Proton transfer in and polarizability of hydrogen bonds in proteins. Tyrosine-lysine and glutamic acid-lysine hydrogen bonds — IR investigations

The OH N O^– H⁺N hydrogen bonds formed between tyrosine and lysine, and between glutamic acid and lysine residues are studied by infrared spectroscopy considering the following systems: (l-lys)_n + phenol, copoly (l-lys, l-tyr)_n, (l-lys)_n + (l-tyr)_n and (l-lys)_n + (l-glu)_n. The phenol-lysine hydrogen bonds are largely symmetrical in the average if the pK_a of the protonated lysine is 2.2 units larger than that of the phenols. In the case of the hydrogen bonds between tyrosine and lysine residues in copoly (l-lys, l-tyr)_n and (l-lys)_n + (l-tyr)_n, the weight of the proton limiting structure OH N is 80–90%, and that of the polar O^– H⁺N structure 10–20%. Double minimum proton potentials occur but the proton is preferentially present at the tyrosine residues. In the (l-lys)_n + (l-glu)_n system, the protons are present at the lysine residues. Thus, these hydrogen bonds have very large dipole moments (about 10 D). With the lysine-phenole hydrogen bonds, hydration shifts the proton transfer equilibrium a little in favour of the polar proton limiting structure O^– H⁺N. These hydrogen bonds are broken to a large extent, however, when only about 3 water molecules are present per lysine residue. When less water is present, as in the copoly (l-lys, l-tyr)_n and (l-lys)_n + (l-tyr)_n systems, these hydrogen bonds are, however, formed quantitatively. Thus — as discussed in this paper — the tyrosine-lysine hydrogen bonds can participate in proton conducting hydrogen bonded systems — as, for instance, present in bacteriorhodopsin — performing the proton transport through hydrophobic regions of biological membranes.     

Ultrastructure of spermatozoa of Peregrinus maidis (Homoptera,Delphacidae)

Summary The spermatozoa of Peregrinus maidis Ashm. are thread-like, approximately 650 long and 1 wide including the head (approximately 28 ).The main part of the spermatozoa consists of two mitochondria derivatives, a central body between them, the axial filament complex, and a newly found element consisting of two wing-shaped bodies. Each mitochondrion derivative shows a peripheral and an inner part. The peripheral part is formed by cristae arranged perpendicularly to the long axis of the spermatozoon. The cristae are approximately 70 Å wide. The dense layers between them measure approximately 280 Å. The inner part of the mitochondrion derivative shows a crystalline array, formed by sub-units of approximately 100 Å diameter. The wing-shaped bodies consist of tubular elements.The head has an elongated nucleus with an electron transparent space inside. At the anterior end of the nucleus lies a tapered acrosome. This appears fibrous and parts of the acrosome fibers seem to run along the nucleus. Acknowledgements. The authors wish to thank Dr. G. H. Bergold for suggestions and support, Drs. J. André, D. W. Fawcett, P. Maillet and G. F. Meyer for very helpful discussion. They are also grateful to Mr. O. Suárez for assistance in the preparation of the organs of P. maidis and to Mrs. M. de Pingarrón for technical assistance.     

Ionic and non-ionic compounds in the germination of spores of bacillus megaterium texas

Summary Germination requirements of suspensions of spores of Bacillus megaterium, Texas strain, an l-alanine-inosine type, have been examined employing a decrease in optical density as the criterion of germination. In deionized water, l-alanine and inosine were devoid of germinative powers. They were effective only in conjunction with any one of a large variety of salts. Data are given for germination by the monovalent and divalent alkali metal chlorides. The potassium halides were germinative; potassium fluoride was the best. Salts of organic acids, including fatty acids and polycarboxylic acids, were germinative. The need for inosine could be bypassed by various salts, e.g., ammonium propionate or salts of dipicolinic acid. Also, l-alanine was replaceable by a variety of amino acids, provided suitable ions were present. In the presence of magnesium chloride, sodium dipicolinate could substitute for either inosine or l-alanine, but not both. Salts of n-hexylamine and n-heptylamine bypassed the need for both l-alanine and inosine. A primary role for ions in germination is proposed and a secondary, augmentative action is attributed to l-alanine and inosine.     

Evaluation of the bioactivities of extracts of endophytes isolated from Taiwanese herbal plants

The endophytic extracts from 19 endophytes, isolated from 13 species of Taiwanese plants, were evaluated for biological activity, including cytotoxicity, anti-platelet aggregation, and anti-inflammatory activity. The extracts of 12 endophytes exhibited inhibitory effects on collagen-induced platelet aggregation with IC₅₀ values of 19.85–87.64 μg/ml. Four strains, Rahnella aquatilis, Pantoea agglomerans, Rhodotorula sp., and Penicillium paxilli, also showed inhibitory effects on thrombin-induced platelet aggregation with IC₅₀ values of 42.80–61.54 μg/ml. Additionally 12 extracts of endophytes exhibited cytotoxicities with IC₅₀ values of 0.12–19.83 μg/ml. However, eight extracts revealed inhibitory effects on superoxide anion generation induced by fMLP (N-formyl-l-methionyl-l-leucyl-l-phenylalanine) in human neutrophils. The extract of Rahnella aquatilis showed anti-platelet aggregation activity, and bioassay-directed fractionation led to the isolation of six compounds, including one isoalloxazine: lumichrome (1); two isoflavones: genistein (2) and daidzein (3); two cyclic peptides: cyclo-Pro-Val (4) and cyclo-Pro-Phe (5); and one benzenoid: methyl 2,4,5-trimethoxybenzoate (6). These results indicated that endophytes from Taiwanese herbal plants could be useful sources for research and development of bioactive lead compounds from nature.     

Magnetic resonance Raman activity: Inverse Faraday effect in hemoglobin

A complete polarization study of human oxy- and carbonmonoxyhemoglobin (carbonylhemoglobin) A and S is reported for backscattered light in the resonance Raman light-scattering situation with excitations in the long-wavelength region, =5682 Å and 5815 Å, and in the short-wavelength region, =4579 Å and 4880 Å, as comparison. All four polarization components of the scattered light with respect to the two conditions of linear and circular polarization of the incident light were measured. These were: (1) parallel; (2) perpendicular; (3) corotating; and (4) contrarotating.This method has been used to characterize the three invariants of the nonsymmetric Raman tensor for randomly oriented molecules. These invariants are based on a model in which the scattered light is dependent on only an induced electric dipole. With no higher moments involved in the scattering process, the amplitude of any band measured under the four conditions should satisfy the relation: (1)+(2)=(3)+(4).The experiments reported here demonstrate that although this relation is satisfied for short-wavelength (4579 Å and 4880 Å) excitation, it does not apply for long-wavelength (5682 Å and 5815 Å) excitation, for which (1)+(2)<(3)+(4) holds.There are larged-d metal-electron influences on the long, but not the short, wavelength absorption region of hemoproteins. A large metal-electron magnetic dipole moment can thus be induced with circularly polarized light of long wavelength, owing to the availability of a transition involving charge transfer interaction coupling between metal orbitals and the porphyrin ^* orbitals.The increase in light scattering is due to aninverse Faraday effect, resulting in magnetic resonance Raman activity. Owing to large crystal field influences and Jahn-Teller instability, the critical zero-field splitting energy is also large. The incident circularly polarized radiation at =5700 Å (=17,544 cm^–1) is approximately the critical cubic splitting parameter, _C , the calculated value for which is 18,500 cm^–1. This incident light equalizes or mixes the energy levels of the¹ A ₁ and⁵ T ₂ states; i.e., it populates magnetic substates. An induced electric dipole-magnetic dipole transition, besides an induced electric dipole transition, is then permitted for circularly polarized light at the Larmor precession frequency, but not permitted for linearly polarized light, as thed-d metal transition is electric dipole disallowed.The hemoglobin S solution and gel scatter longwavelength light in a similar fashion; i.e., a large induced magnetic dipole moment is implicated for the circularly polarized conditions. In addition, there are bands at 750 and 1050 cm^–1 for the hemoglobin S gel not present in dissolved hemoglobin S nor in hemoglobin A. The presence of these bands is attributed to changes in theB _1g mode of porphyrin ring vibrations.     

d-Alanine dehydrogenase

Pseudomonas aeruginosa PA01 was found to utilise both thed- andl-isomers of -alanine and also -alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation ofd-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation ofl-alanine required its conversion to the directly oxidisabled-form by a soluble racemase; (iii) utilisation of -alanine, likel-alanine, involves both the racemase andd-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity, of which is still speculative; (iv)P. aeruginosa, likeEscherichia coli, appears to take upd-alanine andl-alanine by means of two specific permeases.Abbreviation DCPIP 2,6-dichlorophenol-indophenol     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

Lamelläre Einschlußkörper in den Zellkernen vonRhinanthus serotinus Oborny

Zusammenfassung In den Zellkernen der Antherenwand (mit Ausnahme des Tapetums) vonRhinanthus serotinus Oborny treten bis zu vier Einschlußkörper auf, die als Kristalloide vorliegen. Jedes Kristalloid besteht aus einem Stapel ± gleichartig ausgebildeter Lamellen, deren Periode etwa 180 Å beträgt (die Lamellendicke ist etwa 70 Å, der Lamellenabstand rund 110 Å). Die enge Nachbarschaft zwischen Nukleolus und Einschlußkörper scheint nicht zufällig zu sein: vielleicht dienen gewisse Orte am Nukleolus als Kristallisationszentren bei der Stapelbildung.

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Lamellar inclusions in the nuclei ofRhinanthus serotinus oborny

Summary The nuclei of the anthere wall (with the exception of the tapetum) contain inclusions of stacks of lamellae. The thickness of one lamella amounts to 70 Å, the distance from one lamella to the next one measures about 110 Å; the periodicity amounts to 180 Å. The close contact of stack and nucleolus in most nuclei seems to be not accidentically: perhaps locations around the nucleolus are places of cristallization of the stack.

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Herrn Prof. Dr.Karl Carniel danke ich für fruchtbringende Diskussion und FrauChrista Grubmann für technische Mithilfe.     

l-NNA inhibits the histochemical NADPH-d reaction in rat spinal cord neurons

In nerve tissue the histochemical nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction is considered a suitable marker for nitric oxide synthase (NOS) activity. We have previously shown that the NOS-specific inhibitorl-nitroarginine (l-NNA) can block NADPH-d staining in intermediolateral (IML) neurons of the rat spinal cord; such a reaction might serve as a control for the presence of a NOS-related catalytic activity, i.e.,l-NNA-dependent NO synthesis in these neurons. However,l-NNA inhibition of neuronal NADPH-d is inconsistent and is therefore disputed by others. This prompted us to reinvestigate the reaction conditions to provide a standardized protocol for inhibition experiments. In IML neurons of formaldehyde-fixed spinal cord tissue, inhibition of NADPH-d reaction was tested by preincubation of frozen sections with the flavin-binder diphenylene iodonium chloride (DPI, 10 M-1 mM) which blocked the NADPH-d reaction in a concentration-dependent way, suggesting an inverse relationship of inhibitor concentration and final reaction product generated. Preincubation with the NOS-specific inhibitorl-NNA in glycine-NaOH buffer (pH 8.5–9.5) but notl-nitroarginine methyl ester (l-NAME) revealed a concentration-dependent blocking effect on neuronal NADPH-d comparable to the effects seen with DPI, suggesting the existence of al-NNA sensitive NADPH-d activity. Blocking withl-NNA (100 M-10 mM) was prevented by excessl-arginine (10–100 mM), suggesting competitive binding sites. NADPH-d staining was not inhibited by 7-nitro indazole, another NOS inhibitor. Thus, in formaldehyde-fixed nervous tissue both DPI andl-NNA inhibit the NOS-associated catalytic NADPH-d activity, thereby preventing NADPH-dependent conversion of nitroblue tetrazolium to formazan.Presented in the Workshop Detection of NO-synthases at the XXXVI Symposium of the Society for Histochemistry on Oxy Radicals, 20–23 September 1994, Heidelberg, Germany     

Identification of mouse selenomethionine α, γ-elimination enzyme

The purpose of this study was to identify the seleno-l-methionine (l-SeMet) α,γ-elimination enzyme that catalyzes l-SeMet to generate methylselenol (CH₃SeH), a notable intermediate for the metabolism of selenium compounds, in mammalian tissues. The enzyme purified from ICR mouse liver was separated by one-dimensional gel electrophoresis, and the specific band was subjected to in-gel trypsin digestion followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis. In the peptide mass fingerprinting search, the mass numbers of 14 peptides produced by tryptic digestion of the enzyme were consistent with the theoretical mass numbers calculated from the amino acid sequence of murine cystathionine γ-lyase (E.C. 4.4.1.1). The peptide sequence tags search was also performed to obtain the amino acid sequence data of five tryptic peptides. These peptides were significantly identical to the partial amino acid sequences of cystathionine γ-lyase. This enzyme was clearly shown to catalyze the α, γ-elimination reaction of l-cystathionine by the enzymological research. The K _m value for the catalysis of l-cystathionine was 0.81 mM and V _max was. 0.0013 unit/mg protein. These results suggested that cystathionine γ-lyase catalyzes l-SeMet to generate CH₃SeH by its α,γ-elimination reaction.     

Zur chemischen Zusammensetzung der Zellwand der Streptokokken

Zusammenfassung Das Murein (Peptidoglycan) eines aus Faeces isolierten Streptococcus, der in den wichtigsten Merkmalen mit Peptostreptococcus evolutus (Prevot) Smith übereinstimmt, weist folgende Molverhältnisse auf (aufgerundete bzw. abgerundete Zahlen): Mur:GlcNH₂:Ala:Glu:Lys:Gly=1:1:3:1:1:1. Das Verhältnis l-Alanin:d-Alanin=2,15:1. Die Glutaminsäure liegt in der d-Konfiguration und als Amid vor.Durch die Partialhydrolyse der Zellwände und die anschließende Isolierung und Identifizierung der Peptide konnte die Aminosäuresequenz des Mureins geklärt werden. Das Tetrapeptid stimmt mit der üblichen Sequenz l-Ala-d-Glu-NH₂-l-Lys-d-Ala der meisten übrigen Bakterien überein. Die Quervernetzung des Mureins wird durch das Peptid Glycyl-l-Alanin hergestellt, wobei l-Alanin an die -Aminogruppe des Lysins gebunden ist. Die Dinitrophenylierung der Zellwand ergab, daß 35% des Glycins und 6% des Lysins eine freie Aminogruppe aufweisen. Die Quervernetzung ist demnach nur zu höchstens 60% durchgeführt.

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The chemical composition of the cell walls of Streptococci III. The amino acid sequence of a glycine containing murein from Peptostreptococcus evolutus (Prevot) Smith

Summary Peptostreptococcus evolutus was isolated from feces. Its murein containes muramic acid, glucosamine, alanine, d-glutamic acid, lysine and glycine at a molar ratio of about 1:1:3:1:1:1. The ratio of l-alanine: d-alanine is 2,15:1. Glutamic acid is present as an amide.By acid partial hydrolysis of the cell walls and subsequent isolation and identification of the peptides the amino acid sequence of the murein was elucidated. The tetrapeptide is identical with that of most bacteria (l-Ala-d-Glu-NH₂-l-Lys-d-Ala). The crosslinking of the murein is performed by the peptide glycyl-l-alanine. l-alanine is attached to the -amino group of lysine while the amino group of glycine is bound to the carboxyl group of the c-terminal d-alanine of an adjacent tetrapeptide. About 35% glycine and 6% lysine of the murein are dinitrophenylisable indicating that maximally 60% of the possible cross-linkages are realized.

     

Transformation of Nε-CBZ-l-lysine to CBZ-l-oxylysine using l-amino acid oxidase from Providencia alcalifaciens and l-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus

Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O₂ oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O₂ oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions. Correspondence to: R. L. Hanson     

Amino acid transport system y+L of human erythrocytes: Specificity and cation dependence of the translocation step

The transport specificity of system y⁺L of human erythrocytes was investigated and the carrier was found to accept a wide range of amino acids as substrates. Relative rates of entry for various amino acids were estimated from their trans-effects on the unidirectional efflux of l-[¹⁴C]-lysine. Some neutral amino acids, l-lysine and l-glutamic acid induced marked trans-acceleration of labeled lysine efflux; saturating concentrations of external l-leucine and l-lysine increased the rate by 5.3±0.63 and 6.2±0.54, respectively. The rate of translocation of the carrier-substrate complex is less dependent on the structure of the amino acid than binding. Translocation is slower for the bulkier analogues (l-tryptophan, l-phenylalanine); smaller amino acids, although weakly bound, are rapidly transported (l-alanine, l-serine). Half-saturation constants (±sem) calculated from this effect (l-lysine, 10.32±0.49 m and l-leucine, 11.50±0.50 m) agreed with those previously measured in cis-inhibition experiments. The degree of trans-acceleration caused by neutral amino acids did not differ significantly in Na⁺, Li⁺ or K⁺ medium, whereas the affinity for neutral amino acids was dramatically decreased if Na⁺ or Li⁺ were replaced by K⁺. The observation that specificity is principally expressed in substrate binding indicates that the carrier reorientation step is largely independent of the forces of interaction between the carrier and the transport site.We wish to thank Dr C.A.R. Boyd for helpful discussions and Prof. H.N. Christensen for sharing with us very relevant bibliographic material. We are grateful to FONDECYT (1282/91) and DTI (B 2674) (Chile) for financial assistance.     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to l-isoleucine

Summary Corynebacterium glutamicum ATCC 13 032 produces 13 g/l l-isoleucine from 200 mM -ketobutyrate as a synthetic precursor. In fed batch cultures up to 19 g/l l-isoleucine is formed. For optimal conversion the addition of 0.3 mM l-valine plus 0.3 mM l-leucine to the fermentation medium is required. The affinity constants for the acetohydroxy acid synthase (AHAS) were determined. (This enzyme directs the flow of -ketobutyrate plus pyruvate towards l-isoleucine and that of two moles of pyruvate to l-valine and l-leucine, respectively.) For -ketobutyrate the K _m is 4.8×10^-3 M, and V _max 0.58 U/mg, for pyruvate the K _m is 8.4×10^-3 M, and V _max 0.37 U/mg. Due to these characteristics the presence of high -ketobutyrate concentrations apparently results in a l-valine, l-leucine deficiency. This in turn leads to a derepression of the AHAS synthesis from 0.03 U/mg to 0.29 U/mg and high l-isoleucine production is favoured. The derepression of the AHAS synthesis induced by the l-valine, l-leucine shortage was directly proven with a l-valine, l-leucine, l-isoleucine auxotrophic mutant where the starvation of each amino acid resulted in an increased AHAS level. This is in accordance with the fact that only one AHAS enzyme could be verified by chromatographic and electrophoretic separations as being responsible for the synthesis of all three branched-chain amino-acids.     

The properties of l-fucose binding agglutinin associated with the cell wall of Rhizoctonia solani

The adherence of Escherichia coli B cells to cell wall associated-agglutinin of the soil borne plant pathogen Rhizoctonia solani, was inhibited by l-fucose, l-galactose, trypsin, SDS, cycloheximide and Na₂-EDTA. The coiling of the biocontrol agent Trichoderma harzianum around Rhizoctonia hyphae was prevented by SDS, cycloheximide, Na₂-EDTA and methyl--l-fucoside — an inhibitor of Rhizoctonia agglutinin not metabolized by both fungi. The possible role of the agglutinin in Trichoderma-Rhizoctonia interaction is discussed.     

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H 16

Properties and regulation of anthranilate synthase from Alcaligenes eutrophus H 16 were investigated. Anthranilate synthase was partially purified from crude extracts by affinity chromatography on tryptophan-substituted Sepharose, and was used for kinetic measurements. During the purification procedure the enzyme was stabilized by 50 mM l-glutamine or during chromatography on DEAE-cellulose and Sephadex G-200 with 30% glycerol, respectively.The glutamine dependent activity of anthranilate synthase was examined; it showed little change between pH 8.4 and pH 9.1. The Arrhenius plot was broken and the activation energy, H, calculated therefrom amounted to 8.9 kcal/mole up to 30°C and 5.5 kcal/mole at higher temperatures. The molecular weight determined by gelfiltration on Sephadex G-200 and by sucrose density gradient centrifugation resulted in 158000 and 126000, respectively. The K _m -values for the two substrates chorismate and glutamine were found to be 5 M and 560 M, respectively.Anthranilate synthase was strongly inhibited by l-tryptophan; the only amino acid that affected enzyme activity. Homotropic interactions for chorismate (Hill coefficient n=1.4) were obtained in the presence of l-tryptophan. 50% inhibition were caused by 10 M l-tryptophan at 100 M chorismate. The inhibition with respect to l-glutamine was noncompetitive.Anthranilate synthase was not associated to phosphoribosyl transferase and easily separable from the latter by different chromatographic methods.Abbreviation TEA triethanolamine     

Zum Abbau derl-Äpfelsäure durch Milchsäurebakterien

Zusammenfassung Es wird die Aktivität von intaktenL. plantarum-Zellen verschiedener Arten gegenüberl-Äpfelsäure, Oxalessigsäure und Brenztraubensäure getestet. Bei der Dissimilation vonl-Äpfelsäure lassen sich zwei pH-Optima unterscheiden, 2,6–3,0 für eine MDH-Aktivität und 3,6–4,0 für eine Malic-Enzym-Aktivität. Stoffwechselprodukte der Brenztraubensäure-Decarboxylierung sind außer Kohlendioxid Äthylalkohol und Acetoin bzw. Diacetyl.L. plantarum ist außerdem zur Oxydation der Brenztraubensäure befähigt. Ausl-Äpfelsäure entsteht kein Acetoin (Stamm L).

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The dissimilation ofl-malic acid by lactic acid bacteriaIV. The activity of intact cells ofLactobacillus plantarum particularly considering the decarboxylation of pyruvic acid

Summary The decomposition ofl-malic, oxaloacetic and pyruvic acids by intact cells of three strains ofL. plantarum is investigated. The dissimilation ofl-malic acid shows two pH-optima, at pH2.6–3.0 for a malatedehy drogenase activity and at pH 3.6–4.0 for a malic enzyme activity. The decarboxylation of pyruvic acid yields CO₂, ethyl alcohol, acetoin and diacetyl.L. plantarum is also able to oxidize pyruvic acid. The acetoin produced byL. plantarum Strain L does not originate froml-malic acid.

     

Presence of an X AG − carrier in frog (Rana esculenta) red blood cells

Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K _m = 3 m and low capacity (V_max) 0.4 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X _AG ^– family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K _m 39 m but high capacity (V _max) 1.8 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1.     

The differential allosteric regulation of two chorismate-mutase isoenzymes of Nicotiana silvestris

The reaction catalyzed by chorismate mutase (EC 5.4.99.5) is a crucial step for biosynthesis of two aromatic amino acids as well as for the synthesis of phenylpropanoid compounds. The regulatory properties of two chorismate-mutase isoenzymes expressed in Nicotiana silvestris Speg. et Comes are consistent with their differential roles in pathway flow routes ending with l-phenylalanine and l-tyrosine on one hand (isoenzyme CM-1), and ending with secondary metabolites on the other hand (isoenzyme CM-2). Isoenzyme CM-1 was very sensitive to allosteric control by all three aromatic amino acids. At pH 6.1, l-tryptophan was a potent allosteric activator (K _a =1.5 M), while feedback inhibition was effected by l-tyrosine (K _i =15 M) or by l-phenylalanine (K_i=15 M). At pH 6.1, all three effectors acted competitively, influencing the apparent K _m for chorismate. All three allosteric effectors protected isoenzyme CM-1 at pH 6.1 from thermal inactivation at 52° C. l-Tryptophan abolished the weak positive cooperativity of substrate binding found with isoenzyme CM-1 only at low pH. At pH 7.2, the allosteric effects of l-tyrosine and l-tryptophan were only modestly different, in striking contrast to results obtained with l-phenylalanine. At pH 7.2 (i) the K _i for l-phenylalanine was elevated over 30-fold to 500 M, (ii) the kinetics of inhibition became non-competitive, and (iii) l-phenylalanine now failed to protect isoenzyme CM-1 against thermal inactivation. l-Phenylalanine may act at different binding sites depending upon the intracellular pH milieu. In-vitro data indicated that the relative ability of allosteric activation to dominate over allosteric inhibition increases markedly with both pH and temperature. The second isoenzyme, CM-2, was inhibited competitively by caffeic acid (K _i =0.2 mM). Aromatic amino acids failed to affect CM-2 activity over a broad range of pH and temperature. Inhibition curves obtained in the presence of caffeic acid were sigmoid, yielding an interaction coefficient (from Hill plots) of n=1.8.Abbreviation DAHP synthase 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase     

Photoproduction of ammonium by Chlamydomonas reinhardtii cells immobilized in barium alginate: a reactor feasibility study

Summary Chlamydomonas reinhardtii cells immobilized in Ba-alginate beads provide a stable and effective system for photoproducing ammonium from nitrite in a culture medium containing l-methionine-d,l-sulphoximine. The process was studied in either air-lift, fluidized or packed-bed reactors, the last one providing the most suitable system with a volumetric activity of 2700 mol NH _inf+ ^sup4 ·1^–1 per hour.