Biosynthesis of galactan by a particulate enzyme preparation from Phaseolus aureus seedlings.
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ABSTRACT:
SUBMITTER: McNab JM
PROVIDER: S-EPMC1198510 | biostudies-other | 1968 Jan
REPOSITORIES: biostudies-other
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Project description:With a particulate enzyme preparation from Phaseolus aureus hypocotyls, UDP-alpha-d-[U-(14)C]galactose served as a precursor for a number of products. One of these products was characterized as a beta-(1-->4)-linked galactan. The ADP-, GDP-, TDP- and CDP- derivatives of alpha-d-galactose did not serve as biosynthetic precursors for any products insoluble in 70% ethanol, nor as substrates for a sugar nucleotide 4-epimerase which is present in the particulate enzyme preparation. The (14)C-labelled beta-(1-->4)-galactan is alkali-insoluble and was characterized by analysis of partial acetolysis products. The labelling pattern of the [(14)C]oligosaccharides derived from acetolysis indicates that (1) only slightly more than two [(14)C]galactose moieties are added to the growing polysaccharide chain on average, and (2) these additions take place at the reducing end of the polysaccharide chain. The radioactive beta-(1-->4)-linked galactan chain represented 8.5% of the radioactivity initially added, and 20% of the water- and butanol-insoluble products derived from UDP-alpha-d-[(14)C]galactose. Total hydrolysis of the alkali-insoluble fraction of Phaseolus aureus hypocotyl yielded d-glucose and d-mannose in a 5:1 ratio but no detectable quantities of d-galactose. A trace quantity of a radioactive disaccharide, identified as (1-->3)-linked galactobiose, was isolated from the partial acetolysate of the alkali-insoluble [(14)C]polysaccharide material. Also isolated from this partial acetolysate was a C-1 derivative of [(14)C]galactose, which could not be identified. An alkali-soluble galactose-containing polysaccharide was also synthesized in this enzymic reaction, and represented 20% of the water- and butanol-insoluble products derived from UDP-alpha-d-[(14)C]galactose. The spectrum of radioactive oligosaccharides produced by partial acetolysis of this alkali-soluble polysaccharide material was different from that obtained from the alkali-insoluble polysaccharide, indicating a different structure.
| S-EPMC1177695 | biostudies-other
Project description:A soluble enzyme preparation, which catalyses the polymerization of mannose, was obtained by Triton X-100 extraction of a particulate fraction derived from Phaseolus aureus hypocotyls. The product that resulted when GDP-alpha-d-mannose was used as a substrate was a beta-(1-->4)-linked mannan, about three-quarters of which was alkali-insoluble. The mannose-polymerizing enzyme activity was at least as great in the soluble preparation as in the particulate preparation, and the specific activity of the solubilized enzyme was greater by a factor of at least 3.5. Kinetic studies of the soluble enzyme indicate that the apparent K(m) is 55-62mum, and a disproportionate increase in rate is observed at high concentrations. GDP-alpha-d-glucose is a strong competitive inhibitor of the mannose-polymerizing reaction, with an apparent K(i) of 6.2mum. The soluble enzyme is relatively unstable, losing about two-thirds of its original activity in 5h at 0 degrees C or in 24h at -20 degrees C. A solvent (acetone, butanol, diethyl ether)-extracted particulate preparation, which also exhibits the same enzyme activity, is more stable, retaining full activity for at least 5 days at -20 degrees C. There was no polymerizing-enzyme activity in the soluble enzyme preparation when UDP-d-glucose, UDP-d-galactose, UDP-d-xylose, UDP-l-arabinose or UDP-d-glucuronic acid were used as substrates. However, the soluble enzyme preparation would catalyse the polymerization of glucose, with GDP-d-glucose as substrate.
| S-EPMC1173760 | biostudies-other
Project description:1. Fatty acid synthesis was studied in microsomal preparations from germinating pea (Pisum sativum). 2. The preparations synthesized a mixture of saturated fatty acids up to a chain length of C(24) from [(14)C]malonyl-CoA. 3. Whereas hexadecanoic acid was made de novo, octadecanoic acid and icosanoic acid were synthesized by elongation. 4. The products formed during [(14)C]malonyl-CoA incubation were analysed, and unesterified fatty acids and polar lipids were found to be major products. [(14)C]Palmitic acid represented a high percentage of the acyl-carrier protein esters, whereas (14)C-labelled very-long-chain fatty acids were mainly present as unesterified fatty acids. CoA esters were minor products. 5. The addition of exogenous lipids to the incubation system usually resulted in stimulation of [(14)C]malonyl-CoA incorporation into fatty acids. The greatest stimulation was obtained with dipalmitoyl phosphatidylcholine. Both exogenous palmitic acid and dipalmitoyl phosphatidylcholine increased the amount of [(14)C]-stearic acid synthesized, relative to [(14)C]palmitic acid. Addition of stearic acid increased the amount of [(14)C]icosanoic acid formed. 6. [(14)C]Stearic acid was elongated more effectively to icosanoic acid than [(14)C]stearoyl-CoA, and its conversion was not decreased by addition of unlabelled stearoyl-CoA. 7. Incorporation of [(14)C]malonyl-CoA into fatty acids was markedly decreased by iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid). Palmitate elongation was sensitive to arsenite addition, and stearate elongation to the presence of Triton X-100 or fluoride. The action of fluoride was not, apparently, due to chelation. 8. The microsomal preparations differed from soluble fractions from germinating pea in (a) synthesizing very-long-chain fatty acids, (b) not utilizing exogenous palmitate-acyl-carrier protein as a substrate for palmitate elongation and (c) having fatty acid synthesis stimulated by the addition of certain complex lipids.
| S-EPMC1183759 | biostudies-other
Project description:While target-based drug design has proved successful in several therapeutic areas, this approach has not yet provided compelling outcomes in the field of antibacterial agents. This statement remains especially true for the development of novel therapeutic interventions against tuberculosis, an infectious disease that is among the top ten leading causes of death globally. Mycobacterial galactan is an important component of the protective cell wall core of the tuberculosis pathogen and it could provide a promising target for the design of new drugs. In this review, we summarize the current knowledge on galactan biosynthesis in Mycobacterium tuberculosis, including landmark findings that led to the discovery and understanding of three key enzymes in this pathway: UDP-galactose mutase, and galactofuranosyl transferases GlfT1 and GlfT2. Moreover, we recapitulate the efforts aimed at their inhibition. The predicted common transition states of the three enzymes provide the lucrative possibility of multitargeting in pharmaceutical development, a favourable property in the mitigation of drug resistance. We believe that a tight interplay between target-based computational approaches and experimental methods will result in the development of original inhibitors that could serve as the basis of a new generation of drugs against tuberculosis.
| S-EPMC7168186 | biostudies-literature
Project description:1. Carbamoyl phosphate synthetase activity of Phaseolus aureus extracts was assayed by coupling it to the catalytic subunit of Escherichia coli aspartate transcarbamoylase and determining the [(14)C]carbamoylaspartate so formed. The stability of the activity was improved by the addition of ornithine and dimethyl sulphoxide to the extraction medium. 2. The synthetase activity was found to utilize either glutamine or ammonia as amino donor, the Michaelis constants being 0.17+/-0.03mm and 6.1+/-1.0mm respectively. N-Acetylglutamate did not significantly alter the rate with either substrate, and azaserine inhibited the reaction with both amino donors to the same extent. 3. Ornithine was shown to stimulate the activity, and to counteract inhibition by UMP. The purine nucleotides IMP and GMP enhanced carbamoyl phosphate formation, whereas AMP had an inhibitory effect. 4. The Michaelis constant for carbamoyl phosphate was determined in concentrated extracts for both aspartate transcarbamoylase and ornithine transcarbamoylase activities, and was 0.13+/-0.03mm and 1.58+/-0.16mm respectively. The ratio of the activities of these two enzymes, determined at near-saturating substrate concentrations, was 1:3 (aspartate transcarbamoylase/ornithine transcarbamoylase). 5. It is concluded that in this plant tissue there is one enzyme, carbamoyl phosphate synthetase, supplying carbamoyl phosphate to both the pyrimidine and arginine pathways, that the pyrimidine pathway claims most of the available carbamoyl phosphate (depending on the concentration of the nucleotide effectors) when this intermediate is present at low concentrations; and that when the carbamoyl phosphate concentration is increased, possibly by ornithine stimulation, a larger proportion can be taken up by the arginine pathway.
| S-EPMC1174161 | biostudies-other
Project description:1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.
| S-EPMC1264550 | biostudies-other
Project description:1. Aspartate transcarbamoylase from 4-day-old radicles of Phaseolus aureus was purified 190-fold by (NH(4))(2)SO(4) fractionation, DEAE-cellulose and DEAE-Sephadex chromatography and Sephadex-gel filtration. The partially purified enzyme, which required P(i) for maximum stability, had an apparent molecular weight of 83000+/-5000. 2. Uridine nucleotides were found to inhibit the activity; UMP was the most potent inhibitor, followed by UDP and UTP. No other nucleotide was found to affect the enzyme, nor could UMP inhibition be overcome by adding another nucleotide. Aspartate gives a hyperbolic substrate-saturation curve, both with and without UMP. The nucleotide inhibitor is non-competitive with respect to this substrate. Carbamoyl phosphate also yields a hyperbolic substrate-saturation curve in the absence of feedback inhibitor, but when UMP is added a sigmoidal pattern results, and the inhibition is competitive with carbamoyl phosphate. 3. The degree of inhibition by UMP is not affected by p-chloromercuribenzoate, urea, mild heat pretreatment or change in pH over the range 8.5-10.5, but is affected by temperature. 4. The aspartate analogue, succinate, both activates and inhibits the reaction, depending on the concentrations of aspartate and succinate used. 5. Kinetic studies with the partially purified enzyme showed that the K(m) for carbamoyl phosphate (0.091 mm) is much lower than that for aspartate (1.7mm). A sequential reaction mechanism was inferred from product-inhibition kinetics, with carbamoyl phosphate binding to the enzyme before aspartate, and the product, carbamoylaspartate, being released ahead of P(i). Initial-velocity studies gave a set of parallel reciprocal plots, compatible with an essentially irreversible step occurring before the binding of aspartate.
| S-EPMC1174160 | biostudies-other
Project description:The gaseous phytohormone ethylene influences many aspects of plant life, including germination, fruit ripening, senescence, and stress responses. These diverse roles of ethylene occur in part through crosstalk with other phytohormones, which affects ethylene biosynthesis and signaling pathways. We have recently shown that the phytohormones, including gibberellic acid, abscisic acid, auxin, methyl jasmonate, and salicylic acid, regulate the stability of ACC synthases (ACSs), the rate-limiting enzymes in ethylene biosynthesis. Here, we report that treatment of etiolated Arabidopsis seedlings with strigolactone (SL) increases ethylene biosynthesis. SL does not influence ACS stability or ACS gene expression, but it increases the transcript levels of a subset of ACC oxidase (ACO) genes, thereby enhancing ethylene biosynthesis. Taken together with the results of our previous study, these findings demonstrate that most phytohormones differentially regulate ethylene biosynthesis in dark-grown Arabidopsis seedlings by affecting ACS stability and/or the transcript levels of ethylene biosynthesis genes.
| S-EPMC7588227 | biostudies-literature
Project description:Maximum concentrations of microsomal cytochrome P-450 are present in 3-4 day-old mung beans (Phaseolus aureus). On illumination of dark-grown seedlings, cytochrome P-450 and later cytochrome P-450 undergo a rapid decrease in concentration in vivo, with an apparent half-time of about 6 h. Conversely light-grown seedlings, transferred to darkness, show a slow accumulation of cytochrome P-450, doubling time of about 30 h, with a later accumulation of cytochrome P-420. Microsomal cytochromes b559, b560.5 and b562.5 do not significantly alter on light-dark transitions. Possible functions for dark-induced cytochrome P-450 are discussed.
| S-EPMC1163104 | biostudies-other
Project description:D-galactan I is a polysaccharide with the disaccharide repeat unit structure [-->3-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->]. This glycan represents the lipopolysaccharide O antigen found in many Gram-negative bacteria, including several Klebsiella pneumoniae O serotypes. The polysaccharide is synthesized in the cytoplasm prior to its export via an ATP-binding cassette transporter. Sequence analysis predicts three galactosyltransferases in the D-galactan I genetic locus. They are WbbO (belonging to glycosyltransferase (GT) family 4), WbbM (GT-family 8), and WbbN (GT-family 2). The WbbO and WbbM proteins are each predicted to contain two domains, with the GT modules located toward their C termini. The N-terminal domains of WbbO and WbbM exhibit no similarity to proteins with known function. In vivo complementation assays suggest that all three glycosyltransferases are required for D-galactan I biosynthesis. Using a bacterial two-hybrid system and confirmatory co-purification strategies, evidence is provided for protein-protein interactions among the glycosyltransferases, creating a membrane-located enzyme complex dedicated to d-galactan I biosynthesis.
| S-EPMC2885245 | biostudies-literature