Exogenous Lysine Research Articles

High‐lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein accumulation

Corn is one of the major crops in the world, but its low lysine content is often problematic for animal consumption. While exogenous lysine supplementation is still the most common solution for today's feed corn, high-lysine corn has been developed through genetic research and biotechnology. Reducing the lysine-poor seed storage proteins, zeins, or expressing a deregulated lysine biosynthetic enzyme, CordapA, has shown increased total lysine or free lysine content in the grains of modified corn plants, respectively. Here, by combining these two approaches through genetic crosses, the total lysine content has more than doubled in F1 progeny. We also observe a synergy between the transgenic zein reduction and the enhanced lysine biosynthesis by CordapA expression. The zein reduction plants are found to accumulate higher levels of aspartate, asparagine and glutamate, and therefore, provide excess precursors for the enhanced lysine biosynthesis.

CadC-Mediated Activation of the cadBA Promoter in Escherichia coli

The transcriptional activator CadC in Escherichia coli, a member of the ToxR-like proteins, activates transcription of the cadBA operon encoding the lysine decarboxylase CadA and the lysine-cadaverine antiporter CadB. cadBA is induced under conditions of acidic external pH and exogenous lysine; anoxic conditions raise the expression level up to 10 times. To characterize the binding mechanism of CadC, procedures for the purification of this membrane-integrated protein and its reconstitution into proteoliposomes were established. The binding sites of CadC upstream of the cadBA promoter region were determined by in vitro DNaseI protection analysis. Two regions were protected during DNaseI digestion, one from –144 to –112 bp, designated Cad1, and another one from –89 to –59 bp, designated Cad2. Binding of purified CadC to Cad1 and Cad2 was further characterized by DNA-binding assays, indicating that CadC was able to bind to both DNA fragments. Genetic analysis with promoter-lacZ fusions confirmed that both sites, Cad1 and Cad2, are essential for activation of cadBA transcription. Moreover, these experiments revealed that binding of H-NS upstream of the CadC-binding sites is necessary for repression of cadBA expression at neutral pH and under aerobic conditions. Based on these results, a model for transcriptional regulation of the cadBA operon is proposed, according to which H-NS is involved in the formation of a repression complex under non-inducing conditions. This complex is dissolved by binding of CadC to Cad1 under inducing conditions. Upon binding of CadC to Cad2 cadBA expression is activated.

Removing a bottleneck in theBacillus subtilis biotin pathway: BioA utilizes lysine rather thanS-adenosylmethionine as the amino donor in the KAPA-to-DAPA reaction

In biotin biosynthesis, DAPA aminotransferase encoded by the bioA gene catalyzes the formation of the intermediate 7,8-diaminopelargonic acid (DAPA) from 7-keto-8-aminopelargonic acid (KAPA). DAPA aminotransferases from Escherichia coli, Serratia marcescens, and Bacillus sphaericus use S-adenosylmethionine (SAM) as the amino donor. Our observation that SAM is not an amino donor for B. subtilis DAPA aminotransferase led to a search for an alternative amino donor for this enzyme. Testing of 26 possible amino acids in a cell-free extract assay revealed that only l-lysine was able to dramatically stimulate the in vitro conversion of KAPA to DAPA by the B. subtilis DAPA aminotransferase. The K(m) for lysine and KAPA was estimated to be between 2 and 25 mM, which is significantly higher than the K(m) of purified E. coli BioA for SAM (0.15 mM). This higher requirement for lysine resulted in accumulation of KAPA during fermentation of B. subtilis biotin producing strains. However, this pathway bottleneck could be relieved by either addition of exogenous lysine to the medium or by introduction of lysine deregulated mutations into the production strains.

Novel chimeric spermidine synthase-saccharopine dehydrogenase gene (SPE3-LYS9) in the human pathogen Cryptococcus neoformans.

The Cryptococcus neoformans LYS9 gene (encoding saccharopine dehydrogenase) was cloned and found to be part of an evolutionarily conserved chimera with SPE3 (encoding spermidine synthase). spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants were constructed, and these were auxotrophic for lysine and spermidine, spermidine, and lysine, respectively. Thus, SPE3-LYS9 encodes functional spermidine synthase and saccharopine dehydrogenase gene products. In contrast to Saccharomyces cerevisiae spe3 mutants, the polyamine auxotrophy of C. neoformans spe3-LYS9 mutants was not satisfied by spermine. In vitro phenotypes of spe3-LYS9 mutants included reduced capsule and melanin production and growth rate, while SPE3-lys9 mutants grew slowly at 30 degrees C, were temperature sensitive in rich medium, and died upon lysine starvation. Consistent with the importance of saccharopine dehydrogenase and spermidine synthase in vitro, spe3-lys9 mutants were avirulent and unable to survive in vivo and both functions individually contributed to virulence. SPE3-LYS9 mRNA levels showed little evidence of being influenced by exogenous spermidine or lysine or starvation for spermidine or lysine; thus, any regulation is likely to be posttranscriptional. Expression in S. cerevisiae of the full-length C. neoformans SPE3-LYS9 cDNA complemented a lys9 mutant but not a spe3 mutant. However, expression in S. cerevisiae of a truncated gene product, consisting of only C. neoformans SPE3, complemented a spe3 mutant, suggesting possible modes of regulation. Therefore, we identified and describe a novel chimeric SPE3-LYS9 gene, which may link spermidine and lysine biosynthesis in C. neoformans.

The davDT operon of Pseudomonas putida, involved in lysine catabolism, is induced in response to the pathway intermediate delta-aminovaleric acid.

Pseudomonas putida KT2440 is a soil microorganism that attaches to seeds and efficiently colonizes the plant's rhizosphere. Lysine is one of the major compounds in root exudates, and P. putida KT2440 uses this amino acid as a source of carbon, nitrogen, and energy. Lysine is channeled to delta-aminovaleric acid and then further degraded to glutaric acid via the action of the davDT gene products. We show that the davDT genes form an operon transcribed from a single sigma70-dependent promoter. The relatively high level of basal expression from the davD promoter increased about fourfold in response to the addition of exogenous lysine to the culture medium. However, the true inducer of this operon seems to be delta-aminovaleric acid because in a mutant unable to metabolize lysine to delta-aminovaleric acid, this compound, but not lysine, acted as an effector. Effective induction of the P. putida P(davD) promoter by exogenously added lysine requires efficient uptake of this amino acid, which seems to proceed by at least two uptake systems for basic amino acids that belong to the superfamily of ABC transporters. Mutants in these ABC uptake systems retained basal expression from the davD promoter but exhibited lower induction levels in response to exogenous lysine than the wild-type strain.

The Sulfolobus solfataricus Lrp-like Protein LysM Regulates Lysine Biosynthesis in Response to Lysine Availability

Although the archaeal transcription apparatus resembles the eukaryal RNA polymerase II system, many bacterial-like regulators can be found in archaea. Particularly, all archaeal genomes sequenced to date contain genes encoding homologues of Lrp (leucine-responsive regulatory protein). Whereas Lrp-like proteins in bacteria are involved in regulation of amino acid metabolism, their physiological role in archaea is unknown. Although several archaeal Lrp-like proteins have been characterized recently, no target genes apart from their own coding genes have been discovered yet, and no ligands for these regulators have been identified so far. In this study, we show that the Lrp-like protein LysM from Sulfolobus solfataricus is involved in the regulation of lysine and possibly also arginine biosynthesis, encoded by the lys gene cluster. Exogenous lysine is the regulatory signal for lys gene expression and specifically serves as a ligand for LysM by altering its DNA binding affinity. LysM binds directly upstream of the TFB-responsive element of the intrinsically weak lysW promoter, and DNA binding is favored in the absence of lysine, when lysWXJK transcription is maximal. The combined in vivo and in vitro data are most compatible with a model in which the bacterial-like LysM activates the eukarya-like transcriptional machinery. As with transcriptional activation by Escherichia coli Lrp, activation by LysM is apparently dependent on a co-activator, which remains to be identified.

Effect of exogenous lysine on the expression of early cephamycin C biosynthetic genes and antibiotic production in Nocardia lactamdurans MA4213.

In beta-lactam producing microorganisms, the first step in the biosynthesis of the beta-lactam ring is the condensation of three amino acid precursors: alpha-aminoadipate, L-cysteine and D-valine. In Nocardia lactamdurans and other cephamycin-producing actinomycetes, alpha-aminoadipate is generated from L-lysine by two sequential enzymatic steps. The first step involves a lysine-6-aminotransferase activity (LAT), considered to be one of the rate-limiting steps for antibiotic biosynthesis. Here, we report the effect of exogenous lysine on antibiotic production by N. lactamdurans MA4213. Lysine-supplemented cultures showed higher titers of cephamycin C, an effect that was more significant at early fermentation times. The increase in cephamycin C production was not quantitatively correlated with specific LAT activity in lysine-supplemented cultures. Observation of a positive effect of lysine on cephamycin C production by N. lactamdurans was dependent on carbon source availability in the culture media. Supplementation of the culture media with exogenous lysine did not affect the mRNA levels of the early biosynthetic genes controlled by the bidirectional promoter. These results indicate that L-lysine is required not only for antibiotic biosynthesis, but particularly as carbon or nitrogen source.

Unexpected enhancement of β-lactam antibiotic formation in Streptomyces clavuligerus by very high concentrations of exogenous lysine

Unexpected enhancement of β-lactam antibiotic formation in Streptomyces clavuligerus by very high concentrations of exogenous lysine

Unexpected enhancement of beta-lactam antibiotic formation in Streptomyces clavuligerus by very high concentrations of exogenous lysine.

L-Lysine is known to stimulate production of beta-lactam antibiotics by Streptomyces clavuligerus via provision of the lysine breakdown product, L-alpha-aminoadipic acid, which is a limiting precursor. Previous investigations utilized levels of 10-20 mM L-lysine as an addition to chemically-defined media resulting in 50-100% improvement in antibiotic production. We were surprised to note that as the concentration was further increased, the organism responded by producing even higher titers of antibiotics. The optimum concentration of 100 mM L-lysine yielded an approximate 500% increase in production with only minor effects on growth. DL- and D-Lysine also exerted enhancements suggesting the presence of a lysine racemase or some other route from D-lysine to L-alpha-aminoadipate in this organism; D-lysine was considerably less potent than DL- or L-lysine.

Ileal digestibilities and urinary losses of amino acids in pigs fed heat processed soybean products

Twelve crossbred gilts fitted with simple T-cannulas at the distal ileum were fed four soybean products in a replicated 4 × 4 Latin square. Treatments were soybean meal (SBM), extruded (Ex), full-fat soybeans (FFSB), roasted FFSB processed under normal conditions (RoN) and under conditions used to increase rumen bypass protein (RoB). The recovery of endogenous lysine at the distal ileum was determined with the homoarginine technique. Apparent ileal digestibilities of crude protein (CP) and all essential amino acids, with the exception of isoleucine and lysine, were higher ( P < 0.001) in SBM as compared to extruded FFSB but digestibilities in the latter were superior ( P < 0.001) to those in either form of roasted FFSB. Apparent ileal digestibilities of isoleucine and lysine and true digestibility of lysine were similar ( P > 0.05) in SBM and extruded FFSB, but lower ( P < 0.001) in either form of roasted FFSB. When compared to pigs fed extruded FFSB or SBM feeding either form of roasted FFSB resulted in a higher ( P < 0.001) endogenous as well as exogenous flow of lysine. Compared to feeding any other soybean product, RoB FFSB increased ( P < 0.05) urinary losses of lysine when expressed as a percentage of ileal digestible lysine. The present study demonstrated that reductions in apparent ileal lysine digestibilities resulted from higher recoveries of endogenous as well as exogenous lysine. Douze porcs femelles croisés munis d'une canule en T simple dans l'ilön distal ont reçu quatre produits de soja suivant plusieurs dispositifs en carrés latins 4 × 4. Les traitements consistaient en du tourteau de soja (SBM), des graines de soja extradées (Ex) non délipidées (FFSB), du FFSB grillé dans des conditions normales (RoN) ou permettant d'accroître la quantité de protéines non dégradées dans le rumen (RoB). La récupération de la lysine endogéne dans l'illéon distal était déterminée par la technique de l'homoarginine. La digistibilité iléale apparente des protéines (CP) et de tous les acides aminés essentiels, sauf de I'i9soleucine et de la lysine, étail supérieure ( P < 0,001) pour SBM que pour FFSB extradé, mais les digestibilités étaient plus élevées pour ce dernier que pour les deux types de FFSB grillé ( P < 0,001 ). La digestibilité iléale apparente de l'isoleucine el de la lysine et la digestibilité vraie de la lysine étaient similaires ( P > 0,05) pour SBM et FFSB extradé, mais plus faibles ( P < 0,001) pour les 2 types de FFSB grillé. En comparaison du FFSB extrudé ou du SBM, la consommation de FFSB grillé provoquait un flux plus élevé ( P < 0,001) de lysine endogène aussi bien qu'exogène. Par rapport à tous les autres produits du soja, RoB FFSB augmentait ( P < 0,05) les pertes urinaires de lysine experimées en pour cent de la lysine digestible iléale. Cette étude démontrait que la diminution de la digestibilité iléale apparente de la lysine résultait d'une récupération plus importante de lysine endogène aussi bien qu'exogène. Es wurden vier Futtermischungen hergestellt welche 16% Rohprotein aus je einem der untersuchten Sojaprodukte enthielten; extrudierte (Ex) Sojavollbohnen (SVB), SVB welche unier normalen Bedingungen geröstet wurden (RoN), SVB geröstet unter Bedingungen wie sie zur Erhöhung des rumenstabilen Proteins zur Anwendung gelangen (RoB) und Sojaextraktionsschrot (SBM). Zwölf Schweine, versehen mit T-förmigen Kanülen am distalen Ileum, erhielten die verschiedenen Rationen gemäss einem dreifach wiederholten 4 × 4 Lateinischem Quadrat. Die Wiederfindung des endogenen Lysins am distalen Ileum wurde durch den Einsatz der Homoarginintechnik ermöglicht. Die scheinbaren Verdaulichkeit des Rohproteins und aller essentiellen Aminosäuren, mit Ausnahme von Isoleucin and Lysin, waren höher ( P < 0.001) in SBM als in extrudierten SVB. Extrudierte SVB demonstrierten hingegen höhere Verdaulichkeiten als die beiden gerösteten Produkte. Die jeweiligen scheinbaren ilealen Rohproteinverdaulichkeiten für Ex, RoN und RoB SVB und SBM waren 76.2, 63.5, 61.9 und 81.9%. Die scheinbaren Verdaulichkeiten für Isoleucin und Lysin wie auch die wahre Verdaulichkeit des Lysins waren gleichwertig in SBM und Ex SVB, aber tiefer ( P < 0.001) in den beiden gerösteten Produkten. Die Menge des endogenem und exogenem Lysins am distalen Ileum war grösser ( P < 0.001) nach Verfütterung einer der beiden gerösteten Produkte als nach Verfütterung von Ex SVB oder SBM. Die Verfütterung von RoB SVB resultierte, im Unterschied zu allen anderen Sojaprodukten, in höheren ( P < 0.05) Lysinverlusten im Urin, wenn diese in Prozent des absorbierten Lysins errechnet werden. Die observierten tieferen scheinbaren ilealen Lysinverdaulichkeiten in gerösteten SVB sind, gemäss unseren Untersuchungen, das Resultat höherer Lysinverluste aus endogener wie auch exogener Herkunft. Die Hilzebehandlung roher SVB bei hohen Temperaturen, wie sie zum Beispiel zur Erhöhung des rumenslabielen Proteins angewendet werden, resultiert in hitzegeschädigtem Lysin, welches absorbiert aber metabolisch nichl vollständig verfügbar ist.

Altered pH and lysine signalling mutants of cadC, a gene encoding a membrane-bound transcriptional activator of the Escherichia coli cadBA operon.

The Escherichia coli CadC protein is required for activation of cadBA transcription under conditions of low external pH and exogenous lysine. cadBA encodes proteins involved in the decarboxylation of lysine to cadaverine, and cadaverine excretion. Sequence analysis suggested that CadC contains a single transmembrane segment separating a DNA-binding domain in the amino terminus from a periplasmic domain. Western analysis of subcellular fractions demonstrated that CadC is expressed and concentrated in the cytoplasmic membrane in cells grown either at an inducing pH (pH 5.8) or at a non-inducing pH (pH 7.6). Eight cadC mutants were isolated based on their ability to confer expression of a cadA-lacZ fusion independent of low external pH or exogenous lysine. Five of these mutants expressed the cadA-lacZ fusion at both pH 5.8 and pH 7.6, but retained the requirement for the lysine signal while the other three mutants displayed pH independence at pH 5.8 but not at pH 7.6. These results support a model in which CadC is a membrane-bound transcriptional activator of the cadBA operon capable of sensing (directly or indirectly) signals generated outside the cytoplasmic membrane as a consequence of acidic pH and lysine.

Roles of LysP and CadC in mediating the lysine requirement for acid induction of the Escherichia coli cad operon.

Expression of the Escherichia coli cadBA operon, encoding functions required for the conversion of lysine to cadaverine and for cadaverine excretion, requires at least two extracellular signals: low pH and a high concentration of lysine. To better understand the nature of the lysine-dependent signal, mutants were isolated which expressed a cadA-lacZ transcription fusion in the absence of lysine while retaining pH regulation. The responsible mutation in one of these isolates (EP310) was in cadC, a gene encoding a function necessary for transcriptional activation of cadBA. This mutation (cadC310) is in a part of the gene encoding the periplasmic domain of CadC and results in an Arg-to-Cys change at position 265, indicating that this part of the protein is involved in responding to the presence of lysine. Three other mutants had mutations mapping in or near lysP (cadR), a gene encoding a lysine transport protein that has previously been shown to regulate cadA expression. One of these mutations is an insertion in the lysP coding region. Thus, in the absence of exogenous lysine, LysP is a negative regulator of cadBA expression. Negative regulation by LysP was further demonstrated by showing that lysP expression from a high-copy-number plasmid rendered cadA-lacZ uninducible. Expression of cadA-lacZ in a strain carrying the cadC310 allele, however, was not affected by the plasmid-expressed lysP. Cadaverine was shown to inhibit expression of the cadA-lacZ fusion in cadC+ cells but not in a cadC310 background.

Regulation of alpha-aminoadipate reductase from Penicillium chrysogenum in relation to the flux from alpha-aminoadipate into penicillin biosynthesis.

The activity and regulation of alpha-aminoadipate reductase in three Penicillium chrysogenum strains (Q176, D6/1014/A, and P2), producing different amounts of penicillin, were studied. The enzyme exhibited decreasing affinity for alpha-aminoadipate with increasing capacity of the respective strain to produce penicillin. The enzyme from all three strains was inhibited by L-lysine, and the enzyme from the lowest producer, Q176, was least sensitive. Between pH 7.5 and 6.5, inhibition of alpha-aminoadipate reductase by L-lysine was pH dependent, being more pronounced at lower pH. The highest producer strain, P2, displayed the lowest alpha-aminoadipate reductase activity at pH 7.0. In Q176, the addition of 0.5-1 mM of exogenous lysine stimulated penicillin formation, whereas the same concentration was ineffective or inhibitory with strains D6/1014/A and P2. The addition of higher (up to 5 mM) lysine concentrations inhibited penicillin production in all three strains. In mutants of P. chrysogenum D6/1014/A, selected for resistance to 20 mM alpha-aminoadipate, highest penicillin production was observed in those strains whose alpha-aminoadipate reductase was most strongly inhibited by L-lysine. The results support the conclusion that the in vivo activity of alpha-aminoadipate reductase from superior penicillin producer strains of P. chrysogenum is more strongly inhibited by lysine, and that this is related to their ability to accumulate increased amounts of alpha-aminoadipate, and hence penicillin.

Biosynthetic pathway to rhizobitoxine in Pseudomonas andropogonis

When dl-[4- 13C]aspartic acid was provided at regular intervals to Pseudomonas andropogonis cultures between two and three days growth, hydroxythreonine was specifically labelled at C-4 (4-fold enrichment), and rhizobitoxine was specifically labelled at C-4 and C-1 (4-fold and 2-fold enrichments, respectively). The result shows that aspartic acid is converted directly to the 2-amino-4-oxy-but-3-enoic acid moiety of rhizobitoxine. Various l-[U- 14C]amino acids were added to P. andropogonis cultures after two days growth. [ 14C]Aspartic acid was rapidly (<30min) incorporated into rhizobitoxine ( ca 1.5%) and hydroxythreonine ( ca 0.5%). Incorporation from [ 14C]homoserine into rhizobitoxine and hydroxythreonine was 5.7 and 3.2% respectively, whereas from [ 14C]threonine it was 0.05 and 0%. These results suggest that homoserine lies on the pathway to rhizobitoxine and that the biosynthetic pathway to rhizobitoxine branches from the usual aspartic acid→threonine pathway prior to the formation of threonine. Whereas exogenous lysine and methionine had little effect on the biosynthesis of rhizobitoxine, both threonine and homoserine substantially reduced (2.8-fold and 11-fold respectively) the incorporation of 14C from aspartic acid into rhizobitoxine, by P. andropogonis. In the former case this was probably due to feedback inhibition of the aspartic→threonine pathway, and in the latter as a result of competition/dilution. The collective results suggest that the biosynthesis of rhizobitoxine follows the route aspartic acid→homoserine→hydroxythreonine→rhizobitoxine. On the basis of the present results and recent publications, speculation is made concerning the final stages of the biosynthesis of rhizobitoxine, and of the biosynthesis of dihydrorhizobitoxine, a compound that is frequently produced by Bradyrhizobium japonicum.

Erythrocyte rosettes provide an analogue for Schiff base formation in specific T cell activation.

Human T cells spontaneously bind sheep E and this reflects physiologic interactions between specific adhesion molecules, principally T cell CD2, and the sheep equivalent of LFA-3. This interaction is important in T cell adhesion and in transmission of accessory activational signals. In this respect, E rosettes provide a partial analogue for T cell:accessory cell interaction and rosetting induces functional alterations in T cells. In studies of Ag-dependent T cell activation, we have obtained evidence that the formation of covalent Schiff bases between ligands on APC and T cell is an essential element. In our study, the specific chemical criteria defining Schiff base formation were applied to T cell E rosettes formed at room temperature, as follows: 1) Prior formation of Schiff bases on T cell epsilon-amino groups by glutaraldehyde inhibited E rosette formation. 2) Rosette formation was inhibited in the presence of exogenous lysine. 3) Reduction of constitutive T cell aldehydes by NaBH4 inhibited subsequent E rosette formation. In response to these chemical modifications of cellular ligands, T cell E rosette formation and T cell inductive interaction with APC were affected in the same way. 4) Oxidation of NaBH4-treated T cells by NaIO4 or galactose oxidase to regenerate cell-surface aldehydes on N-acetylneuraminic acid or galactose residues respectively, consistently restored E rosette formation. 5) Conversion of reversible Schiff bases to irreversible secondary amines by NaCNBH3 stabilized E rosettes against mechanical disruption. Together, these data demonstrate that E rosettes provide an analogue for the Schiff base-forming reactions that are essential in specific T cell activation.

Dihydrodipicolinate synthase of lysine excreting and non excreting strains of bacillus megaterium

AbstractDihydrodipicolinate synthase activity of lysine producing strain of Bacillus megaterium and its high yielding mutant AECr 60f was highest during the end of the exponential phase and remained at the same level during the stationary phase when major bulk of lysine was accumulated. In comparision in a lysine non producing strain of B. megaterium the activity of the enzyme was low. Lysine producing strains has DHD synthase with two specialities, persistence during the stationary phase and insensitivity to repression by exogenous lysine. The high yielding mutant has increased DHD synthase activity which was not sensitive to feed back inhibition with lysine.

Partially compensated hypoadrenalism presenting with persistent skin pigmentation

A 33-year-old female presented in 1966 with striking pigmentation, typical of Addison's disease, and amenorrhea. Endocrine assessment then showed normal basal serum cortisol and urinary hydroxysteroid levels, but serum cortisol did not respond to stimulation with either exogenous ACTH or lysine vasopressin. Steroid replacement treatment was started. Treatment was discontinued by the patient on her own initiative and after some yr she was lost to follow-up. Reassessment in 1986 showed a pigmented patient who had continued in good health. She had a normal basal serum cortisol level with circadian variation. Plasma ACTH levels were high but showed diurnal rhythmicity and suppressed incompletely with 2 mg or 8 mg of dexamethasone/24 h. Plasma aldosterone levels were normal and showed appropriate postural changes, but plasma renin levels were high. This patient has an immunological profile of autoimmune disease with positive adrenal, thyroid microsomal and gastric parietal cell antibodies with a history of a premature menopause which may also be of autoimmune origin. She has been seen over a 20-yr period and despite her appearance still has no biochemical evidence of glucocorticoid or mineralocorticoid deficiency. It is suggested that the patient had compensated hypoadrenalism, with serum cortisol levels maintained in the normal range by high plasma ACTH levels and serum aldosterone levels maintained by high renin levels. The long term result of the high ACTH levels was increased skin pigmentation.

N6-(1-carboxyethyl)lysine formation by Streptococcus lactis. Purification, synthesis, and stereochemical structure.

During growth in an arginine-deficient (chemically defined) medium, cells of Streptococcus lactis K1 formed significant amounts of a previously undetected ninhydrin-positive compound. This intracellular compound did not cochromatograph with any of a wide range of amino acids or amino acid analogs tested. However, by two-dimensional thin layer chromatography, the unknown compound migrated close to the recently discovered N5-(1-carboxyethyl)ornithine (Thompson, J., Curtis, M. A., and Miller, S. P. F. (1986) J. Bacteriol. 167, 522-529; Miller, S. P. F., and Thompson, J. (1987) J. Biol. Chem. 262, 16109-16115). The purified compound behaved as a neutral amino acid and eluted between valine and methionine in the amino acid analyzer. The results of 1H NMR spectroscopy suggested the presence of a lysine backbone and a coupled methyl-methine unit in the molecule, and 13C NMR showed that there were nine carbon atoms, of which two (C-1 and C-7) were carboxyl carbons. The simplest structure compatible with the physicochemical data was that of an alkylated derivative of lysine. The identity of this new amino acid, N6-(1-carboxyethyl)lysine, was confirmed by chemical synthesis. In vivo labeling experiments conducted using L[U-14C]lysine and [epsilon-15N]lysine showed that exogenous lysine served as the precursor of intracellular N6-(1-carboxyethyl)lysine and that the epsilon-amino N atom was conserved during biosynthesis of the lysine derivative. Of the two possible diastereomers (2S,8S or 2S,8R) of N6-(1-carboxyethyl)lysine, comparative 13C NMR spectroscopy established that the amino acid produced by S. lactis K1 was exclusively of the 2S,8S configuration.

Activity of diaminopimelic acid decarboxylase in bacteria producing lysine

AbstractActivity of DAP‐decarboxylase in lysine overproducing strains of Micrococcus luteus, M. varians and Arthrobacter globiformisLb reached the highest level during the end of the exponetial phase of growth and remained at the same high level during the stationary phase of growth when major bulk of lysine was accumulated. In comparison in a lysine non‐producing strain of Arthrobacter globiformis I 4 the activity of the same enzyme was low. DAP‐decarboxylase of these three lysine overproducers has two specialities, persistence during the stationary phase and insensivity to repression by exogenous lysine.

Vasopressin Blood Pressure Dependency after α-Adrenoceptor Blockade in Normotensive and Mineralocorticoid-Salt Hypertensive Rats

1. The effect of α-adrenoceptor blockade by phentolamine on plasma arginine vasopressin, on vasopressin dependency of blood pressure and on responsiveness to exogenous lysine vasopressin was studied in conscious normotensive and in deoxycorticosterone (DOC)/salt-treated hypertensive rats. 2. Phentolamine infusion reduced blood pressure of DOC-treated hypertensive rats to levels significantly lower than those of similarly treated Wistar control animals. 3. Plasma vasopressin levels of the DOC-treated hypertensive and normotensive control rats were similar. Phentolamine infusion increased plasma arginine vasopressin levels to 109.4 ±21.8 pg/ml in the DOC-treated rats and to 44.4 ± 8.2 pg/ml in the control animals. It also enhanced the pressor response to exogenous lysine vasopressin in both groups. 4. Administration of the vasopressin analogue dPVDAVP had no effect on blood pressure in either the DOC-treated or the control group but induced a similar fall in blood pressure in both groups during α-adrenoceptor blockade with phentolamine.