Parasite sulphur amino acid metabolism

This paper reviews current knowledge regarding the metabolism of the sulphur-containing amino acids methionine and cysteine in parasitic protozoa and helminths. Particular emphasis is placed on the unusual aspects of parasite biochemistry which may present targets for rational design of antiparasite drugs. In general, the basic pathways of sulphur amino acid metabolism in most parasites resemble those of their mammalian hosts, since the enzymes involved in (a) the methionine cycle and S-adenosylmethionine metabolism, (b) the trans-sulphuration sequence, (c) the transminative catabolism of methionine, (d) the oxidative catabolism of cysteine and (e) glutathione synthesis have been demonstrated variously in several helminth and protozoan species. Despite these common pathways, there also exist numerous differences between parasite and mammalian metabolism. Some of these differences are relatively subtle. For example, the biochemical properties (and primary amino acid structures) of certain parasite methionine cycle enzymes and S-adenosylmethionine decarboxylases differ from those of the corresponding mammalian enzymes, and nematodes and trichomonads possess a novel, non-mammalian form of the trans-sulphuration enzyme cystathionine beta-synthase. The most profound differences between parasite and mammalian biochemistry relate to a number of unusual enzymes and thiol metabolites found in parasitic protozoa. In certain protozoa the pathway for methionine recycling from 5'-methylthioadenosine differs markedly from the mammalian route, and involves 2 exclusively microbial enzymes. Trypanosomatid protozoa contain the non-mammalian antioxidant thiol compounds ovothiol A and trypanothione, together with unique trypanothione-linked enzymes. Specific anaerobic protozoa possess another exclusively microbial enzyme, methionine gamma-lyase, which catabolises methionine (and homocysteine); the physiological significance of these non-mammalian activities is not fully understood. These unusual features offer opportunities for chemotherapeutic exploitation, and in some cases represent metabolic similarities with bacteria. Additionally, some anaerobic protozoa contain unidentified thiols and this implies the presence of further unusual enzymes/pathways in these organisms. So far, no truly unique targets for chemotherapy have been found in helminth sulphur amino acid metabolism, and to some degree this reflects the relative lack of detailed study in the area.     

Use of 13C nuclear magnetic resonance to assess fossil fuel biodegradation: fate of [1-13C]acenaphthene in creosote polycyclic aromatic compound mixtures degraded by bacteria

[1-13C]acenaphthene, a tracer compound with a nuclear magnetic resonance (NMR)-active nucleus at the C-1 position, has been employed in conjunction with a standard broad-band-decoupled 13C-NMR spectroscopy technique to study the biodegradation of acenaphthene by various bacterial cultures degrading aromatic hydrocarbons of creosote. Site-specific labeling at the benzylic position of acenaphthene allows 13C-NMR detection of chemical changes due to initial oxidations catalyzed by bacterial enzymes of aromatic hydrocarbon catabolism. Biodegradation of [1-13C]acenaphthene in the presence of naphthalene or creosote polycyclic aromatic compounds (PACs) was examined with an undefined mixed bacterial culture (established by enrichment on creosote PACs) and with isolates of individual naphthalene- and phenanthrene-degrading strains from this culture. From 13C-NMR spectra of extractable materials obtained in time course biodegradation experiments under optimized conditions, a number of signals were assigned to accumulated products such as 1-acenaphthenol, 1-acenaphthenone, acenaphthene-1,2-diol and naphthalene 1,8-dicarboxylic acid, formed by benzylic oxidation of acenaphthene and subsequent reactions. Limited degradation of acenaphthene could be attributed to its oxidation by naphthalene 1,2-dioxygenase or related dioxygenases, indicative of certain limitations of the undefined mixed culture with respect to acenaphthene catabolism. Coinoculation of the mixed culture with cells of acenaphthene-grown strain Pseudomonas sp. strain A2279 mitigated the accumulation of partial transformation products and resulted in more complete degradation of acenaphthene. This study demonstrates the value of the stable isotope labeling approach and its ability to reveal incomplete mineralization even when as little as 2 to 3% of the substrate is incompletely oxidized, yielding products of partial transformation. The approach outlined may prove useful in assessing bioremediation performance.     

Retrobiosynthetic NMR studies with 13C-labeled glucose. Formation of gallic acid in plants and fungi

The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina. Fungal cultures were grown with [1-13C]glucose or with a mixture of unlabeled glucose and [U-13C6]glucose. Young leaves of R. typhina were kept in an incubation chamber and were supplied with a solution containing a mixture of unlabeled glucose and [U-13C6]glucose via the leaf stem. Isotope distributions in isolated gallic acid and aromatic amino acids were analyzed by one-dimensional 1H and 13C NMR spectroscopy. A quantitative analysis of the complex isotopomer composition of metabolites was obtained by deconvolution of the 13C13C coupling multiplets using numerical simulation methods. This approach required the accurate analysis of heavy isotope chemical shift effects in a variety of different isotopomers and the analysis of long range 13C13C coupling constants. The resulting isotopomer patterns were interpreted using a retrobiosynthetic approach based on a comparison between the isotopomer patterns of gallic acid and tyrosine. The data show that both in the fungus and in the plant all carbon atoms of gallic acid are biosynthetically equivalent to carbon atoms of shikimate. Notably, the carboxylic group of gallic acid is derived from the carboxylic group of an early intermediate of the shikimate pathway and not from the side chain of phenylalanine or tyrosine. It follows that the committed precursor of gallic acid is an intermediate of the shikimate pathway prior to prephenate or arogenate, most probably 5-dehydroshikimate. A formation of gallic acid via phenylalanine, the lignin precursor, caffeic acid, or 3,4, 5-trihydroxycinnamic acid can be ruled out as major pathways in the fungus and in young leaves of R. typhina. The incorporation of uniformly 13C-labeled glucose followed by quantitative NMR analysis of isotopomer patterns is suggested as a general method for biosynthetic studies. As shown by the plant experiment, this approach is also applicable to systems with low incorporation rates.     

13C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin's biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin's effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of 13C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate flux into 13C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of long-chain fatty acid synthesis from 13C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.     

Evidence for S-adenosylmethionine independent catabolism of methionine in the rat

Experiments conducted with rats in vivo comparing the metabolism of methionine and S-methyl-L-cysteine and in vitro comparing methionine, S-methyl-L-cysteine and S-adenosyl-L-methionine indicate that a substantial portion of the oxidative metabolism of the methionine methyl group occurs by pathways that are independent of S-adenosylmethionine formation. Inclusion of 1.2% or 2.4% of S-methyl-L-cysteine in a diet containing 3% of L-methionine depressed the conversion of the methionine methyl and carboxyl carbons to CO2 by 39% and 28%, and 52% and 33%, respectively, for the two levels of S-methyl-L-cysteine. Inclusion of 1.65% of methionine in a diet containing 2.4% of S-methyl-L-cysteine did not affect the conversion of the methyl group of S-methylcysteine to CO2, but 3% of methionine depressed the conversion of the S-methylcysteine methyl group to CO2 to 87% of control values. Greater inhibitions were seen when these substrates were compared in a liver homogenate. In a rat liver homogenate system optimized for the conversion of the methyl group of methionine to CO2, the rate of conversion of the methyl group of S-adenosyl-L-methionine to CO2 was less than 1% of that of methionine even when the concentration of S-adenosylmethionine was saturating. Addition of saturating levels of unlabeled S-adenosymethionine to the homogenate system did not effect the rate of conversion of the methionine methyl carbon to CO2. Although S-adenosylmethionine-dependent metabolism of methionine, leading to incorporation of the methyl carbon into sarcosine and serine, could be demonstrated in liver homogenates, essentially all of the CO2 produced from the methionine methyl group was derived by a pathway or pathways independent of S-adenosylmethionine formation. Formaldehyde and formate have been tentatively identified as intermediates in catabolism of the methionine methyl group by this (these) pathway(s).     

Effect of methionine on the metabolism of formate and histidine by rats fed folate/vitamin B-12-methionine-deficient diet

The metabolism of formate and histidine were compared in rats and in perfused livers of rats on diets deficient in vitamin B-12, methionine, and folic acid. Excretion of formate and formiminoglutamic acid, and the oxidation of [2-14C]histidine and [14C]formate to 14CO2 were measured. Liver folate levels decreased to 40% of normal on the vitamin B-12- and methionine-deficient diets but the rate of oxidation of histidine to CO2 in the whole animal decreased to 15% of normal. This indicated a reduction in the metabolic activity of the liver folates in vitamin B-12deficiency. Comparison of formate and histidine catabolism in folic acid deficiency showed that the oxidation of histine was decreased to 5% of normal but formate oxidation was decreased to only 30% of normal. This indicates that 25% of formate oxidation normally proceeds by a non-folate-dependent pathway.     

4-(trans-4-Methylcyclohexyl)-4-oxobutyric acid (JTT-608). A new class of antidiabetic agent

During an investigation of drugs for improving the beta-cell response to glucose, we found that 4-cyclohexyl-4-oxobutyric acid selectively improved glucose-stimulated insulin release and glucose tolerance in both normal and diabetic rats. A series of 4-cycloalkyl-4-oxobutyric acids and related compounds were synthesized and evaluated for their effects on the glucose tolerance test and fasting euglycemia. This study elucidated the structural requirements for drug activity and determined that the optimum compound was 4-(trans-4-methylcyclohexyl)-4-oxobutyric acid 7 (JTT-608). This compound improved glucose tolerance from an oral dose of 3 mg/kg and did not change fasting euglycemia even at an oral dose of 30 mg/kg. Selective improvement of glucose-induced insulin secretion was observed in studies using neonatal streptozotocin rats (nSTZ rats) and perfused pancreases isolated from nSTZ rats.     

A deficiency in aspartate biosynthesis in Lactococcus lactis subsp. lactis C2 causes slow milk coagulation

A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc-) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis.     

5 beta-hydroxylation by the liver. Identification of 3,5,7-trihydroxy nor-bile acids as new major biotransformation products of 3,7-dihydroxy nor-bile acids in rodents

24-Norursodeoxycholic acid (nor-UDCA), when administered into the anesthetized biliary fistula hamster or injected into the perfusate of an isolated liver, was hydroxylated at C-5 to give 5 beta-hydroxynorursodeoxycholic acid 2 (3 alpha,5,7 beta-trihydroxy-24-nor-5 beta-cholan-23-oic acid), which was secreted into bile mainly as such. Similarly, 24-norchenodeoxycholic acid (nor-CDCA) was 5 beta-hydroxylated to give 5 beta-hydroxynor-chenodeoxycholic acid 4 (3 alpha,5,7 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic acid), which was also secreted into bile without appreciable further biotransformation. The site of hydroxylation was assigned by 13C and 1H NMR and mass spectrometry. 5-Hydroxylation was a major biotransformation pathway at physiological bile acid loads. 5-Hydroxylation of UDCA also occurred in the perfused rat liver but to a lesser extent. 5-Hydroxylation of nor-UDCA was not observed in rabbit, dog, or man, indicating that its formation is species-specific. 5-Hydroxylation of nor-CDCA and nor-UDCA is the first reported example of hydroxylation of a tertiary carbon atom of bile acids. Nor-dihydroxy bile acids appear to be useful for the detection of minor hydroxylation pathways, because their prolonged hepatobiliary retention exposes them repeatedly to hydroxylases present in the hepatobiliary system.     

Identification of volatile forms of methyl groups released by Halobacterium salinarium

halobacterium salinarium (formerly H. halobium) is a chemotactic and phototactic archaeon from which volatile methyl groups are released continually, a phenomenon related to its sensory system. We found that released methyl groups comprised two different chemical species, methanol and methanethiol, the sulfur analog of methanol. Radiolabeling experiments showed that the methyl groups of both compounds, as well as the sulfur of methanethiol, were derived from methionine but were donated to cellular components and subsequently cleaved to produce the respective volatile compounds. Previous work had shown that chemostimuli and photostimuli result in transient increases in the rate of release of volatile methyl groups. We found that these increases reflected increased release of methanol but not of methanethiol. Thus, the methyl group chemistry of the H. salinarium sensory system is analogous to the well-studied chemotactic system of Escherichia coli. The reactions that result in methanethiol release are of unknown function and have unusual features. They may involve a methionine-gamma-lyase activity we detected in H. salinarium. Sulfur derived from methionine was found attached to specific proteins in reduction-sensitive disulfide linkages.     

Arginine catabolism and the arginine succinyltransferase pathway in Escherichia coli

Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in Escherichia coli (through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in E. coli and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine succinyltransferase (AST) pathway in E. coli. Nitrogen limitation induced this pathway in both E. coli and Klebsiella aerogenes, but the mechanisms of activation clearly differed in these two organisms. We identified the E. coli gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an astCADBE operon. Its disruption prevented arginine catabolism, impaired ornithine utilization, and affected the synthesis of all the enzymes of the AST pathway. Disruption of astB eliminated succinylarginine dihydrolase activity and prevented arginine utilization but did not impair ornithine catabolism. Overproduction of AST enzymes resulted in faster growth with arginine and aspartate. We conclude that the AST pathway is necessary for aerobic arginine catabolism in E. coli and that at least one enzyme of this pathway contributes to ornithine catabolism.     

Reaction of mucochloric and mucobromic acids with adenosine and cytidine: formation of chloro- and bromopropenal derivatives

Mucochloric (MCA) and mucobromic acid (MBA)--bacterial mutagens and water disinfection byproducts--were reacted with adenosine, cytidine, and guanosine in N,N-dimethylformamide (DMF). In the MCA reaction with adenosine and cytidine and in the MBA reaction with adenosine one major product was formed. In the reactions of MBA with cytidine and in the reactions of MCA and MBA with guanosine only trace levels of products could be detected, and these were not further characterized. The products from the adenosine and cytidine reactions were isolated by preparative chromatography on octadecylsilane columns and structurally characterized by UV absorbance, 1H and 13C NMR spectroscopy, and mass spectrometry. The products were identified as 3-(N6-adenosinyl)-2-chloro-2-propenal (MClA), 3-(N6-adenosinyl)-2-bromo-2-propenal (MBrA), and 3-(N4-cytidinyl)-2-chloro-2-propenal (MClC). The yields of MClA, MBrA, and MClC were 19, 4 and 7 mol %, respectively. These halopropenal derivatives were formed also in reactions carried out in aqueous solutions at pH 7.4 and 37 degrees C at low yields, about 5 x 10(-3)%. The mechanism of formation of the halopropenal derivatives and of the previously identified etheno and ethenocarbaldehyde derivatives was elucidated by reacting 13C-3 labeled MCA with adenosine in DMF and in water. The location of the labeled carbon in the products was determined from the 13 C NMR spectra. It was concluded that the halopropenal derivatives were formed by mechanisms that differ completely from the one responsible for the formation of the propenal adducts (M1A and M1C) previously reported to be formed in reactions of malonaldehyde with adenosine and cytidine.     

A study of branched-chain amino acid aminotransferase and isolation of mutations affecting the catabolism of branched-chain amino acids in Saccharomyces cerevisiae

The specific activity of branched-chain amino acid aminotransferase was highest when S. cerevisiae was grown in minimal medium containing a branched-chain amino acid as nitrogen source. Growth in complex media with glycerol or ethanol gave moderately high levels, whereas with glucose and fructose the specific activity was very low. Mutagenesis defined three genes (BAA1 to BAA3) required for branched-chain amino acid catabolism. The baa1 mutation reduced the specific activity of the aminotransferase, the stationary phase density in YEPD and caused gross morphological disturbance. Branched-chain amino acid aminotransferase is essential for sporulation.     

In vivo 13C NMR measurements of cerebral glutamine synthesis as evidence for glutamate-glutamine cycling

The cerebral tricarboxylic acid (TCA) cycle rate and the rate of glutamine synthesis were measured in rats in vivo under normal physiological and hyperammonemic conditions using 13C NMR spectroscopy. In the hyperammonemic animals, blood ammonia levels were raised from control values of approximately 0.05 mM to approximately 0.35 mM by an intravenous ammonium acetate infusion. Once a steady-state of cerebral metabolites was established, a [1-13C]glucose infusion was initiated, and 13C NMR spectra acquired continuously on a 7-tesla spectrometer to monitor 13C labeling of cerebral metabolites. The time courses of glutamate and glutamine C-4 labeling were fitted to a mathematical model to yield TCA cycle rate (V(TCA)) and the flux from glutamate to glutamine through the glutamine synthetase pathway (V(gln)). Under hyperammonemia the value of V(TCA) was 0.57 +/- 0.16 micromol/min per g (mean +/- SD, n = 6) and was not significantly different (unpaired t test; P > 0.10) from that measured in the control animals (0.46 +/- 0.12 micromol/min per g, n = 5). Therefore, the TCA cycle rate was not significantly altered by hyperammonemia. The measured rate of glutamine synthesis under hyperammonemia was 0.43 +/- 0.14 micromol/min per g (mean +/- SD, n = 6), which was significantly higher (unpaired t test; P < 0.01) than that measured in the control group (0.21 +/- 0.04 micromol/ min per g, n = 5). We propose that the majority of the glutamine synthetase flux under normal physiological conditions results from neurotransmitter substrate cycling between neurons and glia. Under hyperammonemia the observed increase in glutamine synthesis is comparable to the expected increase in ammonia transport into the brain and reported measurements of glutamine efflux under such conditions. Thus, under conditions of elevated plasma ammonia an increase in the rate of glutamine synthesis occurs as a means of ammonia detoxification, and this is superimposed on the constant rate of neurotransmitter cycling through glutamine synthetase.     

Biosynthesis of the diterpene verrucosan-2beta-ol in the phototrophic eubacterium Chloroflexus aurantiacus. A retrobiosynthetic NMR study

The biosynthesis of verrucosan-2beta-ol in the green phototrophic eubacterium Chloroflexus aurantiacus was investigated by in vivo incorporation of singly or doubly 13C-labeled acetate. The 13C labeling of the isolated diterpene was analyzed by one- and two-dimensional NMR spectroscopy. The 13C-labeling patterns of verrucosan-2beta-ol were compared with the labeling patterns of intermediary metabolites (acetyl-CoA, pyruvate, and glyceraldehyde 3-phosphate) which were deduced from amino acids and nucleosides by retrobiosynthetic analysis. The results show that verrucosan-2beta-ol is synthesized via mevalonate and not via the deoxyxylulose pathway, which was discovered recently in some eubacteria, algae, and plants. A scheme for the formation of the unusual tetracyclic ring system is offered. The cyclization process is initiated by the solvolysis of pyrophosphate from geranyllinaloyl pyrophosphate and the mechanism involves a Wagner-Meerwein rearrangement, a 1,5-hydride shift, and a cyclopropylcarbinyl to cyclopropylcarbinyl rearrangement.     

N alpha- and N epsilon-D-galacturonoyl-L-lysine amides: properties and possible occurrence in plant cell walls

Three representatives of a novel class of amide (isopeptide) glycoconjugates have been synthesised: N alpha-D-galacturonoyl-L-lysine and N epsilon-D-galacturonoyl-L-lysine and N epsilon-D-polygalacturonoyl-L-lysine. Galacturonoyl-lysine amide bonds were labile in 2 M trifluoroacetic acid at 120 degrees and in alkali, but relatively stable in cold acid. The amide bonds were resistant to digestion by Driselase, Pronase and trypsin. The polysaccharide backbone of N epsilon-D-polygalacturonoyl-L-lysine was hydrolysed by Driselase to yield two major ninhydrin-positive compounds which were shown by 1H and 13C NMR spectroscopy to be tri- and tetra-alpha-(1-->4)-D-galacturonoyl-L-lysines. To investigate the possible natural occurrence of N-galacturonoyl isopeptide bonds, we fed cell-suspension cultures of spinach and tomato with D-[6-14C]glucuronic acid, which radio-labels pectic polysaccharides. The radioactive cell walls were digested with, sequentially, Driselase, mild acid, and proteinases. On electrophoresis at pH 2.0, several of the radioactive digestion-products were cathodic. Some of the cathodic products yielded [14C]galacturonic acid upon complete acid hydrolysis. The existence of these products is compatible with the presence of novel N-galacturonoyl isopeptide bonds, which could serve as cross-links in plant cell walls.     

Biosynthesis of vitamin B12 in anaerobic bacteria--experiments with Eubacterium limosum on the transformation of 5-hydroxy-6-methyl-benzimidazole, its nucleoside, its cobamide, and of 5-hydroxybenzimidazolylcobamide in vitamin B12

In anaerobic bacteria 5-hydroxybenzimidazole and 5-hydroxy-6-methylbenzimidazole are precursors of the 5,6-dimethylbenzimidazole moiety of vitamin B12. In order to elucidate the pathway from these bases to vitamin B12, experiments on the transformation of 5-hydroxy-6-methylbenzimidazole, of 5-hydroxy-6-methylbenzimidazole-alpha-D-ribofuranoside, of 5-hydroxybenzimidazolylcobamide and of 5-hydroxy-6-methylbenzimidazolylcobamide into vitamin B12 were carried out. The vitamin B12 synthesized by the anaerobe Eubacterium limosum in the presence of 5-hydroxy-6-methylbenzimidazole and L-[methyl-13C]methionine was subjected to NMR spectroscopy. It revealed that the methyl group at C5 of the 5,6-dimethylbenzimidazole moiety was 13C labeled, whereas the methyl group at C6 was unlabeled. This shows that the transformation of 5-hydroxy-6-methylbenzimidazole into the base moiety of vitamin B12 occurs regiospecifically. 5-Hydroxy-6-methylbenzimidazole-alpha-D-ribofuranoside as well as 5-hydroxybenzimidazolylcobamide and 5-hydroxy-6-methylbenzimidazolylcobamide were also transformed into vitamin B12 by E. limosum. When 5-hydroxy-6-methylbenzimidazolylcobamide 13C labeled at C2 of the base part and 14C labeled in the ribose was used for this experiment, the vitamin B12 obtained from this cobamide was 13C and 14C labeled in the same positions. This demonstrates that the alpha-glycosidic bond of the precursor cobamide is not split during the formation of vitamin B12. It can be deduced from these results that the precursor bases are transformed regiospecifically into their alpha-nucleotides, and partially into their cobamides. The alpha-nucleotides are then transformed into alpha-ribazole-5'-phosphate and, subsequently, into vitamin B12. Most likely the cobamides are degraded to the alpha-nucleotides before being used for the biosynthesis of vitamin B12. A pathway for the latter process is suggested.     

Pertussis toxin modification of PC12 cells lowers cytoskeletal F-actin and enhances norepinephrine secretion: involvement of protein kinase C and protein phosphatases

In order to understand the modifications of proteins produced by aldehydes of lipid peroxidation, [1-13C]-2(E)-hexenal, [1-13C]-4-oxopentanal, and a mixture of [1-13C]- and [2-13C]-4-hydroxynon-2(E)-enal were synthesized and the reaction of each of the labeled aldehydes with bovine serum albumin was analyzed by 13C NMR spectroscopy. Protein nucleophiles add to the 3-position of hexenal, and the resulting propanal moieties appear to undergo aldol condensation, form imine cross-links with lysyl residues, or lead to pyridinium rings. During the reaction of 4-oxopentanal with the lysyl residues of bovine serum albumin, only 1-alkyl-2-methylpyrrole and a possible intermediate leading to the pyrrole were observed. Hydroxypyrrolidine cross-links such as 25 could not be detected, leaving the pyrrole as the mediator of protein cross-linking. The Michael adducts are the major products in the reaction between 4-hydroxynon-2-enal and proteins. They exist almost exclusively in the cyclic hemiacetal form and do not appear to cross-link through imine formation with lysyl residues. A minor pathway involves the reaction of 4-hydroxynon-2-enal with the lysyl amino groups of protein resulting in 2-pentylpyrrole adducts that may mediate protein cross-linking. The Michael adducts appear not to be the direct source of the pyrrole, but the imine 32 and the enamine 35 are likely intermediates toward the five-membered ring.     

Dynamic 13C NMR analysis of oxidative metabolism in the in vivo canine myocardium

Oxidative metabolism in the in vivo canine myocardium was studied noninvasively using 13C-enriched acetate and non-steady state 13C NMR techniques. Under low workload conditions, the myocardium oxidized the infused [2-13C]acetate and incorporated the labeled carbon into the glutamate pool as expected. This conclusion stems from the rapid enrichment of the C-2, C-3, and C-4 carbons of glutamic acid both under in vivo conditions and in extracts. Surprisingly, [2-13C]acetate uptake was not observed at high workloads as reflected by an absence of glutamate pool enrichment at these rate pressure products. Rather, the myocardium selected its substrate from an endogenous pool. Since free acetate can directly cross the inner mitochondrial membrane and be converted to acetyl-CoA through acetyl-CoA synthetase, these results support workload-dependent regulation of substrate access to the mitochondrial CoASH pool. As such, we advance the hypothesis that the selection of substrate for condensation with CoASH and subsequent oxidation in the tricarboxylic acid cycle is regulated kinetically through the Km values of the appropriate condensation enzymes and through the absolute levels of free CoASH in the mitochondria.     

Lactate turnover in rat glioma measured by in vivo nuclear magnetic resonance spectroscopy

Elevated tissue lactate concentrations typically found in tumors can be measured by in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, lactate turnover in rat C6 glioma was determined from in vivo 1H NMR measurements of [3-13C]lactate buildup during steady-state hyperglycemia with [1-13C]glucose. With this tumor model, a narrow range of values was observed for the first-order rate constant that describes lactate efflux, k2 = 0.043 +/- 0.007 (n = 12) SD min-1. For individual animals, the standard error in k2 was small (< 18%), which indicated that the NMR data fit the kinetic model well. Lactate measurements before and after infusing [1-13C]glucose showed that the majority of the tumor lactate pool was metabolically active. Signals from 13C-labeled glutamate in tumors were at least 10-fold smaller than the [3-13C]lactate signal, whereas spectra of the contralateral hemispheres revealed the expected labeling of [4-13C]glutamate, as well as [2-13C] and [3-13C]glutamate, which indicates that label cycled through the tricarboxylic acid cycle in the brain tissue. Lack of significant 13C labeling of glutamate was consistent with low respiratory metabolism in this glioma. It is concluded that lactate in rat C6 glioma is actively turning over and that the kinetics of lactate efflux can be quantified noninvasively by 1H NMR detection of 13C label. This noninvasive NMR approach may offer a valuable tool to help evaluate tumor growth and metabolic responsiveness to therapies.