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PubMed2003

Structure and Function of sanV: a gene involved in nikkomycin biosynthesis of Streptomyces ansochromogenes.

A 6.3-kb BamHI- BglII DNA fragment was cloned from cos20 by using chromosome walking strategy. It was partially sequenced with the result that there is a possible ORF of 1272 nucleotides. The ORF designated sanV was deposited in GenBank under accession no. AF469955. Database search indicated that the deduced protein of sanV shows 28% identity and 44% similarity over 405 amino acid residues to the large component (E) of glutamate mutases from Clostridium cochlearium. Gene disruption was performed to study the function of sanV. It was found that sanV disruptants exhibited much poorer inhibition to the test strain than the wild-type S. ansochromogenes 7100. Furthermore, HPLC analysis indicated that the sanV disruptants almost did not produce nikkomycins X and Z, whereas they accumulated new nikkomycins O(x) and O(z), which revealed that sanV is an important gene involved in the biosynthesis of the peptidyl moiety of nikkomycins.

Amino Acid Sequence↗

PubMed2003

Nuclear import of yeast Gcn4p requires karyopherins Srp1p and Kap95p.

The yeast transcription factor Gcn4p contains two stretches of amino acid residues, NLS1 and NLS2, which are independently able to relocate the cytoplasmic protein chorismate mutase into the nucleus. Only NLS2 is conserved among fungi. A truncated version of CPCA (the counterpart of Gcn4p in Aspergillus nidulans), which lacks the conserved NLS, accumulates in the cytoplasm instead of the nucleus. Nuclear uptake mediated by the NLS1 of Gcn4p is impaired by defects in genes for several different karyopherins, whereas NLS2-dependent nuclear import specifically requires the alpha-importin Srp1p and the beta-importin Kap95p. Yeast strains that are defective in either of these two karyopherins are unable to respond to amino acid starvation. We have thus identified Gcn4p as a substrate for the Srp1p/Kap95p transport complex. Our data suggest that NLS2 is the essential and specific nuclear transport signal; NLS1 may play only an unspecific or accessory role.

Active Transport, Cell Nucleus↗

PubMed2005

A genetic linkage map of the soybean cyst nematode Heterodera glycines.

A genetic linkage map of the soybean cyst nematode (SCN) Heterodera glycines was constructed using a population of F2 individuals obtained from matings between two highly inbred SCN lines, TN16 and TN20. The AFLP fingerprinting technique was used to genotype 63 F2 progeny with two restriction enzyme combinations (EcoRI/MseI and PstI/TaqI) and 38 primer combinations. The same F2 population was also genotyped for Hg-cm-1 (H. glycines chorismate mutase-1), a putative virulence gene, using real-time quantitative PCR. Some of the markers were found to be distributed non-randomly. Even so, of the 230 markers analyzed, 131 could be mapped onto ten linkage groups at a minimum LOD of 3.0, for a total map distance of 539 cM. The Hg-cm-1 locus mapped to linkage group III together with 16 other markers. The size of the H. glycines genome was estimated to be in the range of 630-743 cM, indicating that the current map represents 73-86% of the genome, with a marker density of one per 4.5 cM, and a physical/genetic distance ratio of between 124 kb/cM and 147 kb/cM. This genetic map will be of great assistance in mapping H. glycines markers to genes of interest, such as nematode virulence genes and genes that control aspects of nematode parasitism.

Animals↗

PubMed2001

Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety and assembly of the peptidyl nucleoside antibiotic nikkomycin.

Six genes (nikP1, nikP2, nikS, nikT, nikU, and nikV) from Streptomyces tendae Tu901 were identified by analysis of the nucleotide sequence of the nikkomycin gene cluster. These genes, together with the previously described nikQ and nikR, span 9.39 kb and are transcribed as a polycistronic mRNA in a growth-phase-dependent manner. The nikP1 gene encodes a non-ribosomal peptide synthase consisting of an adenylation domain, a thiolation domain, and an N-terminal 70-residue segment of unknown function. The amino acid sequence encoded by the nikP2 gene displays similarity to the sequences of thioesterases, and the nikS product belongs to a superfamily of proteins characterized by a specific ATP-binding fold. The N-terminal 70 amino acids of the predicted nikT gene product show significant sequence similarity to acyl carrier proteins, and the C-terminal 330 amino acids to aminotransferases. The sequences of the deduced proteins NikU and NikV exhibit similarity to components S and E, respectively, of glutamate mutase from Clostridium. Disruption of the nikP1, nikS, nikT, or nikV gene by insertion of a kanamycin resistance cassette abolished formation of nikkomycins I, J, X, and Z, all of which contain hydroxypyridylhomothreonine as the peptidyl moiety. The nikP1 mutants, and the nikS and nikT mutants accumulated the nucleoside moieties nikkomycin Cz, and nikkomycins Cx and Cz, respectively. The nikV mutants formed nikkomycins Ox and Oz, which contain 2-amino-4-hydroxy-4-(3'-hydroxy-6'-pyridyl) butanoic acid as the peptidyl moiety. The nikP2 mutants synthesized nikkomycins I, J, X, and Z, but amounts of nikkomycins I and X, which contain formylimidazolone as the base, were lower. Feeding formylimidazolone to nikP2 mutants restored the ability to form nikkomycins I and X. Our results indicate that nikU and nikV are required for the synthesis of hydroxypyridylhomothreonine, the genes nikP1, nikP2 and nikS are required for the assembly of nikkomycins, and nikT is required for both pathways. The putative activities of each of their products are discussed.

Amino Acid Sequence↗

PubMed2005

Management of methylmalonic acidaemia by combined liver-kidney transplantation.

Methylmalonic acidaemia (MMA) is a rare autosomal recessive inborn error of metabolism that typically presents in infancy with recurrent episodes of metabolic acidosis, developmental delay and failure to thrive. The disease course is complicated by the development of chronic tubulointerstitial nephritis progressing to end-stage renal disease in adolescence. We describe two adolescents with cobalamin-nonresponsive MMA (mut0) who developed polyuria, chronic tubulointerstitial nephritis, dystonia but normal synthetic liver function. Both patients received combined liver-kidney transplantation (CLKT), preceded by a single pretransplant haemodialysis for clearance of methylmalonic acid. Post CLKT there was 95-97% reduction in serum and urine methylmalonic acid, leading to significant liberalization of dietary protein intake and a consequent increase in body mass index, muscle strength and energy. In addition, renal function normalized and clinical neurological status stabilized. We propose that CLKT be considered as a therapeutic option early in the course of cobalamin-nonresponsive MMA. Progressive tubulointerstitial nephritis with disabling polyuria is a confounder in patient management even in the absence of end-stage renal disease. Successful CLKT restores methylmalonyl-CoA mutase enzyme levels in the liver and kidney, improves clearance of methylmalonic acid with resultant dietary protein liberalization, and offers excellent graft and patient outcomes with improvement in quality of life.

Adolescent↗

PubMed1978

The enzymic synthesis of beta-[32P]UDP-N-acetylglucosamine.

Cells of Micrococcus sp. 2102 incorporate inorganic [32P]phosphate from the medium into the sugar-phosphate polymer of the wall. Controlled acid hydrolysis of sodium dodecyl sulphate-extracted cells gives N-acetylglucosamine 6-[32P]-phosphate which can be purified by ion-exchange chromatography and incubated with UTP in the presence of crude preparations of phosphoacetylglucosamine mutase from Neurospora crassa and UTP:N-acetylglucosamine 1-phosphate phosphotransferase from Bacillus licheniformis which act in concert to synthesise beta-[32P]UDP-N-acetylglucosamine.

Acetylglucosamine↗

PubMed1992

Recombination and amplification of multiple portions of genomic DNA by a modified polymerase chain reaction.

A novel method, a modified polymerase chain reaction (mPCR), is reported for in vitro recombination and amplification of two or more regions of DNA located in distinct parts of a genome such as exons of a gene. The reaction consists of three stages. In the first stage, the portions are amplified individually with the aid of four or more primers. Of the primers, two (outside primers) are complementary to 3'-terminal parts of the objective, recombinant DNA, and the others (inside primers) are complementary to 3'-terminal parts of the other portions. Some of the inside primers have extra nucleotide sequences at their 5'-termini which are complementary to 5'-terminal parts of the portions to be recombined. In the second stage, the strands with complementary sequences in their 3'-terminal parts are annealed sequentially to one another and are converted to recombinant DNA with DNA polymerase. In the final stage, the objective DNA is amplified with the excess outside primers. The application of this method for in vitro recombination and amplification of three exons of the gene for human muscle-specific phosphoglycerate mutase is described.

Amino Acid Sequence↗

PubMed1988

Effect of vanadate on the glucose-1,6-bisphosphate synthase and glucose-1,6-bisphosphatase activities of phosphoglucomutase.

Phosphoglucomutase, in addition to catalyzing the interconversion of glucose 1-P and glucose 6-P, catalyzes both the synthesis of glucose 1,6-P2 from glucose monophosphate and either fructose 1,6-P2 or glycerate 1,3-P2, and the hydrolysis of glucose 1,6-P2. Vanadate inhibits the mutase activity, activates the synthase activities, and does not affect the phosphatase activity. These effects suggest that the "exchange" step postulated for the phosphoglucomutase pathway is specifically inhibited by vanadate.

Animals↗

PubMed1985

A futile cycle in erythrocyte glycolysis.

The storage lesion which limits the shelf life of human blood in blood banking is associated with a metabolic loss of 2,3-diphosphoglycerate and ATP. This metabolic loss is driven by intracellular ATPase which are usually considered to include the ion pumps and the reactions which maintain the discoid shape of the human erythrocyte. Under the acidic conditions of blood storage, the energy-yielding reactions of the glycolytic pathway are restricted at the hexokinase and phosphofructokinase steps. We show here that under such circumstances the enzyme of the diphosphoglycerate shunt, diphosphoglycerate mutase/phosphatase and the glycolytic enzyme phosphoglycerate kinase can form a futile cycle with ATPase activity. This ATPase activity responds to 2-phosphoglycolate which is known to activate both diphosphoglycerate mutase and diphosphoglycerate phosphatase reactions. When the enzymes of the futile cycle are combined with the enzymes of the lower glycolytic pathway in a reconstitution experiment designed to represent conditions within the stored erythrocyte, the futile cycle does provide an ATPase activity which results in the metabolic loss of 2,3-diphosphoglycerate. An isotope incorporation experiment demonstrates that the futile cycle is active in glucose-depleted erythrocytes.

2,3-Diphosphoglycerate↗

PubMed1987

Determination of erythrocyte glucose 1,6-bisphosphate--a comparison of two methods using a centrifugal analyzer.

Glucose 1,6-bisphosphate is a key effector of human erythrocyte glycolysis, yet to date its assay has been problematical. Two methods for measuring glucose 1,6-bisphosphate were modified and adapted to a centrifugal analyser and the inaccuracy and imprecision of each method were compared. One assay, based on stimulation of phosphoglucomutase, was shown to underestimate the erythrocyte levels by approximately 5% due to inhibition of the mutase by endogenous 2,3-bisphosphoglycerate. An alternative chemical/enzymic method, consisting of acid hydrolysis of glucose 1,6-bisphosphate to glucose 6-phosphate and subsequent determination of the monophosphate was modified by omitting an initial alkaline hydrolysis step and by increasing the duration of acid hydrolysis. The modified method also enabled the determination of erythrocyte glucose 6-phosphate. The normal concentration of glucose 1,6-bisphosphate in whole blood and in washed human erythrocytes, determined using the more accurate chemical/enzymic method was 83 +/- 5 mumol/l cells and 86 +/- 4 mumol/l cells, respectively; the corresponding concentrations of glucose 6-phosphate were 26 +/- 2 mumol/l cells and 15 +/- 3 mumol/l cells.

Centrifugation↗

PubMed1991

Lysosomal cobalamin accumulation in fibroblasts from a patient with an inborn error of cobalamin metabolism (cblF complementation group): visualization by electron microscope radioautography.

Cobalamin (Cbl, vitamin B12) bound to transcobalamin II (TCII) enters cultured fibroblasts by receptor-mediated endocytosis. Following degradation of the TCII, Cbl is subsequently found in either the cytoplasm bound to methionine synthase or in the mitochondria bound to methylmalonyl CoA mutase. In fibroblasts from patients belonging to the cblF complementation group, Cbl is found free in the cell and is not transferred to the above two target enzymes. Quantitative EM radioautography was utilized to visualize intracellular Cbl in fibroblasts from cblF patients and from normal subjects. In cblF cells, 60% of all silver grains were assigned to lysosomes, with only 12.6% over cytoplasm and 1.2% over mitochondria. In contrast, in control cells, only 4.7% were assigned to lysosomes, with 47% to cytoplasm and 23.4% to mitochondria. Subcellular fractionation showed that in cblF cells, the majority of label was associated with clearly recognizable lysosomes. These studies conclusively demonstrate that secondary lysosomes accumulate Cbl in cblF disease.

Autoradiography↗

PubMed1985

Leiomyosarcoma of the vagina.

A 46-year-old woman, who had very interesting tumor of the vagina microscopically, was presented. The removed tumor (40 g) in the upper lateral wall of the vagina was composed of two nodules. The cut surface of one nodule (20 g) was separated into two parts and they were diagnosed as necrosis and borderline case of leiomyomata microscopically. The other nodule (20 g) was diagnosed as leiomyosarcoma with interlacing bundles of spindle-shaped cells with large hyperchromatic, irregularly shaped nuclei and abundant mutases. These bindings suggest a possibility that leiomyoma of the vagina may undergo malignant change to become leiomyosarcoma.

Female↗

PubMed1987

Accumulation of CK-MM is impaired in innervated and contracting cultured muscle fibers of Duchenne muscular dystrophy patients.

No specific abnormalities have been reproducibly manifested in aneurally cultured muscle of Duchenne muscular dystrophy (DMD) patients. We now report that the accumulation of the muscle-"specific" isozyme of creatine kinase (CK-MM) was significantly and preferentially impaired in long-term innervated contracting muscle fibers cultured from 4 DMD patients (DMD-InnCMFs) compared to: i) their noninnervated sister-cultured muscle fibers, and ii) innervated contracting control cultured human muscle fibers (Control-InnCHMFs). Accumulation of other muscle-"specific" isozymes (MSIs), viz. glycogen phosphorylase, phosphoglycerate mutase, and lactic dehydrogenase, was not significantly impaired. We have not observed preferentially-impaired CK-MM accumulation in any Control-InnCHMFs from 22 patients (children and adults) with a variety of neuromuscular diseases. There was no apparent difference between DMD-InnCMFs and Control InnCHMFs regarding: acceptance of innervation; neuronally-driven, virtually continuous muscle-fiber contractions; characteristic myofiber organization by phase-contrast microscopy, and increased longevity of the innervated fibers.

Animals↗

PubMed1981

Uptake and metabolism of free cyanocobalamin by cultured human fibroblasts from controls and a patient with transcobalamin II deficiency.

We have investigated the uptake and metabolism of free cyanocobalamin (CN-Cbl; vitamin B12) by intact cultured human skin fibroblasts. Monolayers of control fibroblasts take up free CN-[57Co]Cbl via a saturable, calcium-independent process that is inhibited by sulfhydryl reagents, inhibitors of protein synthesis, and inhibitors of electron transport, but not by inhibitors of glycolysis. CN-Cbl taken up in this manner is converted to active cobalamin (Cbl) coenzymes (adenosylcobalamin and methylcobalamin) and becomes associated with intracellular Cbl-dependent apoenzymes (methylmalonyl CoA mutase and homocysteine:methyltetrahydrofolate methyltransferase). Since fibroblasts from controls were also found to synthesize transcobalamin II (TC II), a plasma protein shown previously to facilitate the cellular uptake of Cbl, it seemed possible that the observed uptake of free CN-Cbl was TC II-mediated. This thesis was rejected by demonstrating that cells from a patient with complete TC II deficiency took up free CN-Cbl as well as control cells did. Finally, we propose a mechanism by which an uptake process for free Cbl might serve a function in intracellular metabolism of Cbl.

Blood Proteins↗

PubMed1992

Galactosamine-synthesizing enzymes are induced when Giardia encyst.

Galactosamine, a Giardia filamentous cyst wall specific-sugar, is below the limits of detection in non-encysting trophozoites. Radiolabeling studies suggest that Giardia synthesize galactosamine primarily from endogenous glucose rather than salvage it from the environment. Enzymes responsible for galactosamine synthesis from glucose are induced during encystment and have been characterized in crude homogenates and in supernatant (soluble) fractions. These enzymes (specific activity; time after encystment is induced for maximal activity; x-fold increase) include glucosamine 6-phosphate isomerase (in the deaminating direction, 167 mU mg protein-1; 20 h; x 182-fold; in the aminating direction, 258 mU mg protein-1; 20 h; x 13-fold), glucosamine 6-phosphate N-acetylase (11 mU mg protein-1; 20 h; x 20-fold), phosphoacetylglucosamine mutase (160 mU mg protein-1; 20 h; x 12-fold), UDP-N-acetylglucosamine pyrophosphorylase (22 mU mg protein-1; 48 h; x 8-fold), and UDP-N-acetylglucosamine 4'-epimerase (13 mU mg protein-1; 48 h; x 4000-fold). This represents the first report of these enzymes and of an inducible carbohydrate-synthesizing pathway in any protozoan.

Animals↗

PubMed1986

Kinetic properties and essential amino acids of the 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle.

Histidine, arginine and lysine residues are essential for the multifunctional 2,3-bisphosphoglycerate synthase-phosphatase purified from pig skeletal muscle. The synthase, phosphatase and phosphoglycerate mutase activities of the enzyme are concurrently lost upon treatment with diethylpyrocarbonate, phenylglyoxal and trinitrobenzenesulfonate. The phosphatase activity shows hyperbolic kinetics. In contrast, the synthase activity shows a nonhyperbolic pattern which fits to a second-degree polynomial. The Km values for glycerate 1,3-P2, glycerate 3-P and glycerate 2,3-P2 are similar to those of the enzyme from mammalian erythrocytes.

Amino Acids↗

PubMed1988

Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol).

Interactions of glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1), phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.3), enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11), pyruvate kinase (ATP:Pyruvate O2-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase [S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with F-actin, among the glycolytic enzymes listed above, and with phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) were studied in the presence of poly(ethylene glycol). Both purified rabbit muscle enzymes and rabbit muscle myogen, a high-speed supernatant fraction containing the glycolytic enzymes, were used to study enzyme-F-actin interactions. Following ultracentrifugation, F-actin and poly(ethylene glycol) tended to increase and KCl to decrease the pelleting of enzymes. In general, the greater part of the pelleting occurred in the presence of both F-actin and poly(ethylene glycol) and the absence of KCl. Enzymes that pelleted more in myogen preparations than as individual purified enzymes in the presence of poly(ethylene glycol) and the absence of F-actin were tested for specific enzyme-enzyme associations, several of which were observed. Such interactions support the view that the internal cell structure is composed of proteins that interact with one another to form the microtrabecular lattice.

Actins↗

PubMedDate not supplied

Human erythrocyte phosphoglucomutase: comparison of the kinetic properties of PGM1 and PGM2 isoenzymes.

Kinetic properties of PGM1 and PGM2 phosphoglucomutase "primary" isoenzymes from human erythrocytes were studied. The two enzyme forms share a "ping-pong" kinetic mechanism and show similar Km for substrate (glucose 1-P) and cofactor (glucose 1,6-P2). Micromolar concentrations of fructose 1,6-P2 and glycerate 2,3-P2 inhibit both PGM1 and PGM2 isoenzymes to a similar extent. The sole PGM2 form is affected by ribose monophosphates (ribose 1-P and ribose 5-P) that act as mutase inhibitors vs. glucose 1,6-P2 and as apparent activators vs. glucose 1-P. The interaction between PGM2 isoenzyme and ribose monophosphates is discussed in the light of the ability of this form to also display phosphoribomutase activity.

2,3-Diphosphoglycerate↗