A blue native polyacrylamide gel electrophoretic technology to probe the functional proteomics mediating nitrogen homeostasis in Pseudomonas fluorescens.

As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This electrophoretic technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.

Characterization of the ornithine aminotransferase from Plasmodium falciparum.

The ornithine aminotransferase from Plasmodium falciparum 3D7 was cloned, functionally expressed, and characterized. The gene exists as a single copy in the malarial genome and is located on chromosomes 6/7/8. The deduced amino acid sequence was found to be 85% identical to a similar sequence discovered in Plasmodium yoelii, 82% identical to a partial sequence from Plasmodium vivax, and 42-53% identical to ornithine aminotransferases from other eukaryotes. The enzyme had a very narrow substrate specificity, and could only catalyze the transamination of alpha-ketoglutarate with ornithine or N-acetylornithine, and of glutamate-5-semialdehyde with glutamate and alanine. The aminooxy analogue of ornithine, canaline, was found to inhibit the ornithine aminotransferase uncompetatively with a Ki of 492+/-98 nM. As the enzyme effectively catalyzed both ornithine catabolism and formation, its potential role in ornithine biosynthesis from glutamine, via glutamate, glutamate-5-phosphate, and glutamate-5-semialdehyde, was examined. Over the course of a 3.5 h incubation, P. falciparum converted 34% of exogenous, radiolabeled glutamine to glutamate and 0.68% to ornithine. This low level of conversion suggests that the parasite may have alternative mechanisms for obtaining ornithine for polyamine biosynthesis.

Isolation of the ornithine-delta-aminotransferase cDNA and effect of salt stress on its expression in Arabidopsis thaliana.

To evaluate the relative importance of ornithine (Orn) as a precursor in proline (Pro) synthesis, we isolated and sequenced a cDNA encoding the Orn-delta-aminotransferase (delta-OAT) from Arabidopsis thaliana. The deduced amino acid sequence showed high homology with bacterial, yeast, mammalian, and plant sequences, and the N-terminal residues exhibited several common features with a mitochondrial transit peptide. Our results show that under both salt stress and normal conditions, delta-OAT activity and mRNA in young plantlets are slightly higher than in older plants. This appears to be related to the necessity to dispose of an easy recycling product, glutamate. Analysis of the expression of the gene revealed a close association with salt stress and Pro production. In young plantlets, free Pro content, Delta1-pyrroline-5-carboxylate synthase mRNA, delta-OAT activity, and delta-OAT mRNA were all increased by salt-stress treatment. These results suggest that for A. thaliana, the Orn pathway, together with the glutamate pathway, plays an important role in Pro accumulation during osmotic stress. Conversely, in 4-week-old A. thaliana plants, although free Pro level also increased under salt-stress conditions, the delta-OAT activity appeared to be unchanged and delta-OAT mRNA was not detectable. Delta1-pyrroline-5-carboxylate synthase mRNA was still induced at a similar level. Therefore, for the adult plants the free Pro increase seemed to be due to the activity of the enzymes of the glutamate pathway.

A variant of ornithine aminotransferase from mouse small intestine.

The ornithine aminotransferase (OAT) activity of mouse was found to be highest in the small intestine. The mitochondrial OAT from mouse small intestine was purified to homogeneity by the procedures including heart treatment, ammonium sulfate fractionation, octyl-Sepharose chromatography, and Sephadex G-150 gel filtration. Comparing to the amino acid sequence of mouse hepatic OAT, six N-terminal amino acid residues have been deleted in intestinal OAT. However, the subsequent sequence was identical with that of hepatic OAT. The molecular weights of both intestinal and hepatic OAT were estimated as 46 kDa by SDS-gel electrophoresis and as 92 kDa by gel filtration, indicating that both native OATs are dimeric. Biochemical properties of intestinal OAT, such as molecular weight, pH optimum and K(m) values for L-ornithine and alpha-ketoglutarate, were similar to those of hepatic OAT. However, intestinal OAT was more labile than hepatic OAT to tryptic digestion.

A variant of ornithine aminotransferase from mouse small intestine

The ornithine aminotransferase (OAT) activity of mouse was found to be highest in the small intestine. The mitochondrial OAT from mouse small intestine was purified to homogeneity by the procedures including heart treatment, ammonium sulfate fractionation, octyl-Sepharose chromatography, and Sephadex G-150 gel filtration. Comparing to the amino acid sequence of mouse hepatic OAT, six N-terminal amino acid residues have been deleted in intestinal OAT. However, the subsequent sequence was identical with that of hepatic OAT. The molecular weights of both intestinal and hepatic OAT were estimated as 46 kDa by SDS-gel electrophoresis and as 92 kDa by gel filtration, indicating that both native OATs are dimeric. Biochemical properties of intestinal OAT, such as molecular weight, pH optimum and K(m) values for L-ornithine and alpha-ketoglutarate, were similar to those of hepatic OAT. However, intestinal OAT was more labile than hepatic OAT to tryptic digestion.

Thermostable ornithine aminotransferase from Bacillus sp. YM-2: purification and characterization.

Thermostable L-ornithine: alpha-ketoglutarate delta-aminotransferase (L-ornithine: 2-oxo-acid 5-aminotransferase) [EC 2.6.1.13] was purified to homogeneity from Bacillus sp. YM-2. The enzyme has a molecular weight of about 82,000 and consists of two subunits with identical molecular weights. The enzyme catalyzes transamination from L-ornithine to alpha-ketoglutarate, producing L-glutamate and L-glutamate gamma-semialdehyde, which is spontaneously dehydrated to L-delta 1-pyrroline-5-carboxylate, and the enzyme is most active at 70 degrees C. In addition to L-ornithine, the enzyme unexpectedly acts on D-ornithine, the reaction rate being 6% of that for L-ornithine. The enzyme contains 1 mol each of pyridoxal 5'-phosphate and another vitamin B6 compound per mol. The enzyme released the bound pyridoxal 5'-phosphate, as judged from the absorption at 425 nm on incubation with 2.0 M guanidine hydrochloride. The resultant inactive enzyme still gave a 340-nm peak and contained 1 mol of the vitamin B6 compound. The partial amino acid sequence shows high homology with those of mammalian and yeast ornithine delta-aminotransferases.

Purification and properties of L-ornithine delta-aminotransferase from gramicidin S-producing Bacillus brevis.

In gramicidin S-producing Bacillus brevis, the addition of L-ornithine to the minimal medium with L-glutamate as the sole carbon and nitrogen source caused an 8-fold induction of L-ornithine delta-aminotransferase [EC 2.6.1.13]. The enzyme was purified to homogeneity. The native enzyme had a molecular weight of about 88,000 after gel filtration and consisted of two subunits with an identical in molecular weight of about 45,000. The enzyme was specific for L-ornithine (Km = 1.05 mM) as an amino donor and for 2-oxoglutarate (Km = 6.25 mM) as an amino acceptor, and catalyzed the conversion of L-ornithine and 2-oxoglutarate, respectively, to glutamic-gamma-semialdehyde, which is spontaneously cyclized to delta 1-pyrroline-5-carboxylate and L-glutamate. The enzyme exhibits an absorption maximum at 425 nm at neutral pH, and 1 mol of pyridoxal phosphate is bound per subunit. The enzyme activity was irreversibly inhibited by gabaculine, and L-ornithine protected the enzyme from the inhibition. The N-terminal amino acid sequence revealed a noteworthy similarity between human and yeast L-ornithine delta-aminotransferases in residues 17-28 of the B. brevis enzyme.

Interaction of L-canaline with ornithine aminotransferase of the tobacco hornworm, Manduca sexta (Sphingidae).

Ornithine aminotransferase (L-ornithine:2-oxo-acid aminotransferase (EC 2.6.1.13)) has been purified to homogeneity from last instar larvae of the tobacco hornworm, Manduca sexta (Sphingidae). This enzyme is a 144,000-Da tetramer constructed from 36,000-Da protomeric units. It has a high aspartate/asparagine and glutamate/glutamine content and 2 cysteine residues/subunit. All 8 cysteine residues can react with N-ethylmaleimide to inactivate the enzyme. Maintenance of the enzyme in the presence of 2-mercaptoethanol and dithiothreitol maximizes enzymatic activity and improves storage conditions, presumably by protecting these sulfhydryl groups. The apparent Km values for L-ornithine and 2-oxoglutaric acid are 2.3 and 3.2 mM, respectively. The turnover number is 2.0 +/- 0.1 mumol min-1 mumol-1. L-Canaline (L-2-amino-4-(aminooxy)butyric acid) is a potent ornithine aminotransferase inhibitor. Reaction of the enzyme with L-[U-14C]canaline produces an enzyme-bound, covalently linked, radiolabeled canaline-pyridoxal phosphate oxime. The L-[U-14C]canaline-pyridoxal phosphate oxime has been isolated from canaline-treated enzyme. Dialysis of canaline-inactivated ornithine aminotransferase against free pyridoxal phosphate slowly reactivates the enzyme as the oxime is replaced by pyridoxal phosphate. Analysis of L-[U-14C]canaline binding to ornithine aminotransferase reveals the presence of 4 mol of pyridoxal phosphate/mol of enzyme.

Regulation of ornithine aminotransferase in retinoblastomas.

Ornithine-delta-aminotransferase (OAT) is a nuclear-encoded, mitochondrial enzyme that converts ornithine to glutamate semialdehyde. Although OAT is expressed in most tissues of the rat, liver, kidney, and retina have the highest levels of OAT activity. Studies of OAT regulation in liver and kidney have indicated transcriptional and translational controls for the enzyme in a tissue-specific manner. Little is known about OAT modulation in retinal tissue, although chorio-retinal degeneration is the predominant feature in a hereditary disorder of OAT deficiency, gyrate atrophy. To characterize OAT regulation in retinal lines, we studied its synthesis in two retinoblastoma strains, Y79 and RB355. Baseline OAT mRNA levels were similar in the two cell lines, yet Y79 expressed 3-fold more immunoreactive OAT protein and enzyme activity than RB355; this finding suggested the presence of a post-transcriptional mechanism for the regulation of steady-state OAT levels. Treatment of the two strains with estradiol or thyroid hormone for 24 h resulted in approximately 5-fold increases in OAT protein and activity. Since similar increases in OAT mRNA levels were observed in both strains after Northern blotting, it is likely that these hormones exert their effects at the transcriptional level. Finally, primer extension analysis revealed two OAT mRNA species in both strains, due to the presence of an additional exon (exon 2) in one of the transcripts. The absence of this exon in other tissues reflects the unique mechanisms which govern OAT in retinoblastomas.

Purification of ornithine aminotransferase by immunoadsorption.

Ornithine aminotransferase was purified by conventional biochemical methods from rat kidney, rat liver, and human liver. Affinity-purified antibodies raised to the rat kidney enzyme were used to produce an immunoadsorbent enabling a one-step purification of ornithine aminotransferase to be made from crude human liver extracts. The harsh chemical conditions often required to desorb immunoadsorbents were avoided by isolating antibodies with low functional affinity and employing an electrophoretic desorption method which allowed the enzyme activity to be retained. The close structural similarity between human and rat ornithine aminotransferase was demonstrated by immunodiffusion reactions. It was therefore possible to purify the enzyme from human liver using immobilized antibodies raised against rat kidney ornithine aminotransferase. Furthermore, desorption was more readily achieved due to the lower affinity for the human enzyme.

A comparison of ornithine aminotransferase from human and rat sources.

Ornithine aminotransferase was purified from human liver, rat liver and rat kidney. Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a subunit molecular weight of 45,000 in all three cases. Estimations of the native molecular weights of ornithine aminotransferase were determined by Sephadex G-200 chromatography in the presence and absence of 0.1% (w/v) Triton X-100. Human and rat enzymes were tetrameric in the presence of detergent but the rat subunits aggregated further in its absence. Characterisation of ornithine aminotransferase from the two rat sources indicated that they were the same protein. The human and rat enzymes were similar but not identical.

Quaternary structure of ornithine aminotransferase in solution and preliminary crystallographic data.

Ornithine aminotransferase was purified from rat liver and crystallized in the presence of ammonium sulphate and poly(ethylene glycol) (PEG 4000). The crystallographic threefold symmetry observed for the resulting two crystal forms stimulated a re-examination of the enzyme's quaternary structure in solution by analytical ultracentrifugation and chemical cross-linking. The results indicate that the oligomeric state or ornithine aminotransferase, under conditions similar to those used in crystallization experiments, is a hexamer (Mr = 256,000) rather than a tetramer or higher oligomers as reported previously. The subunits of the enzyme are identical (Mr = 45,000). Only the hexagonal prismatic crystals obtained with PEG 4000 were suitable for crystallographic studies and diffracted X-rays to a resolution of at least 0.16 nm. However, these crystals contained an unusual element of disorder which was persistent under a variety of conditions and was only noticeably diminished in the presence of the non-ionic detergent octyl beta-glucoside. The crystals apparently belong to the trigonal space group P3(1)12 (or enantiomorph) with axial lengths of a = 19.5 nm, c = 5.9 nm and contain three monomers per asymmetric unit.

Purification and properties of ornithine aminotransferase from rat brain.

Ornithine aminotransferase (E.C. 2.6.1.13) from rat brain was purified 100-fold by ammonium sulphate fractionation, DEAE cellulose chromatography, calcium phosphate gel and alumina C gamma gel. Pyridoxal phosphate was essential for maximum activity of the enzyme. The brain enzyme did not differ from liver and kidney enzymes in properties such as pH optimum, Km, substrate specificity and the inhibition by branched chain amino acids. Unlike rat liver enzyme, brain ornithine aminotransferase was able to catalyze the reaction between L-lysine and 2-oxoglutarate. Spermidine and spermine inhibited brain ornithine aminotransferase activity.

Crystallization and properties of human liver ornithine aminotransferase.

Ornithine aminotransferase [EC 2.6.1.13] was purified and crystallized from human liver by a procedure involving heat treatment, chromatographies on DEAE-cellulose, Octyl-Sepharose CL-4B and Sephadex G-200, and crystallization. The purified enzyme appeared to be homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated as 44,000 by sodium dodecyl sulfate electrophoresis and as 177,000 by sucrose density gradient centrifugation, indicating that the enzyme is tetrameric. Various properties of the enzyme from human liver are similar to those of the enzyme from rat liver, including its molecular weight, pH optimum, Km values for ornithine, alpha-ketoglutarate and pyridoxal phosphate and specificity for amino acceptor from ornithine. The amino acid compositions of the two enzymes also have certain similarities, but the enzymes differ in electrophoretic mobility and antigenicity: the human enzyme moved more slowly to the anode, and on immunodiffusion analysis, the single precipitin lines formed between anti-human enzyme serum or anti-rat liver enzyme and the enzyme from human liver or lymphoblastoid cells and the rat liver enzyme fused with spur formation.

Ornithine delta-transaminase activity in Escherichia coli: its identity with acetylornithine delta-transaminase.

Procedures that have been developed for the purification of acetylornithine delta-transaminase from Escherichia coli W also lead to the simultaneous purification of ornithine delta-transaminase. These two enzymatic activities have the same electrophoretic mobility and are identical immunochemically. Studies of inhibition kinetics demonstrate that the two substrates, acetylornithine and ornithine, compete for the same active site of acetylornithine delta-transaminase; thus, the ornithine delta-transaminase activity in E coli is due to acetylornithine delta-transaminase and not to a separate specific ornithine delta-transaminase.