Effects of exogenous amino acids on growth and activity of four aspartate pathway enzymes in barley

Excised barley embryos were grown in the presence of 1 mM lysine, threonine, methionine and isoleucine, alone and in combinations. Growth was similar in all treatments except lysine plus threonine, where growth was severely inhibited. Activities of four regulatory biosynthetic enzymes were measured and expressed on a protein or fresh weight basis to assess possible repression/derepression under these conditions. Aspartate kinase (EC 2.7.2.4) (AK) activity and sensitivity to feedback regulators did not vary greatly between treatments. The activity and feedback sensitivity of homoserine dehydrogenase (EC 1.1.1.3) (HSDH) also showed little variation. Cystathionine synthase (EC 4.2.99.x) (CS) activity was markedly reduced in plants grown in the presence of methionine, and increased nearly 4-fold in the presence of lysine plus threonine, a condition in which methionine is limiting. Activity increased to a lesser extent in plants grown in the presence of threonine alone. Threonine synthase (EC 4.2.99.2) (TS) activity in the seedlings was reduced by up to one half in the presence of methionine, and to a smaller degree in the presence of isoleucine. None of the treatments led to increased activity of this enzyme.     

Nutritional studies of some members of mucorales IV. 1. Sugars,amino- and organic acids of the mycelium

Summary and Conclusions The sugars and amino acids of the fungal mycelium of six species of the order Mucorales were determined with the use of two-dimensional as well as circular paper chromatography. A critical survey of the above conclusions revealed that aspartic acid, glutamic acid, glycine and serine and -alanine were found in either free and bound form. Glutamine and arginine were absent in bound form. Threonine, histidine, isoleucine and tryptophane were absent in the ethanol soluble fraction. The presence and absence of certain amino acids in these species are good subsidiary characters for distinguishing one of them from the other.In the majority of the species three sugars viz., glucose, fructose and sucrose were found. InM. indica andC. bertholletiae sucrose was absent. Amongst the organic acid only tartaric and malic acids were detected in all the organisms.     

Membrane fatty acids as phenotypic markers in the polyphasic taxonomy of methylotrophs within the Proteobacteria.

A polyphasic approach to bacterial taxonomy attempts to integrate phylogenetic relationships with phenotypic marker analysis. This study describes the application of membrane fatty acids as a phenotypic marker for methylotrophs. Detailed phospholipid, ester-linked fatty acid (PLFA) profiles are reported for 17 methylotrophic eubacterial strains. These profiles included verification of double bond positions and geometries, both critical features for this analysis. Multivariate cluster analysis was used to indicate groupings of these strains along with literature values of both methylotrophs and non-methylotrophs based on the PLFA phenotype. Like many phenotypic characteristics, PLFA profiles were influenced by environmental conditions. The instabilities displayed, however, were predictable from physiological studies including increased trans/cis and cyclopropyl/cis ratios. Cluster analysis of PLFA profiles generated by separate investigators with different culture conditions indicated reproducibility by strain and species. The PLFA phenotype relationships compare favourably with phylogenetic associations based on 16S rRNA data for methylotrophs and will continue to be a valuable phenotypic marker for Proteobacteria taxonomy.     

Metabolism and the triggering of germination of Bacillus megaterium. Concentrations of amino acids, organic acids, adenine nucleotides and nicotinamide nucleotides during germination.

A considerable amount of evidence suggests that metabolism of germinants or metabolism stimulated by them is involved in triggering bacterial-spore germination. On the assumption that such a metabolic trigger might lead to relatively small biochemical changes in the first few minutes of germination, sensitive analytical techniques were used to detect any changes in spore components during the L-alanine-triggered germination of Bacillus megaterium KM spores. These experiments showed that no changes in spore free amino acids or ATP occurred until 2-3 min after L-alanine addition. Spores contained almost no oxo acids (pyruvate, alpha-oxoglutarate, oxaloacetate), malate or reduced NAD. These compounds were again not detectable until 2-3 min after addition of germinants. It is suggested, therefore, that metabolism associated with these intermediates is not involved in the triggering of germination of this organism.     

Influence of organic acids on the growth ofThiobacillus neapolitanus

An ether-soluble fraction of yeast extract was found to bring about the reduction of cytochromes of thec type in crude cell-free extracts of autotrophically grown cells ofT. neapolitanus. Re-extraction of the ether-soluble fraction with petroleum ether resulted in marked decrease in its ability to reduce the cytochromes ofT. neapolitanus. Among several organic acids tested only succinate was effective in reducing the cytochromes. Acetate at 20mm concentration was a potent inhibitor of growth, although thiosulfate oxidation by intact cells was only slightly affected. No poly-β-hydroxybutyric acid was found in cells grown on thiosulfate in the presence or absence of non-inhibitory concentrations of acetate. Glycollic acid was not inhibitory to growth at concentrations up to 60mm. Glycollic oxidase could not be demonstrated in crude extracts, however glyoxylic reductase was present in high concentrations. In cells fixing¹⁴CO₂ in the presence or α-hydroxypyridyl-methanosulfonic acid, (HPMSA), a specific inhibitor of glycollic acid oxidase, 18% of the total radioactivity extracted by 95% alcohol was present in glutamate, whereas in the absence of HPMSA, glutamate accounted only for about 9% of the total radioactivity. No accumulation of glycollic acid, glycine or serine was observed in the presence of HPMSA. It appears that glycollic acid formation does not participate to a significant extent in CO₂ fixation by this organism. α-Hydroxypyridylmethanosulfonic acid appears to act differently on this obligate autotroph than on photosynthetic organisms.     

Effect of some amino acids and complex organic nitrogenous substances on vegetative growth and gametangial formation in the female clone of Riccia frostii aust.

Investigations in vitro have been carried out with the female clone of Riccia frostii to study the effect of some amino acids and complex organic nitrogenous substances on vegetative growth and gametangial formation. Of the three amino acids tried, aspartic acid and threonine stimulated vegetative growth as well as archegonial production at all levels, whereas tryptophan did so only at lower levels. Aspartic acid was most effective in promoting vegetative growth and threonine the least. At optimum levels addition of threonine initiated maximum archegonia and was followed, in order of effectiveness, by addition of aspartic acid and tryptophan. Complex organic nitrogenous substances (casein hydrolysate, peptone and yeast extract), in general, did not favour normal growth and development, especially at higher levels. Peptone proved inhibitory for archegonial formation, but casein hydrolysate and yeast extract stimulated production of archegonia at 50 ppm and 0.5% (w/v), respectively.     

Effect of exogenous fatty acids on growth,membrane fluidity,and phospholipid fatty acid composition in yeast

The growth response of a double-mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis-Δ¹¹- eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity. There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 1 5:0, and 1 6:0. We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism.     

Enzymological aspects of the pathways for trimethylamine oxidation and C1 assimilation of obligate methylotrophs and restricted facultative methylotrophs.

Extracts of trimethylamine-grown W6A and W3A1 (type M restricted facultative methylotrophs) contain trimethylamine dehydrogenase whereas similar extracts of Bacillus PM6 and Bacillus S2A1 (type L restricted facultative methylotrophs) contain trimethylamine mono-oxygenase and trimethylamine N-oxide demethylase but no trimethylamine dehydrogenase. Extracts of the restricted facultatives and of the obligate methylotroph C2A1 contain hexulose phosphate synthase-hexulose phosphate isomerase activity; hydroxypyruvate reductase was not detected. Neither the restricted facultatives nor the obligates 4B6 and C2A1 contain all the enzymes of the hexulose phosphate cycle of formaldehyde assimilation as originally proposed by Kemp & Quayle (1967). Organisms PM6 and S2A1 lack transaldolase and use a modified cycle involving sedoheptulose 1,7-diphosphate and sedoheptulose diphosphatase. The obligates 4B6 and C2A1, and the type M organisms W6A and W3A1, use a different modification of the assimilatory hexulose phosphate cycle involving the Entner-Doudoroff-pathway enzymes phosphogluconate dehydratase and phospho-2-keto-3-deoxygluconate aldolase. The lack of fructose diphosphate aldolase and hexose diphosphatase in these organisms may be a partial explanation of their restricted growth-substrate range. Enzymological evidence suggests that all the obligates and the restricted facultatives use a dissimilatory hexulose phosphate cycle to accomplish the complete oxidation of formaldehyde to CO2 and water.     

Mechanism of racemization of amino acids by aspartate aminotransferase.

Aspartate aminotransferase (mitochondrial isoenzyme from chicken) has been found to racemize very slowly dicarboxylic amino acid substrates in the presence of their cognate oxo acids [Kochhar, S. & Christen, P. (1988) Eur. J. Biochem. 175, 433-438]. Tyrosine, phenylalanine and alanine are racemized at the same rate although they undergo the transamination reaction 3-5 orders of magnitude more slowly than the dicarboxylic substrates. Similarly, the truncated enzyme aspartate aminotransferase-(27/32-410) catalyzes the racemization at the same rate as the native enzyme, while its rate of transamination is decreased to 3% of that of the native enzyme. Apparently, the rate-limiting step in racemization is not immediately linked to the transamination cycle. Decreasing the water concentration in the reaction medium by adding methanol at 0 degrees C drastically reduces the rate of racemization without affecting the rate of transamination. On the basis of these and additional kinetic data and the model of the three-dimensional structure of the active site, we conclude that a water molecule is responsible for the protonation of C alpha of the coenzyme-substrate intermediate from the wrong side. The diffusion of the water molecule into the interior of the enzyme appears to be the rate-limiting step in aspartate-aminotransferase-catalyzed racemization.     

Radiorespirometric study of the utilization of exogenous sucrose,glucose and fructose by germinating apple pollen

Po?adí intensity, s jakou prodýchávají pylové lá?ky testované cukry z 0,3 M roztok?, je sacharosa> glukosa> invertní cukr> fruktosa. Stejné po?adí je zaehováno na cukr-agarových mediích s výjimkou prvých dvou hodin inkubace, během kterých jsou sacharosa, glukosa a fruktosa prodýchávány témě? stejnou rychlostí. Během této doby se v prost?edí fruktosy, stejně jako v kontrole bez cukru, nevytvo?ily pylové lá?ky, zatím za p?itomnosti sacharosy dosahovaly délky a? 450 µ. Jestli?e byla pou?ita pro radioaktivní cukry jako nosi? sacharosa, byla fruktosa-¹⁴C prodýchávána a? 12krát, glukosa-¹⁴C a? 6krât intensivnëji neá sacharosa-¹⁴C. Za pou?ití nosi?e sacharosa+glukosa ?i sacharosa+fruktosa (molárni poměry 1:1), prodýchávaji pylové lácky sacharosu-¹⁴C pomaleji ne? p?íslu?ny monosaeharid a rovně? pomaleji ne? z prost?edí samotnéé sacharosy. Jestli?e byla nosi?em sacharosy-¹⁴C glukosa nebo fruktosa, byla (v některých ?asových úsecíeh pokusu) produkce¹⁴CO₂ pylovými láckami několik desítek procent mohutněj?í ne? za pou?ití nosi?e sacharosového. Z prost?edí invertního cukru je p?ednostně prodýchäväna fruktosa. Je tedy kapacita pylových enzymových systém? za?leňujících sledované cukry do jejich dýchacích cest pro fruktosu>glukosu> sacharosu, co? je opa?né po?adí ne? platí pro intensitu r?stového ú?inku těchto cukr? a ne? jaké bylo zji?těno pro rychlost jejich prodýchávání, jestli?e nebyly navzájem kombinovány. Ve specifickém r?stovém efektu sacharosy nem??e tedy být primárním faktorem ani rychlost její absorpce, ani intensita jejího prodýcháváni. Rychlá utilisace samotné sacharosy je následkem intensivněj?ího r?stu v jejím prost?edí. Získané výsledky dále ukazují, ?e sacharosa je vyu?ívána p?edev?ím cestou její inverse, p?i ?em? je p?ednostně prodýchávána fruktosová slo?ka.     

Trans fatty acids promote the growth of some Lactobacillus strains

Five Lactobacillus strains (2 L. gasseri, 2 L. plantarum and 1 L. reuteri) were cultured in modified MRS medium containing fatty acids (FAs) instead of Tween 80 for 24 h at 37 degrees C, to learn the effect of saturated and unsaturated FAs on the Lactobacillus growth. Free FAs included palmitic (16:0), palmitoleic (c9-16:1), stearic (18:0), oleic (c9-18:1), elaidic (t9-18:1), cis-vaccenic (c11-18:1), vaccenic (t11-18:1), linoleic (c9, c12-18:2), conjugated linoleic (c9, t11- and t10, c12-18:2), alpha-linolenic (c9, c12, c15-18:3), alpha-eleostearic (c9, t11, t13-18:3), eicosapentaenoic (20:5), and docosahexaenoic (22:6) acids. Among free FAs, oleic acid stimulated the growth of all Lactobacillus strains, whereas palmitoleic acid had almost no affect on the Lactobacillus growth. Saturated FAs such as stearic and palmitic acids inhibited or did not affect the Lactobacillus growth. Polyunsaturated FAs such as alpha-linolenic, eicosapentaenoic and docosahexaenoic acids strongly inhibited the Lactobacillus growth at 7.6 x 10(-4) m. Octadecenoic acids such as oleic, elaidic, cis-vaccenic and vaccenic acids remarkably promoted the growth of L. gasseri, regardless of the different double bond positions and configurations. When oleic or cis-vaccenic acid was incubated with L. gasseri, the FAs was transformed to cyclopropane FAs (methyleneoctadecanoic acids) after incorporation into the cells. On the other hand, trans FAs such as elaidic and vaccenic acids incorporated into the cells were not converted to another FAs. Conjugated linoleic and alpha-eleostearic acids having a trans double bond promoted the Lactobacillus growth. The growth of L. gasseri was also stimulated by trans-rich free FAs from hydrogenated canola and fish oils. These results showed that octadecenoic acid and trans FAs had strong promotion activities for the Lactobacillus growth due to their incorporation into membrane lipids.     

Changes in colour,polygalacturonase monosaccharides and organic acids during storage of tomatoes

The appearance of polygalacturonase and red pigmentation in mature-green tomato fruit was prevented by storing the fruit in 5% O₂, 5% CO₂ and 90% N₂. However, the breakdown of starch to give monosaccharides and the change in concentration of organic acids which is normally associated with ripening still took place. On removal of the fruit to ambient conditions, polygalacturonase was synthesized and the fruit changed colour but monosaccharide and organic acid concentrations did not change.