Formation and degradation of Δ9-1,18-octadecenedioic acid from oleic acid by Candida tropicalis

Summary The formation and degradation of ⁹-1,18-octadecenedioic acid from oleic acid by mutant S ₇₆ of Candida tropicalis was studied. It was found that the mutant could convert oleic acid to ⁹-1,18-octadecenedioic acid by -oxidation of the terminal methyl group. The substance was identified by various analytical methods and could be degraded to unsaturated dioic acids of shorter chain length with even numbers of carbon atoms from 6 to 16, and saturated dioic acids with 6, 8 and 10 carbon atoms. Neither unsaturated nor saturated dioic acids with odd numbers of carbon atoms were found in the media. From these results some metabolic pathways of the degradation of this unsaturated dioic acid are proposed. Some mass spectra of unsaturated dioic acids are presented.     

Degradation of trans-1,2-dichloroethene by mixed and pure cultures of methanotrophic bacteria

Summary Out of seven chlorinated aliphatic hydrocarbons tested, only trans-1,2-dichloroethene was relatively non-toxic for a mixed methanotrophic culture. The compound was degraded at a rate of 0.4 mol/mg protein·h^-1 and liberation of inorganic chloride was observed. Trans-2,3-dichlorooxirane was formed as an intermediate which was converted further only by chemical transformation with a half life of 31 h. From the consortium, a pure culture was isolated and found to be capable of degradation of trans-1,2-dichloroethene when grown in the presence of methane or methanol. The ability of cometabolic degradation of this compound was not specific for this isolate, since Methylomonas methanica NCIB11130 and Methylosinus trichosporium OB3b also showed degradation of trans-1,2-dichloroethene when grown with methane as sole carbon source.Abbreviations t12DCE trans-1,2-dichloroethene - t23DCO trans-2,3-dichlorooxirane - c23DCO cis-2,3-dichlorooxirane - ¹H-NMR proton nuclear magnetic resonance     

Desaturation of oxygenated fatty acids in Lesquerella and other oil seeds

Species of the genus Lesquerella, within the Brassicaceae family, have seed oils containing hydroxy fatty acids. In most Lesquerella species, either lesquerolic (14-hydroxy-eicosa-11-enoic), auricolic (14-hydroxy-eicosa-11,17-dienoic) or densipolic (12-hydroxy-octadeca-9,15-dienoic) acid dominates in the seed oils. Incubations of developing seed from Lesquerella species with 1-¹⁴C-fatty acids were conducted in order to study the biosynthetic pathways of these hydroxylated fatty acids. [¹⁴C]Oleic (octadeca-9-enoic) acid, but not [¹⁴C]linoleic (octadeca-9,12-dienoic) acid, was converted into the hydroxy fatty acid, ricinoleic (12-hydroxy-octadeca-9-enoic) acid, which was rapidly desaturated to densipolic (12-hydroxy-octadeca-9,15-dienoic) acid. In addition, [¹⁴C] ricinoleic acid added to Lesquerella seeds was efficiently desaturated at the 15 carbon. A pathway for the biosynthesis of the various hydroxylated fatty acids in Lesquerella seeds is proposed. The demonstration of desaturation at position 15 of a fatty acid with a hydroxy group at position 12 in Lesquerella prompted a comparison of the substrate recognition of the desaturases from Lesquerella and linseed. It was demonstrated that developing linseed also was able to desaturate ricinoleate at position 15 into densipolic acid. In addition, the linseed 15 desaturase was able to desaturate vernolic (12,13-epoxy-octadeca-9-enoic) acid and safflower microsomal 12 desaturase was able to desaturate 9-hydroxy-stearate. Thus, hydroxy and epoxy groups may substitute for double bonds in substrate recognition for oil-seed 12 and 15 desaturases.Abbreviations GLC gas-liquid chromatography - lysoPC palmitoyl-lysophosphatidylcholine - PC phosphatidylcholine This work was supported by grants from Stifteisen Svensk Oljeväxtforskning, Skanska Lantmännen Foundation, Swedish Farmers Foundation for Agricultural research, The Swedish Natural Science Research Council and The Swedish Council for Forestry and Agricultural Research. Nicki Engeseth was supported by the National Science Foundation under a grant award in 1992.     

Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles

Summary We have confirmed previous demonstrations of sodium gradient-stimulated transport ofl-alanine, phenylalanine, proline, and -alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these¹⁴C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability ofd-glucose to inhibit tracer uptake.l-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the¹⁴C-l-alanine uptake could be inhibited by proline and -alanine, while lysine and MeAIB were no more effective thand-glucose. Roughly 50% of the¹⁴C-phenylalanine uptake could be inhibited by proline and -alanine; lysine was as effective as proline and -alanine, and the effects of pairs of these amino acids at 50mm each were not cumulative. MeAIB was no more effective thand-glucose. We conclude that three pathways mediate the uptake of neutral,l, -amino acids. One system is inaccessible to lysine, proline, and -alanine. The second system carries a major fraction of thel-alanine flux; it is sensitive to proline and -alanine, but not to lysine. The third system carries half the¹⁴C-phenylalanine flux, and it is sensitive to proline, lysine, and -alanine. Since the neutral,l, -amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classicalA system, and since all of thel-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classicalASC system.l-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective thand-glucose in inhibiting lysine uptake, while proline and -alanine appear to inhibit a component of the lysine flux. We conclude that the¹⁴C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, -alanine, andl-alanine, and one which is inhibited byl-alanine and phenylalanine but inaccessible to proline, -alanine, and MeAIB. Fluxes of¹⁴C-proline and¹⁴C-MeAIB are completely inhibited byl-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit¹⁴C--alanine uptake. However, -alanine inhibits only 38% of the¹⁴C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports -alanine.     

Intermediary metabolism of carbon compounds by nitrifying bacteria

Summary The assimilation of¹⁴CO₂ and [2-¹⁴C] acetate, [3-¹⁴C] pyruvate, [5-¹⁴C] -ketoglutarate, [2,3-¹⁴C] succinate, [U-¹⁴C] glutamate and [U-¹⁴C] aspartate was followed in cell suspensions ofNitrosomonas europaea andNitrobacter agilis respectively. There was appreciable incorporation of these substrates even without adding the inorganic nitrogen compounds that are oxidized by these bacteria yielding ATP. In the soluble amino acid fraction most of¹⁴C label was recovered in glutamate while in the protein amino acids a more uniform distribution was found. Acetate was rapidly incorporated to a high level in both nitrifying bacteria while inNitrobacter there was a relatively lower uptake of the other substrates especially succinate. High levels of the NAD malate dehydrogenase and NADP isocitrate dehydrogenase were measured but no significant amounts of the other tricarboxylic acid cycle enzymes or NADH oxidase were found. Glutamate decarboxylase was detected in both organisms and the transferase assay for glutamine synthetase indicated a 30-fold higher activity for this enzyme inNitrobacter. The amino acid composition of the water soluble fraction was determined in both bacteria.     

Metabolism of α-aminoisobutyric acid in mungbean hypocotyls in relation to metabolism of 1-aminocyclopropane-1-carboxylic acid

1-Aminocyclopropane-1-carboxylic acid (ACC) is known to be converted to ethylene and conjugated into N-malonyl-ACC in plant tissues. When -amino[1-¹⁴C]isobutyric acid (AIB), a structural analog of ACC, was administered to mungbean (Vigna radiata L.) hypocotyl segments, it was metabolized to ¹⁴CO₂ and conjugated to N-malonyl-AIB (MAIB). -Aminoisobutyric acid inhibited the conversion of ACC to ethylene and also inhibited, to a lesser extent, N-malonylation of ACC and d-amino acids. Although the malonylation of AIB was strongly inhibited by ACC as well as by d-amino acids, the metabolism of AIB to CO₂ was inhibited only by ACC but not by d-amino acids. Inhibitors of ACC conversion to ethylene such as anaerobiosis, 2,4-dinitrophenol and Co²⁺, similarly inhibited the conversion of AIB to CO₂. These results indicate that the malonyalation of AIB to MAIB is intimately related to the malonylation of ACC and d-amino acids, whereas oxidative decarboxylation of AIB is related to the oxidative degradation of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyric acid - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid - MAIB -(malonylamino)-isobutyric acid - Mes 2-(N-morpholino)ethanesulfonic acid     

Degradation of diphenylether by Pseudomonas cepacia Et4: enzymatic release of phenol from 2,3-dihydroxydiphenylether

2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation. The enzyme was purified and characterized. It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa). The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols. The K _m-value of 1 M for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate. The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates. Isotope labeling experiments carried out with ¹⁸O₂ and H₂ ¹⁸O indicated the incorporation of ¹⁸O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate. Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al. (1982).Non-standard abbreviations DPE diphenylether - 2,3-dihydroxy-DPE 2,3-dihydroxydiphenylether - PCA 2-pyrone-6-carboxylic acid - 2,3-dihydroxy-BP dioxygenase 2,3-dihydroxybiphenyl dioxygenase - GC gas chromatography     

Chemical composition of the lipopolysaccharides of Ectothiorhodospira shaposhnikovii,Ectothiorhodospira mobilis,and Ectothiorhodospira halophila

Lipopolysaccharides were isolated from the moderate halophilic Ectothiorhodospira shaposhnikovii slight to and Ectothiorhodospira mobilis and from the extremely halophilic Ectothiorhodospira halophila by the hot phenol-water and purified by the phenol-chloroform-petroleum ether methods. The isolated lipopolysaccharides of all three species contained 3-deoxy-d-manno-octulosonic acid and d-glycero-d-mannoheptose indicating the existence of a core. They contained additionally glucose and uronic acids (E. shaposhnikovii and E. mobilis) or glucose, uronic acids and threonine (E. halophila). Sodium deoxycholate gel-electrophoresis of the three lipopolysaccharides, each showing only one major band, indicated R-type character of the lipopolysaccharides of the three Ectothiorhodospira species.The lipid A fractions of the lipopolysaccharides from E. shaposhnikovii and E. mobilis represented phosphorylated mixed lipid A types with both 2,3-diamino-2,3-dideoxy-d-glucose and d-glucosamine. The lipid A from E. halophila contained also phosphate and 2,3-diamino-2,3-dideoxy-d-glucose but only traces of d-glucosamine, which would indicated lipid A_DAG. The fatty acid spectra were characterized by amide-bound 3-OH-10:0 and 3-OH-12:0 (E. shaposhnikovii), 3-OH-10:0 (E. mobilis), or 3-OH-10:0,3-OH-14:0, and 3-oxo-14-0 (E. halophila). The predominant ester-bound fatty acids were 14:0 and 16:0 (E. shaposhnikovii and E. mobilis), or 12:0 and 14:1 (E. halophila).Abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 3-deoxy-d-manno-octulosonic acid - GlcA glucuronic acid - GalA galacturonic acid - GC-MS combined gas liquid chromatographymass spectrometry - GlcN Glucosamine - DOC sodium deoxycholate - LPS lipopolysaccharide - PAGE polyacrylamide gel electrophoresis - PCP phenol-chloroform-petroleum ether     

Positional specificity in the incorporation of isomeric cis- and trans-octadecenoic acids into glycerolipids of cultured soya cells

Heterotrophically grown cell suspension cultures of soya (Glycine max L.) were incubated with two different mixed substrates consisting of positional isomers of either cis-[1-¹⁴C]octadecenoic acids (8 to 15) or trans-[1-¹⁴C]octadecenoic acids (8 to 16), each with known composition. With both substrates, about one-fourth of the radioactivity supplied was incorporated into the diacylglycerophosphocholines, while another one-fourth of the radioactivity was almost equally distributed between diacylglycerophos-phoethanolamines and triacylglycerols. All the positional isomers of cis-and trans-octadecenoic acids supplied to the cells were readily incorporated into various classes of glycerolipids. None of the octadecenoic acids was isomerized, elongated or desaturated during incubation. From the cis-octadecenoic acids, only the naturally occurring 9-isomer (oleic acid) was preferentially incorporated into position 2 of diacylglycerophosphocholines, diacylglycerophospho-ethanolamines, and triacyglycerols; all the other isomers exhibited a strong affinity for position 1 of the glycerophospholipids and positions 1 and 3 of the triacylglycerols. From the trans-octadecenoic acids, only the 9-isomer (elaidic acid) was preferentially incorporated into position 2 of diacylglycerophospho-cholines and triacylglycerols; all the other isomers preferred position 1 and positions 1 and 3, respectively, of these lipids. In diacylglycerophospho-ethanolamines, however, each of the trans-octadecenoic acids, including the 9-isomer, exhibited a strong affinity for position 1. Apparently, the enzymes involved in the incorporation of exogenous monounsaturated fatty acids into membrane lipids of plant cells can recognize the preferred substrate in a mixture of closely related isomers.     

Salinity dependence,uptake kinetics,and specificity of amino-acid absorption across the body surface of the oligochaete annelidEnchytraeus albidus

Enchytraeus albidus is able to absorb dissolved¹⁴C-labeled neutral amino acids (glycine, L-alanine, L-valine,-aminoisobutyric acid) and an amino-acid mixture from ambient water across the body surface against considerable concentration gradients. Saturation kinetics and susceptibility of glycine uptake to competitive inhibition by alanine suggest mediated transport. Absorption of neutral amino acids is an active process. Exchange diffusion of preloaded-aminoisobutyric acid against external glycine or-aminoisobutyric acid could not be detected. Results on inhibition of glycine uptake by a variety of low-molecular-weight substances indicate that glycine absorption is highly specific for neutral amino acids and somewhat less for basic amino acids; it is unspecific for non--amino acids, acidic amino acids, carbohydrates, and organic acids. Rates of transintegumentary net influx of glycine are nearly identical to¹⁴C-glycine influx, suggesting that only small amounts of amino acids are released, as compared with the capacity for uptake. Thus,¹⁴C-amino-acid influx data are used for characterization of the uptake system. Glycine uptake is positively correlated to external salinity. In fresh water, absorption is nearly zero; between 10 and 20 S, uptake increases markedly reaching maximum values at 30 S; these remain almost constant at 40 S. Transport constants and maximum uptake rates increase with rising salinities. Since absorption of glycine and L-valine is susceptible to sodium depletion, similar mechanisms presumably underly salinity-dependent uptake of amino acids and sodium-dependent solute transport. Oxygen consumption is not significantly modified by different external salinities. Estimates of nutritional profit gained from absorption of amino acids vary between 4 and 15 % of metabolic rate for glycine absorption and between 10 and 39 % for uptake of an amino-acid mixture, according to external concentrations (10 and 50 µM) and salinities (20 and 30 S).     

Involvement of the glutamate receptor AtGLR3.3 in plant defense signaling and resistance to Hyaloperonospora arabidopsidis

Like their animal counterparts, plant glutamate receptor‐like (GLR) homologs are intimately associated with Ca²⁺ influx through plasma membrane and participate in various physiological processes. In pathogen‐associated molecular patterns (PAMP)‐/elicitor‐mediated resistance, Ca²⁺ fluxes are necessary for activating downstream signaling events related to plant defense. In this study, oligogalacturonides (OGs), which are endogenous elicitors derived from cell wall degradation, were used to investigate the role of Arabidopsis GLRs in defense signaling. Pharmacological investigations indicated that GLRs are partly involved in free cytosolic [Ca²⁺] ([Ca²⁺]_cyt) variations, nitric oxide (NO) production, reactive oxygen species (ROS) production and expression of defense‐related genes by OGs. In addition, wild‐type Col‐0 plants treated with the glutamate‐receptor antagonist 6,7‐dinitriquinoxaline‐2,3‐dione (DNQX) had a compromised resistance to Botrytis cinerea and Hyaloperonospora arabidopsidis. Moreover, we provide genetic evidence that AtGLR3.3 is a key component of resistance against H. arabidopsidis. In addition, some OGs‐triggered immune events such as defense gene expression, NO and ROS production are also to different extents dependent on AtGLR3.3. Taken together, these data provide evidence for the involvement of GLRs in elicitor/pathogen‐mediated plant defense signaling pathways in Arabidopsis thaliana.     

Untersuchungen zur Biosynthese eines Cytokinins in Calluszellen von Laubmoossporophyten

Summary When callus cells derived from the sporogon of the hybrid Funaria hygrometrica x Physcomitrium piriforme are supplied with adenine-8-¹⁴C, they produce a labelled cytokinin which has the same chromatographic behavior as N⁶--(dimethylallyl)aminopurine. The cytokinin is the first radioactive product that can be detected in the culture medium. It is formed as long as labelled adenine is available. When callus cells are grown in an optimum culture medium containing amino acids, about 10% of the radioactivity supplied as adenine is found in the cytokinin. When the cells are grown in a medium without amino acids, the RNA-content of the cells and the total yield of cytokinin decrease, but about 18% of the radioactivity is taken up into RNA as adenine and guanine, which are both degraded to allantoin and urea but not converted to cytokinin. Free guanine is converted neither to adenine nor to cytokinin.Weak cytokinin activity can be detected in hydrolysates of sRNA, but no radioactive cytokinin can be isolated from sRNA of adenine-labelled callus cells. it is assumed that free cytokinin is not a degradation product of tRNA.     

Incorporation of [214C]malonyl CoA into fatty acids by a cell-free extract of Catharanthus roseus suspension culture cells

A cell-free extract containing the enzymes for de-novo synthesis, elongation and desaturation of fatty acids was prepared from cultured cells of Catharanthus roseus G. Don. ¹⁴C-Fatty acids synthesized by the extract from [2-¹⁴C]malonyl CoA substrate were palmitic (16:0), stearic (18:0) and oleic (18:1). Dialyzed extract was active and stable at room temperature and at 4° C, but was inactivated on boiling. There was an absolute requirement for NADPH for incorporation of [2-¹⁴C]malonyl CoA into total fatty acids. Escherichia coli acyl carrier protein stimulated total fatty-acid synthesis without affecting the relative ratio of individual fatty acids. Total fatty-acid synthesis at a rate of 45 nmol·mg^-1 protein·h^-1 occurred at a substrate level of 73 M malonyl CoA, cofactor levels of 500 M NADPH, 30 g·ml^-1 E. coli ACP, and 1.0 mg·ml^-1 extract protein. Total fatty acid synthesis was also sensitive to cerulenin and CoA levels. Variations in the relative abundance of individual ¹⁴C-fatty acids were regulated by concentrations of [¹⁴C]malonyl CoA. NADPH and ferredoxin, as well as by pH, temperature and length of incubation. Fatty-acid synthetase enzymes responsible for [¹⁴C]palmitic acid were rapidly saturated at a low substrate level (0.3 M malonyl CoA). Increasing the level of [2-¹⁴C]malonyl CoA permitted further synthesis of [¹⁴C]stearate and [¹⁴C]oleate. Desaturation of [¹⁴C]stearate to [¹⁴C]oleate was stimulated by increasing the levels of NADPH and ferredoxin. The desaturase and elongase enzymes were sensitive to acidic pH. The desaturase was also unstable at 41° C, although fatty acid synthetase and elongase were unaffected by this temperature.Abbreviation ACP Acyl carrier protein     

Cell wall teichoic acids of actinomycetes of three genera of the order actinomycetales

The structures of cell wall teichoic acids of the members of newly recognized genera of the order Actinomycetales were studied. Planotetraspora mira VKM Ac-2000^T contains two types of teichoic acids: 2,3-poly(glycerol phosphate) substituted with -D-Galp at C-1 of glycerol and 1,3-poly(glycerol phosphate) substituted with -L-Rhap at OH-2 of glycerol (60%). Herbidospora cretacea VKM Ac-1997^T contains the chains of 1,3-poly(glycerol phosphate) partially substituted with -D-Galp and -D-GalpNAc at C-2 of glycerol. The majority of -D-galactopyranosyl residues are substituted at OH-3 with a sulfate. The aforementioned teichoic acids have not been found in bacteria thus far. Actinocorallia herbida VKM Ac-1994^T contains poly(galactosylglycerol phosphate), with the -Galp-(12)-Gro-P repeating units being linked via the phosphodiester bonds between the OH-3 of glycerol and OH-6 of galactose. Earlier, this structure was found in the cell wall of Actinomadura madura. The polymer structures were determined by chemical analysis and using ¹³C-NMR spectroscopy. The results show that teichoic acids are widespread in the order Actinomycetales.     

Heterogeneous Long Chain Acyl-CoA Synthetases Control Distribution of Individual Fatty Acids in Newly-Formed Glycerolipids of Neuronal Cells Undergoing Neurite Outgrowth

Using PC12 cells undergoing neurite outgrowth, we studied the activation of various fatty acids, of different chain lengths and degrees of saturation, by long chain acyl-CoA synthetases (LCASs). Cells treated with nerve growth factor (NGF) were labeled with [³H]glycerol, [³H]oleic acid (OA) or [³H]arachidonic acid (AA) in the presence of other unlabeled fatty acids of endogenous or exogenous origin. Triacsin C (4.8 M), an inhibitor of acyl-CoA synthetase, decreased the incorporation of exogenous [³H]OA into glycerolipids by 30–90%, and increased by about 60% the accumulation of free [³H]OA in the cells. However it did not affect the incorporation of endogenous fatty acids nor of exogenous [³H]AA into phospholipids, suggesting that LCASs which activate exogenous AA and at least some endogenous fatty acids are relatively insensitive to this drug. Activities of the LCAS that is specific for AA (ACS), or of the non-specific LCAS which activates OA and other fatty acids (OCS), were much higher in microsomal and cytoplasmic fractions than in mitochondria or nuclei. The Vmax and Km values of ACS and OCS in microsomes were 12 and 0.7 nmol/min/mg protein and 70 and 37 M, respectively; and in cytoplasm, 6 and 0.6 nmol/ min/mg protein and 38 and 60 M, respectively. Triacsin C (2–33 M) did not affect ACS activity in microsomal or cytoplasmal fractions, but inhibited OCS activities dose-dependently and competitively: IC₅₀ and apparent Ki values were 13.5 M and 14 M in microsomes, and 3.8 M and 4 M in cytoplasm. NGF stimulated the activities of the LCASs, and, consistently, the incorporation of the various fatty acids into glycerolipids. These data indicate that LCASs are heterogeneous with respect to their intracellular locations, substrate specificities, kinetic characteristics and sensitivities to triacsin C; and that this heterogeneity affects the extents to which individual fatty acids are utilized to form glycerolipids.     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

In diatoms, the siliceous cell walls are enveloped by an organic component which includes 4-hydroxyproline and 3,4-dihydroxy-L-proline. The formation of these two amino acids were studied in Nitzschia angularis in Si-starvation synchrony. Both appear to arise from peptidyl proline. Its conversion to peptidyl hydroxyproline was shown in cell-free extracts and in kinetic studies using [¹⁴C]proline. Two lines of evidence indicate that dihydroxyproline does not arise from the further hydroxylation of peptidyl hydroxyproline: First, there was a lag of several minutes between the incorporation of [¹⁴C]proline into protein and the appearance therein of [¹⁴C]hydroxyproline but no such lag for the appearance of dihydroxyproline. Second, ,-dipyridyl blocked the formation of hydroxyproline, but not of dihydroxypyroline, from peptidyl proline. Cell walls made in the presence of dipyridyl differed little in overall chemical composition from walls made in its absence and were morphologically identical. [¹⁴C]dehydroproline was rapidly metabolized in the cells, with [¹⁴C]dihydroxyproline a prominent product. Studies of the conversion of [¹⁴C]proline to [¹⁴C]hydroxyproline at various stages of wall formation showed an increased synthesis of [¹⁴C]dihydroxyproline at the end of cell separation.     

Regulation of Phosphatidylethanolamine Degradation by Enzyme(s) of Subcellular Fractions from Cerebral Cortex

Hydrolysis of 1-acyl-2-[¹⁴C]arachidonoyl-sn-glycero-3-phosphoethanolamine was studied in cerebral cortex homogenate and subcellular fractions. The enzyme(s) confined to the synaptic plasma membrane (SPM) hydrolyze(s) [¹⁴C-arachidonoyl]phosphatidylethanolamine (PE) in the presence of EGTA to [¹⁴C-arachidonoyl]diacylglycerol (DAG) and a small amount of [¹⁴C]arachidonic acid (AA). Degradation of PE is time-, protein- and substrate-dependent with a pH optimum of 7.8. The highest activity of PE degradation was observed in the presence of 10 mM EGTA. Under this condition GTPS has no effect on PE hydrolysis. In the presence of Ca²⁺ ions degradation of PE was significantly lower as compared to the conditions with EGTA. However, the percentage distribution of free AA in the sum of both products of PE hydrolysis (AA + DAG) increases from 16 and 20% observed in the presence of EGTA 2 mM and 10 mM to 34% and 43% in the presence of 0.5 mM CaCl₂ alone and together with GTPS, respectively. Cytosolic enzymes also degrade PE in the presence of 2 mM EGTA with the formation of DAG and AA. Radioactivity in the AA represents about 80% of the total radioactivity of the products of PE degradation. The hydrolysis of PE by cytosolic enzymes is almost completely inhibited by neomycin but the hydrolysis by the SPM-bound enzyme(s) is inhibited only 70%. Other studies with quinacrine indicated that only a small pool of PE is degraded by SPM-bound Ca²⁺-independent phospholipase A₂ (PLA₂). All of these data suggest that PE in cerebral cortex is mainly degraded by cytosolic and SPM-bound Ca²⁺-independent phospholipase C. Further studies towards a better understanding of the mechanisms of cerebral degradation and the physiological significance of Ca²⁺-independent pathways of PE hydrolysis are necessary.     

Bacterial siderophores : structure and NMR assignment of pyoverdins Pa,siderophores ofPseudomonas aeruginosa ATCC 15692

Summary In iron-deficient conditions,Pseudomonas aeruginosa ATCC 15692 synthesizes two major siderophores, pyoverdins Pa and pyoverdin Pa B. Two other compounds, pyoverdin Pa A (occurring from hydrolysis of pyoverdin Pa during the culture) and pyoverdin Pa C (occurring artifactually during the purification procedure) were also isolated. All these compounds possess the same partly cyclic peptide chain wherel-Orn(OH · HCO) isN -formyl,N -hydroxy-l-ornithine. The chain is bound to a chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and having the (S) configuration. The four pyoverdins differ only in the acyl substituent bound to the nitrogen atom bound to carbon C3 of the chromophore. This is succinamide (pyoverdin Pa), succinic acid (pyoverdin Pa A), methyl succinate (pyoverdin Pa C) and 2-oxoglutaric acid (pyoverdin Pa B). The complete¹H- and¹³CNMR assignments, using two-dimensional total correlation NMR spectroscopy (TOCSY) and rotating-frame Overhauser enhancement spectroscopy (ROESY) procedures, as well as¹H-¹³C correlations, are reported. The complete sequence of the peptide using CH-NH correlations was achieved by NMR and confirmed the partly cyclic structure earlier reported using fast-atom-bombardment mass spectrometry (FAB-MS) on the siderophores and their dansylated fragments [Briskot G, Taraz K, Budzikiewicz H (1989)Liebigs Ann Chem: 375–384]. The use of these NMR procedures appears to be a tool of choice and a complementary approach to FAB-MS in the structure determination of some complex pyoverdins.Abbreviations Ser serine - Arg arginine - Thr ethreonine - Lys lysine - OHOrn N -hydroxyornithine - Chr chromophore     

Degradation of long-chain n-alkanes by the yeast Lodderomyces elongisporus

Summary Cells of the yeast Lodderomyces elongisporus, precultured on glycerol, were incubated with long-chain n-alkanes. The results whow that monoterminal alkane oxidation is the main pathway of alkane degradation in the investigated yeast. The amount of diterminal activity is negligible, while subterminal degradation did not occur at all.Fatty acids were the first detectable intermediates. Using different n-alkanes, in every case the fatty acids with substrate chain length predominated in the cells. The formation of radioactive fatty acids from (1-¹⁴C)-hexadecane was time-dependent and indicated that desaturation elongation and -oxidation occurred.Extracellularly, the fatty acid pattern was similar, except for the additional presence of fatty acid methyl esters and the prevalence of octadecenoic acid after growth of cells on n-hexadecane.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.