Production of delta-aminolevulinate: subcellular localization and purification of murine hepatic L-alanine: 4,5-dioxovaleric acid aminotransferase

1. L-Alanine: 4,5-dioxovaleric acid aminotransferase (DOVA transaminase) activity was measured in murine liver, kidney and spleen homogenates. 2. Among the organs examined, the specific activity of the enzyme was highest in kidney, followed by liver then spleen. 3. No differences in DOVA transaminase activity in kidney, liver and spleen homogenates were detected between mouse strains C57BL/6J and DBA/2J. 4. Based on enzyme activity, the capacity of DOVA transaminase to catalyze the formation of delta-aminolevulinic acid (ALA) in liver appeared much greater than the capacity of ALA synthase. 5. In DBA/2J animals, DOVA transaminase activity in liver mitochondrial fractions prepared by differential centrifugation was 24 nmol ALA formed/hr/mg protein compared with 0.63 nmol ALA formed/hr/mg protein for ALA synthase. 6. Cell fractionation analyses indicated that liver DOVA transaminase is located in the mitochondrial matrix. 7. The liver enzyme was purified from mitoplasts by chromatography on DEAE-Sephacel followed by affinity chromatography on L-alanine-AH-Sepharose. 8. The specific activity of the purified DOVA transaminase was 1600 nmol ALA formed/hr/mg protein. 9. The yield of the purification was ca 90 micrograms of protein per gram liver wet weight. 10. The purified enzyme had a subunit mol. wt of 146,000 +/- 5000 as determined by electrophoresis under denaturing conditions.     

Production of delta-aminolevulinic acid: characterization of murine liver 4,5-dioxovaleric acid: L-alanine aminotransferase.

1. We report on the kinetic properties of murine liver 4,5-dioxovaleric acid:L-alanine aminotransferase (DOVA transaminase). 2. The transamination of 4,5-dioxovaleric acid (DOVA) led to the production of delta-aminolevulinic acid. 3. L-Alanine was the preferred amino group donor among the common 20 amino acids. 4. The optimum pH of the reaction was 7-8. 5. A Km of 220 microM for DOVA and a Km of 970 microM for L-alanine were obtained. 6. The reaction was inhibited by each of the following: glyoxylate, beta-chloroalanine, methylglyoxal, delta-aminolevulinate, pyruvate, heme, and gabaculine. 7. None of several xenobiotic inducers of microsomal mixed function oxidases tested had a significant effect on DOVA transaminase activity in studies performed with murine primary hepatocyte cultures.     

The determination of 4,5-dioxovaleric acid in plant preparations and a procedure for the assay of l-alanine:4,5-dioxovaleric acid aminotransferase (EC 2.6.1.43) activity

Usually, 4,5-dioxovaleric acid (DOVA) is determined in biological materials by measuring the absorption at 269 nm of its benzoquinoxaline derivative which is formed by condensation with 2,3-diaminonaphthalene (DAN). Not only must this benzoquinoxaline be separated from unreacted DAN and flavins which have interfering uv absorption but, when working with higher-plant tissues, additional interfering compounds with uv absorption, ionic and solubility properties similar to polyphenols must also be removed. The separation of the DOVA-derived benzoquinoxaline from all these interfering compounds by a series of solvent extractions utilizing the difference in ionic behaviour of the benzoquinoxaline and the interfering contaminants is described.It was found that a small but significant amount of a benzoquinoxaline is formed when 5-aminolaevulinic acid (ALA) was incubated with DAN at pH 8 at 60°C and was due to the prior non-enzymic deamination of a small portion of ALA to DOVA: this benzoquinoxaline was spectrophotometrically, spectrofluorimetrically, and chromatographically indistinguishable from that formed by the condensation of DOVA and DAN. Since formation of this benzoquinoxaline interferes with the assay of l-alanine:4,5-dioxovaleric acid aminotransferase (EC 2.6.1.43), a procedure to measure DOVA formed by this enzyme from ALA and pyruvate is described in which the DOVA is first separated from the ALA by ion-exchange chromatography prior to condensation with DAN: this method permits the separate determination of both DOVA and ALA concentrations in the aminotransferase reaction mixture.     

Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves : I. Formation from 2-Ketoglutarate via 4,5-Dioxovaleric Acid

Cell-free extracts from greening maize (Zea mays L.) leaves catalyze the conversion of [¹⁴C]2-ketoglutarate (KG) to [¹⁴C]5-aminolevulinic acid (ALA) in a reaction which requires NADH and an amino donor and shows maximal activity around pH 6.5. The enzymic system is located in the cytosol. This cell fraction contains a low level of `KG dehydrogenase' activity and a transaminase which catalyzes the conversion of 4,5-dioxovaleric acid (DOVA) to ALA. The transaminase can use glutamate, aspartate, or alanine as amino donor. It is effectively inhibited by aminooxyacetate and ethylenediamine tetraacetate and shows maximal activity at pH 6.7. The activity of DOVA transaminase is only slightly affected by preillumination of leaves and can also be detected in green leaves and in roots.

DOVA was isolated from leaves and roots and determined as its benzoquinoxaline derivative. Significant amounts were found only in tissues in which ALA had accumulated or after it was exogenously supplied. DOVA was labeled in vivo by both [¹⁴C]ALA and [¹⁴C]KG. Small amounts were also formed from ALA in a cell-free system.

It is suggested that DOVA may be an intermediate in the diversion of ALA to respiratory metabolism and that it is not involved in the biosynthesis of this porphyrin precursor.

     

The effect of glyoxalase I on the metabolism of 4,5-dioxovaleric acid

Biosynthesis of 5-aminolevulinic acid in mammalian cells is catalyzed by aminolevulinic acid synthase in a condensation reaction utilizing glycine and succinyl X coenzyme A. An alternate pathway in mammalian cells may involve the biosynthesis of aminolevulinic acid via a transamination reaction in which L-alanine is the amino donor and 4,5-dioxovaleric acid is the acceptor. This transamination reaction, or one very similar, is employed by plants for the biosynthesis of aminolevulinic acid which is ultimately converted to chlorophyll. The effect of glyoxalase I on the diversion of dioxovaleric acid to other products was tested using both purified glyoxalase I and crude tissue homogenates. Glyoxalase I is a metalloenzyme and glutathione is a co-substrate. Purified glyoxalase I reduced the amount of aminolevulinic acid formed in the presence of dioxovaleric acid, L-alanine, glutathione, and purified L-alanine: 4,5-dioxovaleric acid aminotransferase (dioxovalerate transaminase). The conversion of dioxovaleric acid to aminolevulinic acid was inhibited by the addition of glutathione when a dialyzed bovine liver homogenate served as the source of both glyoxalase I and dioxovalerate transaminase. Removal of metals from bovine liver homogenates produced an 85% decrease in glyoxalase I activity. These 'metal-free' homogenates still affected the conversion of dioxovaleric acid to aminolevulinic acid after preincubation with MgSO4. The effect of glyoxalase I on the metabolism of dioxovaleric acid was also studied using a fluorometric enzyme assay for the quantification of dioxovaleric acid via a coupled enzyme reaction converting it to uroporphyrin. Homogenates of both liver and barley diminished the amount of dioxovaleric acid detected by the coupled assay, but this effect could be prevented by dialysis of the homogenates. Addition of glutathione to dialyzed homogenates markedly reduced the amount of uroporphyrin generated from dioxovaleric acid. Metal-free homogenates supplemented with glutathione reduced the conversion of dioxovaleric acid to uroporphyrin in the coupled assay, but preincubation with MgSO4 greatly augmented this effect. These studies point out the difficulty in evaluating dioxovaleric acid as a heme precursor using whole cell homogenates.     

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.     

Measurement of 4,5-dioxovaleric acid by high-performance liquid chromatography and fluorescence detection

In this work we describe a sensitive method for the detection of 4,5-dioxovaleric acid (DOVA). 4,5-Dioxovaleric acid is derivatized with 2,3-diaminonaphthalene to form 3-(benzoquinoxalinyl-2)propionic acid (BZQ), a product with favorable UV absorbance and fluorescence properties. The high-performance liquid chromatographic method with UV absorbance and fluorescence detection is simple and its detection limit is approximately 100 fmol. This method was used to detect 4,5-dioxovaleric acid formation during metal-catalyzed 5-aminolevulinic acid (ALA) oxidation. Iron and ferritin were active in the formation of 4,5-dioxovaleric acid in the presence of 5-aminolevulinic acid. In addition, HPLC–MS–MS assay was used to characterize BZQ. The determination of 4,5-dioxovaleric acid is of great interest for the study of the mechanism of the metal-catalyzed damage of biomolecules by 5-aminolevulinic acid. This reaction may play a role in carcinogenesis after lead intoxication. The high frequency of liver cancer in acute intermittent porphyria patients may also be due to this reaction.     

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.     

5-Aminolevulinic acid synthesis in epimastigotes of Trypanosoma cruzi

BACKGROUND AND AIMS: Trypanosoma cruzi is the causative agent of Chagas disease or American trypanosomiasis. The parasite manifests a nutritional requirement for heme compounds because of its biosynthesis deficiency. The aim of this study has been to investigate the presence of metabolites and enzymes of porphyrin pathway, as well as ALA formation in epimastigotes of T. cruzi, Tulahuén strain, Tul 2 stock. METHODS: Succinyl CoA synthetase, 5-aminolevulinic acid (ALA) synthetase, 4,5-dioxovaleric (DOVA) transaminase, ALA dehydratase and porphobilinogenase activities, as well as ALA, porphobilinogen (PBG), free porphyrins and heme content were measured in a parasite cells-free extract. Extracellular content of these metabolites was also determined. RESULTS: DOVA, PBG, porphyrins and heme were not detected in acellular extracts of T. cruzi. However ALA was detected both intra- and extracellularly This is the first time that the presence of ALA (98% of intracellularly formed ALA) is demonstrated in the extracellular medium of a parasite culture. Regarding the ALA synthesizing enzymes, DOVA transaminase levels found were low (7.13+/-0.49EU/mg protein), whilst ALA synthetase (ALA-S) activity was undetectable. A compound of non-protein nature, low molecular weight, heat unstable, inhibiting bacterial ALA-S activity was detected in an acellular extract of T. cruzi. This inhibitor could not be identified with either ALA, DOVA or heme. CONCLUSIONS: ALA synthesis is functional in the parasite and it would be regulated by the heme levels, both directly and through the inhibitor factor detected. ALA formed can not be metabolized further, because the necessary enzymes are not active, therefore it should be excreted to avoid intracellular cytotoxicity.     

Purification and Characterization of 4,5-Dioxovalerate Reductase (NADPH) from Chlorella regularis

Two types of 4,5-dioxovalerate reductases (NADPH) were partiallypurified and characterized from green alga, Chlorella regularis.The enzyme was separated by DEAE-Sephacel chromatography intotwo peaks: type I (first peak) and type II (second peak). Theactivity ratio of the type II to type I enzyme varied between5 to 7 with a starting cell material. Both enzymes had the samepH optimum at 6.0 and pI value of 4.9. The molecular weightestimated by gel filtration was 33,000 for type I and 99,000for type II enzyme. Both enzymes used only NADPH, but were notspecific for 4,5-dioxovaleric acid (DOVA). Type I enzyme reducedglyoxylate 68-fold faster than DOVA, whereas type II enzymeacted more specifically on a variety of aldehydes than DOVA.It is suggested that these enzymes may not function primarilyas NADPH-DOVA reductases in the metabolic pathway of DOVA. (Received June 15, 1985; Accepted October 14, 1985)     

Glutamate:4,5-dioxovaleric acid transaminase from Euglena gracilis. Kinetic studies

The kinetic properties of the enzyme L-glutamate:4,5-dioxovaleric acid aminotransferase (Glu:DOVA transaminase) from Euglena gracilis have been studied. 5-Aminolevulinic acid formation was linear with time for at least 45 min at 37 degrees C and L-glutamate was the most effective amino-group donor. Lineweaver-Burk double-reciprocal plots suggested a ping-pong reaction mechanism, with Km values for L-glutamate and DOVA of 1.92 mM and 0.48 mM respectively. Competitive parabolic substrate inhibition by DOVA at concentrations greater than 3.5-4.5 mM was observed. Glyoxylate (4-10 mM) was found to be a competitive inhibitor with respect to DOVA, whereas at low concentrations (0-4 mM) noncompetitive plots were obtained. An analysis of the possible enzyme forms involved, was carried out. In more crude preparations most of the enzyme is found to be in the form of an enzyme-glutamate complex.     

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.     

delta-Aminolevulinic Acid Formation from gamma,delta-Dioxovaleric Acid in Extracts of Euglena gracilis

γ,δ-Dioxovaleric acid (DOVA) has been proposed as a precursor to heme and chlorophyll in plants and algae. DOVA transaminase activity was found in extracts of the unicellular green alga Euglena gracilis Klebs strain Z Pringsheim. Optimum conversion of DOVA to δ-aminolevulinic acid (ALA) occurred at pH 6.8. ALA formation was linear with time for at least 30 minutes at 37° C and was proportional to amount of cell extract in the incubation mixture. Boiled cell extract was inactive. DOVA transaminase from either wild-type or aplastidic derivative strain W₁₄ZNaIL ran as a single band in agarose gel permeation chromatography, with a calculated molecular weight of 98,000 ± 3,000. l-Glutamic acid was the most effective amino donor. d-Glutamic acid was inactive. K_m values for l-glutamic acid and DOVA were 11 and 1.1 millimolar, respectively. Pyridoxal phosphate stimulated activity maximally at 30 micromolar, and (aminooxy)acetate was strongly inhibitory. Glyoxylic acid was a competitive inhibitor with respect to DOVA, with an inhibition constant of 0.62 millimolar. Wild-type and aplastidic cells vielded equal activity, 31 ± 1 nanomoles ALA per 30 minutes per 10⁷ cells, whether grown in light or dark. DOVA transaminase could not be separated from glyoxylate transaminase activity by agarose gel permeation or diethylaminoethyl-cellulose column chromatography. In all fractions, glyoxylate transaminase activity was at least 75 times greater than DOVA transaminase activity. DOVA transamination appears to be catalyzed by glyoxylate transaminase, and not to be of physiological significance with respect to chlorophyll synthesis in Euglena.     

Activity and spectroscopic properties of the Escherichia coli glutamate 1-semialdehyde aminotransferase and the putative active site mutant K265R.

Glutamate 1-semialdehyde aminotransferase (glutamate 1-semialdehyde 2,1-aminomutase; EC 5.4.3.8; GSA-AT) catalyzes the transfer of the amino group on carbon 2 of glutamate 1-semialdehyde (GSA) to the neighboring carbon 1 to form delta-aminolevulinic acid (ALA). To gain insight into the mechanism of this enzyme, possible intermediates were tested with purified enzyme and the reaction sequence was followed spectroscopically. While 4,5-dioxovaleric acid (DOVA) was efficiently converted to ALA by the pyridoxamine 5'-phosphate (PMP) form of the enzyme, 4,5-diaminovaleric acid (DAVA) was a substrate for the pyridoxal 5'-phosphate (PLP) form of GSA-AT. Thus, both substances are reaction intermediates. The purified enzyme showed an absorption spectrum with a peak around 338 nm. Addition of PLP led to increased absorption at 338 nm and a new peak around 438 nm. Incubation of the purified enzyme with PMP resulted in an additional absorption peak at 350 nm. The reaction of the PLP and PMP form of the enzyme with GSA allowed the detection of a series of peaks which varied in their intensities in a time-dependent manner. The most drastic changes to the spectrum that were observed during the reaction sequence were at 495 and 540 nm. Some of the detected absorption bands during GSA-AT catalysis were previously described for several other aminotransferases, indicating the relationship of the mechanisms. The reaction of the PMP form of the enzyme with DOVA resulted in a similar spectrum as described above, while the spectrum for the conversion of DAVA by the PLP form of the enzyme indicated a different mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA damage by 5-aminolevulinic and 4,5-dioxovaleric acids in the presence of ferritin

Cellular accumulation of 5-aminolevulinic acid (ALA), the first specific intermediate of heme biosynthesis, is correlated in liver biopsy samples of acute intermittent porphyria affected patients with an increase in the occurrence of hepatic cancers and the formation of ferritin deposits in hepatocytes. 5-Aminolevulinic acid is able to undergo enolization and to be subsequently oxidized in a reaction catalyzed by iron complexes yielding 4,5-dioxovaleric acid (DOVA). The released superoxide radical (O(*-)(2)) is involved in the formation of reactive hydroxyl radical ((*)OH) or related species arising from a Fenton-type reaction mediated by Fe(II) and Cu(I). This leads to DNA oxidation. The metal catalyzed oxidation of ALA may be exalted by the O(*-)(2) and enoyl radical-mediated release of Fe(II) ions from ferritin. We report here the potentiating effect of ferritin on the ALA-mediated cleavage of plasmid DNA and the enhancement of the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodGuo). Plasmid pBR322 was incubated with ALA and varying amounts of purified ferritin. DNA damage was assessed by gel electrophoresis analysis of the open and the linear forms of the plasmid from the native supercoiled structure. Addition of either the DNA compacting polyamine spermidine or the metal chelator ethylenediaminetetraacetic acid (EDTA) inhibited the damage. It was also shown that ALA in the presence of ferritin is able to increase the oxidation of the guanine moiety of monomeric 2'-deoxyguanosine (dGuo) and calf thymus DNA (CTDNA) to form 8-oxodGuo as inferred from high performance liquid chromatography (HPLC) measurements using electrochemical detection. The formation of the adduct dGuo-DOVA was detected in CTDNA upon incubation with ALA and ferritin. In a subsequent investigation, the aldehyde DOVA was also able to induces strand breaks in pBR322 DNA.     

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.     

Chemical synthesis of 4,5-dioxovaleric acid and its nonenzymatic transamination to 5-aminolevulinic acid

4,5-Dioxovaleric acid (DOVA) was synthesized from 5-bromolevulinic acid via formation of the pyridinium bromide of 5-bromolevulinic acid, followed by nitrone formation with p-nitrosodimethylaniline, and hydrolysis of the nitrone to yield DOVA. Partial purification of DOVA was obtained by passage of the reaction mixture through a cation exchange column. DOVA was identified by paper electrophoresis and by a specific fluorometric assay. DOVA was nonenzymatically transaminated to 5-aminolevulinic acid (ALA) with glycine serving as the amino donor. Other compounds tested were less effective amino donors. Glyoxylic acid was identified as a reaction product by paper electrophoresis and a specific calorimetric test. ALA was identified by paper electrophoresis, paper chromatography of a pyrrole derivative, reaction with Ehrlich reagent, and by its enzymatic conversion by a barley extract to porphobilinogen and uroporphyrin. The nonenzymatic transamination was inhibited by Tris and was stimulated by high pH. The existence of this nonenzymatic activity is discussed in relation to previous reports of dova transaminase activity in cell extracts.     

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.     

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.