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PubMed1988

Ontogeny and estrogen responsiveness of creatine kinase and glycolytic enzymes in brain and uterus of rat.

We have studied the postnatal ontogeny of creatine kinase (CK) and the glycolytic enzymes phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM), enolase (En), and pyruvate kinase (PK) in rat brain and uterus. In 30-day-old rat, brain and uterus express the fetal isoforms CK-B, PGK-A, PGM-B, En-alpha and PK-M2, and the differentiated isoforms En-gamma and PK-M1. The activity of glycolytic enzymes in uterus of two-day-old rat is as in brain, while CK activity is 3 times higher in brain. The activity of the glycolytic enzymes in brain began to increase (3-4-fold) 10 days after birth, in a coordinated manner. CK activity began to increase 5 days after birth in both brain (4.2-fold) and uterus (4.5-fold), suggesting the dissociation of glycolytic enzyme ontogeny from CK. In contrast to brain, the levels of glycolytic enzymes in uterus were highest at birth, suggesting the action of a tissue-specific mechanism for regulation of the constitutive levels of glycolytic isozymes. Except for PGM, all enzymes showed an increase in total activity, in response to estrogen, in uterus but not in whole brain.

Animals↗

PubMed1975

On the association of glycolytic enzymes with structural proteins of skeletal muscle.

1. The effects of protein concentration and ionic strength on the adsorption of the individual glycolytic enzymes to F-actin and F-actin--trypomyosin--troponin have been studied. 2. Appreciable association was demonstrated under conditions of physiological ionic strength and high protein concentration, and tropomyosin--troponin established as an important and generalized component of these interactions. 3. Phosphofructokinase, aldolase, pyruvate kinase, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate isomerase were strongly bound under these conditions, while triosephosphate isomerase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and hexokinase displayed less adsorption to the structural proteins. 4. The influence of a number of parameters on the adsorption phenomena was examined. Ca2+ and fructose 1,6-diphosphate increased the adsorption of aldolase, lactate dehydrogenase and pyruvate kinase, while decreasing the adsorption of the enzymes of the constant-proportion group. 5. Of the other major enzymic components of skeletal muscle, creatine kinase, adenylate kinase and malate dehydrogenase showed no adsorption to F-actin--tropomyosin--troponin under the experimental conditions. Some adsorption was evident, however, in the case of aspartate aminotransferase, (NADP) isocitrate dehydrogenase and alpha-glycerolphosphate dehydrogenase. 6. These results have been discussed in relation to their functional significance and the roles of enzyme compartmentation in the cell.

Animals↗

PubMed1993

Identification and characterization of two enzymes involved in the intracellular metabolism of cobalamin. Cyanocobalamin beta-ligand transferase and microsomal cob(III)alamin reductase.

Two enzymes involved in the intracellular metabolism of cobalamin have been identified and characterized: cyanocobalamin beta-ligand transferase and microsomal cob(III)alamin reductase. The beta-ligand transferase is a cytosolic enzyme utilizing FAD, NADPH and reduced glutathione. The product of the reaction has been identified as glutathionyl-cobalamin. NADH-linked cob(III)alamin reductase has been found in two subcellular fractions: microsomal and inner mitochondrial membrane. The product of the reduction catalyzed by the microsomal enzyme has been identified as cob(II)alamin. In cbl C mutant fibroblasts, the specific activities of cyanocobalamin beta-ligand transferase and cob(III)alamin reductase were markedly decreased and have varied from 3%-30% and 36%-42% of normal, respectively. The specific activity of mitochondrial cob(III)alamin reductase was only 30% of normal in two cbl C mutants and normal in remaining mutant cell lines. In the cbl D cells, the specific activities were 33% and 55%. Mitochondrial cob(III)alamin reductase was not affected by cbl D mutation. Methionine synthase, L-methylmalonyl-CoA mutase and microsomal cytochrome c and b5 reductases are not affected by both mutations. The cbl E mutation affects only the activity of methionine synthase. These results support the hypothesis that the early enzymatic steps of intracellular metabolism of cobalamin are similar in the synthesis of both methylcobalamin and adenosylcobalamin and these steps are altered by the cbl C and cbl D mutations.

Animals↗

PubMed1984

Glycolytic enzymes of fowl and turkey spermatozoa.

It was confirmed that, under anaerobic conditions, fowl spermatozoa formed lactate from glucose thirteen times faster than turkey spermatozoa. The profiles of glycolytic enzyme activities were similar for spermatozoa from both species; however fowl spermatozoal activities were generally 2- to 4-fold higher. Exceptions were glycerophosphate mutase and lactate dehydrogenase activities which were respectively 9.5 and 41 times greater in fowl spermatozoa. In both species, spermatozoal glyceraldehyde-3-phosphate dehydrogenase had the lowest activity of the glycolytic enzymes.

Anaerobiosis↗

PubMed1989

Comparative aspects of propionate metabolism.

1. The catabolism of propionate has been studied extensively in vertebrates and the major pathway has been shown to be its derivatization to propionyl-CoA, carboxylation to D-methylmalonyl-CoA, isomerization to L-methylmalonyl-CoA and then conversion to succinyl-CoA via a vitamin B12 dependent methylmalonyl-CoA mutase. 2. By contrast, in all insect species studied to date, many of which do not contain detectable levels of vitamin B12, the major metabolic pathway of propionate is its conversion to 3-hydroxypropionate and then to acetate. Carbon-3 of propionate becomes the carboxyl carbon of acetate and carbon-2 of propionate becomes the methyl carbon of acetate. 3. A number of species of non-insect arthropods and other invertebrates contain relatively high levels of vitamin B12 and catabolize propionate by the same pathway as that of vertebrates. Under anoxic conditions, some invertebrates, including bivalves, convert succinate to propionate. 4. In plants, evidence has been presented for the metabolism of propionate to both acetate and succinate. Micro-organisms possess a myriad of pathways by which they produce and catabolize propionate.

Animals↗

PubMed1995

A putative pathway for perosamine biosynthesis is the first function encoded within the rfb region of Vibrio cholerae O1.

The first four genes (rfbA,B,D,E) of the rfb region of Vibrio cholerae O1 are predicted to encode the enzymes required for the biosynthesis of perosamine, which constitutes the backbone structure of the O-antigen of the lipopolysaccharide. Based on homology to known proteins/protein families, the following functions are predicted: RfbA, phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase; RfbB, phosphomanno-mutase; RfbD, oxido reductase and RfbE, perosamine synthetase (amino-transferase). Thus, perosamine is synthesized from fructose 6-phosphate via the intermediates mannose 6-phosphate by RfbA, to mannose 1-phosphate by RfbB, to GDP-mannose by RfbA, to GDP-4-keto-6-dideoxymannose by RfbD and to GDP-perosamine by RfbE. This final product would then serve as the substrate for the addition of the tetronate, which could then be polymerized into the O-antigen for transfer to the lipid A plus core oligosaccharide and export to the cell surface. The organization of these genes are such that one would expect them to be translationally coupled as part of the rfb operon. However, the absence of readily detectable promoter sequences suggests low levels of transcription, in line with other studies. The nucleotide sequence of these genes is absolutely conserved in the two isolates 569B (classical, Inaba) and O17 (El Tor, Ogawa) which were expected to show maximal sequence variation. This suggests very tight constraints on the micro-evolution within these sequences.

Amino Acid Sequence↗

PubMed1990

Expression of muscle-specific isozymes of phosphorylase and creatine kinase in human muscle fibers cultured aneurally in serum-free, hormonally/chemically enriched medium.

Primary cultures of muscle cells derived from a biopsied adult human skeletal muscle were grown up to 6 weeks in a hormonally/chemically enriched serum-free medium. The expression of muscle-specific isozymes of creatine kinase, glycogen phosphorylase, and phosphoglycerate mutase, indicative of muscle cell maturation, was studied after 1, 4 and 6 weeks of growth. The maturation of muscle fibers cultured in serum-free medium was comparable to that achieved by muscle fibers cultured in medium containing 10% serum and supplemented with growth factors (insulin, epidermal growth factor, and fibroblastic growth factor) and was greater than that achieved in medium containing 10% serum only. Our study demonstrates that adult human muscle can be cultured aneurally for a long period of time in a serum-free medium, and that it can achieve a high degree of maturation. This study provides an important basis for investigations related to: (1) assessment of the influence of individual components of the medium on human muscle maturation in culture; (2) studies of regulation of abnormal gene expression in diseased human muscle cultured in serum-fre medium.

Adult↗

PubMed1992

Heterogeneity in cblG: differential retention of cobalamin on methionine synthase.

Cultured fibroblasts from patients with functional methionine synthase deficiency have been shown to belong to two complementation classes, cblE and cblG. Both are associated with decreased intracellular levels of methylcobalamin (MeCbl) and decreased incorporation of label from 5-methyltetrahydrofolate into macromolecules. Methionine synthase specific activity is normal or near normal in cell extracts from cblE patients under standard reducing conditions, whereas specific activity is low in cblG extracts. Seven of 10 cblG cell lines accumulated [57Co]CN-Cbl equivalent to control cells and showed similar proportions of label associated with the two intracellular cobalamin binders, methionine synthase and methylmalonyl-CoA mutase. The remaining three cblG lines showed reduced accumulation of labeled Cbl and virtually none associated with methionine synthase. The specific activity of methionine synthase was decreased in cell extracts from both cblG subgroups, being almost undetectable in extracts from the latter three lines. Incorporation of label from [14C]MeTHF into either macromolecules or into methionine was decreased in both cblG groups, but was paradoxically higher in the three lines with very low in vitro methionine synthase activity. These results demonstrate further heterogeneity within cblG and suggest that the defect in the three variant lines affects the ability of methionine synthase to retain Cbl.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2003

Enzymatic synthesis of radiolabeled phosphonoacetaldehyde.

Phosphonoacetaldehyde (Pald) is formed in a variety of biosynthetic pathways leading to natural phosphonates and is an intermediate in the degradation pathway of the natural product 2-aminoethylphosphonate. To facilitate the investigation of the enzymes catalyzing these pathways, a method for the synthesis of radiolabeled Pald was developed. The enzyme pyruvate phosphate dikinase was used to prepare phosphoenolpyruvate (PEP) from pyruvate, adenosine triphosphate (ATP), and orthophosphate. Then PEP was converted to phosphonopyruvate (Ppyr) with PEP mutase and then to Pald with Ppyr decarboxylase. By using [beta-32P]ATP or [2-14C]pyruvate as precursor, [32P]Pald or [1-14C]Pald was obtained, respectively. The utilization of the synthetic, radiolabeled Pald as a probe of enzyme mechanism was demonstrated with the enzyme phosphonoacetaldehyde hydrolase (trivial name phosphonatase). The single turnover time course for the formation and consumption of radiolabeled covalent enzyme species evidenced a kinetically competent covalent intermediate.

Acetaldehyde↗

PubMed2005

Characterization of new D-beta-aspartate-containing proteins in a lens-derived cell line.

Although proteins are generally composed of l-alpha-amino acids, biologically uncommon D-beta-aspartic acid (Asp)-containing proteins have been reported in various tissues from elderly individuals. Our previous study indicated that the N/N1003A cell line, derived from rabbit lens, includes D-beta-Asp-containing proteins of approximately 50 kDa by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for D-beta-Asp-containing proteins. In this study, we identified the D-beta-Asp-containing proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. The results indicate that one of these 50 kDa proteins is an enolase showing homology with tau-crystallin. Other D-beta-Asp-containing proteins, which we have recently discovered include lamin A/C, cytoplasmic NADP+-dependent isocitrate dehydrogenase, fructose-bisphosphate aldolase A, aldose reductase, L-lactate dehydrogenase A or calponin H2, phosphoglycerate mutase 1, phosphatidylethanolamine-binding protein, alpha-B-crystallin, and peptidyl-prolyl cis-trans isomerase A (PPlase).

Animals↗

PubMed2006

Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis.

To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 degrees C (nonpermissive) to 32 degrees C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of approximately 40kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.

Animals↗

PubMed2004

Cloning, sequencing, and functional analysis of the biosynthetic gene cluster of macrolactam antibiotic vicenistatin in Streptomyces halstedii.

Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. The vicenistatin biosynthetic gene cluster (vin) spanning approximately 64 kbp was cloned and sequenced. The cluster contains putative genes for the aglycon biosynthesis including four modular polyketide synthases (PKSs), glutamate mutase, acyl CoA-ligase, and AMP-ligase. Also found in the cluster are genes of NDP-hexose 4,6-dehydratase and aminotransferase for vicenisamine biosynthesis. For the functional confirmation of the cluster, a putative glycosyltransferase gene product, VinC, was heterologously expressed, and the vicenisamine transfer reaction to the aglycon was chemically proved. A unique feature of the vicenistatin PKS is that the loading module contains only an acyl carrier protein domain, in contrast to other known PKS-loading modules containing certain activation domains. Activation of the starter acyl group by separate polypeptides is postulated as well.

Aminoglycosides↗

PubMed2006

Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation.

An operon consisting of three open reading frames, annotated in silico as methylmalonyl-CoA (mm-CoA) epimerase, mm-CoA mutase (MCM), and meaB, was identified in the sequencing project of the myxobacterium Sorangium cellulosum So ce56. This putative MCM pathway operon was subcloned from a bacterial artificial chromosome by Red/ET recombineering onto a minimal replicon derived from p15A. This plasmid was modified for integration and heterologous expression in Pseudomonas putida to enable the production of complex secondary metabolites requiring mm-CoA as precursor. Methylmalonate was identified in the recombinant P. putida strain by an analysis method based on gas chromatography/mass spectrometry. The engineered strain is able to synthesize polyketides requiring mm-CoA as an extender unit, which was demonstrated by the production of myxothiazol after integration of the biosynthetic gene cluster into the chromosome, followed by induction of expression.

Acyl Coenzyme A↗

PubMed2006

The role of nitric oxide on the convulsive behavior and oxidative stress induced by methylmalonate: an electroencephalographic and neurochemical study.

Methylmalonic acidemias consist of a group of inherited metabolic disorders caused by deficiency of methylmalonyl-CoA mutase activity and biochemically characterized by methylmalonate (MMA) accumulation, impairment mitochondrial oxidative metabolism and reactive species production. Preliminary studies with nitric oxide synthase (NOS) inhibitors have suggested that nitric oxide (NO) plays a role in the convulsant effect of MMA. However, definitive biochemical and electrophysiological evidence of the involvement of NO in the convulsions induced by MMA are lacking. In this study, we investigated whether the inhibition of NOS by 7-nitroindazole (7-NI, 3-60mg/kg, i.p.) altered the convulsions, protein oxidative damage, NO(x) (NO(2) plus NO(3)) production and Na(+),K(+)-ATPase activity inhibition induced by MMA. 7-NI decreased striatal NO(x) content, but increased seizures and protein carbonylation induced by MMA (6mumol/striatum). The intrastriatal injection of l-arginine (50nmol/0.5mul), but not of d-arginine (50nmol/0.5mul), increased striatal NO(x) content and protected against MMA-induced electroencephalographic seizures, striatal protein carbonylation and Na(+),K(+)-ATPase inhibition. Furthermore, l-arginine (50nmol/0.5mul) and MMA had no additive effect on NO(x) increase. These results are experimental evidence that endogenous NO plays a protective role in the convulsions and acute neurochemical alterations induced by this organic acid.

Animals↗

PubMed2006

Three-dimensional structure of iminodisuccinate epimerase defines the fold of the MmgE/PrpD protein family.

Iminodisuccinate (IDS) epimerase catalyzes the epimerisation of R,R-, S,S- and R,S- iminodisuccinate, one step in the biodegradation of the chelating agent iminodisuccinate by Agrobacterium tumefaciens BY6. The enzyme is a member of the MmgE/PrpD protein family, a diverse and little characterized class of proteins of prokaryotic and eukaryotic origin. IDS epimerase does not show significant overall amino acid sequence similarity to any other protein of known three-dimensional structure. The crystal structure of this novel epimerase has been determined by multi-wavelength diffraction to 1.5 A resolution using selenomethionine-substituted enzyme. In the crystal, the enzyme forms a homo-dimer, and the subunit consists of two domains. The larger domain, not consecutive in sequence and comprising residues Met1-Lys266 and Leu400-Pro446, forms a novel all alpha-helical fold with a central six-helical bundle. The second, smaller domain folds into an alpha+beta domain, related in topology to chorismate mutase by a circular permutation. IDS epimerase is thus not related in three-dimensional structure to other known epimerases. The fold of the IDS epimerase is representative for the whole MmgE/PrpD family. The putative active site is located at the interface between the two domains of the subunit, and is characterized by a positively charged surface, consistent with the binding of a highly negatively charged substrate such as iminodisuccinate. Docking experiments suggest a two-base mechanism for the epimerisation reaction.

Agrobacterium tumefaciens↗

PubMed2006

Identification of genes differentially expressed during ripening of banana.

The banana (Musa acuminata, subgroup Cavendish 'Grand Nain') is a climacteric fruit of economic importance. A better understanding of the banana ripening process is needed to improve fruit quality and to extend shelf life. Eighty-four up-regulated unigenes were identified by differential screening of a banana fruit cDNA subtraction library at a late ripening stage. The ripening stages in this study were defined according to the peel color index (PCI). Unigene sequences were analyzed with different databases to assign a putative identification. The expression patterns of 36 transcripts confirmed as positive by differential screening were analyzed comparing the PCI 1, PCI 5 and PCI 7 ripening stages. Expression profiles were obtained for unigenes annotated as orcinol O-methyltransferase, putative alcohol dehydrogenase, ubiquitin-protein ligase, chorismate mutase and two unigenes with non-significant matches with any reported sequence. Similar expression profiles were observed in banana pulp and peel. Our results show differential expression of a group of genes involved in processes associated with fruit ripening, such as stress, detoxification, cytoskeleton and biosynthesis of volatile compounds. Some of the identified genes had not been characterized in banana fruit. Besides providing an overview of gene expression programs and metabolic pathways at late stages of banana fruit ripening, this study contributes to increasing the information available on banana fruit ESTs.

DNA, Complementary↗

PubMed2005

The crystal structure of putative precorrin isomerase CbiC in cobalamin biosynthesis.

The leptospira cbiC encodes the enzyme catalyzing the methyl rearrangement reaction of the cobalamin biosynthesis pathway. The protein has been cloned and overexpressed as a His-tagged recombinant protein in Escherichia coli. The crystal structures have been solved in two crystal forms (P4(2)2(1)2 and P3(1)21) diffracting to 3.0 and 2.3A resolution, respectively. The structures are similar to the precorrin-8x methyl mutase (CobH), an enzyme of the aerobic pathway to vitamin B12.

Amino Acid Sequence↗

PubMed2006

Proteomic analysis effects of ginsenoside Rg1 on human umbilical vein endothelial cells stimulated by tumor necrosis factor-alpha.

Ginsenoside Rg1 (derived from ginseng root) has been found to have many vasoprotective activities. The present study was undertaken to examine effect of ginsenoside Rg1 on the secretion of nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs) stimulated with or without tumor necrosis factor-alpha (TNF-alpha). We showed here that ginsenoside Rg1 can increase the basal and TNF-alpha-attenuated NO production in a dose-dependent manner. As little is known regarding the vascular molecular mechanism of ginsenoside Rg1 on HUVECs and proteomic technique has more advantages in molecular identification, we attempted to use proteomic analysis to explain vascular molecular mechanism of ginsenoside Rg1 on HUVECs. Proteomic analytical result showed that 21 protein spots were changed in TNF-alpha stimulated HUVECs, including 9 up-regulated spots, 11 down-regulated spots, and 1 spot detected in TNF-alpha stimulated group only. The expression level of proteins such as MEKK3, phosphoglycerate mutase was increased, and nitric-oxide synthase, mineralocorticoid receptor were decreased in TNF-alpha stimulated HUVECs, while ginsenoside Rg1 could prevent this change or reverse to some degree. This study suggested that NO production increased via ginsenoside Rg1 played an important role in the protective effect on TNF-alpha stimulated HUVECs and was helpful to deeply understand the active mechanism of ginsenoside Rg1 to HUVECs at the molecular level.

Blotting, Western↗