Investigation Of Metabolism Research Articles

Stereoselective syntheses of deuterium labelled marmesins; valuable metabolic probes for mechanistic studies in furanocoumarin biosynthesis

Stereoselective syntheses of deuterium labelled marmesins 3a-3e as metabolic probes for biosynthetic studies are described. Starting from iodo-umbelliferone ( 5), the title compounds were readily available by copper catalysed alkylation-cyclisation and subsequent transfer-hydrogenation of the resulting furanocoumarins 6a-6e. The bioconversion of marmesin by a microsomal preparation of A. majus yielded psoralene ( 4) and acetone in a single step and proceeded as a syn-elimination of the alkoxy-substituent together with a single hydrogen atom from a vicinal carbon atom (β-cleavage).

Highly Efficient and Versatile Synthesis of Isotopically Labelled 1-Deoxy-D-xylulose

An efficient and versatile synthesis of 1-deoxy-[4,4- 2H 2]- d-xylulose 1b (= 1-deoxy-[4,4- 2H 2]- d- threopentulose) from dimethyl 2,3- O-isopropylidene- d-tartrate in 31% overall yield is described. The synthetic protocol allows a flexible adaptation to other labelling patterns and isotopes. Labelled 1-deoxy-xylulose is of high value as a metabolic probe in biosynthetic studies towards terpenoids and prenylated compounds following the Rohmer pathway. © 1997 Published by Elsevier Science Ltd.

Effects of ketone bodies on astrocyte amino acid metabolism.

The effects of acetoacetate and 3-hydroxybutyrate on glial amino acid metabolism were studied in primary cultures of astrocytes. The exchange of nitrogen among amino acids was measured with 15N as a metabolic probe and gas chromatography-mass spectrometry as a tool with which to quantify isotope abundance. Addition of either acetoacetate or 3-hydroxybutyrate (5 mM) to the incubation medium did not alter the initial rate of appearance of [15N]glutamate in the glia, but it did inhibit transamination of glutamate to [15N]aspartate. Addition of acetoacetate also inhibited formation of [2-(15)N]glutamine, but 3-hydroxybutyrate had a stimulatory effect. The presence in the medium of sodium acetate (5 mM) was also associated with diminished production of [15N]aspartate and [2-(15)N]glutamine with [15N]glutamate as precursor. Studies with [2-(15)N]glutamine as precursor indicated that treatment of the astrocytes with ketone bodies did not alter flux through the glutaminase pathway. Nor did the presence of the ketone bodies reduce significantly the flux of nitrogen from [15N]GABA to [2-(15)N]glutamine when the former species served as a metabolic tracer. The concentration of internal citrate increased in the presence of acetoacetate, 3-hydroxybutyrate, and acetate. Studies with purified sheep brain glutamine synthetase showed that citrate inhibited this enzyme. These findings are considered in terms of the known anticonvulsant effect of a ketogenic diet.

Assessment of cytochrome P450 3A4 activity during the menstrual cycle using alfentanil as a noninvasive probe.

Gender-dependent differences in cytochrome P450 activity, drug metabolism, drug elimination, and their clinical consequences are increasingly apparent. P450 3A4 is the most abundant P450 isoform in the human liver and is responsible for metabolizing a vast and diverse assortment of therapeutic agents, including opioids, benzodiazepines, and local anesthetics. P450, 3A4 activity is higher in women, influenced by steroid hormone levels, and is speculated to vary during the menstrual cycle. This investigation tested the hypothesis that P450 3A4 activity varies during the menstrual cycle. Alfentanil clearance was used as a metabolic probe for P450 3A4 activity. Alfentanil (20 micrograms/kg bolus) was administered to nine nonsmoking, nonpregnant female volunteers (age, 26 +/- 5 yr) with normal menstrual cycles on three separate occasions during the same menstrual cycle: days 2 (menstrual phase), 13 (estrogen peak), and 21 (progesterone peak). Venous plasma alfentanil concentrations were determined by gas chromatography-mass spectrometry. Alfentanil clearance was determined by noncompartmental methods and by a three-compartment model with both pooled population and two-stage analysis. There was no significant difference in any measure of alfentanil clearance. Noncompartmental clearances (mean +/- SD) were 3.62 +/- 0.76, 3.81 +/- 0.96, and 3.60 +/- 0.84 ml/kg/ min, respectively, on days 2, 13, and 21 of the menstrual cycle. Alfentanil clearances were not different on menstrual cycle days 2, 13, and 21, strongly suggesting no change in P450 3A4 activity. Menstrual cycle differences in alfentanil clearances do not contribute to interindividual variability in alfentanil disposition in women. If other P450 3A4 substrates are comparable, then menstrual cycle variability in their metabolism may not be a consideration in dosing or in the design of pharmacokinetic investigations.

Comparison of metabolic ratios to assess CYP1A2, xanthine oxidase and N-acetyltransferase activities using caffeine as a metabolic probe

Comparison of metabolic ratios to assess CYP1A2, xanthine oxidase and N-acetyltransferase activities using caffeine as a metabolic probe

Comparison of metabolic ratios to assess CYP1A2, Xanthine oxidase and N-acetyltransferase activities using caffeine as a metabolic probe

Comparison of metabolic ratios to assess CYP1A2, Xanthine oxidase and N-acetyltransferase activities using caffeine as a metabolic probe

Antipyrine as a probe for human oxidative drug metabolism: identification of the cytochrome P450 enzymes catalyzing 4-hydroxyantipyrine, 3-hydroxymethylantipyrine, and norantipyrine formation.

Antipyrine has been widely used as a probe drug for human oxidative drug metabolism. To evaluate the role of antipyrine as a model drug, we have identified the cytochrome P450 enzymes involved in 4-hydroxyantipyrine, 3-hydroxymethylantipyrine, and norantipyrine formation. We used the following methods for this study: (1) determination of enzyme kinetics for antipyrine metabolite formation in human liver microsomes, (2) inhibition studies with antibodies and inhibitors, and (3) formation of metabolites by stable expressed human P450 enzymes. Antipyrine biotransformation could be described by Michaelis-Menten kinetics: norantipyrine: maximum rate of metabolite formation (Vmax), 0.91 +/- 0.04 nmol . mg-1 . min-1; Michaelis-Menten constant (Km), 19.0 +/- 0.8 mmol/L; 4-hydroxyantipyrine: Vmax, 1.54 +/- 0.08 nmol . mg-1 . min-1;Km,39.6 +/- 2.5 mmol/L. Antibodies against CYP3A4 inhibited the formation of 4-hydroxyantipyrine by 25% to 65%. LKM-2 antibodies (anti-CYP2C) caused a 75% to 100% inhibition of norantipyrine and a 58% to 80% inhibition of 3-hydroxymethylantipyrine formation. Sulfaphenazole inhibited the formation of 3-hydroxymethylantipyrine and norantipyrine by about 50%. Furafylline and fluvoxamine inhibited norantipyrine, 4-hydroxyantipyrine, and 3-hydroxymethylantipyrine formation by about 30%, 30%, and 50%, respectively. Ketoconazole reduced formation of norantipyrine, 3-hydroxymethylantipyrine, and 4-hydroxyantipyrine by up to 80%. Formation in stable expressed enzymes indicated involvement of CYP1A2, CYP2B6, CYP2C, and CYP3A4 in metabolite formation. Antipyrine metabolites are formed by at least six hepatic cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18, and CYP3A4). 4-Hydroxylation is mainly catalyzed by CYP3A4 and, to a lesser extent, by CYP1A2. The CYP2C subfamily contains the predominant enzymes for norantipyrine formation, and CYP1A2 is also involved. Formation of 3-hydroxymethylantipyrine is mediated by CYP1A2 and CYP2C9. Because several cytochrome P450 enzymes are involved in the formation of each metabolite, antipyrine is not well suited as a probe for distinct human cytochrome P450 enzymes.

Simple and efficient synthesis method of radioiodine-phenyl-pentadecanoic acid and uptake of tissues of animals

Fatty acid are the major energy source for the myocardium, thus radiolabeled fatty have often employed as heart imaging agent to localize ischemia and as potential probes of myocardial metabolism. This paper presents the synthesis of phenyl-pentadecanoic acid (PPA) by alkylation reaction and Wollf-Kishner method. Finally, PPA was iodinated with thallium trifluoroacetate on localizing para-position of phenyl ring and radioiodine was exchanged. The overall chemical yield was 20%, labelling yield 95%, radiochemical purity 98%. Tissues distribution of mice shows that IPPA was very high activity in mice heart muscle. Autoradiogram of mouse section at 2 min is clear.

Identification of the enzyme responsible for oxidative halothane metabolism: implications for prevention of halothane hepatitis

SummaryBackground Fulminant hepatic necrosis ("halothane hepatitis") is an unusual and often fatal complication of halothane anaesthesia. It is mediated by immune sensitisation in susceptible individuals to trifluoroacetylated liver protein neoantigens, formed by oxidative halothane metabolism. The seminal event in halothane hepatitis is hepatic metabolism, yet the enzyme responsible for oxidative halothane metabolism and trifluoroacetylated neoantigen formation remains unidentified. This investigation tested the hypothesis that cytochrome P450 2E1 (CYP2E1) is responsible for human halothane metabolism in vivo.Methods 20 elective surgical patients received either disulfiram (500 mg orally, n=10) or nothing (controls, n=10) the night before surgery. Disulfiram, converted in vivo to an effective inhibitor of P450 2E1, was used as a metabolic probe for P450 2E1. All patients received standard halothane anaesthesia (1·0% end-tidal, 3 h). Blood halothane and plasma and urine trifluoroacetic acid, bromide, and fluoride concentrations were measured for up to 96 h postoperatively.Findings Total halothane dose, measured by cumulative end-tidal (3·8 SE 0·1 minimum alveolar concentration hours) and blood halothane concentrations, was similar in the two groups. Plasma concentrations and urinary excretion of trifluoroacetic acid and bromide, indicative of oxidative and total (oxidative and reductive) halothane metabolism, respectively, were significantly diminished in disulfiram-treated patients. In control and disulfiram-treated patients cumulative 96 h postoperative trifluoroacetic acid excretion was 12 900 (SE 1700) and 2010 (440) μmol, respectively (p<0·001) while that of bromide was 1720 (290) and 160 (70) μmol (p<0·001).Interpretation The substantial attenuation of trifluoroacetic acid production by disulfiram after halothane anaesthesia suggests that P450 2E1 is a predominant enzyme responsible for human oxidative halothane metabolism. Inhibition of P450 2E1 by a single preoperative oral disulfiram dose greatly diminished production of the halothane metabolite responsible for the neoantigen formation that initiates halothane hepatitis. Single-dose disulfiram may provide effective prophylaxis against halothane hepatitis.

Caffeine as a metabolic probe: NAT2 phenotyping.

1. Caffeine has been used to determine acetylator phenotype for some 15 years but the interpretation of metabolic ratios with this substance raises theoretical and methodological issues. 2. N-acetyltransferase type 2 (NAT2) status was assessed in 23 young healthy subjects using both caffeine overnight and spot urine samples, and sulphadimidine. 3. Frequency distribution analysis of the metabolic ratios of NAT2, indicated two distinct groups for sulphadimidine, and for caffeine spot but not overnight samples. Spearman's rank correlation values were low indicating differences between the data sets for sulphadimidine and spot caffeine samples. Correlation between the two urine collection periods for caffeine was poor. 4. The complex metabolism of caffeine may compromise its value as a probe for determining acetylator phenotype until the effects of important variables are better understood.

The use of caffeine as a metabolic probe for human drug metabolizing enzymes

1. 1. Caffeine (CA) is metabolized extensively and at least 17 metabolites arising from primary and secondary biotransformation pathways are found in urine following CA ingestion. The enzymes responsible for the formation of most of the metabolites derived from CA have been identified. 2. 2. Given the near ubiquitous consumption of CA, this compound potentially constitutes a useful substrate probe for assessment of certain xenobiotic metabolizing enzyme activities in vivo. Indeed, various ratios of CA metabolites excreted in urine (urinary metabolic ratios; MRs) are now utilized widely for the population screening of enzyme activities. 3. 3. Excretion of the acetylated secondary metabolite 5-actylamino-6-formylamino-3-methyluracil (AFMU) is dependent on the activity of the polymorphic N-acetyltransferase (NAT2), and certain MRs incorporating AFMU may be used for NAT2 phenotyping. 4. 4. The conversion of 1-methylxanthine (1-MX), another secondary metabolite of CA, to 1-methyluric acid (1-MU) is catalyzed by xanthine oxidase (XO), and the urinary 1-MU to 1MX ratio reflects XO activity. 5. 5. N3-demethylation to form paraxanthine (PX), a reaction mediated by cytochrome P4501A2 (CYP1A2), is the dominant primary metabolic pathway of CA. CA N3-demethylation activity may be used as a measure of human hepatic CYPIA2 in vitro. 6. 6. Plasma CA clearance is considered to reflect CYP1A2 activity in vivo. Although a number of MRs are based on the excretion of PX metabolites (PX derived from CA is employed for the assessment of CYP1A2 activity in vivo), factors other than enzyme activity may affect these ratios.

Evaluation of nitroxides for the study of myocardial metabolism and oxygenation

AbstractElectron paramagnetic resonance spectroscopy at the L‐band (1.2 GHz) was used to investigate the redox metabolism and oxygenation within ischemic rat hearts loaded with a variety of six‐ and five‐membered carbocyclic nitroxide spin labels. Both cardiopleged and non‐cardiopleged hearts were studied at room temperature and at physiological temperatures. The results indicate that nitroxides that have greater intracellular partitioning are reduced faster than those that are extracellular. In addition, the six‐membered rings are metabolized much faster than the five‐membered rings. Increased nitroxide radical metabolism was observed in non‐cardiopleged compared with cardiopleged hearts. The oxygen utilization in the ischemic hearts was studied by using the oxygen‐dependent broadening of the nitroxide signals that occurs due to Heisenberg spin exchange. These structurally different nitroxides were evaluated for their sensitivity and suitability as probes of myocardial redox metabolism and oxygenation in the heart.

Clinical Sevoflurane Metabolism and Disposition

Sevoflurane is metabolized to free fluoride and hexafluoroisopropanol (HFIP). Cytochrome P450 2E1 is the major isoform responsible for sevoflurane metabolism by human liver microsomes in vitro. This investigation tested the hypothesis that P450 2E1 is predominantly responsible for sevoflurane metabolism in vivo. Disulfiram, which is converted in vivo to a selective inhibitor of P450 2E1, was used as a metabolic probe for P450 2E1. Twenty-one patients within 30% of ideal body weight, who provided institutional review board-approved informed consent and were randomized to receive disulfiram (500 mg oral, n = 11) or nothing (control, n = 10) the night before surgery, were evaluated. All patients received sevoflurane (2.7% end-tidal, 1.3 MAC) in oxygen for 3 h after propofol induction. Thereafter, sevoflurane was discontinued, and anesthesia was maintained with propofol, fentanyl, and nitrous oxide. Blood sevoflurane concentrations during anesthesia and for 8 h thereafter were measured by gas chromatography. Plasma and urine fluoride and total (unconjugated plus glucuronidated) HFIP concentrations were measured by an ion-selective electrode and by gas chromatography, respectively, during anesthesia and for 96 h postoperatively. Patient groups were similar with respect to age, weight, sex, case duration, and intraoperative blood loss. The total sevoflurane dose, measured by cumulative end-tidal sevoflurane concentrations (3.7 +/- 0.1 MAC-h; mean +/- SE), total pulmonary uptake, and blood sevoflurane concentrations, was similar in both groups. In control patients, plasma fluoride and HFIP concentrations were increased compared to baseline values intraoperatively and postoperatively for the first 48 and 60 h, respectively. Disulfiram treatment significantly diminished this increase. Plasma fluoride concentrations increased from 2.1 +/- 0.3 microM (baseline) to 36.2 +/- 3.9 microM (peak) in control patients, but only from 1.7 +/- 0.2 to 17.0 +/- 1.6 microM in disulfiram-treated patients (P < 0.05 compared with control patients). Peak plasma HFIP concentrations were 39.8 +/- 2.6 and 14.4 +/- 1.1 microM in control and disulfiram-treated patients (P < 0.05), respectively. Areas under the plasma fluoride- and HFIP-time curves also were diminished significantly to 22% and 20% of control patients, respectively, by disulfiram treatment. Urinary excretion of fluoride and HFIP was similarly significantly diminished in disulfiram-treated patients. Cumulative 96-h fluoride and HFIP excretion in disulfiram-treated patient was 1,080 +/- 210 and 960 +/- 240 mumol, respectively, compared to 3,950 +/- 560 and 4,300 +/- 540 mumol in control patients (P < 0.05). Disulfiram, an effective P450 2E1 inhibitor, substantially decreased fluoride ion and HFIP production during and after sevoflurane anesthesia. These results suggest that P450 2E1 is a predominant P450 isoform responsible for human sevoflurane metabolism in vivo.

Interaction between two probes used for phenotyping cytochromes P4501A2 (caffeine) and P4502E1 (chlorzoxazone) in humans.

The first steps in the metabolism of caffeine and chlorzoxazone are primarily catalysed by CYP1A2 and CYP2E1, respectively. Accordingly, these compounds have been developed as metabolic probes for non-invasive phenotyping of these two P450s. Their specificities, however, have been shown to overlap. In this study, 140 mg of caffeine and 500 mg of chlorzoxazone were administered alone or together in 16 healthy subjects under standardized conditions. The metabolites of these two probes were measured in the blood and also in the urine for caffeine. CYP1A2 activity was determined either by the paraxanthine/caffeine ratio in the blood or by the usual caffeine metabolic ratio in the urine. The CYP2E1 activity was determined by the 6-OH-chlorzoxazone/chlorzoxazone ratio in blood. CYP1A2 activities measured in blood and urine were highly significantly correlated. CYP2E1 activity was not modified when chlorzoxazone was given together with caffeine. In contrast, an inhibition of CYP1A2 by chlorzoxazone was demonstrated by a 16% decrease in the caffeine metabolic ratio in urine when both caffeine and chlorzoxazone were given together. Under the same conditions, the paraxanthine/caffeine ratio in plasma also decreased by about 20%. These results were confirmed in vitro by the incubation of 1 mM caffeine with human hepatic liver microsomes in the presence of 0.4 mM chlorzoxazone. The overall metabolism of caffeine decreased by 38% compared to controls incubated without chlorzoxazone.(ABSTRACT TRUNCATED AT 250 WORDS)

Iodine-123-labelled fatty acids for myocardial single-photon emission tomography: current status and future perspectives

Renewed interest in the clinical use of iodine-123-labelled fatty acids is currently primarily focused on the use of iodine-123-labelled 15-(p-iodophenyl)pentadecanoic acid (IPPA) and "modified" fatty acid analogues such as 15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP) which show delayed myocardial clearance, thus permitting single-photon emission tomographic imaging. Interest in the use of BMIPP and similar agents results from the differences which have often been observed in various types of heart disease between regional myocardial uptake patterns of [123I]BMIPP and flow tracer distribution. Although the physiological basis is not completely understood, differences between regional fatty acid and flow tracer distribution may reflect alterations in important parameters of metabolism which can be useful for patient management or therapy planning. These tracers may also represent unique metabolic probes for correlation of energy substrate metabolism with regional myocardial viability. The two agents currently most widely used clinically are 123I-labelled IPPA and BMIPP. While [123I]IPPA is commercially available as a radiopharmaceutical in Europe (Cygne) and Canada (Nordion), multicenter trials are in progress in the United States as a prelude to approval for broad use. [123I]BMIPP was recently introduced as Cardiodine for commercial distribution in Japan (Nihon Medi-Physics, Inc.). [123I]BMIPP is also being used in clinical studies on an institutional approval basis at several institutions in Europe and the United States. In this review, the development of a variety of radioiodinated fatty acids is discussed. The results of clinical trials with [123I]IPPA and [123I]BMIPP are discussed in detail, as are the future prospects for fatty acid imaging.

Stable Isotopes as Metabolic Probes for Nutritional Studies in Neonates

Metabolic and nutritional studies with stable isotope tracers have proved valuable in the human newborn. This technique is acceptable ethically, because it does not expose the infant to radioactivity, and it is sensitive enough for the analysis of very small samples. The stable isotope method has been especially applied to studies of neonatal carbohydrate, protein, and energy metabolism.

Metabolism of cytochrome P4503A substrates in vivo administered by the same route: Lack of correlation between alfentanil clearance and erythromycin breath test

Because there is considerable interindividual variation in both microsomal CYP3A4 activity and CYP3A4 substrate disposition, an established probe of in vivo CYP3A4 activity would represent an important advance in clinical practice. In a previous study, no correlation was found between the 14C-erythromycin breath test and urinary dapsone recovery ratio. However these drugs were administered by different routes, with the orally administered dapsone being exposed to presystemic metabolism by the gut and renal metabolism before the measurement of the urinary ratio. To overcome the variable of route of administration, the aim of this study was to determine whether the elimination of two intravenously administered CYP3A4 substrates (alfentanil and erythromycin) correlate. We compared the metabolism of alfentanil to its CYP3A4-dependent metabolite, noralfentanil, with the erythromycin breath test in 14 young healthy white men. No significant correlation was found between alfentanil metabolism and the erythromycin breath test: alfentanil clearance versus erythromycin breath test, r = 0.45, p = 0.1; partial metabolic clearance to noralfentanil versus erythromycin breath test, r = 0.35, p = 0.23. Because these two CYP3A4 substrates were administered by the same (intravenous) route, we conclude that differences in the route of administration do not explain the lack of correlation between the erythromycin breath test and other probes of CYP3A4 metabolism.

Metabolism and genotoxicity of the halogenated alkyl compound tris(2,3-dibromopropyl)phosphate.

1. The genotoxicity of the flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) was studied in vivo. Results showed that Tris-BP was highly clastogenic, but it could only initiate a low number of preneoplastic foci in the rat liver in vivo. In Drosophila, Tris-BP could be classified as a cross-linking agent, because it was more clastogenic than mutagenic. The use of completely deuterated Tris-BP as a metabolic probe revealed that cytochrome P450 and most likely the formation of 2-bromoacrolein (2BA) from Tris-BP is important for the observed genotoxic effects. 2. In contrast to the high mutagenicity of Tris-BP and 2BA in Salmonella typhimurium, we were unable to detect an increase in mutation frequency of 2BA on the hprt locus of human TK6 cell line. In another system, using a shuttle vector modified with 2BA:DNA-adducts, also no increase in mutation frequency could be detected in human cells. This low mutagenicity of 2BA corresponds with its low mutagenicity in Drosophila and its low induction of preneoplastic foci in the rat liver. 3. Several DNA adducts of 2BA have been identified, including an unstable 3-(bromooxypropyl)thymidine adduct which has the potential to form cross-links and a cyclic 3,N4-(bromo)propeno-deoxycytidine adduct which can possibly be involved in the clastogenicity of Tris-BP. 4. Taken together, these data indicate that Tris-BP and 2BA may not effectively induce gene mutations in eukaryotic systems, but rather be potent clastogens. Risk assessment of these and related compounds should therefore be based on the knowledge of clastogens rather than mutagens.

Two saturable mechanisms of iron uptake from transferrin in human melanoma cells: the effect of transferrin concentration, chelators, and metabolic probes on transferrin and iron uptake.

The mechanisms of iron (Fe) and transferrin (Tf) uptake by the human melanoma cell line, SK-MEL-28, have been investigated using chelators and metabolic probes. These data provide evidence for two saturable processes of Fe uptake from Tf, namely, specific receptor-mediated endocytosis and a second nonspecific, non-receptor-mediated mechanisms which saturated with respect to Fe uptake at a Tf concentration of approximately 0.3 mg/ml. In contrast to Fe uptake, Tf uptake increased linearly up to at least 1 mg/ml. Furthermore, under the culture conditions used, the second nonspecific, non-receptor-mediated mechanism was the most important process in terms of quantitative Fe uptake. Two concentrations of Tf-125I-59Fe (0.01 and 0.1 mg/ml) were used in order to characterise the specific and nonspecific Fe uptake pathways. Membrane permeable chelators were equally effective at both Tf concentrations, whereas membrane impermeable chelators were significantly (P < 0.001) more effective at reducing the internalisation of Fe at the higher Tf concentration, consistent with a mechanism of Fe uptake which occurred at a site in contact with the extracellular medium. The oxidoreductase inhibitor, amiloride, only slightly inhibited Fe uptake at the higher Tf concentration, suggesting that the second nonspecific process was not mediated by a diferric Tf reductase. Three lysosomotrophic agents and the endocytosis inhibitor, phenylglyoxal, markedly reduced Fe uptake at both Tf concentrations, and it is concluded that a saturable process consistent with receptor-mediated endocytosis of Tf occurred at the lower Tf concentration, while the predominant mechanism of Fe uptake at high Tf concentrations was a second saturable process consistent with adsorptive pinocytosis.

Biosynthesis of Rhizobium meliloti lipooligosaccharide Nod factors: NodA is required for an N-acyltransferase activity.

Rhizobium bacteria synthesize N-acylated beta-1,4-N-acetylglucosamine lipooligosaccharides, called Nod factors, which act as morphogenic signal molecules to legume roots during development of nitrogen-fixing nodules. The biosynthesis of Nod factors is genetically dependent upon the nodulation (nod) genes, including the common nod genes nodABC. We used the Rhizobium meliloti NodH sulfotransferase to prepare 35S-labeled oligosaccharides which served as metabolic tracers for Nod enzyme activities. This approach provides a general method for following chitooligosaccharide modifications. We found nodAB-dependent conversion of N-acetylchitotetraose (chitotetraose) monosulfate into hydrophobic compounds which by chromatographic and chemical tests were equivalent to acylated Nod factors. Sequential incubation of labeled intermediates with Escherichia coli containing either NodA or NodB showed that NodB was required before NodA during Nod factor biosynthesis. The acylation activity was sensitive to oligosaccharide chain length, with chitotetraose serving as a better substrate than chitobiose or chitotriose. We constructed a putative Nod factor intermediate, GlcN-beta 1,4-(GlcNAc)3, by enzymatic synthesis and labeled it by NodH-mediated sulfation to create a specific metabolic probe. Acylation of this oligosaccharide required only NodA. These results confirm previous reports that NodB is an N-deacetylase and suggest that NodA is an N-acyltransferase.