthesis or the repressor, hemin. ADT activity was not induced by the porphyrinogenic agents nor reduced by hemin, indicating that ADT probably has no regulatory role in the heme synthesis pathway. The same conclusion was drawn from similar experiments performed in monolayers of chick embryo liver cells.     

Characterization of chicken liver L-alanine:4,5-dioxovaleric acid aminotransferase

1. A procedure is described for purifying the enzyme L-alanine:4,5-dioxovaleric acid aminotransferase (DOVA transaminase) from chicken liver. The enzyme catalyzes a transamination reaction between L-alanine and 4,5-dioxovaleric acid (DOVA), yielding delta-aminolevulinic acid (ALA). 2. In cell fractionation studies, DOVA transaminase activities were detected in mitochondria and in the post-mitochondrial supernatant fraction from liver homogenates. 3. For the mitochondrial enzyme, any of most L-amino acids could serve as a source for the amino group transferred to DOVA, but L-alanine appeared the preferred substrate. At pH 7.0, the enzyme had an apparent Km of 60 microM for DOVA and of 400 microM for L-alanine. 4. The enzyme was purified from disrupted mitoplasts in three steps: chromatography on DEAE-Sephacel, gel filtration through Sephadex G-150, and chromatography on hydroxyapatite. The yield was approx. 100 micrograms of enzyme protein per 10 g wet wt of liver. 5. The purified enzyme had a subunit mol. wt of 63,000 as determined by gel electrophoresis under denaturing conditions. 6. The activity of DOVA transaminase was also measured in embryonic chicken liver, and based on activity, the enzyme's capacity to produce ALA was significantly greater than that of ALA synthase. Unlike ALA synthase, however, DOVA transaminase activity did not increase in liver mitochondria of chicken embryos exposed for 18 hr to two potent porphyrogenic agents.     

Increased liver alanine in the tumour-bearing host. Altered levels of some enzymes involved in alanine metabolism

A 2-fold increase in hepatic alanine concentration was observed in rats bearing a Walker 256 carcinoma growing sub-cutaneously. Decreases were observed in the activities of both cytosolic and mitochondrial isozyme forms of L-alanine-2-oxoglutarate aminotransferase. Activities of two enzymes involved in a secondary pathway of haem synthesis involving alanine, L-alanine-4,5-dioxovalerate aminotransferase and the NADP-requiring isozyme form of 4-oxo-5-hydroxyvalerate dehydrogenase were also reduced but there was no change in liver porphyrin concentration. L-alanine-glyoxalate aminotransferase activity was unaffected. The results are discussed in relation to the utilisation of alanine as a gluconeogenic substrate in the tumor-bearing host.     

Trout (Salmo gairdneri R.) liver 5-aminolevulinate synthetase: subcellular distribution and properties

The subcellular distribution of trout liver 5-aminolevulinate synthetase has been studied. A cytosolic form of the enzyme has been found. Its activity was a 30% of the mitochondrial enzyme. The cytosolic form has a molecular weight of 110,000, larger than the mitochondrial enzyme (70,000). The two enzyme forms showed a pH optimum of 7.5. The kinetic characteristic of both forms suggest that the cytosolic form is a precursor of the mitochondrial ALA-synthetase.     

Isolation of glyoxalase II from bovine liver mitochondria

Bovine liver mitochondria contain about 10% of the total glyoxalase II activity in the homogenate. Electrophoresis and isoelectric focussing of either crude mitochondrial extract or the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms (pl 6.3, 6.7, 7.1, 7.7, and 7.9). Since bovine liver cytosol contains a single form of glyoxalase II (pl 7.5), at least four forms are exclusively mitochondrial with no counterpart in the cytosol. The relative molecular mass of mitochondrial glyoxalase II is about 23-24 kDa, similar to the cytosolic form. The kinetic constants obtained using S-D-lactoyl, S-acetyl-, S-acetoacetyl-, and S-succinyl-glutathione as substrates are similar to those reported for glyoxalase II from rat liver mitochondria. S-D-Lactoyl- and S-acetoacetyl-glutathione are the best substrates. S-Acetylglutathione is the poorest substrate with respect to both Vmax and Km values.     

The role of 4,5-dioxovaleric acid in porphyrin and vitamin B12 formation by clostridia

4,5-Dioxovalerate, which has been proposed as an intermediate in the newly discovered so-called C₅ pathway that leads from L-glutamate to δ-aminolevulinate, strongly inhibits uroporphyrin formation from δ-aminolevulinate in cells of $\text{Clostridium}\text{tetanomorphum}$ and in cell-free extracts of this organism, in spite of the presence of L-alanine: 4,5-dioxovalerate aminotransferase (aminolevulinate aminotransferase, EC 2.6.1.43). The interference by 4,5-dioxovalerate with porphyrin formation is due to strong inhibition of δ-aminolevulinate dehydratase (EC 4.2.1.24). Since 4,5-dioxovalerate hence effectively prevents the operation of the reaction sequence from L-glutamate to porphyrin, it is concluded that 4,5-dioxovalerate does not function as a physiological δ-aminolevulinate precursor.     

Isolation and characterization of mitochondrial alanine aminotransferase from porcine tissue.

Mitochondrial alanine aminotransferase L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) has been isolated in homogeneous form from both porcine liver and kidney cortex, but in low yield. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate or 8 M urea gave a single band. An isoelectric point of 8.5 +/- 0.5 and a molecular weight of 75--80 000 were obtained. The enzyme is specific for L-alanine and is inhibited by D-alanine, aminooxyacetate and cyclosterine. The Km for pyruvate and glutamate is 0.4 mM and 32 mM, respectively. These values are similar to those determined for the cytoplasmic enzyme; however, at high concentrations, both compounds strongly inhibit the mitochondrial enzyme, an inhibition not observed with cytosolic alanine aminotransferase. These characteristics and the fact that the mitochondrial alanine aminotransferase was inactivated by procedures effective in the preparation of the cytosolic enzyme, clearly differentiate the two proteins and further support different roles for the two alanine aminotransferases in vivo.     

Purification,characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17

A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37 degrees C, respectively. Pyruvate and pyridoxal 5'-phosphate increased enzyme stability whereas (S)-alpha-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues) showed significant homology with omega-amino acid:pyruvate transaminases (omega-APT) from various bacterial strains (80 identical residues with four omega-APTs). However, of 159 conserved residues in the four omega-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward beta-alanine (a typical amino donor for the omega-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date.