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PubMed2005

Experimental and in silico analyses of glycolytic flux control in bloodstream form Trypanosoma brucei.

A mathematical model of glycolysis in bloodstream form Trypanosoma brucei was developed previously on the basis of all available enzyme kinetic data (Bakker, B. M., Michels, P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. Biol. Chem. 272, 3207-3215). The model predicted correctly the fluxes and cellular metabolite concentrations as measured in non-growing trypanosomes and the major contribution to the flux control exerted by the plasma membrane glucose transporter. Surprisingly, a large overcapacity was predicted for hexokinase (HXK), phosphofructokinase (PFK), and pyruvate kinase (PYK). Here, we present our further analysis of the control of glycolytic flux in bloodstream form T. brucei. First, the model was optimized and extended with recent information about the kinetics of enzymes and their activities as measured in lysates of in vitro cultured growing trypanosomes. Second, the concentrations of five glycolytic enzymes (HXK, PFK, phosphoglycerate mutase, enolase, and PYK) in trypanosomes were changed by RNA interference. The effects of the knockdown of these enzymes on the growth, activities, and levels of various enzymes and glycolytic flux were studied and compared with model predictions. Data thus obtained support the conclusion from the in silico analysis that HXK, PFK, and PYK are in excess, albeit less than predicted. Interestingly, depletion of PFK and enolase had an effect on the activity (but not, or to a lesser extent, expression) of some other glycolytic enzymes. Enzymes located both in the glycosomes (the peroxisome-like organelles harboring the first seven enzymes of the glycolytic pathway of trypanosomes) and in the cytosol were affected. These data suggest the existence of novel regulatory mechanisms operating in trypanosome glycolysis.

Animals↗

PubMed2005

New insights into the evolutionary links relating to the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase subfamilies.

Bacterial 3-deoxy-d-arabino-heptulosonate 7-phosphate synthases (DAHPSs) have been divided into either of two classes (Class I/Class II) or subfamilies (AroAI(alpha)/AroAI(beta)). Our investigation into the biochemical properties of the unique bifunctional DAHPS from Bacillus subtilis provides new insight into the evolutionary link among DAHPS subfamilies. In the present study, the DAHPS (aroA) and chorismate mutase (aroQ) activities of B. subtilis DAHPS are separated by domain truncation. Detailed enzymatic studies with the full-length wild-type protein and the truncated domains led to our hypothesis that the aroQ domain was fused to the N terminus of aroA in B. subtilis during evolution for the purpose of feedback regulation and not for the creation of a bona fide bifunctional enzyme. In addition, examination of aroA and aroQ fusion proteins from Porphyromonas gingivalis, in which the aroQ domain is fused to the C terminus of aroA, further supports the hypothesis. These results, along with sequence structure analysis of the DAHPS families suggest that "feedback regulation" may indeed be the evolutionary link between the two classes/subfamilies. It is likely that DAHPSs evolved from a primitive unregulated member of the AroAI(beta) subfamily. During evolution, some members of the AroAI(beta) subfamily remained unregulated, whereas other members acquired an extra domain for feedback regulation. The AroAI(alpha) subfamilies, however, evolved in a more complex manner to acquire insertions/extensions in the (beta/alpha)(8) barrel to function as regulatory elements.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed2006

Two crystal structures of the isochorismate pyruvate lyase from Pseudomonas aeruginosa.

Enzymatic systems that exploit pericyclic reaction mechanisms are rare. A recent addition to this class is the enzyme PchB, an 11.4-kDa isochorismate pyruvate lyase from Pseudomonas aeruginosa. The apo and pyruvate-bound structures of PchB reveal that the enzyme is a structural homologue of chorismate mutases in the AroQalpha class despite low sequence identity (20%). The enzyme is an intertwined dimer of three helices with connecting loops, and amino acids from each monomer participate in each of two active sites. The apo structure (2.35 A resolution) has one dimer per asymmetric unit with nitrate bound in an open active site. The loop between the first and second helices is disordered, providing a gateway for substrate entry and product exit. The pyruvate-bound structure (1.95 A resolution) has two dimers per asymmetric unit. One has two open active sites like the apo structure, and the other has two closed active sites with the loop between the first and second helices ordered for catalysis. Determining the structure of PchB is part of a larger effort to elucidate protein structures involved in siderophore biosynthesis, as these enzymes are crucial for bacterial iron uptake and virulence and have been identified as antimicrobial drug targets.

Amino Acid Sequence↗

PubMed2006

PGAM5, a Bcl-XL-interacting protein, is a novel substrate for the redox-regulated Keap1-dependent ubiquitin ligase complex.

Keap1 is a BTB-Kelch substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex that functions as a sensor for thiol-reactive chemopreventive compounds and oxidative stress. Inhibition of Keap1-dependent ubiquitination of the bZIP transcription factor Nrf2 enables Nrf2 to activate a cyto-protective transcriptional program that counters the damaging effects of oxidative stress. In this report we have identified a member of the phosphoglycerate mutase family, PGAM5, as a novel substrate for Keap1. The N terminus of the PGAM5 protein contains a conserved NXESGE motif that binds to the substrate binding pocket in the Kelch domain of Keap1, whereas the C-terminal PGAM domain binds Bcl-X(L). Keap1-dependent ubiquitination of PGAM5 results in proteasome-dependent degradation of PGAM5. Quinone-induced oxidative stress and the chemopreventive agent sulforaphane inhibit Keap1-dependent ubiquitination of PGAM5. The identification of PGAM5 as a novel substrate of Keap1 suggests that Keap1 regulates both transcriptional and post-transcriptional responses of mammalian cells to oxidative stress.

Amino Acid Motifs↗

PubMed2006

Characterization of the 2-hydroxy-acid dehydrogenase McyI, encoded within the microcystin biosynthesis gene cluster of Microcystis aeruginosa PCC7806.

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thio-template function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of d-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce alpha-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role.

Alcohol Oxidoreductases↗

PubMed2005

Concentrations of glycolytic enzymes and other cytosolic proteins in the diffusible fraction of a vertebrate muscle proteome.

We used a novel microvolumetric technique based on protein diffusion to characterize the subproteome of muscle that consists of diffusible proteins, including those involved in cell metabolism. Muscle fiber segments were mechanically demembranated under mineral oil and transferred into drops of relaxing solution. After the fiber segment was depleted of diffusible proteins, the content of each drop and residual segment was analyzed by one-dimensional polyacrylamide gel electrophoresis. Proteins were identified through peptide mass fingerprinting and quantified using purified protein standards. Ten of the most abundant cytosolic proteins, distinguished by their ability to readily diffuse out of the skinned fiber, were glycolytic enzymes whose concentrations ranged from 2.6+/-1.0 g liter-1 (phosphoglucose isomerase) to 12.8+/-1.1 g liter-1 fiber volume (pyruvate kinase). The concentrations of the other five most abundant cytosolic proteins were as follows: glycogen phosphorylase, 6.0+/-2.3 g liter-1; phosphoglucose mutase, 2.2+/-0.2 g liter-1; adenylate kinase, 1.6+/-1.3 g liter-1; phosphocreatine kinase, 6.6+/-2.6 g liter-1; and parvalbumin, 0.7+/-0.4 g liter-1. Given the molecular weight and subunit number of each enzyme, the combined concentration of the 15 most abundant cytosolic proteins was 82.3 g liter-1; the volume fraction was 0.093. The large volume fraction of diffusible proteins favors nonspecific interactions and associations, particularly if the glycolytic enzymes and diffusible phosphocreatine kinase are restricted to the I-band as previous studies suggest. The relative molar concentration of glycolytic enzymes is roughly consistent with a stoichiometry of 1:2 for enzymes catalyzing the hexose and triose sugar reactions, respectively, a stoichiometry that may favor metabolic channeling of intermediates during glycolysis. Our results indicate that subcellular fractionation of muscle proteins, in which cytosolic constituents are distinguished by their ability to diffuse readily from demembranated cells, is a promising microvolumetric technique that allows conclusions to be drawn about native protein-protein interactions based on concentration and stoichiometry.

Animals↗

PubMed2002

The structure and function of novel proteins of Bacillus anthracis and other spore-forming bacteria: development of novel prophylactic and therapeutic agents.

The overall goal of this review is to summarize the current body of knowledge about the structure and function of major proteins of Bacillus anthracis and/or similar spore-forming organisms. B. anthracis is a key spore-forming biological threat agent, as well as human and animal Gram-positive bacterial pathogen. The structural information described here is limited to approximately the last 5 years. This information is then related to the role of the selected proteins in pathogenesis and in the possible development of novel vaccine and/or other antimicrobial agents against spore-forming organisms, including anthrax, a disease caused by B. anthracis. Among spore-forming bacteria, Bacillus and Clostridium species are the predominant spore-forming bacilli that cause serious diseases. The biochemical properties and mechanism of catalysis of the novel spore germination protease that degrades small, acid-soluble proteins protecting DNA against damage, a cofactor independent phosphoglycerate mutase, NAD+ synthetase, and the three know B. anthracis toxins, protective antigen, lethal factor, and edema factor are described. The studies described in this work review and unify selected information critical for the prevention of microbial diseases such as anthrax. A strategy for the structure-guided development of new prophylactic and therapeutic agents is discussed.

Adenylyl Cyclases↗

PubMed2004

Over-expression of gastrin-releasing peptide in human esophageal squamous cell carcinomas.

Gastrin-releasing peptide (GRP) is known as an autocrine growth factor for a number of gastrointestinal cancers. There is, however, little information on the expression of GRP in the squamous epithelia and squamous cell carcinoma, particularly in the esophagus. With a differential display approach, up-regulated GRP was observed in human esophageal squamous cell carcinoma (ESCC) samples obtained from a high-risk area for esophageal cancer, Linzhou in northern China. Up-regulation of phosphoglycerate mutase and P311 HUM (3.1) and down-regulation of keratin 13, cystatin B, endoglin and annexin I were observed. Using a reverse transcription-polymerase chain reaction (RT-PCR) method, significant over-expression of GRP was observed in 10 out of 12 ESCC samples (83.3%) and all four ESCC cell lines. With in situ hybridization, GRP mRNA expression was detected in nine out of 21 (42.8%) samples with basal cell hyperplasia (BCH), five out of seven (71.4%) samples with dysplasia (DYS) and 17 out of 24 (70.9%) ESCC samples. In contrast, GRP was expressed only in three out of 16 (18.7%) normal epithelium. Digital image analysis revealed that the mean value of GRP expression index, determined by intensity and area ratio of staining, was 0.19 in normal epithelium, 1.23 in BCH, 2.94 in DYS and 2.38 in ESCC, showing a progressive increase. Studies on ESCC cell lines showed GRP increased cell growth in a dose-dependent pattern in GRP receptor-positive ESCC cells, but not in GRP receptor-negative ESCC cells. GRP (1 mM) also increased cyclooxygenase-2 protein expression by 3.4-fold. This is the first demonstration that GRP is over-expressed in ESCC, and its over-expression may play a role in ESCC development and growth.

Base Sequence↗

PubMed2001

V-shaped structure of glutamyl-tRNA reductase, the first enzyme of tRNA-dependent tetrapyrrole biosynthesis.

Processes vital to life such as respiration and photosynthesis critically depend on the availability of tetrapyrroles including hemes and chlorophylls. tRNA-dependent catalysis generally is associated with protein biosynthesis. An exception is the reduction of glutamyl-tRNA to glutamate-1-semialdehyde by the enzyme glutamyl-tRNA reductase. This reaction is the indispensable initiating step of tetrapyrrole biosynthesis in plants and most prokaryotes. The crystal structure of glutamyl-tRNA reductase from the archaeon Methanopyrus kandleri in complex with the substrate-like inhibitor glutamycin at 1.9 A resolution reveals an extended yet planar V-shaped dimer. The well defined interactions of the inhibitor with the active site support a thioester-mediated reduction process. Modeling the glutamyl-tRNA onto each monomer reveals an extensive protein-tRNA interface. We furthermore propose a model whereby the large void of glutamyl-tRNA reductase is occupied by glutamate-1-semialdehyde-1,2-mutase, the subsequent enzyme of this pathway, allowing for the efficient synthesis of 5-aminolevulinic acid, the common precursor of all tetrapyrroles.

Aldehyde Oxidoreductases↗

PubMed1998

Reversion of the tyrosine ochre strain Escherichia coli WU3610 under starvation conditions depends on a new gene tas.

When 3 x 10(8) bacteria of the Escherichia coli tyrA14(oc) leu308(am) strain WU3610 are plated on glucose salts agar supplemented with leucine only, colonies of slow-growing Tyr+ suppressor mutants begin to appear after about a week and increase in numbers roughly linearly with time thereafter (stationary phase or starvation-associated mutation). From a library constructed from two of these mutants, a clone was obtained that suppressed the tyrosine requirement of WU3610 when present on a multicopy plasmid. The activity was identified to an open reading frame we call tas, the sequence for which has homology with a variety of known genes with aldo-keto reductase activity. The activity of tas complements the prephenate dehydrogenase dysfunction of tyrA14 (the chorismate mutase activity of tyrA possibly being still functional). A strain deleted for tas showed no spontaneous mutation under starvation conditions. Whereas neither tas+ nor tas bacteria showed any increase in viable or total count when plated under conditions of tyrosine starvation at 3 x 10(8) cells per plate, at lower density (approximately 10(7) per plate) tas+ but not tas bacteria showed considerable residual growth. We suggest that the single copy of tas present in WU3610 allows cryptic cell or DNA turnover under conditions of tyrosine starvation and that this is an essential prerequisite for starvation-associated mutation in this system. The target gene for mutation is not tas, although an increase in the expression of this gene, for example, resulting from a suppressor mutation affecting supercoiling, could be responsible for the slow-growing Tyr+ phenotype.

Alcohol Oxidoreductases↗

PubMed2001

Expression and identification of the RfbE protein from Vibrio cholerae O1 and its use for the enzymatic synthesis of GDP-D-perosamine.

The 4-amino-6-deoxy-monosaccharide D-perosamine is an important element in the glycosylation of interesting cell products, such as antibiotics and lipopolysaccharides (LPS) of Gram-positive and Gram-negative bacteria. The biosynthetic pathway of the precursor molecule, GDP-D-perosamine, in Vibrio cholerae O1 starts with an isomerisation of fructose-6-phosphate catalyzed by the bifunctional enzyme phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase (RfbA; E.C. 2.7.7.22) creating the intermediate mannose-6-phosphate, which is subsequently converted by the phosphomanno-mutase (RfbB; E.C. 5.4.2.8) and further by RfbA to GDP-D-mannose, to GDP-4 keto-6-deoxymannose by a 4,6-dehydratase (RfbD; E.C. 4.2.1.47) and finally to GDP-D-perosamine by an aminotransferase (RfbE; E.C. not yet classified). We cloned the rfbD and the rfbE genes of V. cholerae O1 in Escherichia coli expression vectors. Both biosynthetic enzymes were overproduced in E. coli BL21 (DE3) and their activities were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-D-perosamine was optimized and the final product, GDP-D-perosamine, was purified and identified by nuclear magnetic resonance, mass spectrometry, and chromatography. The catalytically active form of the GDP-4-keto-6-deoxy-D-mannose-4-aminotransferase seems to be a tetramer of 170 kDa. The His-tag RfbE fusion protein has a Km of 0.06 mM and a Vmax value of 38 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose. The Km and Vmax values for the cosubstrate L-glutamate were 0.1 mM and 42 nkat/mg protein, respectively. The intention of this work is to establish a basis for both the in vitro production of GDP-D-perosamine and for an in vivo perosaminylation system in a suitable bacterial host, preferably E. coli.

Amino Acid Sequence↗

PubMed2000

Tn551-mediated insertional inactivation of the fmtB gene encoding a cell wall-associated protein abolishes methicillin resistance in Staphylococcus aureus.

A Tn551 insert in a gene termed fmtB was shown to reduce oxacillin as well as Triton X-100 resistance in highly methicillin-resistant Staphylococcus aureus (MRSA) COL. Backcrosses of fmtB::Tn551 into S. aureus COL and into two genetically distinct MRSA strains, KSA8 and NCTC10443, confirmed the linkage of fmtB::Tn551 with loss of oxacillin resistance. The fmtB gene codes for a protein of a deduced molecular mass of 263 kDa that contains 17 tandem repeats of 75 amino acids and a C-terminal LPXTG cell wall-sorting motif. Immunoblots with anti-FmtB antibodies confirmed its localization in the cell wall fraction. The fmtB gene was mapped downstream of the phosphoglucosamine mutase operon glmM which catalyses formation of glucosamine-1-phosphate. Oxacillin resistance was not restored in fmtB mutants by trans-complementation with fmtB. However, although GlmM production was not affected by fmtB inactivation, oxacillin resistance was increased in fmtB mutants by introducing a plasmid-borne glmM gene, presumably by GlmM overexpression. Interestingly, a similar phenotypic complementation was obtained in fmtB mutants by including substrate level concentrations of N-acetylglucosamine or glucosamine in the growth medium. Inactivation of the fmtB gene seems therefore to have an indirect effect on methicillin resistance which can be relieved by increasing the production of the cell wall precursor glucosamine-1-phosphate.

Bacterial Proteins↗

PubMed1989

Effect of carnitine on propionate metabolism in the vitamin B-12--deficient rat.

Acyl-CoA thioesters are generated during the oxidation of organic acids in mammalian systems. Vitamin B-12 deficiency is associated with decreased L-methylmalonyl-CoA mutase activity, and consequent accumulation of propionyl-CoA and methylmalonyl-CoA. The formation of propionylcarnitine from propionyl-CoA and carnitine provides an alternative pathway to remove propionyl-CoA from cells. Hepatocytes isolated from vitamin B-12--deficient rats metabolized propionate (1 mM) to CO2 and glucose at only 23% and 12%, respectively, of the rates observed in hepatocytes from control animals. In contrast, no difference was seen in rates of pyruvate metabolism by hepatocytes from control and vitamin B-12--deficient rats. Addition of carnitine (10 mM) to hepatocyte incubations increased the rate of propionylcarnitine formation 10- to 20-fold without altering conversion of propionate to CO2 or glucose. The rate of propionylcarnitine formation was not affected by vitamin B-12 deficiency. When carnitine (10 mM) was added, propionylcarnitine generation represented 65-71% of total propionate utilization in hepatocytes isolated from vitamin B-12--deficient rats. Gluconeogenesis from [1-14C]pyruvate was inhibited by 1 mM propionate in hepatocytes from vitamin B-12--deficient rats. No effect of 1 mM propionate on glucose formation from pyruvate was seen using hepatocytes from control rats. Intraperitoneal administration of L-carnitine resulted in a significant increase in urinary propionylcarnitine excretion from vitamin B-12--deficient rats, but not from control animals. The results demonstrate that exogenous carnitine can significantly enhance propionyl-group utilization via the formation of acylcarnitines under the conditions of impaired acyl-CoA metabolism associated with vitamin B-12 deficiency.

Animals↗

PubMed1990

Coenzyme A metabolism in vitamin B-12-deficient rats.

Vitamin B-12 (cobalamin) deficiency results in decreased L-methylmalonyl-coenzyme A (CoA) mutase activity. The consequence of this defect on the cellular CoA pool was studied in rats with functional vitamin B-12 deficiency induced by administration of the cobalamin analogue hydroxy-cobalamin [c-lactam] or by dietary vitamin B-12 deficiency. Both types of vitamin B-12 deficiency were associated with methylmalonic acidemia (100-300-fold increases in plasma methylmalonic acid concentration compared with controls), but overall fuel homeostasis was intact. Liver from rats treated with hydroxy-cobalamin [c-lactam] contained a threefold greater concentration of total CoA (free CoA plus all acyl-CoA) compared with saline-treated rats. Fractionation of the CoA pool revealed higher levels of CoA, propionyl-CoA, methyl-malonyl-CoA, acid-insoluble CoA, as well as total CoA in the rats treated with hydroxy-cobalamin [c-lactam] compared with controls. Similar increases in liver CoA content were seen in dietary vitamin B-12 deficiency in both the fed and fasted states. To examine the hypothesis that sequestration of hepatic CoA as propionyl-CoA and methylmalonyl-CoA could increase CoA biosynthesis, the effect of propionate on CoA biosynthesis was studied in hepatocytes isolated from control rats. Propionate (1 mM) increased the formation of 14C-CoA from [14C]pantothenate (10 microM) by 27% in the hepatocyte system. When butyrate (1 mM) was provided as substrate, propionate (10 mM) increased [14C]CoA formation by 63%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Rat hepatic coenzyme A is redistributed in response to mitochondrial acyl-coenzyme A accumulation.

Coenzyme A without an acyl-thioester (CoASH) is required for numerous cellular reactions, and sequestration of CoASH as acyl-CoAs may impair metabolic function. Increased total CoA protects the cell from acyl-CoA accumulation, and enhanced CoA biosynthesis may represent a compensatory response in metabolic disease. To test the hypothesis that cellular CoA is redistributed from the cytosol to the mitochondria in response to mitochondrial acyl-CoA accretion, the subcellular distribution of hepatic CoA was determined by differential centrifugation and measurement of the mitochondrial marker enzyme citrate synthase. Liver from control, clofibrate-treated and hydroxycobalamin[c-lactam] (HCCL)-treated rats were used. Clofibrate increased total hepatic CoA concentration 2.2-fold, whereas HCCL (which causes inhibition of L-methylmalonyl-CoA mutase and consequent propionyl- and methylmalonyl-CoA accumulation) increased it threefold. However, clofibrate did not affect the percentage of total CoA in the mitochondria (control: 44 +/- 3%, clofibrate: 49 +/- 5%), and HCCL-treatment induced a marked redistribution of CoA into the mitochondria (HCCL: 78 +/- 8%). Redistribution of total CoA was also induced acutely by incubation of hepatocytes from control rats with 10 mmol/L propionate. Thus, redistribution of the cellular CoA pool can help maintain CoASH availability as mitochondrial acyl-CoA accumulation occurs and may be an important compensatory response to metabolic injury.

Acyl Coenzyme A↗

PubMed1993

Hydroxycobalamin[c-lactam] increases total coenzyme A content in primary culture hepatocytes by accelerating coenzyme A biosynthesis secondary to Acyl-CoA accumulation.

Hydroxycobalamin[c-lactam] (HCCL) treatment in rats results in decreased hepatic L-methylmalonyl-CoA mutase activity and increased hepatic total CoA content. To test the hypothesis that HCCL increases hepatic CoA biosynthesis secondary to propionyl- and methylmalonyl-CoA accumulation. CoA homeostasis was studied in primary culture rat hepatocytes. Conversion of [14C]pantothenic acid to [14C]CoA in the primary culture system was accelerated to rates 3-5 times control by acute incubation with the acyl-CoA-generating carboxylic acids pivalate (10 mmol/L) or propionate (10 mmol/L). HCCL (1 mg/L included from 24 to 72 h of culture) had no affect on the distribution of the hepatocyte CoA pool or total CoA content. However, culture in the presence of HCCL and propionate (2 mmol/L) resulted in accumulation of methylmalonyl-CoA and a 59% increase in total CoA content. The combination of HCCL (1 mg/L) and propionate (0.5 mmol/L), but not HCCL alone, increased the rate of [14C]pantothenic acid conversion to [14C]CoA by 150%. Degradation of [14C]CoA in the primary culture hepatocyte system was unaffected by chronic exposure to 2 mmol/L propionate, but was increased 90% by the combination of HCCL and 2 mmol/L propionate. Thus, in the presence of a source of propionyl-CoA, HCCL treatment results in methylmalonyl-CoA accumulation and accelerated CoA biosynthesis. The increased CoA biosynthesis leads to increased hepatocyte total CoA content, which may contribute to cellular metabolic homeostasis under conditions of acyl-CoA accretion.

Acyl Coenzyme A↗

PubMed1996

The use of homocysteine and other metabolites in the specific diagnosis of vitamin B-12 deficiency.

Vitamin B-12 (cobalamin) is a cofactor for only two enzymes, methionine synthase and L-methylmalonyl-CoA mutase. The serum vitamin B-12 concentration has been shown to have limitations in specificity and sensitivity in diagnosing vitamin B-12 deficiency and predicting response to therapy in subjects with clinical deficiency syndromes. Serum methylmalonic acid and/or total homocysteine concentrations have been shown to be elevated in almost every patient who has a clinical response to vitamin B-12. In elderly populations serum methylmalonic acid concentrations are elevated in the majority (60-66%) of subjects who have elevated total homocysteine concentrations, suggesting that vitamin B-12 deficiency (with or without associated folate deficiency) and/or chronic renal insufficiency may be the primary cause of most of the elevated total homocysteine concentrations in elderly populations. In such subjects vitamin B-12 and folate concentrations are both frequently in the low or low normal range, making differentiation of the clinical syndromes by use of serum vitamin concentrations problematic. Elevations of 2-methylcitric acid and cystathionine also result from vitamin B-12 deficiency. Serum N-methylglycine concentrations are normal in cobalamin deficiency but are increased in 40% of patients deficient in folate. In conclusion, elevations of methylmalonic acid and total homocysteine are very sensitive and specific in diagnosing vitamin B-12 deficiency and can be used to help differentiate vitamin B-12 deficiency from folate deficiency. Elevated total homocysteine concentrations that may have been attributed to folate deficiency in elderly subjects may in many instances be the result of vitamin B-12 deficiency even though serum vitamin B-12 concentrations are within normal limits.

Adolescent↗

PubMed2004

Determining protein identity from sieve element sap in Ricinus communis L. by quadrupole time of flight (Q-TOF) mass spectrometry.

The phloem transport system is a complex tissue that primarily carries photoassimilate from source to sink. Its function depends on anucleate sieve elements (SE) supported by companion cells (CC). In this study, SE sap was sampled and the protein identity of soluble proteins was determined with the aim of understanding the function of proteins within the conduit. Unlike many plants, SE sap exudes from incisions in the bark of Ricinus communis and, although there is a greater possibility of contamination from tissues other than SE, sap can be obtained in sufficient quantities to separate proteins using 2D electrophoresis. Spots were excised for trypsin digest, then analysed by quadrupole time of flight (Q-TOF) mass spectrometry (MS) and database searched to determine sequence identity. Overall, 18 proteins were identified in the SE-enriched sap. Proteins identified that have not previously been identified directly from SE sap included a glycine-rich RNA-binding protein, metallothionein, phosphoglycerate mutase, and phosphopyruvate hydratase. The potential role of the identified protein in SE function is discussed. The protein identification in this study provides a first step towards the goal of a greater understanding of the function of proteins within the SE.

Amino Acid Sequence↗