Tauropine dehydrogenase from the starfish Asterina pectinifera (Echinodermata: Asteroidea): presence of opine production pathway in a deuterostome invertebrate

Tauropine dehydrogenase (tauropine:NAD oxidoreductase; TaDH) was purified to homogeneity from the body wall of the starfish Asterina pectinifera Müller at Troschel(Echinodermata: Asteroidea) by means of (NH4)2SO4 precipitation followed by column chromatographies in DEAE-cellulose, Sephadex G75, Macro-prep ceramic hydroxyapatite, PBE 94, and Toyopearl HW50S. The enzyme was a monomeric protein of approximately 42000 Da and pI 5.2. The maximum rate of the tauropine biosynthetic reaction was observed at pH 6.0, and that of the tauropine catabolic reaction was at pH 8.7-9.2. Taurine and pyruvate were the preferred substrates. The tauropine catabolic reaction was inhibited by the substrate tauropine: the peak rate was observed at 12.5 mM. Apparent Km values for NADH, taurine, and pyruvate were 0.036 +/- 0.002, 21.3 +/- 1.6, and 0.46 +/- 0.02 mM, respectively, and for tauropine and NAD+ were 2.64 +/- 0.73 and 0.068 +/- 0.005 mM, respectively. The molecular and catalytic properties of the starfish TaDH were basically similar to those of TaDH from other species belonging to the lower invertebrate phyla and the middle phyla of Prostostomia. Tauropine accumulation in vivo during experimental anoxia was also demonstrated. These results gave clear evidence of opine production pathway in deutrostome invertebrate.     

Purification and properties of tauropine dehydrogenase from a red alga,Rhodoglossum japonicum

Tauropine dehydrogenase which is a member of opine dehydrogenases and catalyzes the reductive condensation of taurine with pyruvate was purified from a red alga, Rhodoglossum japonicum using a combination of ammonium sulfate fractionation, gel filtration, affinity, and ion exchange chromatography. The molecular mass of this enzyme, obtained by HPLC using TSK SW2000G in its native form and SDS-PAGE in its denatured form, was 39000 and 42000, respectively. This means tauropine dehydrogenase has monomeric structure like other opine dehydrogenases. The relative activities for amino acids as substrate were 100 for taurine, 17 for valine and 12 for homotaurine. The apparent Km values for taurine, pyruvate and NADH were 15.0 mM, 0.80 mM and 0.04 mM, and for tauropine and NAD⁺ were 30 mM and 0.12 mM, respectively. Diurnal change of tauropine content was observed in R. japonicum, tauropine increased in the daytime and decreased in the nighttime.     

Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa

Tauropine dehydrogenase (tauropine:NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co-enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N-(D-1-carboxyethyl)-taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion-exchange and affinity chromatography. The relative molecular mass was 38,000 +/- 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42,000 +/- 150 by electrophoresis on 5-10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the oxo acids tested, 2-oxobutyrate and 2-oxovalerate were also found to be substrates. Apparent Km values for the substrates NADH, pyruvate and taurine are 0.022 +/- 0.003 mM, 0.64 +/- 0.07 mM and 64.7 +/- 5.4 mM, respectively, at pH 7.0 and for the products, NAD+ and tauropine, are 0.29 +/- 0.01 mM and 9.04 +/- 1.27 mM, respectively, at pH 8.3. Apparent Km values for both pyruvate and taurine decrease with increasing co-substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD+ was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non-competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L-lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.     

A biocatalyst for pyruvate preparation from -lactate: lactate oxidase in a Pseudomonas sp.

For the purpose of producing pyruvate from -lactate by enzymatic methods, four microorganism strains that produce lactate oxidase (LOD) were screened and isolated from many soil samples. Among them, strain SM-6, which showed high potential for pyruvate production, was chosen for further research. Physiological studies and 16S rDNA relationship reveal that SM-6 belongs to Pseudomonas putida. The optimized pH and temperature of the enzyme-catalyzed reaction were pH 7.2, and 39 °C, respectively. Low-concentration EDTA (1 mM) could improve the stability of pyruvate and conversion ratio of lactate oxidase. V_max and K_m value for -lactate were 2.46 μmol/(min mg) protein and 9.53 mM, respectively. On preparation scale, cell-free extract from SM-6, containing 300 mg/l of crude enzyme (4037 U/ml lactate oxidase), could convert 66% of 116 mM of -lactate into 76.6 mM pyruvate in 18 h, and 82% of substrate was transformed after 48 h, giving 95.0 mM (10.5 mg/ml) of pyruvate. The ratio of product to biocatalyst was 34.8:1 (g/g).     

Morpholine-induced formation of l-alanine dehydrogenase activity in Mycobacterium strain HE5

An NAD-dependent, morpholine-stimulated l-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4.6-fold increase of l-alanine dehydrogenase activity when l-alanine was supplied at suboptimal concentration. l-Alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of l-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent K _m values for l-alanine and NAD were 1.0 and 0.2 mM, respectively. K _m values of 0.6, 0.02, and 72 mM for pyruvate, NADH, and NH₄ ⁺, respectively, were estimated at pH 8.7 for the reductive amination reaction. Received: 25 September 1998 / Accepted: 11 March 1999     

Alanine dehydrogenase of the β-lactam antibiotic producer Streptomyces clavuligerus

l-Alanine dehydrogenase was found in extracts of the antibiotic producer Streptomyces clavuligerus. The enzyme was induced by ammonia, and the level of induction was dependend on the extracellular concentration. l-Alanine was the only amino acid able to induce alanine dehydrogenase. The enzyme was characterized from a 38-fold purified preparation. Pyruvate (K _m =1.1 mM), ammonia (K _m =20 mM) and NADH (K _m =0.14 mM) were required for the reductive amination, and l-alanine (K _m =9.1 mM) and NAD (K _m =0.5 mM) for the oxidative deaminating reaction. The aminating reaction was inhibited by alanine, serine and NADPH. Alanine inhibited uncompetitively with respect to NADH (K _i =1.6 mM) and noncompetitively with respect to ammonia (K _i =2.0 mM) and pyruvate (K _i =3.0 mM). In the aminating reaction 3-hydroxypyruvate, glyoxylate and 2-oxobutyrate could partially (6–7%) substitute pyruvate. Alanine dehydrogenase from S. clavuligerus differed with respect to its molecular weight (92000) and its kinetic properties from those described for other microorganisms.Abbreviation Alanine-DH l-alanine:NAD oxidoreductase     

Purification and partial characterization ofd-(−)-lactate dehydrogenase fromLactobacillus helveticus CNRZ 32

Summary d-(–)-Lactate dehydrogenase (LDH) was purified to homogeneity from a cell-free extract ofLactobacillus helveticus CNRZ 32. The native enzyme was determined to have a molecular weight of 152 000 and consisted of four identical subunits of 38 000. This enzyme was NAD dependent fructose 1,6-diphosphate (FDP) and ATP independent. It was most active on pyruvate followed by -hydroxypyruvate as substrates. TheK _m values for pyruvate andd-(–)-lactate were 0.64 and 68.42 mM respectively, indicating that the enzyme has a higher affinity for pyruvate. The enzyme activity was completely inhibited byp-chloromercuribenzoate (1 mM) and partially by iodoacetate, suggesting the involvement of the sulfhydryl group (-SH) in catalysis. Optima for activity by the purified enzyme were pH 4.0 and 50–60°C. Limited inhibition ofd-(–)-LDH was observed with several divalent cations. Additionally, HgCl₂ was observed to strongly inhibit enzyme activity. The purified enzyme was not affected by dithiothreitol or any of the metal chelating agents examined.     

Biochemical and molecular characterization of taurine:pyruvate aminotransferase from the anaerobe Bilophila wadsworthia.

Bilophila wadsworthia RZATAU is a Gram-negative bacterium which converts the sulfonate taurine (2-aminoethanesulfonate) to ammonia, acetate and sulfide in an anaerobic respiration. Taurine:pyruvate aminotransferase (Tpa) catalyses the initial metabolic reaction yielding alanine and sulfoacetaldehyde. We purified Tpa 72-fold to apparent homogeneity with an overall yield of 89%. The purified enzyme did not require addition of pyridoxal 5'-phosphate, but highly active enzyme was only obtained by addition of pyridoxal 5'-phosphate to all buffers during purification. SDS/PAGE revealed a single protein band with a molecular mass of 51 kDa. The apparent molecular mass of the native enzyme was 197 kDa as determined by gel filtration, which indicates a homotetrameric structure. The kinetic constants for taurine were: Km = 7.1 mM, Vmax = 1.20 nmol.s-1, and for pyruvate: Km = 0.82 mM, Vmax = 0.17 nmol.s-1. The purified enzyme was able to transaminate hypotaurine (2-aminosulfinate), taurine, beta-alanine and with low activity cysteine and 3-aminopropanesulfonate. In addition to pyruvate, 2-ketobutyrate and oxaloacetate were utilized as amino group acceptors. We have sequenced the encoding gene (tpa). It encoded a 50-kDa peptide, which revealed 33% identity to diaminopelargonate aminotransferase from Bacillus subtilis.     

NADP+-specific 2-oxoglutarate dehydrogenase in denitrifying Pseudomonas species

Several denitrifying Pseudomonas strains contained an NADP⁺-specific 2-oxoglutarate dehydrogenase, in contrast to an NAD⁺-specific pyruvate dehydrogenase, if the cells were grown anaerobically with aromatic compounds. With non-aromatic substrates or after aerobic growth the coenzyme specificity of 2-oxoglutarate dehydrogenase changed to NAD⁺-specificity. The reaction stoichiometry and the apparent K _m-values of the enriched enzymes were determined: pyruvate 0.5 mM, coenzyme A 0.05 mM, NAD⁺ 0.25 mM; 2-oxoglutarate 0.6 mM, coenzyme A 0.05 mM, NADP⁺ 0.03 mM. Isocitrate dehydrogenase was NADP⁺-specific. The findings suggest that these strains contained at least two lipoamide dehydrogenases, one NAD⁺-specific, the other NADP⁺-specific.     

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K _M values were 5.0 mM for L-alanine and 0.11 mM for NAD⁺. K _M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH₄Cl.Abbreviation AlaDH alanine dehydrogenase     

Unusually stable NAD-specific glutamate dehydrogenase from the alkaliphile Amphibacillus xylanus

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GDH; EC 1.4.1.3) from Amphibacillus xylanus DSM 6626 was enriched 100-fold to homogeneity. The molecular mass was determined by native polyacrylamide electrophoresis and by gel filtration to be 260 kDa (±25 kDa); the enzyme was composed of identical subunits of 45 (±5) kDa, indicating that the native enzyme has a hexameric structure. NAD-GDH was highly specific for the coenzyme NAD(H) and catalyzed both the formation and the oxidation of glutamate. Apparent K _m -values of 56 mM glutamate, 0.35 mM NAD (oxidative deamination) and 6.7 mM 2-oxoglutaric acid, 42 mM NH₄Cl and 0.036 mM NADH (reductive amination) were measured. The enzyme was unusually resistant towards variation of pH, chaotropic agents, organic solvents, and was stable at elevated temperature, retaining 50% activity after 120 min incubation at 85°C.     

Kinetic Studies on the Inhibition of GABA-T by γ-Vinyl GABA and Taurine

γ-Aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of γ-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: γ-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by γ-vinyl GABA and taurine at concentrations of 51.0 and 78.5?mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (K_i) values for γ-vinyl GABA and taurine were found to be 26±3?mM and 68±7?mM respectively. The transamination process of α-ketoglutarate was not affected by the presence of γ-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when α-ketoglutarate was the substrate. The inhibition dissociation constant (K_ii) for this system was found to be 96±10?mM. The Michaelis-Menten constant (K_m) in the absence of inhibition, was found to be 0.79±0.11?mM, and 0.47±0.10?mM for GABA and α-ketoglutarate respectively.     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.     

Purification and properties of the coenzyme A-linked acetaldehyde dehydrogenase of Acetobacterium woodii

Coenzyme A-linked acetaldehyde dehydrogenase (ACDH) of ethanol-grown cells of Acetobacterium woodii was purified to apparent homogeneity; a 28-fold purification was achieved with 13% yield. The enzyme proved to be oxygen-sensitive and was inactive in the absence of dithioerythritol. During the purification procedure addition of 1 mM MgCl₂ was necessary to maintain enzyme activity. Alcohol dehydrogenase (ADH) activity was separated from ACDH during anion exchange chromatography using DEAE Sephacel. A part of the ACDH activity coeluted with ADH, but both could be separately eluted from a Cibacron Blue 3GA-Agarose column, revealing the same subunit structure and activity band for ACDH as found before and, thus, indicating an aggregation of the enzyme. The remaining ADH activity could be separated by gel filtration. For the native ACDH a molecular mass of 255 kDa was determined by polyacrylamide gel electrophoresis and of 272 kDa by gel filtration using Superose 12. The enzyme subunit sizes were 28 kDa and 40 kDa, respectively, indicating a ₄₄ structure for the active form. The enzyme catalyzed the oxidation of several straight chain aldehydes although it was most active with acetaldehyde. NADH strongly inhibited oxidation of acetaldehyde whereas NADPH had no effect. The inhibition was noncompetitive.Non-standard abbrevations ACDH acetaldehyde dehydrogenase - ADH alcohol dehydrogenase - CHES 2-(N-cyclohexylamino)-ethanesulfonate - DTE dithioerythritol - KP-buffer 25 mM K-PO₄, pH 7.5, containing, 4 mM DTE - MES 2-(N-morpholino)-ethanesulfonate - TAPS N-Tris-(hydroxymethyl)-methyl-3-aminopropa-nesulfonate     

Purification and kinetic properties of skeletal muscle lactate dehydrogenase from the lizard Agama stellio stellio

Lactate dehydrogenase isoenzyme LDH-5 (M4) was purified to homogeneity from the skeletal muscle of lizard Agama stellio stellio as a poikilothermic animal, using colchicine-Sepharose chromatography and heat inactivation. The purified enzyme showed a single band after SDS-PAGE, corresponding to a molecular weight of 36 kD. The K _m values for pyruvate, NADH, lactate, and NAD⁺ were 0.020, 0.040, 8.1, and 0.02 mM, respectively. Pyruvate showed maximum activity at about 180 M, with a decline at higher concentrations. The enzyme was stable at 70°C for 30 min, but was rapidly inactivated at 90°C. The optimum pH for the forward reaction (pyruvate to lactate) was 7.5, and for the reverse reaction (lactate to pyruvate) was 9.2. Oxalate, glutamate, Cu²⁺, Co²⁺, Mn²⁺, and Mg²⁺ were inhibitory in both forward and reverse reactions.     

Purification and characterization of pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an αβγδ-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80° C. The pH optimum was 7.6–7.8. The apparent K _m values for pyruvate and coenzyme A at 70° C were 3.45 mM and 54 μM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t _50%) at 70° C was approximately 8 h. Received: 9 September 1996 / Accepted: 27 December 1996     

Purification and substrate characterization of α-ketobutyrate decarboxylase from Pseudomonas putida

α-Ketobutyrate decarboxylase encoded in the -methionine catabolism operon of Pseudomonas putida is homologous with the E1 component of pyruvate dehydrogenase complex from gram-negative bacteria. The enzyme was purified to homogeneity from the cell extract of an Escherichia coli transformant. The purified enzyme was homodimeric with a subunit of M_r 93,000 on SDS-PAGE. The enzyme activity was activated by the addition of both thiamine pyrophosphate (TPP) and a divalent cation, such as Mg²⁺, Mn²⁺, and Co²⁺. The enzyme showed high activity for α-ketobutyrate and α-keto-n-valerate rather than pyruvate, but the α-keto acids with increasing length of the side chain as well as branching, such as α-keto-n-caproate and α-keto-3-methylvalerate, were not used by the enzyme. The K_m values for α-ketobutyrate and pyruvate were 0.016 and 0.147 mM, respectively, and the k_cat/K_m value (10.69 s⁻¹ mM⁻¹) for α-ketobutyrate was 29-fold greater than that for pyruvate. Thus, α-ketobutyrate decarboxylase is distinguished from the pyruvate dehydrogenase E1 component with respect to the substrate specificity, although their structural and enzymological properties were similar. These results suggest that the unique substrate specificity of α-ketobutyrate decarboxylase is due to a slight difference in the highly conserved active sites of both enzymes.     

Novel halophilic 2-aminobutyrate dehydrogenase from Halobacterium saccahrovorum DSM 1137

A halophilic NAD⁺-dependent 2-aminobutyrate dehydrogenase (EC1.4.1.1) was purified to homogeneity from a crude extract of an extreme halophile, Halobacterium saccharovorum DSM 1137, with a 30% yield. The enzyme had a molecular mass of about 160 kDa and consisted of four identical subunits. It retained more than 70% of the activity after heating at 60 °C for 1 h and kept it at 30 °C for 8 months in the presence of 2 M NaCl. The enzyme showed maximum activity in the presence of 2 M RbCl or KCl. The enzyme required NAD⁺ as a coenzyme and used -2-aminobutyrate, -alanine, and -norvaline as substrates. The best substrate was -2-aminobutyrate. The optimum pH was 9.3 for the oxidative deamination of -2-aminobutyrate and 8.6 for the reductive amination of 2-ketobutyrate. The Michaelis constants were 1.2 mM for -2-aminobutyrate, 0.16 mM for NAD⁺, 0.012 mM for NADH, 0.78 mM for 2-ketobutyrate, and 500 mM for ammonia in the presence of 2 M KCl. The K_m values for the substrates depended on the concentration of KCl, and the K_m values decreased under high salt conditions.     

Pyruvate,orthophosphate dikinase from Acetobacter aceti

A pyruvate, orthophosphate dikinase (EC 2.7.9.1) has been isolated from Acetobacter aceti grown on pyruvate as the only source of carbon and energy. The enzyme was purified 65-fold, and its molecular weight was determined to be about 330,000 by gel filtration.The optimum pH was 8.0 in the forward direction [phosphoenolpyruvate (PEP) formation] and 7.1 for the backward reaction (pyruvate production). In both directions Mg²⁺ was required (forward K _m 1.70 mM; reverse K _m 0.87 mM) and no other divalent cation was able to replace it. The K _m values for pyruvate, ATP, and P_i were 27 M, 0.20 mM, and 0.83 mM, respectively, in the forward direction. The K _m values for PEP, AMP, and PP_i were 0.13 mM, 6 M, and 62 M, respectively, for the reverse reaction. The substrate-product pairs pyruvate-PEP, ATP-AMP, P_i-PP_i were competitive inhibitors to each other in both directions. These product inhibition studies suggest for the enzyme from A. aceti nonclassical three-site Tri (Uni Uni) Ping-Pong kinetics.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - SDS sodium dodecyl sulphate - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM glutathione     

Biochemical characterization and kinetic properties of alanine aminotransferase homologues partially purified from wheat (Triticum aestivum L.)

Four homologues of alanine aminotransferase have been isolated from shoots of wheat seedlings and purified by saline precipitation, gel filtration, preparative electrophoresis and anion exchange chromatography on Protein-Pak Q 8HR column attached to HPLC. Alanine aminotransferase 1 (AlaAT1) and 2 (AlaAT2) were purified 303- and 452-fold, respectively, whereas l-glutamate: glyoxylate aminotransferase 1 (GGAT1) and 2 (GGAT2) were purified 485- and 440-fold, respectively. Consistent inhibition of AlaAT (EC 2.6.1.2) and GGAT (EC 2.6.1.4) activities by p-hydroxymercuribenzoate points on participation of cysteine residues in the enzyme activity. The molecular weight of AlaAT1 and AlaAT2 was estimated to be 65 kDa and both of them are monomers in native state. Nonsignificant differences between K_m using alanine as substrate and catalytic efficiency (k_cat/K_m) for l-alanine in reaction with 2-oxoglutarate indicate comparable kinetic constants for AlaAT1 and AlaAT2. Similar kinetic constants for l-alanine in reaction with 2-oxoglutarate and for l-glutamate in reaction with pyruvate for all four homologues suggest equally efficient reaction in both forward and reverse directions. GGAT1 and GGAT2 were able to catalyze transamination between l-glutamate and glyoxylate, l-alanine and glyoxylate and reverse reactions between glycine and 2-oxoglutarate or pyruvate. Both GGATs also consisted of a single subunit with molecular weight of about 50 kDa. The estimated K_m for GGAT1 (3.22 M) and GGAT2 (1.27 M) using l-glutamate as substrate was lower in transamination with glyoxylate than with pyruvate (9.52 and 9.09 mM, respectively). Moreover, distinctively higher values of catalytic efficiency for l-glutamate in reaction with glyoxylate than for l-glutamate in reaction with pyruvate confirm involvement of these homologues into photorespiratory metabolism.