Biosynthesis of marsupial milk oligosaccharides. II: Characterization of a beta 6-N-acetylglucosaminyltransferase in lactating mammary glands of the tammar wallaby, Macropus eugenii.

Tammar wallaby (Macropus eugenii) mammary glands contain a UDP-GlcNAc:Gal beta 1----3Gal beta 1----4Glc beta 1----6-N-acetylglucosaminyltransferase (GlcNAcT) whose activity has been characterized with respect to the effect of pH, apparent Km for acceptor, effects of bivalent metal ions, acceptor specificity and identity of products. The enzyme did not show an absolute requirement for any bivalent metal ion but its activity was increased markedly by Mg2+, Ca2+ and Ba2+ and, to a lesser extent, by Mn2+. When Gal beta 1----3Gal beta 1----4Glc was used as acceptor, the product was Gal beta 1----3[GlcNAc beta 1----6]Gal beta 1----4Glc. With Gal beta 1----3Gal beta 1----3Gal beta 1----4Glc as acceptor, the product was shown, by 1H-NMR spectroscopy and exo-beta-galactosidase digestion, to be a novel pentasaccharide with the structure Gal beta 1----3[GlcNAc beta 1----6]Gal beta 1----3Gal beta 1----4Glc, suggesting that the enzyme recognises the non-reducing end of the acceptor substrate, rather than the reducing end.

Synthesis and enzymic evaluation of 4-mercapto-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide as an inhibitor of mammalian dihydroorotase.

The design, synthesis, and enzymic evaluation of cis- and trans-4-mercapto-6-oxo-1,4-azaphosphinane-2-carboxylic acid 4-oxide 5 against mammalian dihydroorotase is presented. The design strategy for 5 was based on the strong affinity of phosphinothioic acids for zinc and that 5 also resembles the postulated tetrahedral transition state for the enzyme-catalyzed reaction. The synthesis of 5 utilized a novel protection/deprotection sequence upon 4-hydroxy-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide 4, followed by incorporation of alpha-phenyl benzenemethanethiol and exhaustive deprotection to afford 5 in 40% overall yield from 4. The activities of both isomers of 5 as inhibitors of mammalian dihydroorotase were marginally greater than that of the parent phosphinic acid 4, indicating a weak binding enhancement due to the phosphinothioic acid moiety.

Experiments to detect long-range heteronuclear shift correlations: LR-J-HSMQC.

The utility of the J-HSMQC experiment to detect long-range CH correlations was investigated. Two new long-range J-compensated pulse sequences, LR-J-HSMQC(80,27) and LR-J-HSMQC(27,80), were developed using the (3beta(x))beta(y) composite 90 degrees pulse sequence. These two experiments were shown to be effective for long-range coupling constants, (n)J(CH), that were greater than 3 Hz. Although the overall sensitivities of the long-range J-HSMQC experiments were slightly lower than that of the conventional decoupled HMBC experiment, their 2D maps showed additional cross peaks that could be useful in structure elucidation. LR-J-HSMQC(27,80) was very efficient in yielding two- and four-bond relay correlations. The utility of the new sequences is demonstrated with strychnine as the sample.

A triterpenoid saponin from Maesa ramentacea.

The structure of a piscicidal triterpenoid saponin (saponin A) isolated from the leaves of Maesa ramentacea has been shown to be 3-O-[[(alpha-rhamnopyranosyl (1-->2)-alpha-L-rhamnopyranosyl(1-->2)-beta-D-galactopyranosyl (1-->3)]-[beta-D-glucopyranosyl(1-->2)]-beta-D-glucuronopyranosyl] barringtogenol C21, 22-O-diangeloate. Extensive use was made of homo- and heteronuclear 2D NMR techniques.

Strategies for studies of neurotoxic mechanisms involving deficient transport of L-glutamate: antisense knockout in rat brain in vivo and changes in the neurotransmitter metabolism following inhibition of glutamate transport in guinea pig brain slices.

This communication briefly reviews characteristics of glutamate transport in the central nervous system and is involved in the aetiology of slow neurodegenerative diseases. Data in the literature suggest that antisense oligonucleotides targeted against glutamate transporters and administered in vivo over a period of days could be used to test the hypothesis. Data from our laboratory have indicated that single intraventricular doses of antisense oligonucleotides can also results in significant reductions in the numbers of substrate binding sites associated with glutamate transporters and may even cause subtle changes in their characteristics. In order to study metabolism in brain tissue, we have used 13C-nuclear magnetic resonance spectroscopy to analyse extracts of slices of guinea pig cerebral cortex exposed to glutamate transport inhibitor L-anti,endo-methanopyrrolidine dicarboxylate (L-a,e-MPDC). The results have shown-for the first time in an experimental model that preserves the relationship between glia and neurones within the context of brain tissue-that inhibition of L-glutamate transport can exert a significant influence on neurotransmitter-related metabolism. These findings suggest that metabolic disturbances caused by deficient glutamate transport could play a significant role in the death of neurones under pathological conditions in vivo.

Dichloroacetate (DCA) reduces brain lactate but increases brain glutamine in experimental cerebral malaria: a 1H-NMR study.

Recent findings that levels of brain lactate and alanine were elevated in murine cerebral malaria led us to investigate the effect of dichloroacetate (DCA; 60 mg/kg), an activator of pyruvate dehydrogenase, on the levels of brain metabolites, and on the survival of mice infected with Plasmodium berghei ANKA which normally causes lethal cerebral malaria. DCA significantly reduced brain lactate and alanine levels when administered to infected mice, had no effect on the TCA cycle-related metabolites glutamate, GABA and aspartate and was associated with increased brain glutamine levels: 40% of mice thus treated survived the normally lethal infection.

Structural characterisation of the exocellular polysaccharide produced by Streptococcus thermophilus OR 901.

The exocellular polysaccharide of Streptococcus thermophilus OR 901, isolated from partially deproteinised whey, is a heteropolymer of D-galactopyranose and L-rhamnopyranose residues in the molar ratio 5:2. The structure was established by methylation analysis and 1D and 2D NMR spectroscopy of the native polysaccharide, in combination with characterisation of oligosaccharide fragments, obtained by partial acid hydrolysis, using methylation analysis and 1D 1H NMR spectroscopy. The polysaccharide has a branched heptasaccharide repeating unit with the following structure: [sequence: see text]

An NMR study of the lactonization of alpha-N-acetylneuraminyl-(2 --> 3)-lactose.

The composition of the products formed by treatment of commercial alpha-Neu5Ac-(2 --> 3)-beta-D-Galp-(1 --> 4)-D-Glc (3'-sialyllactose) with glacial acetic acid was investigated by 1H-13C one- and two-dimensional NMR spectroscopy and fast atom bombardment-mass spectrometry. The data confirmed that the major product of the reaction was alpha-Neu5Ac-(2 --> 3)-beta-D-Galp-(1 --> 4)-D-Glc-(1c --> 2b)-lactone, which reverted to the starting material on standing in aqueous solution at ambient temperature, but for which complete NMR assignments are reported. The NMR data led to the tentative conclusion that the reaction also yielded small amounts of lactose, and alpha-Neu5Ac-(2 --> 3)-beta-D-Galp-(1 --> 4)-D-Glc-(1c --> 4b)-lactone which was stable in aqueous solution.

Studies of the neutral trisaccharides of goat (Capra hircus) colostrum and of the one- and two-dimensional 1H and 13C NMR spectra of 6'-N-acetylglucosaminyllactose.

Four neutral trisaccharides were isolated from goat colostrum by dialysis, and ion-exchange, activated charcoal column, preparative paper, and Bio-Gel P-4 column chromatography. The following structures were elucidated by GC analysis of hydrolysis products and by 400-MHz 1H NMR spectroscopy: alpha-L-Fuc p-(1-->2)-beta-D-Gal p-(1-->4)-D-Glc, alpha-D-Gal p- (1-->3)-beta-D-Gal p-(1-->4)-D-Glc, beta-D-Gal p-(1-->3)-beta-D-Gal p-(1-->4)-D-Glc, and beta-D-Gal p-(1-->6)-beta-D-Gal p-(1-->4)-D-Glc. beta-D-Glc pNAc-(1-->6)-beta-D-Gal p-(1-->4)-D-Glc, previously reported to be present in goat milk, was not detected in this study. beta-D-Glc pNAc-(1-->6)-beta-D-Gal p-(1-->4)-D-Glc (6'-N- acetylglucosaminyllactose) was prepared by beta-D-galactosidase digestion of beta-D-Gal p-(1-->3)[beta-D-Glc pNAc-(1-->6)]-beta-D-Gal p-(1-->4)-D-Glc (lacto-N-novotetraose) and characterized by one- and two-dimensional NMR spectroscopy at 600 MHz.

Occurrence of an unusual lactose sulfate in dog milk.

The milk of a beagle dog (Canis familiaris) was extracted and fractionated to yield, inter alia, beta-D-Galp3S-(1-->4)-D-Glc (lactose 3'-sulfate), which does not appear to have previously been isolated from milk or other natural sources. The structure was established by 2D NMR spectroscopy and mass spectrometry. By contrast with the milk of some closely related Carnivora, the major constituent of the dog milk was lactose, with minor amounts of 2'-fucosyllactose and sialyl oligosaccharides.

Improved J-compensated sequences based on short composite pulses.

Efficient J-compensated sequences that are shorter in duration and use less RF pulses have been created from short but very efficient composite 90 degrees RF pulses. The improved J-compensation transforms in-phase into antiphase magnetization and can be incorporated in any pulse sequence that involves evolution of heteronuclear J-couplings. The compensated sequences were tested and incorporated into an HMBC sequence. J-compensated experiments referred to as HMBC-J45 + 90A and HMBC-J45 + 90B, were found to be effective over a wide range of J values.

Effect of caspofungin on metabolite profiles of Aspergillus species determined by nuclear magnetic resonance spectroscopy.

Invasive aspergillosis remains a potentially life-threatening infection, the incidence of which is increasing. Current methods used to determine the susceptibilities of Aspergillus strains to antifungal drugs are often unreliable. Nuclear magnetic resonance (NMR) spectroscopy can identify the metabolic complement of microorganisms while monitoring nutrient utilization from the incubation medium. We used 600-MHz (1)H NMR spectroscopy to monitor the metabolic responses of five Aspergillus species cultured in RPMI 1640-2% glucose-morpholinepropanesulfonate buffer to various concentrations of the antifungal drugs amphotericin B (AMB) and caspofungin. The metabolic endpoint (MEP) was determined from nutrient and metabolite resonances, measured as a function of the drug concentration, and was defined as a > or =50% reduction in nutrient consumption or metabolite production. MICs were evaluated by a modification of Clinical and Laboratory Standards Institute broth microdilution method M27-A, and minimal effective concentrations (MECs) were determined by microscopic examination of fungal hyphae. For AMB, the MEPs coincided with the MICs. For caspofungin, the MEPs agreed with the MECs for several Aspergillus strains, but the effect of drug pressure was more complex for others. Expansion of the MEP definition to include any significant changes in metabolite production resulted in agreement with the MEC in most cases. Paradoxical metabolic responses were observed for several Aspergillus strains at either high or low caspofungin concentrations and for one Aspergillus terreus strain with AMB. NMR spectroscopy proved to be a powerful tool for detecting the subtle effects of drug pressure on fungal metabolism and has the potential to provide an alternative method for determining the susceptibilities of Aspergillus species to antifungal drugs.

Modelling Staphylococcus aureus-induced septicemia using NMR.

We present a novel NMR-based study of the molecular aspects of the "attack" on human red blood cells (RBCs) by growing bacteria. Staphylococcus aureus expresses virulence factors, including alpha-hemolysin, which contribute to the clinical condition known as septic shock. alpha-Hemolysin is a pore-forming toxin and its secretion increases the permeability of a range of mammalian cell types infected with S. aureus. (31)P NMR spectra of the probe molecules dimethyl methylphosphonate (DMMP) and hypophosphite (HPA) in RBC suspensions show separate intra- and extracellular resonances. These resonances coalesced over time in RBC suspensions inoculated with S. aureus or pure alpha-hemolysin, due to increasing permeability of the RBC membrane. Increased RBC permeability resulted in leakage of intracellular proteins, plus an increase in the exchange rate of the solutes between the intra- and extracellular compartments, both effects contributing to the coalescence of the split peaks. The addition of antibiotics prevented peak coalescence and enabled the minimal inhibitory concentration (MIC) for eight strains of S. aureus to be determined for oxacillin and erythromycin. The MIC values obtained by using (31)P NMR spectroscopy were within one dilution of the MICs obtained using the standard National Committee for Clinical Laboratory Standards (NCCLS) method. The results are encouraging for the use of NMR spectroscopy in clinical microbiology.

Substrates for endogenous metabolism by mature boar spermatozoa.

Washed boar spermatozoa incubated in the absence of exogenous substrates maintained a high energy charge potential (ECP) for at least 10 h. Addition of bromopyruvate, an inhibitor of stage 2 of the glycolytic pathway, at any time during the incubation caused an immediate decrease in the ECP, indicating that the mobilization of endogenous compounds requires this section of the pathway for the production of lactate, the major mitochondrial substrate for ATP production. Some of the sources of the metabolic substrates have been identified, by NMR and metabolic studies, as di- or triglycerides, to produce glycerol, and membrane phospholipids for the production of glycerol 3-phosphate. Acetylcarnitine contributes acetyl groups early in the incubation; glycerylphosphorylcholine is degraded to glycerol 3-phosphate and choline after about 5 h, and acetate also accumulates after about 5 h. The presence of phosphorylcholine and phosphorylethanolamine later in the incubation indicates that phospholipids are also degraded to glycerol.

Aldolase-catalyzed diketone phosphate formation from oxoaldehydes. NMR studies and metabolic significance.

NMR spectroscopy showed fructose-1,6-bisphosphate aldolase from rabbit muscle accepts as substrates, in lieu of glyceraldehyde 3-phosphate, the oxoaldehydes methylglyoxal and phenylglyoxal but not hydroxymethylglyoxal. The enzyme catalyzed an aldol condensation between the oxoaldehyde and dihydroxyacetone phosphate to form a monophosphorylated diketone and was inactivated in the process. Circumvention of this reaction, by metabolism of oxoaldehydes to hydroxy acids, may be a metabolic role for the glyoxalase enzyme system. Transketolase and transaldolase were found not to accept oxoaldehydes as substrates in place of glyceraldehyde 3-phosphate.

13C n.m.r. isotopomer and computer-simulation studies of the non-oxidative pentose phosphate pathway of human erythrocytes.

13C double-quantum filtered correlation spectroscopy (DQF-COSY) provides a novel method for the detection of reactions involving carbon-bond scissions. We report the use of this technique to investigate isotopic exchange reactions of the non-oxidative pentose phosphate pathway in human erythrocytes. These exchange reactions resulted in the formation of a range of isotopic isomers (isotopomers) of glucose 6-phosphate after incubation of a mixture of universally 13C-labelled and unlabelled glucose 6-phosphate with fructose 1,6-bisphosphate and haemolysates. These isotopomers were detected in the coupling patterns of cross-peaks within the DQF-COSY spectrum of the deproteinized sample. A computer model which fully describes the reactions of the non-oxidative pentose phosphate pathway in human erythrocytes has previously been constructed and tested with 31P n.m.r. time-course data in our laboratory. This model was refined using 13C n.m.r. time-course data and extended to include the range of isotopomers which may be formed experimentally by the reactions of the non-oxidative pentose phosphate pathway. The isotopomer ratios obtained experimentally from the DQF-COSY spectrum were consistent with simulations generated by this model.

Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: in vivo kinetic characterization of 2,3-bisphosphoglycerate synthase/phosphatase using 13C and 31P NMR.

This is the first in a series of three papers [see also Mulquiney and Kuchel (1999) Biochem. J. 342, 579-594; Mulquiney and Kuchel (1999) Biochem. J. 342, 595-602] that present a detailed mathematical model of erythrocyte metabolism which explains the regulation and control of 2,3-bisphosphoglycerate (2,3-BPG) metabolism. 2,3-BPG is a modulator of haemoglobin oxygen affinity and hence plays an important role in blood oxygen transport and delivery. This paper presents an in vivo kinetic characterization of 2,3-BPG synthase/phosphatase (BPGS/P), the enzyme that catalyses both the synthesis and degradation of 2,3-BPG. Much previous work had indicated that the behaviour of this enzyme in vitro is markedly different from that in vivo. (13)C and (31)P NMR were used to monitor the time courses of selected metabolites when erythrocytes were incubated with or without [U-(13)C]glucose. Simulations of the experimental time courses were then made. By iteratively changing the parameters of the BPGS/P part of the model until a good match between the NMR-derived data and simulations were achieved, it was possible to characterize BPGS/P kinetically in vivo. This work revealed that: (1) the pH-dependence of the synthase activity results largely from a strong co-operative inhibition of the synthase activity by protons; (2) 3-phosphoglycerate and 2-phosphoglycerate are much weaker inhibitors of 2,3-BPG phosphatase in vivo than in vitro; (3) the K(m) of BPGS/P for 2,3-BPG is significantly higher than that measured in vitro; (4) the maximal activity of the phosphatase in vivo is approximately twice that in vitro, when P(i) is the sole activator (second substrate); and (5) 2-phosphoglycollate appears to play no role in the activation of the phosphatase in vivo. Using the newly determined kinetic parameters, the percentage of glycolytic carbon flux that passes through the 2, 3-BPG shunt in the normal in vivo steady state was estimated to be 19%.

Comparison of computer simulations of the F-type and L-type non-oxidative hexose monophosphate shunts with 31P-NMR experimental data from human erythrocytes.

Mathematical modelling was used to predict the behaviour of the two most favoured schemes for the operation of the non-oxidative hexose monophosphate shunt (HMS), the F-type and the L-type pathways. The models simulate the time courses of sugar-phosphate concentrations when various substrates are metabolized via each pathway. A 31P-NMR technique, with which to observe time courses of concentrations of sugar phosphates in a human red cell lysate, was developed. The accuracy of each hypothesised scheme was then evaluated by comparing predicted with observed data. The results were more consistent with time courses of sugar-phosphate levels predicted by the F-type (classical) pathway than those predicted by the L-type model. However, the accumulation of sedoheptulose 1,7-bisphosphate when a haemolysate was incubated with ribose 5-phosphated showed that the F-type pathway is not a complete description of the system of reactions. Transaldolase was demonstrated to be essential for the normal metabolism of sugar phosphates by haemolysates. The effects of the heat-inactivation of transaldolase on the metabolism of sugar phosphates were accurately predicted by the F-type model. The relevance of attempting to describe the reaction of the non-oxidative HMS as a distinct 'pathway' or 'cycle' is discussed.

Inhibition of fructolytic enzymes in boar spermatozoa by (S)-alpha-chlorohydrin and 1-chloro-3-hydroxypropanone.

When boar spermatozoa were incubated with the (S)-isomer of the male antifertility agent alpha-chlorohydrin the activity of glyceraldehyde-3-phosphate dehydrogenase was inhibited. The (R)-isomer had no significant effect on the activity of this enzyme whereas (R,S)-3-chlorolactaldehyde caused an inhibition of its activity and also in that of lactate dehydrogenase. The in vitro production of (S)-3-chlorolactaldehyde, the active metabolite of (S)-alpha-chlorohydrin, was attempted by incubating boar spermatozoa with 1-chloro-3-hydroxypropanone. Preliminary results lead us to propose that this compound is converted into (S)-3-chlorolactaldehyde as well as to another metabolite which is an inhibitor of other enzymes within the fructolytic pathway.

Stereospecificity of substrate usage by glyoxalase 1: nuclear magnetic resonance studies of kinetics and hemithioacetal substrate conformation.

The specificity of glyoxalase 1 for the diastereomers of its hemithioacetal substrate [which forms spontaneously between an alpha-keto aldehyde and reduced glutathione (GSH)] was investigated by exploiting the differences between their 1H NMR spectra at pH* 4.4. The 1H NMR spectra of the hemithioacetals of glutathione with phenylglyoxal or methylglyoxal were assigned with the aid of conventional decoupling and two-dimensional NMR spectroscopic techniques. The rate of interconversion of the diastereomers was determined at 30 degrees C from the results of an inversion-transfer technique and found to be 0.30 +/- 0.04 s-1 (+/- sd) in the case of phenylglyoxal and 0.15 +/- 0.02 s-1 in the case of methylglyoxal. Stereopreference of the enzyme was tested by the addition of large amounts of yeast glyoxalase 1 to a reaction mixture; glyoxalase 1 preferentially operated on one diastereomer of the phenylglyoxal hemithioacetal but the diastereomers of methylglyoxal appeared to be operated upon indiscriminately. From computer models of the kinetics of possible reaction schemes, a mechanism involving glyoxalase 1 catalysis of both diastereomers of the hemithioacetals was shown to be the most consistent with the experimental data. Estimates of internuclear distances in the diastereomers, obtained from 2D NMR spectra were used in "dynamical simulated annealing" calculations to generate likely structures of the substrates. Relative ring-current shifts obtained from 1D NMR spectra were used, together with a ring-current shift algorithm, to select structures with compatible conformations. We conclude that the rate of conversion of substrate by the enzyme is dependent upon the overall conformation of the substrate molecule, rather than merely its stereochemical configuration (R or S).