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Kynurenic acid (KA) is an endogenous glutamate receptor antagonist at the level of the different ionotropic glutamate receptors. One of the enzymes responsible for the production of KA, kynurenine aminotransferase I (KATI), also catalyses the reversible transamination of glutamine to oxoglutaramic acid (GTK, EC 2.6.1.15). The enzyme exists in a cytosolic and in a mitochondrial form because of the presence of two different KATI mRNAs coding for a protein respectively with and without leader sequence targeting the protein into mitochondria. We have cloned from a phage library of rat kidney cDNA four new KATI cDNAs containing different 5' untranslated regions (UTRs). One of the transcripts (+14KATI cDNA) contains an alternative site of initiation of translation. The tissue distribution of the different transcripts was studied by RT-PCR. The study demonstrated that several KATI mRNAs are constitutively expressed in ubiquitous manner, while +14KATI mRNA is present only in kidney. The translational efficiency of the different transcripts was studied in vitro and enzymatic activities were measured in transiently transfected Cos-1 cells. Each KATI mRNA exhibits a different in vitro translational efficiency, which corresponds to different levels of KAT enzymatic activity in transfected cells. Both findings correlate with the predicted accessibility of the ribosomal binding sites of the different mRNAs. The structure of the rat KATI/GTK gene was also studied. The expression of several KATI mRNAs with different 5'UTRs represents an interesting example of transcriptional/translational control on the expression of pyridoxal phosphate (PLP)-dependent aminotransferases.

A simultaneous synthesis of (R)-1-phenylethanol and (R)-alpha-methylbenzylamine from racemic alpha-methylbenzylamine was achieved using an omega-transaminase, alcohol dehydrogenase, and glucose dehydrogenase in a coupled reaction. Racemic alpha-methylbenzylamine (100 mM) was converted to 49 mM (R)-1-phenylethanol (> 99% ee) and 48 mM (R)-alpha-methylbenzylamine (> 98% ee) in 18 h at 37 degrees C. This method was also used to overcome product inhibition of omega-TA by the ketone product in the kinetic resolution of racemic amines at high concentration.

The neurons of the transient subplate zone, considered important for the prenatal development of the cerebral cortex, were shown here to express kynurenine aminotransferase (KAT)-I from embryonic day (E) 16 until postnatal day (P) 7 in the rat. No other cells of brain tissue exerted KAT-I immunoreactivity during this period. From P3 on, the neurons of the subplate gave rise to KAT-I immunoreactive, varicose axons, which entered the thalamus and terminated around thalamic nerve cells that are devoid of KAT-I immunoreactivity. Other subplate markers displayed a different expression pattern during development. Thus, subplate neurons displayed parvalbumin (PV) immuno-reactivity from E16 to P10 and an intense NPY immunoreaction from P7 to P1. They also exhibited nitric oxide synthase immunoreactivity from E16 to P10, whereas on the surface of the subplate neurons, the alpha7 subunit of the nicotinic acetylcholine receptor (nAChR) was present from P1 to P10. The cells of Cajal-Retzius were nAChR-immunoreactive during this period. Between P1 and P7, the perikarya of subplate neurons also showed an intense immuno-reaction with the N-methyl-D-aspartate (NMDA) receptor subtype R2A. After the first postnatal week, many of the KAT-I positive subplate neurons display a gradual decrease of immunoreactivity and undergo programmed cell death. Since KAT-I persists in the subplate through the period E16-P7, we conclude that KAT-I is a useful and reliable subplate marker in the rat. Since it is assumed that migration of nerve cells is regulated by NMDA receptors, and since kynurenic acid--the only naturally occurring NMDA receptor antagonist--is synthesized by KAT, we suggest that a temporary breakdown of the delicate equilibrium between NMDA and KAT might induce abnormal neuronal migration, giving rise to developmental abnormalities.

The aim of the present study was to evaluate the effect of mitochondrial inhibitors, 1-methyl-4-phenylpyridinium (MPP(+)) and 3-nitropropionic acid (3-NPA), on the brain production of endogenous glutamate antagonist, kynurenic acid (KYNA). MPP(+) and 3-NPA dose-dependently impaired the synthesis of KYNA in rat cortical slices. Enzymatic studies revealed that MPP(+) inhibits in a concentration-dependent manner the activity of kynurenine aminotransferase II (KAT II), but not the activity of kynurenine aminotransferase I (KAT I). 3-NPA impaired the activity of both enzymes, KAT I and KAT II. Thus, MPP(+)- and 3-NPA-evoked neurotoxicity may be at least partially associated with the depletion of KYNA.

The 2-aminoethylphosphonate transaminase (AEPT; the phnW gene product) of the Salmonella enterica serovar Typhimurium 2-aminoethylphosphonate (AEP) degradation pathway catalyzes the reversible reaction of AEP and pyruvate to form phosphonoacetaldehyde (P-Ald) and L-alanine (L-Ala). Here, we describe the purification and characterization of recombinant AEPT. pH rate profiles (log V(m) and log V(m)/K(m) versus pH) revealed a pH optimum of 8.5. At pH 8.5, K(eq) is equal to 0.5 and the k(cat) values of the forward and reverse reactions are 7 and 9 s(-1), respectively. The K(m) for AEP is 1.11 +/- 0.03 mM; for pyruvate it is 0.15 +/- 0.02 mM, for P-Ald it is 0.09 +/- 0.01 mM, and for L-Ala it is 1.4 +/- 0.03 mM. Substrate specificity tests revealed a high degree of discrimination, indicating a singular physiological role for the transaminase in AEP degradation. The 40-kDa subunit of the homodimeric enzyme is homologous to other members of the pyridoxalphosphate-dependent amino acid transaminase superfamily. Catalytic residues conserved within well-characterized members are also conserved within the seven known AEPT sequences. Site-directed mutagenesis demonstrated the importance of three selected residues (Asp168, Lys194, and Arg340) in AEPT catalysis.

Background and Aim: Acute alcohol withdrawal causes changes in hepatic blood flow and metabolism that may result in liver damage. This study aims to assess liver function tests and markers of hepatic fibrogenesis following alcohol withdrawal in alcoholics with clinically compensated liver disease., Methods: Serial liver function tests and clinical assessments were performed on 22 male alcoholics during alcohol withdrawal. Plasma tissue inhibitor of metalloproteinase 1 (TIMP1), an inhibitor of collagen degradation, and plasma amino-terminal procollagen III peptide (PIIINP), a collagen precursor molecule, were measured in these alcoholics and in 11 control subjects., Results: Transaminase levels did not change significantly over 7 days when all subjects were analyzed together. However, 32% of subjects showed a marked transaminase rise. These subjects did not differ from the others in baseline characteristics or short-term outcome, but had a greater benzodiazepine requirement. Only one subject consumed paracetamol (acetaminophen; 1-2 g/day). He had the largest transaminase rise. By comparing PIIINP assays, intact PIIINP concentration appears to increase following alcohol withdrawal. The TIMP1 levels were elevated in alcoholic subjects, but did not change following withdrawal., Conclusions: Increasing PIIINP suggests that hepatic fibrogenesis increases, or hepatic clearance falls, during acute alcohol withdrawal. The TIMP1 elevation in these alcoholics suggests that the inhibition of collagen degradation occurs while liver disease is still compensated. The period following alcohol withdrawal may be a time of marked increased susceptibility to paracetamol. The biochemical changes we observed were not associated with adverse short-term outcome, but the cumulative effect after repeated episodes of abrupt withdrawal may be of concern.

The 4-amino-6-deoxy-monosaccharide D-perosamine is an important element in the glycosylation of interesting cell products, such as antibiotics and lipopolysaccharides (LPS) of Gram-positive and Gram-negative bacteria. The biosynthetic pathway of the precursor molecule, GDP-D-perosamine, in Vibrio cholerae O1 starts with an isomerisation of fructose-6-phosphate catalyzed by the bifunctional enzyme phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase (RfbA; E.C. 2.7.7.22) creating the intermediate mannose-6-phosphate, which is subsequently converted by the phosphomanno-mutase (RfbB; E.C. 5.4.2.8) and further by RfbA to GDP-D-mannose, to GDP-4 keto-6-deoxymannose by a 4,6-dehydratase (RfbD; E.C. 4.2.1.47) and finally to GDP-D-perosamine by an aminotransferase (RfbE; E.C. not yet classified). We cloned the rfbD and the rfbE genes of V. cholerae O1 in Escherichia coli expression vectors. Both biosynthetic enzymes were overproduced in E. coli BL21 (DE3) and their activities were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-D-perosamine was optimized and the final product, GDP-D-perosamine, was purified and identified by nuclear magnetic resonance, mass spectrometry, and chromatography. The catalytically active form of the GDP-4-keto-6-deoxy-D-mannose-4-aminotransferase seems to be a tetramer of 170 kDa. The His-tag RfbE fusion protein has a Km of 0.06 mM and a Vmax value of 38 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose. The Km and Vmax values for the cosubstrate L-glutamate were 0.1 mM and 42 nkat/mg protein, respectively. The intention of this work is to establish a basis for both the in vitro production of GDP-D-perosamine and for an in vivo perosaminylation system in a suitable bacterial host, preferably E. coli.

The innate immune response to intraportally infused adenoviral vector was evaluated in rhesus monkeys. A first-generation adenovirus-expressing lacZ (Ad-lacZ) was administered at a dose just below that which causes severe morbidity. The response to vector was evaluated for the initial 24 h following infusion. Clinical findings during this time were primarily limited to petechiae, consistent with the development of thrombocytopenia and biochemical evidence of disseminated intravascular coagulation. Serum transaminases were elevated and a lymphopenia developed. Tracking of fluorescent-labeled vector demonstrated distribution to macrophages and dendritic cells of the spleen and Kupffer cells of the liver. A systemic release of the cytokine IL-6 occurred soon after vector infusion. Analysis of splenic cells revealed acute activation of macrophages and dendritic cells followed by massive apoptosis. Bone marrow cultures demonstrated normal erythroid and primitive progenitors with a significant decrease in myeloid progenitors. Similar findings, except the abnormality in bone marrow cultures, were observed in monkeys who received an identical dose of Ad-lacZ in which vector genes were inactivated with psoralen and UV irradiation. These data suggest that inadvertent targeting of antigen-presenting cells following intraportal infusion of vector leads to a systemic cytokine syndrome which may be triggered by the viral capsid proteins.

The effects of 1,8-cineole on D-galactosamine/lipopolysaccharide (GalN/LPS)-induced shock model of liver injury was investigated in mice. The co-administration of GalN (700 mg kg(-1), i.p.) and LPS (5 microg kg(-1), i.p.) greatly elevated serum concentrations of tumour necrosis factor-alpha (TNF-alpha), alanine aminotransferase and aspartate aminotransferase, and induced massive hepatic necrosis and lethality in 100% of control mice. Pretreatment with 1,8-cineole (400 mg kg(-1), p.o.) and dexamethasone (1 mg kg(-1), s.c.), 60 min before GalN/LPS, offered complete protection (100%) against the lethal shock and acute elevation in serum TNF-alpha and serum transaminases. Hepatic necrosis induced by GalN/LPS was also greatly reduced by both 1,8-cineole and dexamethasone treatment. The results indicate that 1,8-cineole protects mice against GalN/LPS-induced liver injury through the inhibition of TNF-alpha production, and suggest that 1,8-cineole may be a promising agent to combat septic-shock-associated pathologies.

This study was designed to determine the effect of lactation and weaning on the gene expression of branched-chain aminotransaminase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD) in different tissues of the lactating rat. BCAT activity increased in mammary tissue during lactation and was 6-fold higher than in virgin rats. This increase was associated with an increase in protein levels measured by immunoblot analysis, and with an increase in BCAT mitochondrial (BCATm) mRNA concentration. Twenty-four hours after weaning, BCAT activity, protein concentration, and mRNA levels in the dam decreased. BCAT activity, protein enzyme levels, and BCATm mRNA concentration in muscle were higher in weaning rats than in lactating rats. BCAT cytosolic (BCATc) mRNA was not expressed in mammary tissue, and there was no BCATc enzyme detected by Western blot in any physiological state. Mammary tissue BCKD activity increased and was active (dephosphorylated) during the lactation period. The level of enzyme also increased and the mRNA level for the E2 subunit in mammary tissue was 10-fold higher than the virgin values. Hepatic enzyme activity increased during weaning, and this was associated with the protein level and with the mRNA level of the E2 subunit. Muscle BCKD activity and protein content were the lowest of all tissues, and the E2 subunit mRNA level was barely detected by Northern blot analysis. The results suggest gene regulation of the two main catabolic enzymes of the branched-chain amino acid metabolism during lactation.

The autosomal recessive disorder primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver-specific pyridoxal-phosphate-dependent enzyme alanine:glyoxylate aminotransferase (AGT). Numerous mutations and polymorphisms in the gene encoding AGT have been identified, but in only a few cases has the causal relationship between genotype and phenotype actually been demonstrated. In this study, we have determined the effects of the most common naturally occurring amino acid substitutions (both normal polymorphisms and disease-causing mutations) on the properties, especially specific catalytic activity, of purified recombinant AGT. The results presented in this paper show the following: 1) normal human His-tagged AGT can be expressed at high levels in Escherichia coli and purified in a correctly folded, dimerized and catalytically active state; 2) presence of the common P11L polymorphism decreases the specific activity of purified recombinant AGT by a factor of three; 3) AGTs containing four of the most common PH1-specific mutations (G41R, F152I, G170R, and I244T) are all soluble and catalytically active in the absence of the P11L polymorphism, but in its presence all lead to protein destabilization and aggregation into inclusion bodies; 4) naturally occurring and artificial amino acid substitutions that lead to peroxisome-to-mitochondrion AGT mistargeting in mammalian cells also lead to destabilization and aggregation in E. coli; and 5) the PH1-specific G82E mutation abolishes AGT catalytic activity by interfering with cofactor binding, as does the artificial K209R mutation at the putative site of cofactor Shiff base formation. These results are discussed in the light of the high allelic frequency ( approximately 20%) of the P11L polymorphism and its importance in determining the phenotypic manifestations of mutations in PH1.

To examine normal and aberrant translation initiation in Saccharomyces cerevisiae mitochondria, we fused the synthetic mitochondrial reporter gene ARG8m to codon 91 of the COX2 coding sequence and inserted the chimeric gene into mitochondrial DNA (mtDNA). Translation of the cox2(1-91)::ARG8m mRNA yielded a fusion protein precursor that was processed to yield wild-type Arg8p. Thus mitochondrial translation could be monitored by the ability of mutant chimeric genes to complement a nuclear arg8 mutation. As expected, translation of the cox2(1-91)::ARG8m mRNA was dependent on the COX2 mRNA-specific activator PET111. We tested the ability of six triplets to function as initiation codons in both the cox2(1-91)::ARG8m reporter mRNA and the otherwise wild-type COX2 mRNA. Substitution of AUC, CCC or AAA for the initiation codon abolished detectable translation of both mRNAs, even when PET111 activity was increased. The failure of these mutant cox2(1-91)::ARG8m genes to yield Arg8p demonstrates that initiation at downstream AUG codons, such as COX2 codon 14, does not occur even when normal initiation is blocked. Three mutant triplets at the site of the initiation codon supported detectable translation, with efficiencies decreasing in the order GUG, AUU, AUA. Increased PET111 activity enhanced initiation at AUU and AUA codons. Comparisons of expression, at the level of accumulated product, of cox2(1-91)::ARG8m and COX2 carrying these mutant initiation codons revealed that very low-efficiency translation can provide enough Cox2p to sustain significant respiratory growth, presumably because Cox2p is efficiently assembled into stable cytochrome oxidase complexes.

The diamines putrescine, cadaverine, and diaminopropane stimulate cephamycin biosynthesis in Nocardia lactamdurans, in shake flasks and fermentors, without altering cell growth. Intracellular levels of the P7 protein (a component of the methoxylation system involved in cephamycin biosynthesis) were increased by diaminopropane, as shown by immunoblotting studies. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase activities involved in biosynthesis of the alpha-aminoadipic acid precursor were also greatly stimulated. The diamine stimulatory effect is exerted at the transcriptional level, as shown by low-resolution S1 protection studies. The transcript corresponding to the pcbAB gene and to a lesser extent also the lat transcript were significantly increased in diaminopropane-supplemented cultures, whereas transcription from the cefD promoter was not affected. Coupling of the lat and pcbAB promoters to the reporter xylE gene showed that expression from the lat and pcbAB promoters was increased by addition of diaminopropane in Streptomyces lividans. Intracellular accumulation of diamines in Nocardia may be a signal to trigger antibiotic production.

Fibrates modify the expression of genes implicated in lipoprotein and fatty acid metabolism via the peroxisome proliferator-activated receptor alpha(PPARalpha), leading to reductions in serum triglycerides and cholesterol. The expression of certain genes regulated by PPARalpha have been shown to be modified in a species dependent manner. Aspartate aminotransferase (AspAT or GOT) and alanine aminotransferase (AlaAT or GPT) are enzymes involved in intermediate metabolism in all cells and in hepatic gluconeogenesis. These enzymes are also widely used as serum markers of possible tissue damage. This study investigated whether fenofibrate could modify the expression of liver AspAT and/or AlaAT and thus possibly alter transaminase levels independently of a cytotoxic effect. In human Hep G2 cells, fenofibrate increased cytosolic AspAT (cAspAT) activity by 40% and AlaAT activity by 100%, as well as both mRNAs. Nuclear run on assays showed that this effect was, at least in part, transcriptional. Increases in mRNA were also observed in human hepatocyte cultures at concentrations of the drug attained in patients. In C57BL/6 mice, fenofibrate decreased cAspAT and cAlaAT mRNA, while these effects were abolished in PPARalpha knock-out mice. In conclusion, fenofibrate has been shown to modify cAspAT and AlaAT gene expression in a species and PPARalpha dependent manner. This is the first demonstration that cAspAT and AlaAT activities may be pharmacologically altered, independently of a toxic phenomenon.

A Nicotiana tabacum cDNA sequence encoding histidinol phosphate aminotransferase (HPA) was isolated by functional complementation of an Escherichia coli histidine auxotroph (UTH780). The enzymatic assay has confirmed that the isolated cDNA encodes a functional HPA protein. Amino acid sequence alignment of the HPA protein from N. tabacum, Saccharomyces cerevisiae and E. coli revealed that, despite the low degree of identity, some residues were found to be highly conserved. The predicted protein contains a transit peptide sequence at the amino-terminal end, suggesting a chloroplastic localization of the HPA enzyme. Western blot analysis demonstrated that the deduced HPA protein and the mature HPA protein have an apparent molecular mass of about 45 kDa and 40 kDa respectively. Gene copy number estimation by Southern analysis indicates the presence of at least two genes per haploid genome coding for this protein in Nicotiana sp. From northern analysis results, the gene seems to be highly expressed in green tissues and the detected transcript showed a single band of expected molecular size.

During the preclinical, early clinical, late-stage clinical, and postmarketing phases of the pharmaceutical discovery and development process, one important aspect of drug safety assessment involves monitoring for possible drug-induced hepatic injury. Hepatic injuries vary in nature from direct, intrinsic effects that are observed in most recipients and more than one species to rare idiosyncratic responses seen only in a few clinical subjects. Histological types of injuries vary from hepatocellular to hepatobiliary with multiple cellular effects characteristic of each type. Of the various clinical laboratory markers for hepatic injury, serum transaminases, especially alanine aminotransferase (ALT), are the most universally important indicators for studies ranging from early preclinical animal testing to postmarketing patient monitoring. This review examines the characteristics of hepatic toxicity that result in serum ALT changes, the differences in the etiology of hepatic responses which govern when liver injury is most likely to be detected during the four phases of the drug discovery and development process, and those modulating factors which affect the utility of ALT as a dependable marker of hepatic injury in clinical populations. The paper concludes with a summary of some ancillary methods for early preclinical screening such as in vitro metabolism and toxicity assays, gene and protein expression analysis, and some strategies for enhancing the probability for the early detection of idiosyncratic hepatotoxic responses which are infrequent but significant factors in the safety assessment process., (Copyright 1998 Academic Press.)

In rat liver, a single serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) gene is transcribed from two transcription initiation sites. Transcription from the upstream site generates the mRNA encoding the precursor for mitochondrial SPT (pSPTm) and is markedly enhanced by the administration of glucagon or cAMP. In this report we show the increase in the downstream transcript, the peroxisomal SPT (SPTp) mRNA, caused by peroxisome proliferators and triiodothyronine (T3). In the case of T3, the pSPTm mRNA was also increased 72 h after a single administration of the hormone in addition to an earlier increase in SPTp mRNA.

Cysteine conjugate beta-lyase/glutamine transaminase K/kynurenine aminotransferase (CS-lyase/GTK/KAT) is a tri-functional enzyme found in several organs, including the brain. Kynurenine aminotransferase is important in tryptophan metabolism in the CNS, producing kynurenic acid, a NMDA receptor antagonist and neuroprotective. Tryptophan not metabolised via kynurenine aminotransferase may form quinolinic acid, a NMDA receptor agonist and neurotoxin. Kynurenic acid co-treatment blocks quinolinic acid induced lesions in the CNS in rat. In many conditions exhibiting neurodegeneration (i.e. Huntington's, Parkinsonism, Down's syndrome) quinolinic acid and/or kynurenic acid concentrations are altered, suggesting the ratio of these chemicals may be important in neurodegeneration. We have investigated the developmental modulation of CS-lyase/GTK/KAT mRNA in rat brain. CS-lyase/GTK/KAT mRNA was measured in 14, 21, 28, 35, 42 day post-natal and adult rats. While many regions demonstrated a steady increase to adult levels, two other profiles were seen. Five regions rapidly reached adult levels of the mRNA, while two peaked above the adult level before falling back. This provides evidence that expression of the CS-lyase/GTK/KAT gene is physiologically modulated, and provides the basis for further investigation into the mechanism of control. Artificial modulation could possibly be used to alter levels of the neuroprotectant kynurenic acid in neurodegeneration.

Polyamine aminotransferase of Arthrobacter sp. TMP-1 was induced by 1,3-diaminopropane (DAP), N-3-aminopropyl-1,3-diaminopropane (norspermidine), spermidine, and spermine, but not by putrescine. The enzyme was purified to homogeneity. Its molecular weight and subunit size were 129,000 and 64,000, respectively. Its absorption spectrum had maxima at 280 and 420 nm and a shoulder at about 350 nm, and changes were observed upon the addition of DAP, putrescine, and sodium borohydride. The spectrum and its changes indicated that the enzyme contained pyridoxal-5'-phosphate as the coenzyme. The coenzyme content was found to be 1 mol per mol of subunit. DAP, putrescine, norspermidine, spermidine, and spermine were active amino donors and gave relative rates of 100, 73, 24, 30, and 23%, respectively. Pyruvate was the most active amino acceptor, while 2-ketoglutarate and oxaloacetate were inert. The equilibrium constant of the DAP-pyruvate transamination was 0.34. DAP was suggested to be a minor product of the norspermidine-pyruvate reaction.

Eight new genes, strO-stsABCDEFG, were identified by sequencing DNA in the gene cluster that encodes proteins for streptomycin production of Streptomyces griseus N2-3-11. The StsA (calculated molecular mass 43.5 kDa) and StsC (45.5 kDa) proteins - together with another gene product, StrS (39.8 kDa), encoded in another operon of the same gene cluster - show significant sequence identity and are members of a new class of pyridoxal-phosphate-dependent aminotransferases that have been observed mainly in the biosynthetic pathways for secondary metabolites. The aminotransferase activity was demonstrated for the first time by identification of the overproduced and purified StsC protein as the L-glutamine:scyllo-inosose aminotransferase, which catalyzes the first amino transfer in the biosynthesis of the streptidine subunit of streptomycin. The stsC and stsA genes each hybridized specifically to distinct fragments in the genomic DNA of most actinomycetes tested that produce diaminocyclitolaminoglycosides. In contrast, only stsC, but not stsA, hybridized to the DNA of Streptomyces hygroscopicus ssp. glebosus, which produces the monoaminocyclitol antibiotic bluensomycin; this suggests that both genes are specifically used in the first and second steps of the cyclitol transamination reactions. Sequence comparison studies performed with the deduced polypeptides of the genes adjacent to stsC suggest that the enzymes encoded by some of these genes [strO (putative phosphatase gene), stsB (putative oxidoreductase gene), and stsE (putative phosphotransferase gene)] also could be involved in (di-)aminocyclitol synthesis.

Kynurenic acid (KYNA) is a tryptophan metabolite and represents the only known endogenous compound acting as an antagonist to excitatory amino acid receptors in the mammalian CNS. Blocking of these receptors in CNS by KYNA affects cardiac function. As it is not known whether human heart is able to synthesize this neuromodulatory amino acid, we investigated the biosynthesizing enzyme of kynurenine aminotransferase (KAT) in the human heart and compared the activity with that of the human brain. The activities of heart and brain KATs were assayed by the conversion of L-kynurenine (L-KYN) to KYNA and quantitated by HPLC with fluorescence detection. Using either pyruvate or 2-oxoglutarate as cosubstrates, heart KAT was found to have a shallow pH optimum between 8 and 9. Highest heart KAT activity was seen in the presence of 2-oxoglutarate, followed by pyruvate. 2-oxoadipate, and 2-oxoisocaproate. Kinetic analyses, performed at pH 8.5, and using various concentrations of L-KYN (from 0.125 to 22.8 mM) in the presence of 2-oxoglutarate (1 and 5 mM) or pyruvate (5 mM) revealed apparent K(m) values in the millimolar range, for L-KYN 1.5, 27, and 20 mM, respectively. Heart KAT activities were compared with those in human brain KAT I and KAT II showing different pH optima 7.4 and 9.6, respectively. In contrast to brain KAT I, heart KAT activity was not inhibited by an excess of 2 mM L-tryptophan, L-glutamine, or L-phenylalanine at pH 9.6, as well as at pH 8 or 7.4. Our study demonstrates that human heart is capable of synthesizing KYNA from low concentrations of L-KYN selectively. A shallow pH optimum of KAT activity, i.e. between 8.0 and 9.0, pronounced 2-oxoacid specificity, and a lack of sensitivity to inhibition by L-glutamine, L-phenylalanine, and L-tryptophan indicate that the heart KAT system displays enzymatic characteristics different from those of human brain KAT I or KAT II. Fluctuation of L-KYN and 2-oxoacid levels may markedly influence the KYNA synthesis and subsequent KYNA effect on cardiac activity. KYNA synthesis in the human heart suggests a neurophysiologic role. Our studies from the basis for purification and further characterization of KAT protein in human heart as well as for physiologic studies.

The gene encoding the D-aminotransferase from Bacillus licheniformis was cloned and the complete DNA sequence was determined. The deduced D-aminotransferase protein sequence, consists of 283 amino acids and shows a high degree of homology with other Bacillus D-aminotransferases, branched chain aminotransferase of Escherichia coli and the 4-amino-benzoate-4-deoxychorismate lyase of Bacillus subtilis and Escherichia coli.

Phosphoserine aminotransferase (PSA) catalyzes the conversion of phosphohydroxypyruvate to phosphoserine in the phosphorylated pathway of serine biosynthesis. A cDNA clone encoding PSA was isolated from the cDNA library of spinach (Spinacia oleracea L.) green leaves. Determination of the nucleotide sequence revealed the presence of an open reading frame encoding 430 amino acids, exhibiting 38-50% homology with the amino acid sequences of bacterial, yeast and animal PSA. It contains an N-terminal extension of ca. 60 amino acids in addition to the sequences from other organisms. The general features of plastidic transit peptide are observed in this N-terminal sequence, suggesting the plastid localization of the PSA protein encoded by this cDNA. The bacterial expression of the cDNA could functionally rescue the auxotrophy of serine in the serC- mutant, Escherichia coli KL282. The enzymatic activity of PSA was demonstrated in vitro in the extracts of E. coli over-expressing the cDNA. Southern blot analysis indicated the presence of a couple of related genes (Psa) in the spinach genome. RNA blot hybridization suggested the preferential expression of the Psa gene in the roots of green seedlings and in the suspension cells cultured under a dark condition.

L-Lysine epsilon-aminotransferase (LAT) catalyzes the first reaction in the two-step conversion of L-lysine (Lys) to 1-alpha-aminoadipic acid (Aaa), a direct precursor of cephalosporins (including cephamycin C) in Streptomyces clavuligerus. Previous work showed that addition of Lys to chemically defined medium improved antibiotic production. We show that in S. clavuligerus cultures supplemented with high concentrations of Lys, Lys enhances antibiotic production by a dual effect, i.e. as a substrate of LAT thus providing Aaa and also as an inducer of LAT yielding even more Aaa. On the other hand, LAT is not induced by Aaa.

Genetic code differences prevent expression of nuclear genes within Saccharomyces cerevisiae mitochondria. To bridge this gap a synthetic gene, ARG8m, designed to specify an arginine biosynthetic enzyme when expressed inside mitochondria, has been inserted into yeast mtDNA in place of the COX3 structural gene. This mitochondrial cox3::ARG8m gene fully complements a nuclear arg8 deletion at the level of cell growth, and it is dependent for expression upon nuclear genes that encode subunits of the COX3 mRNA-specific translational activator. Thus, cox3::ARG8m serves as a mitochondrial reporter gene. Measurement of cox3::ARG8m expression at the levels of steady-state protein and enzymatic activity reveals that glucose repression operates within mitochondria. The levels of this reporter vary among strains whose nuclear genotypes lead to under- and overexpression of translational activator subunits, in particular Pet494p, indicating that mRNA-specific translational activation is a rate-limiting step in this organellar system. Whereas the steady-state level of cox3::ARG8m mRNA was also glucose repressed in an otherwise wild-type strain, absence of translational activation led to essentially repressed mRNA levels even under derepressing growth conditions. Thus, the mRNA is stabilized by translational activation, and variation in its level may be largely due to modulation of translation.

The biosynthesis of beta-lactams is one of the most thoroughly studied antibiotic pathways. The availability of the characteristics and the time profiles of activities of enzymes involved in the biosynthesis allows one to critically evaluate the potential rate-limiting steps in its production. Our approach to understanding the control of beta-lactam biosynthesis has been pursued using a two-stage strategy: (1) to predict the rate-limiting steps using a kinetic model and (2) to relax the rate-limiting steps by engineering the biosynthetic pathway or by altering the kinetic parameters of the predicted key rate-limiting enzyme. Kinetic analysis of the pathway dynamics of cephamycin C production in Streptomyces clavuligerus was performed using data obtained from wild type. Sensitivity analysis revealed that the availability of precursor alpha-aminoadipic acid and activity of ACV synthetase were the potential rate-limiting steps. Relaxation of the precursor limitation was accomplished by integration of an additional copy of the gene encoding lysine-epsilon-aminotransferase (lat) into the chromosome. The recombinant strain showed an increased level of cephamycin C production as expected. The intracellular levels of different intermediates in the pathway in batch cultures were analyzed.

The cDNA for the rat cytosolic branched chain aminotransferase (BCATc) has been cloned. The BCATc cDNA encodes a polypeptide of 410 amino acids with a calculated molecular mass of 46.0 kDa. By Northern blot analysis, BCATc message of approximately 2.7 kilobases was readily detected in rat brain, but was absent from liver, a rat hepatoma cell line, kidney, and skeletal muscle. When expressed in COS-1 cells, the enzyme is immunologically indistinguishable from the native enzyme found in rat brain cytosol. Comparison of the rat BCATc sequence with available data bases identified the Escherichia coli (and Salmonella typhimurium) branched chain aminotransferase (BCAT) and revealed a Haemophilus influenzae BCAT, a yeast BCAT, which is hypothesized to be a mitochondrial form of the enzyme, and the murine BCATc (protein ECA39). Calculated molecular masses for the complete proteins are 33.9 kDa, 37.9 kDa, 42.9 kDa, and 43.6 kDa, respectively. The rat BCATc sequence was 84% identical with murine BCATc, 45% identical with yeast, 33% identical with H. influenzae, 27% identical with the E. coli and S. typhimurium BCAT, and 22% identical with the evolutionary related D-amino acid aminotransferase (D-AAT) (Tanizawa, K., Asano, S., Masu, Y., Kuramitsu, S., Kagamiyama, H., Tanaka, H., and Soda, K. (1989) J. Biol. Chem. 264, 2450-2454). Amino acid sequence alignment of BCATc with D-AAT suggests that the folding pattern of the overlapping mammalian BCATc sequence is similar to that of D-AAT and indicates that orientation of the pyridoxal phosphate cofactor in the active site of the eukaryotic BCAT is the same as in D-AAT. Thus, BCAT are the only eukaryotic aminotransferases to abstract and replace the proton on the re face of the pyridoxal phosphate cofactor. Finally, requirements for recognition of substrate L-amino acid and alpha-carboxylate binding are discussed.

Insertion mutations that suppress some or all the pleiotropic effects of HisH and HisF overproduction were obtained by using transposons Tn10dTet and Tn10dCam. All suppressor mutations proved to be recessive, indicating that their effects were caused by loss of function; thus, the suppressors identify genes that are necessary to trigger the pleiotropic response when HisH and HisF are overproduced. Genetic mapping of the suppressor mutations identifies four novel loci on the Salmonella typhimurium genetic map. Mutations in osmH (min 49) behave as general suppressors that abolish all manifestations of the pleiotropic response. Mutations in sfiY (min 83) suppress cell division inhibition and thermosensitivity but not osmosensitivity. Mutations that suppress only cell division inhibition define another locus, sfiX (min 44). A fourth novel locus, sfiW (min 19), is also involved in cell division inhibition. The phenotype of sfiW mutations is in turn pleiotropic: they suppress cell division inhibition, make S. typhimurium unable to grow in minimal media, and cause slow growth and abnormal colony and cell shape. The inability of sfiW mutants to grow in minimal medium cannot be relieved by any known nutritional requirement or by the use of carbon sources other than glucose. The hierarchy of suppressor phenotypes and the existence of epistatic effects among suppressor mutations suggest a pathway-like model for the Hisc pleiotropic response.

Induction of mitochondrial serine: pyruvate aminotransferase (SPT) in rat liver by administration of glucagon was studied quantitatively by immunoblot analysis and cryoimmunogold electron microscopy. Immunoblot analysis revealed that two daily injections of glucagon produced marked increase of SPT protein mass to a level as much as 18 times that of the untreated rat. Cryoimmunogold electron microscopic analysis showed that the labeling density of the mitochondria increased in a parallel manner. Thus the induction of SPT analyzed by two methods showed an excellent correlation with a relative correlation coefficient of 0.98, indicating that the induction of SPT can be analyzed quantitatively by immunogold electron microscopy on cryoultrathin sections.

Two D-glutamic acid biosynthetic activities, glutamate racemase and D-amino acid transaminase, have been described previously for bacteria. To date, no bacterial species has been reported to possess both activities. Genetic complementation studies using Escherichia coli WM335, a D-glutamic acid auxotroph, and cloned chromosomal DNA fragments from Staphylococcus haemolyticus revealed two distinct DNA fragments containing open reading frames which, when present, allowed growth on medium without exogenous D-glutamic acid. Amino acid sequences of the two open reading frames derived from the DNA nucleotide sequences indicated extensive identity with the amino acid sequence of Pediococcus pentosaceous glutamate racemase in one case and with that of the D-amino acid transaminase of Bacillus spp. in the second case. Enzymatic assays of lysates of E. coli WM335 strains containing either the cloned staphylococcal racemase or transminase verified the identities of these activities. Subsequent DNA hybridization experiments indicated that Staphylococcus aureus, in addition to S. haemolyticus, contained homologous chromosomal DNA for each of these genes. These data suggest that S. haemolyticus, and probably S. aureus, contains genes for two D-glutamic acid biosynthetic activities, a glutamate racemase (dga gene) and a D-amino acid transaminase (dat gene).

The polycistronic mRNA of the histidine operon is subject to a processing event that generates a rather stable transcript encompassing the five distal cistrons. The molecular mechanisms by which such a transcript is produced were investigated in Escherichia coli strains carrying mutations in several genes for exo- and endonucleases. The experimental approach made use of S1 nuclease protection assays on in vivo synthesized transcripts, site-directed mutagenesis and construction of chimeric plasmids, dissection of the processing reaction by RNA mobility retardation experiments, and in vitro RNA degradation assays with cellular extracts. We have found that processing requires (1) a functional endonuclease E; (2) target site(s) for this activity in the RNA region upstream of the 5' end of the processed transcript that can be substituted by another well-characterized rne-dependent cleavage site; (3) efficient translation initiation of the first cistron immediately downstream of the 5' end; and (4) a functional endonuclease P that seems to act on the processing products generated by ribonuclease E. This is the first evidence that ribonuclease P, an essential ribozyme required for the biosynthesis of tRNA, may also be involved in the segmental stabilization of a mRNA.

Previous investigations in seizure-prone mice have suggested that an abnormally elevated production of the astrocyte-derived neuroexcitant, quinolinic acid (QUIN), plays a role in seizure susceptibility. In order to evaluate further the role of QUIN metabolism in genetic murine seizure models, the activities of its biosynthetic enzyme 3-hydroxyanthranilic acid oxygenase (3HAO), and of two other astrocytic enzymes, kynurenine aminotransferase (KAT) and glutamine synthetase (GS), were measured in the brains of seizure-prone EL and DBA/2 mice and two non-epileptic strains (BALB/c and Swiss-Webster). 3HAO activity was found to be markedly higher in both EL and DBA/2 mice than in the non-epileptic strains in all brain regions examined. The activity of 3HAO was not modified by the tossing procedure employed to promote seizures in EL mice. While some strain differences were noted in the activities of KAT and GS, these enzymes did not distinguish seizure-prone from the non-epileptic mice. In order to delineate better the relationship between glial activation and 3HAO, KAT and GS, further studies were performed in the ibotenate-lesioned hippocampus. In mice (but not in rats), the activity of 3HAO was selectively increased in gliotic tissue. These data demonstrate substantial species and strain differences in astroglial enzymes and in their response to brain injury. The observation of widespread abnormally high 3HAO activity in two distinct seizure-prone mouse strains strengthens the hypothesis that enhanced production of QUIN contributes to seizure susceptibility in mice.

A mutant of the enterobacterium Klebsiella pneumoniae with a defect in the hisF gene (in the histidine biosynthesis pathway) was isolated, which can only grow with high but not low ammonia concentrations. The mutated hisF product can use ammonia for the formation of the imidazole ring of histidine but not glutamine provided by the hisH product. Site-directed insertional mutagenesis of hisH led to the same dependence of prototrophic growth on high ammonia levels. The nucleotide sequence of K. pneumoniae hisF is almost identical to that of hisF from other enterobacteria. Similarities of the hisF product with the hisA product and of HisH sequences with the glutamine binding domains of TrpG-type amidotransferases provide additional evidence for the functions of the hisF and hisH products in histidine biosynthesis, namely that HisF catalyzes the ammonolytic cleavage of N'-(5'-phosphoribulosyl)-formimino-5- aminoimidazole-4-carboxamide ribonucleotide either utilizing free ammonia or deriving the ammonia moiety from glutamine bound to HisH.

Glutamine transaminase K and omega-amidase activities are present in the chick brain and in the brains of adult mice, rats, and humans. However, the activity of glutamine transaminase K in adult mouse brain is relatively low. In the chick embryo, cerebral glutamine transaminase K activity is low between embryonic days 5 and 17, but by day 23 (day of hatching) activity rises dramatically (> 15-fold). Cerebral omega-amidase activity is relatively high at embryonic day 5 but lower between days 5 and 17; at embryonic day 23 the activity rises to a maximum. Both glutamine transaminase K and omega-amidase are present in cultured chick, rat, and mouse astrocytes and neurons. For each species, the activity of glutamine transaminase K is higher in the astrocytes than in the neurons. The activity of omega-amidase is about the same in the cultured chick astrocytes and neurons but significantly higher in rat astrocytes than in rat neurons. The data suggest that the rise in brain glutamine transaminase K activity in the chick embryo at hatching correlates with maturation of astrocytes. Glutamine transaminase K may be involved in glutamine cycling in astrocytes. Glutamine transaminase K appears to be a major cysteine S-conjugate beta-lyase of the brain and may play a role in the neurotoxicity associated with exposure to dichloroacetylene and perhaps to other toxins.

Targeted gene insertion methodology was used to study the effect of perturbing alpha-aminoadipic acid precursor flux on the overall production rate of beta-lactam biosynthesis in Streptomyces clavuligerus. A high-copy-number plasmid containing the lysine epsilon-aminotransferase gene (lat) was constructed and used to transform S. clavuligerus. The resulting recombinant strain (LHM100) contained an additional complete copy of lat located adjacent to the corresponding wild-type gene in the chromosome. Biological activity and production levels of beta-lactam antibiotics were two to five times greater than in wild-type S. clavuligerus. Although levels of lysine epsilon-aminotransferase were elevated fourfold in LHM100, the level of ACV synthetase, whose gene is located just downstream of lat, remained unchanged. These data strongly support the notion that direct perturbation of alpha-aminoadipic acid precursor flux resulted in increased antibiotic production. This strategy represents a successful application of metabolic engineering based on theoretical predictions of precursor flux in a secondary metabolic pathway.

The role of rat kidney cysteine conjugate beta-lyase in the production of nephrotoxic thiols from S-cysteine conjugates of xenobiotics has been well established. However, the factors controlling the cellular distribution and substrate specificity of the enzyme have yet to be elucidated. As an approach to this we have isolated a cDNA for cysteine conjugate beta-lyase from a rat kidney cDNA library, using a combination of immunological and hybridization screening. A full length cDNA was sequenced and its identity was confirmed by deduced molecular weight, deduced amino acid composition, the presence of a consensus pyridoxal phosphate (PLP) binding site in the deduced amino acid sequence, kidney-specific expression of the corresponding mRNA, and the expression of beta-lyase and glutamine transaminase K activities in tissue culture cells transfected with the cDNA. The cDNA coded for a protein of 48 kDa containing the sequence Ser-Ala-Gly-Lys-Ser-Phe, which corresponds closely to the PLP binding site in other PLP-containing enzymes. Use of the cDNA to detect beta-lyase mRNA sequences in rat liver and kidney RNA demonstrated that expression was kidney specific and that the mRNA size (2.1 kilobases) was in good agreement with the size of the cDNA. When the cDNA was inserted into the expression vector pUS1000 and transfected into COS-1 tissue culture cells, a 7-10-fold increase in cytosolic beta-lyase and glutamine transaminase K activities could be detected. The use of beta-lyase cDNA for the elucidation of the mechanism of action of this enzyme and for the development of in vitro systems to examine xenobiotic cysteine conjugate toxicity is discussed.

A gene (lat) encoding lysine 6-aminotransferase was found upstream of the pcbAB (encoding alpha-aminoadipylcysteinyl-valine synthetase) and pcbC (encoding isopenicillin N synthase) genes in the cluster of early cephamycin biosynthetic genes in Nocardia lactamdurans. The lat gene was separated by a small intergenic region of 64 bp from the 5' end of the pcbAB gene. The lat gene contained an open reading frame of 1,353 nucleotides (71.4% G + C) encoding a protein of 450 amino acids with a deduced molecular mass of 48,811 Da. Expression of DNA fragments carrying the lat gene in Streptomyces lividans led to a high lysine 6-aminotransferase activity which was absent from untransformed S. lividans. The enzyme was partially purified from S. lividans(pULBS8) and showed a molecular mass of 52,800 Da as calculated by Sephadex gel filtration and polyacrylamide gel electrophoresis. DNA sequences which hybridized strongly with the lat gene of N. lactamdurans were found in four cephamycin-producing Streptomyces species but not in four other actinomycetes which are not known to produce beta-lactams, suggesting that the gene is specific for beta-lactam biosynthesis and is not involved in general lysine catabolism. The protein encoded by the lat gene showed similarity to ornithine-5-aminotransferases and N-acetylornithine-5-aminotransferases and contained a pyridoxal phosphate-binding consensus amino acid sequence around Lys-300 of the protein. The evolutionary implications of the lat gene as a true beta-lactam biosynthetic gene are discussed.

Lysine epsilon-aminotransferase (LAT) in the beta-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a beta-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an approximately Mr 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies approximately 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the beta-lactam biosynthetic cluster between pcbC and cefE (approximately 25 kbp) is nearly complete.

A 13.1-kb DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment contained five different cob genes named cobN to cobQ and cobW. Based on the similarity of NH2-terminal sequences and molecular weights of the purified Cob proteins, CobQ was identified as cobyric acid synthase, CobP was identified as a bifunctional enzyme exhibiting both cobinamide kinase and cobinamide phosphate guanylyltransferase activities, and CobO was identified as cob(I)alamin adenosyltransferase. CobN is proposed to play a role in cobalt insertion reactions. Four other open reading frames were identified on the 13.1-kb fragment, but their chromosomal inactivation did not lead to a cobalamin-minus phenotype.

Escherichia coli pabA encodes the glutamine amidotransferase subunit of p-aminobenzoate synthase. p-Aminobenzoate synthase catalyzes the conversion of chorismate and glutamine to 4-amino-4-deoxychorismate, which is then converted to p-aminobenzoate by a 4-amino-4-deoxychorismate lyase. The 5'-terminal segment of pabA was previously shown to be transcribed from two different promoters, one near the pabA coding sequence (P1) and one preceding fic (P2). However, a pabA-lacZ translational fusion was expressed only from the mRNA originating at P1. We have determined that expression of a pabA-lacZ chromosomal fusion is not changed by p-aminobenzoate limitation, growth rate, catabolite repression, overexpression of either p-aminobenzoate synthase subunit, or gene dosage of pabA and pabB. The lack of pabA expression from P2 appears to be the result of a stable secondary structure in the intergenic space preceding pabA that sequesters the pabA ribosome binding site. Disruption of the secondary structure by mutation allowed expression of pabA from P2, as did translation of ribosomes into the fic-pabA intergenic region.

The efficiency of protein synthesis is discussed with the main emphasis on the accuracy. The error increase by erroneous synthesizing proteins is studied by a theoretical model which includes the coupling of the amino acylation step to the protein synthesis at the ribosomes. Depending on parameter values, the model yields a stable point of relatively high accuracy, a point where most of the accuracy is lost and virtually no functioning proteins are produced (error catastrophe), or both. The effect of an error enhancing drug such as streptomycin can be considered, and it is found that one can have a situation where small amounts of the drug yields a decreased but stable accuracy, and where this possibility disappears at a certain drug level. At higher drug levels, the cells cannot attain any accuracy. The transition is quite drastic, and analogous to a phase transition.

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase are homologous proteins. Both are encoded by nuclear DNA and synthesized on free polysomes. The organization of their genes is very similar, five out of a total of eight introns are located at the same nucleotide position. A variant consensus sequence was observed at the 3' splice site of introns of genes of imported mitochondrial proteins which may reflect the existence of splicing factors specific for the genes of this particular group of nuclear-encoded proteins. To date the amino acid sequences of 22 aminotransferases are known. A rigorous analysis yielded clear evidence that aspartate, tyrosine, and histidinol-phosphate aminotransferases are homologous proteins despite their low degree of sequence identity. The evolutionary relationship among the vitamin B6-dependent enzymes in general appears less clear. Conceivably, their common structural and mechanistic features are dictated by the chemical properties of pyridoxal 5'-phosphate rather than being due to a common ancestor of their protein moieties. In agreement with this notion, the ubiquitous active-site lysine residue that forms a Schiff base with the coenzyme can be replaced in the case of aspartate aminotransferase by a histidine residue without complete loss of catalytic competence.

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.

Mitochondrial ornithine aminotransferase was synthesized in a rabbit reticulocyte cell-free system using polysomal mRNA from rat liver. Upon analyzing the primary translation product by electrophoresis and fluorography, we found that ornithine aminotransferase is synthesized not as a large precursor but as one with its authentic size. Free ribosomes were found to be the main site of synthesis of ornithine aminotransferase.