Polyprenol phosphates and mannosyl transferases in Phaseolus aureus

The transfer of mannose from GDP[¹⁴C]mannose to lipid and to insoluble polymer by a particulate preparation of Phaseolus aureus has been investigated. The evidence favours the lipid being a prenol phosphate mannose. Of a range of prenol phosphates tried, betulaprenol phosphate was the most effective exogenous acceptor of mannose. Most of the insoluble [¹⁴C]polymer formed was glycoprotein in nature although small quantities of ¹⁴C were associated with glucomannan and galactoglucomannan fractions. Time studies failed to reveal a typical precursor-product relationship between the lipid and polymer fractions but on incubation of [¹⁴C]mannolipid with the particulate fraction a small transfer (0·5–0·7%) of [¹⁴C] to polymer was detected. p-Hydroxymercuribenzoate inhibited (by 90%) the transfer of [¹⁴C] from GDP[¹⁴C]-mannoseto polymer and simultaneously increased (3-fold) the [¹⁴C] recovered in the lipid fraction. The effect was nullified by mercaptoethanol. Attempts to solubilize the transfer system were only partially successful. The formation of a chromatographically identical mannolipid was demonstrated in particulate fractions of Codium fragile and tomato roots.     

Announcement

Phosphate concentration was found to control the biosynthesis of the antibiotic candicidin by resting cells of Streptomyces griseus. Phosphate concentrations above 1 mM decreased the rate of incorporation of [¹⁴C]propionate and [¹⁴C]p-aminobenzoic acid into candicidin in relation to the concentration of phosphate. The inhibitory effect of phosphate on incorporation of labeled precursors into candicidin was not caused by inhibition of cellular uptake of precursors. Protein synthesis, sensitive to chloramphenicol, was not affected by phosphate levels that inhibit antibiotic synthesis. Similarly, phosphate concentrations inhibitory to antibiotic synthesis did not affect rifampinsensitive RNA synthesis.     

The pentose phosphate pathway as a source of NADPH for lignin synthesis

The aim of this work was to investigate whether the pentose phosphate pathway provides reducing power for lignin synthesis. Explants of the stem of Coleus blumei and the storage tissue of Helianthus tuberosus were cultured for 4 days on media which caused extensive lignification. [3-³H]-glucose and either [3-¹⁴C]- or [U-¹⁴C]-glucose were supplied to such 4-day-cultured explants, and also to the roots of 5-day-old seedlings of Pisum sativum. Significant amounts of ³H and ¹⁴C were recovered in syringaldehyde, vanillin, p-hydroxybenzaldehyde, and ligothio-glycollic acid from the explants of Coleus and Helianthus; and in vanillin, p-hydroxybenzaldehyde, and milled-wood lignin from pea roots. The ³H/¹⁴C ratios in these derivatives and preparations of lignin are held to indicate that much of the reducing power for lignin synthesis comes from the pentose phosphate pathway.     

Biosynthesis of p-hydroxybenzoic acid in poplar lignin

Biosynthetic pathways to p-hydroxybenzoic acid in polar lignin were examined by tracer experiments. High incorporation of radioactivity to the acid was observed when shikimic acid-[1-¹⁴C], phenylalanine-[3-¹⁴C], trans-cinnamic acid-[3-¹⁴C], p-coumaric acid-[3-¹⁴C] and p-hydroxybenzoic acid-[COOH-¹⁴C] were administered, while incorporation was low from shikimic acid-[COOH-¹⁴C], phenylalanine-[1-¹⁴C], phenylalanine-[2-¹⁴C], tyrosine-[3-¹⁴C], benzoic acid-[COOH-¹⁴C], sodium acetate-[1-¹⁴C] and d-glucose-[U-¹⁴C]. Thus p-hydroxybenzoic acid in poplar lignin is formed mainly via the pathway: shikimic acid → phenylalanine → trans-cinnamic acid → p-coumaric acid → p-hydroxybenzoic acid.     

Microbial growth on C1 compounds: Uptake of [14C]formaldehyde and [14C]formate by methane-grown Pseudomonas methanica and determination of the hexose labelling pattern after brief incubation with [14C]methanol

1. A study has been made of the incorporation of carbon from [¹⁴C]formaldehyde and [¹⁴C]formate by cultures of Pseudomonas methanica growing on methane. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled compounds for periods of up to 1min., has been analysed by chromatography and radioautography. 3. Radioactivity was fixed from [¹⁴C]formaldehyde mainly into the phosphates of the sugars, glucose, fructose, sedoheptulose and allulose. 4. Very little radioactivity was fixed from [¹⁴C]formate; after 1min. the only products identified were serine and malate. 5. The distribution of radioactivity within the carbon skeleton of glucose, obtained from short-term incubations with [¹⁴C]methanol of Pseudomonas methanica growing on methane, has been investigated. At the earliest time of sampling over 70% of the radioactivity was located in C-1; as the time increased the radioactivity spread throughout the molecule. 6. The results have been interpreted in terms of a variant of the pentose phosphate cycle, involving the condensation of formaldehyde with C-1 of ribose 5-phosphate to give allulose phosphate.     

Incorporation of various putative precursors into cuticular 3,4-dihydroxyphenylacetic acid of adult Tenebrio molitor

Post-emergence levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and ketocatechol were determined in cuticle from adult Tenebrio molitor. Possible pathways for biosynthesis of DOPAC were studied by comparing the incorporation of injected [U-¹⁴C]tyrosine, [7-¹⁴C]dopamine, [7-¹⁴C]DOPA, [7-¹⁴C]tyramine, [U-¹⁴C]p-hydroxyphenylpyruvic acid (p-HPPA) and [ring-³H]p-hydroxyphenylacetic acid (p-HPAA) into cuticular DOPAC during its period of maximal increase 1–3 days after adult emergence. Increased incorporation of [U-¹⁴C]tyrosine between days 0 and 3 suggests rapid de novo biosynthesis of DOPAC from this primary precursor. Of the putative intermediates tested, only p-HPPA had a pattern of incorporation similar to that seen with tyrosine. Since p-HPAA was poorly incorporated into both cuticle and DOPAC, a tentative pathway tyrosine → p-HPPA → 3,4-dihydroxyphenylpyruvic acid → DOPAC is proposed.     

The mode of condensation of aspartic acid and dihydroxyacetone phosphate in quinolinate synthesis in escherichia coli

Dihydroxy[3-¹⁴C]acetone phosphate was prepared enzymatically from [1-¹⁴C]glucose and used as a substrate in a partially purified quinolinate synthetase system prepared from Escherichia coli mutants. Carbon-by-carbon degradation of the resulting [¹⁴C]quinolinate showed that 96% of the ¹⁴C was located in carbon-4, indicating that carbon-3 of dihydroxyacetone phosphate condenses with carbon-3 of aspartate in quinolinate synthesis in E. coli.     

A procedure for the synthesis of p-fluorosulfonyl[14C]-benzoyl-5′-adenosine with [14C] in the benzoyl moiety

Carboxy-p-fluorosulfonyl[¹⁴C]benzoyl-5′-adenosine has been synthesized with the radiolabel ultimately derived from carboxy-p-amino[¹⁴C]benzoic acid by a synthetic route employing four reaction steps. Starting with 1 mmol of p-amino[¹⁴C]benzoic acid, p-fluorosulfonyl[¹⁴C]benzoyl-5′-adenosine is obtained with an overall yield of 25–30%.     

The fate of [14C]glucose during cold-hardening in Eurosta solidaginis (Fitch)

Glucose catabolism in overwintering larvae Eurosta solidaginis was examined to determine the relative contributions of glycolysis and the pentose phosphate pathway to polyol synthesis at different temperatures. Rates of ¹⁴CO₂ evolution were determined after injection of [¹⁴C]1-glucose, [¹⁴C]6-glucose, and [¹⁴C]3,4-glucose. In addition incorporation of label from each isotope into sorbitol and glycerol was monitored. The respirometric studies showed a relative increase in pentose phosphate activity between 10 and 5°C. Similar results were obtained from the changes of radioactivities incorporated into glycerol, although the activation of the pentose phosphate pathway was low. The conversion of [¹⁴C]glucose to glycerol was highest at 10°C, suggesting that maximum glycerol synthesis may occur at this temperature. Radioactivity appeared in the sorbitol fraction of larvae incubated at temperatures below 5°C. Late autumn larvae converted more [¹⁴C]glucose than did early autumn larvae.     

Multi-step metabolic activation of benzene. Effect of superoxide dismutase on covalent binding to microsomal macromolecules,and identification of glutathione conjugates using high pressure liquid chromatography and field desorption mass spectrometry

Incubation of [¹⁴C]benzene or [¹⁴C]phenol with liver microsomes from untreated rats, in the presence of a NADPH-generating system, gave rise to irreversible binding of metabolites to microsomal macromolecules. For both substrates this binding was inhibited by more than 50% by addition of superoxide dismutase to the incubation mixtures. The decrease in binding was compensated for by accumulation of [¹⁴C]hydroquinone, indicating superoxide-mediated oxidation of hydroquinone as one step in the activation of benzene to metabolites binding to microsomal macromolecules. Since our previous work had shown that binding occurred mainly with protein rather than ribonucleic acid and was virtually completely prevented by glutathione, suggesting identity of metabolite(s) responsible for binding to protein and glutathione, a conjugate was chemically prepared from p-benzoquinone and reduced glutathione (GSH) and identified by field desorption mass spectrometry (FDMS) as 2-(S-glutathionyl) hydroquinone. Microsomal incubations, containing an NADPH-generating system, with benzene, phenol, hydroquinone or p-benzoquinone in the presence of [³H]glutathione or, alternatively, with [¹⁴C]benzene or [¹⁴C]phenol in the presence of unlabeled glutathione, were performed. All of these incubations gave rise to a peak of radioactivity eluting from the high pressure liquid chromatograph (HPLC) at a retention time identical to that of the chemically prepared 2-(S-glutathionyl) hydroquinone, whilst microsomal incubation of catechol in the presence of [³H]glutathione led to a conjugate with a very different retention time which was not observed after incubation of benzene or phenol. The microsomal metabolites of p-benzoquinone, hydroquinone and phenol thus eluting from the HPLC were further identified as the 2-(S-glutathionyl) hydroquinone by field desorption mass spectrometry. The glutathione adduct formed from benzene during microsomal activation eluted from HPLC with the same retention time and its mass spectrum also contained the molecular ion (MH⁺) (m/e 416) of this conjugate as an intense peak, but the fragmentation patterns did not allow definite assignments probably due to the considerably smaller amounts of ultimate reactive metabolites formed from this pre-precursor and thus relatively larger amounts of impurities.The results indicate that rat liver microsomes activate benzene via phenol and hydroquinone to p-benzosemiquinone and/or p-benzoquinone as quantitatively important reactive metabolites.     

A technique for the isolation of yeast alcohol dehydrogenase mutants with altered substrate specificity.

α-Amylases have been found to convert starch and glycogen, in part, to products other than hemiacetal-bearing entities (maltose, maltodextrins, etc.)—hitherto, the only products obtained from natural α-glucans by α-amylolysis. Glycosides of maltosaccharides were synthesized by purified α-amylases acting on starch or bacterial glycogen in the presence of p-nitrophenyl α- or β-d-glucoside. From a digest with crystallized B. subtilis var. amyloliquefaciens α-amylase, containing 4 mg/ml of [¹⁴C]glycogen and 40 mmp-NP β-d-glucoside, three pairs of correspondingly labeled glycosides and sugars were recovered: p-NP α-d-[¹⁴C]glucopyranosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]glucose; p-NP α-[¹⁴C]maltosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]maltose; p-NP α-[¹⁴C]maltotriosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]maltotriose. The three glycosides accounted for 11.4% of the [¹⁴C]glycogen donor substrate; the three comparable sugars, for 30.4%; higher maltodextrins, for 58.2%. Calculations based on the molar yields of all reaction products show that [¹⁴C]glycosyl moieties were transferred from donor to p-NP β-d-glucoside with a frequency of 0.234 relative to all transfers to water. This is a very high value considering the minute molar ratio (0.0007) of β-d-glucoside-to-water concentration. Less striking but similar findings were obtained with cryst. hog pancreatic and Aspergillus oryzae α-amylases. The results extend earlier findings (Hehre et al., Advan. Chem. Ser. (1973) 117, 309) in showing that α-amylases have a substantial capacity to utilize the C₄-carbinols of certain d-glucosyl compounds as acceptor sites.     

A method for the determination of glucose-6-phosphatase activity in rat liver with [U-14C]glucose 6-phosphate as substrate.

A method is described for measuring the activity of glucose-6-phosphatase (EC 3.1.3.9) in rat liver. [U-¹⁴C]Glucose 6-phosphate, as substrate, is converted by the enzyme to [¹⁴C]glucose and inorganic phosphate. The addition of ZnSO₄ and Ba(OH)₂ at the end of the reaction precipitates phosphate and the unreacted [¹⁴C]glucose 6-phosphate, whereas [¹⁴C]glucose is not precipitated. After centrifugation, the amount of [¹⁴C]glucose formed is determined in a liquid scintillation counter.     

Transport of dicarboxylic acids in castor bean mitochondria

Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent K_m values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.

The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [¹⁴C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [¹⁴C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4°C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [¹⁴C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent K_m of 2.5 millimolar and V_max of 250 nanomoles per minute per milligram protein at 4°C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.

     

Regional distribution study of brain perfusion and metabolic agents in normal and tumour bearing rat brains using autoradiographic technique

Regional distribution of brain perfusion imaging agents, [¹³¹I]N,N,N′-trimethyl-N′-[2-hydroxy-3-methyl-5-iodobenzyl]1,3 propanediamine (HIPDM) and [¹³¹I]N-isopropyl-p-iodoamphetamine (IMP), was compared with the distribution of patterns of [¹⁴C]l-methionine and [¹⁴C]d-glucose in normal and tumour bearing rat brains using autoradiographic technique. There was higher concentration of the radiopharmaceutical in grey than white matter in normal rat brain. Autoradiographs of brain tumour sections showed very low uptake of [¹³¹I]HIPDM and [¹³¹I]IMP as compared to normal brain tissue. There was moderate concentration of [¹⁴C]d-glucose and avid uptake of [¹⁴C]l-methionine in tumours. Autoradiographic study is useful for evaluating distribution patterns of radiopharmaceuticals.     

Degradation of phenylalanine and tyrosine by Sporobolomyces roseus

Ammonia-lyase activity for l-phenylalanine, m-hydroxyphenylalanine and l-tyrosine was demonstrated in cell-free extracts of Sporobolomyces roseus. Cultures of this organism converted dl-[ring-¹⁴C]phenylalanine and l-[U-¹⁴C]tyrosine into the corresponding cinnamic acid. Tracer studies showed that these compounds were further metabolized to [¹⁴C]protocatechuic acid. Benzoic acid and p-hydroxybenzoic acid were intermediates in this pathway. Washed cells of the organism readily utilized cinnamic acid, p-coumaric acid, caffeic acid, benzoic acid and p-hydroxybenzoic acid. Protocatechuic acid was the terminal aromatic compound formed during the metabolism of these compounds. The cells of S. roseus were able to convert m-coumaric acid into m-hydroxybenzoic acid, but the latter compound, which accumulated in the medium, was not further metabolized. 4-Hydroxycoumarin was identified as the product of o-coumaric acid metabolism by this organism.     

The biosynthesis of ubiquinone in isolated rat heart cells

Beating heart cells were isolated from the adult rat and the biosynthesis of ubiquinone was studied. These cells were able to incorporate p-hydroxy[U-¹⁴C]benzoate into ubiquinone and some unidentified compounds, presumably intermediates in the biosynthesis of ubiquinone. The unidentified compounds were labile to alkali and were also labeled by [5-³H]-mevalonate and [methyl-³H]methionine, but not by p-hydroxy[carboxy-¹⁴C]benzoate. They appear to be chromatographically different from 5-demethoxy ubiquinone and 5-desmethyl ubiquinone. Addition of unlabeled mevalonate stimulated the incorporation of p-hydroxy [U-¹⁴C]benzoate into ubiquinone and the other compounds. The addition of dimethylsulfoxide to the isolated cells or the isolation medium caused inhibition of ubiquinone biosynthesis. Adriamycin was not inhibitory to the biosynthesis of ubiquinone in the cells. The advantages of these cells are the rapidity and ease in studying the biosynthesis of ubiquinone from various precursors and its regulation.     

Heritability and Clinical Determinants of Serum Indoxyl Sulfate and p-Cresyl Sulfate,Candidate Biomarkers of the Human Microbiome Enterotype

Background

Indoxyl sulfate and p-cresyl sulfate are unique microbial co-metabolites. Both co-metabolites have been involved in the pathogenesis of accelerated cardiovascular disease and renal disease progression. Available evidence suggests that indoxyl sulfate and p-cresyl sulfate may be considered candidate biomarkers of the human enterotype and may help to explain the link between diet and cardiovascular disease burden.

Objective and Design

Information on clinical determinants and heritability of indoxyl sulfate and p-cresyl sulfate serum is non-existing. To clarify this issue, the authors determined serum levels of indoxyl sulfate and p-cresyl sulfate in 773 individuals, recruited in the frame of the Flemish Study on Environment, Genes and Health Outcomes (FLEMENGHO study).

Results

Serum levels of indoxyl sulfate and p-cresyl sulfate amounted to 3.1 (2.4–4.3) and 13.0 (7.4–21.5) μM, respectively. Regression analysis identified renal function, age and sex as independent determinants of both co-metabolites. Both serum indoxyl sulfate (h² = 0.17) and p-cresyl sulfate (h² = 0.18) concentrations showed moderate but significant heritability after adjustment for covariables, with significant genetic and environmental correlations for both co-metabolites.

Limitations

Family studies cannot provide conclusive evidence for a genetic contribution, as confounding by shared environmental effects can never be excluded.

Conclusions

The heritability of indoxyl sulfate and p-cresyl sulfate is moderate. Besides genetic host factors and environmental factors, also renal function, sex and age influence the serum levels of these co-metabolites.     

Dietary effects on the formation of dolichyl monophosphate mannose by microsomal preparations of rat adipose tissue

Microsomal preparations from rat adipose tissue catalyse the transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to an endogenous acceptor forming a [¹⁴C]mannosyl lipid. The mannosyl lipid co-chromatographs with hen oviduct dolichyl monophosphate β-mannose on three solvent systems. It is stable to mild alkaline hydrolysis, but strong alkaline treatment yields a compound that co-migrates with mannose 1-phosphate. The mannosyl lipid is labile to mild acid hydrolysis, yielding [¹⁴C]mannose. Formation of the compound is reversible by GDP, but not GMP, and is stimulated by exogenous dolichyl phosphate.

The kinetics of transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to form dolichyl monophosphate mannose were studied by using preparations derived from rats fed on one of four diets: G (high glucose), L (high lard), F (fructose) or GC (high glucose, 0.9% cholesterol). The K_m and V_max. values for transfer from GDP-mannose were virtually indistinguishable in the four preparations.

In the absence of exogenous dolichyl phosphate, the largest amount of transfer of [¹⁴C]mannose into the mannosyl lipid was observed with preparations from fructose-fed animals. Preparations from glucose-fed animals showed about 60% as much transfer, whereas membranes from rats fed the other diets showed intermediate values between the fructose- and glucose-fed animals. The inclusion of cholesterol in the glucose diet elicited an increase in transfer of mannose.

Under conditions of saturating exogenous dolichyl phosphate, preparations from lard-fed animals have 1.5 times as much enzyme activity as do preparations from animals fed the other three diets.

     

Phosphatidylcholine Synthesis in Castor Bean Endosperm : Occurrence of an S-Adenosyl-l-Methionine:Ethanolamine N-Methyltransferase

The methylation steps in the biosynthesis of phosphatidylcholine by castor bean (Ricinus communis L.) endosperm have been studied by pulse-chase labeling. Endosperm halves were incubated with [methyl-¹⁴C]S-adenosyl-l-methionine, [2-¹⁴C]ethanolamine, [¹⁴C]ethanolamine phosphate, or [¹⁴C]serine phosphate. The kinetics of appearance were followed in the free, phospho-, and phosphatidyl-bases. The initial methylation utilized ethanolamine as a substrate to form methylethanolamine, which was then converted to dimethylethanolamine, choline, and phosphomethylethanolamine. Subsequent methylations occurred at the phospho-base and, to a lesser extent, the phosphatidyl-base levels, after which the radioactivity either remained constant or decreased in these compounds and accumulated in phosphatidylcholine. Although the precursors tested did support the synthesis of choline, the kinetics of the labeling make them unlikely to be the major sources of free choline to be utilized for the nucleotide pathway. A model with two pools of choline is proposed, and the implications of these results for the pathways leading to phosphatidylcholine biosynthesis are discussed.     

Sucrose Phosphate Is Not Transported into Vacuoles or Tonoplast Vesicles from Red Beet (Beta vulgaris) Hypocotyl

Tonoplast vesicles and vacuoles isolated from red beet (Beta vulgaris L.) hypocotyl accumulated externally supplied [¹⁴C]sucrose but not [¹⁴C]sucrose phosphate despite the occurrence of sucrose phosphate phosphohydrolytic activity in the vacuole. The activities of sucrose synthase and sucrose phosphate synthase in whole cell extracts were 960 and 30 nanomoles per milligram protein per minute, respectively; whereas, no sucrose synthesizing activity was measured in tonoplast preparations. The results obtained in this investigation are incompatible with the involvement of sucrose phosphate synthase in the process of sucrose synthesis and accumulation in the storage cells of red beet.