Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.1.18 (branching enzyme)
628 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A branching enzyme was extracted from the mycelia of Neurospora crassa and was purified to electrophoretic homogeneity by procedures including DEAE-Sephacel column chromatography, 6-aminohexyl-Sepharose 4B column chromatography and gel filtration on Toyopearl HW-55S. The final yield of the branching enzyme activity was 15.1%, and the final purified enzyme preparation showed a specific activity of 702 units per mg of protein. The molecular weight of this enzyme was estimated to be 80,000 by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. The amino acid composition and the carbohydrate content of this enzyme were analyzed. The isoelectric point of this enzyme determined by polyacrylamide gel isoelectrofocusing was 5.6. The branching activity of the enzyme was confirmed by its action on amylopectin as well as by the combined action of this enzyme and N. crassa glycogen synthase. The action of this enzyme on amylopectin decreased the wavelength of the absorption maximum of the glucan-iodine complex, and increased the amount of the short unit chains of the debranched product. The product obtained by the combined action yielded beta-limit dextrin upon hydrolysis with beta-amylase. No multiplicity was found for the branching activity either by chromatography or by electrophoresis.
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PMID:Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the branching enzyme. 622 52

The structure of alpha-glucan, isolated from wild-type Escherichia coli B, a glycogen branching enzyme (BE)-deficient E. coli AC71 (glgB-), or from AC71 transformed with genes coding for maize BEI and BEII individually as well as with both genes, was analyzed by high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection. Transformation of the maize BE gene(s) in AC71 (glgB-) showed complementation in branching activity. Analysis by HPAEC revealed different structures between glycogen of E. coli B and alpha-glucan of AC71 transformed with a different maize BE gene(s). The individual chains of the alpha-glucan debranched with isoamylase were distributed between chain length (CL) 3 and > 30 and the chain with CL 6 was the most abundant. In comparison with the glycogen of E. coli B, the alpha-glucan of AC71 transformed with the maize BE gene(s) consisted of a lesser amount of chains with CL 7-9 and a larger amount of chains with CL > 14. It also showed a broad peak with chains of CL 9-12 as in maize amylopectin. This study provides in vivo evidence that glycogen BE and maize BE isozymes may have different specificities in the length of chain transferred. Furthermore, this study suggests that the specificity of glycogen synthase and starch synthase and their concerted action with BE play an important role in determining the structure of the polysaccharide synthesized.
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PMID:Maize branching enzyme catalyzes synthesis of glycogen-like polysaccharide in glgB-deficient Escherichia coli. 786 74

The synthesis of glycogen in Saccharomyces cerevisiae is stimulated by nutrient limitation and requires both glycogen synthase and the glycogen branching enzyme. Of the two glycogen synthase genes present in yeast, GSY2 appears to be more important for the accumulation of glycogen upon entry into stationary phase. In cells grown on glucose, GSY2 mRNA levels increased approximately 10-fold during the transition from logarithmic to stationary phase. Growth of cells in glycerol, however, resulted in constitutive expression of GSY2 mRNA and the corresponding protein, GS-2, suggestive of glucose repression of GSY2. Mutants defective in the SNF1 gene, which encodes a protein kinase important in glucose repression mechanisms, are known not to accumulate glycogen. A modest 2-4-fold decrease in total GS-2 level was observed, and upon entry into stationary phase, the enzyme was blocked in the inactive, phosphorylated state in snf1 strains. The GS-2 protein is thought to be regulated by covalent phosphorylation of three COOH-terminal sites (Hardy, T.A., and Roach, P.J. (1993) J. Biol. Chem. 268, 23799-23805), removal of which results in constitutively active glycogen synthase that bypasses phosphorylation controls. Expression of COOH-terminally truncated GS-2 in snf1 cells restored glycogen accumulation, and so we propose that the SNF1 kinase controls the phosphorylation state of GS-2. Cyclic AMP pathways also exert control over glycogen accumulation. In bcy1 cells, which have constitutively active cyclic AMP-dependent protein kinase, greatly reduced levels of both GS-2 message and protein were observed. With wild type GSY2 placed under control of the ADH1 promoter, bcy1 cells did not accumulate glycogen despite increased GS-2. Overexpression of truncated GS-2, however, resulted in definite though reduced glycogen accumulation; the glycogen synthesized was structurally distinct from wild type with properties characteristic of less branched polysaccharide. We conclude that the cAMP pathway controls both the expression and the phosphorylation state of GS-2. Furthermore, other factor(s) necessary for glycogen biosynthesis, such as the branching enzyme GLC3, must also be under negative control by the cAMP pathway. The results demonstrate interactive controls of GS-2 by the cAMP-dependent and SNF1 protein kinases.
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PMID:Interactions between cAMP-dependent and SNF1 protein kinases in the control of glycogen accumulation in Saccharomyces cerevisiae. 796 23

High glycogen content and abnormal mitochondria have been seen in muscles from RN- carrier pigs in a previous work. Glycogen synthase, branching enzyme, phosphorylase and debranching enzyme activities, and mitochondrial characteristics were studied in normal and RN- carrier pigs. Branching enzyme activity was higher (P < 0.01) and glycogen synthase activity tended to be higher in longissimus dorsi muscle from RN- carrier pigs compared to normal pigs. There were no differences in the activities of either phosphorylase and debranching enzyme between both types of pigs. Citrate synthase activity and mitochondrial respiration were slightly higher in muscle from RN- pigs compared to normal pigs. Glycogen content in muscle from RN- pigs could result from the imbalance between anabolic and catabolic enzyme activities of glycogen metabolism. The higher specific activity in mitochondria of RN- pigs muscle might be the compensatory effect of an abnormal glycolytic metabolism.
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PMID:Enzyme activities of glycogen metabolism and mitochondrial characteristics in muscles of RN- carrier pigs (Sus scrofa domesticus). 808 56

Consistent with previous results, overexpression of rabbit skeletal muscle glycogen synthase in COS cells did not lead to overaccumulation of glycogen unless activating Ser-->Ala mutations were present at key regulatory phosphorylation sites 2 (Ser7) and 3a (Ser644) in the enzyme. In addition, we found that expression of glycogenin, glycogen branching enzyme, or UDP-glucose pyrophosphorylase alone in COS cells had no effect on the glycogen level. However, coexpression of the hyperactive 2,3a glycogen synthase mutant with either glycogenin or UDP-glucose pyrophosphorylase led to higher glycogen accumulation than that obtained from the expression of glycogen synthase alone. Coexpression of glycogenin with the 2,3a mutant of glycogen synthase led to the appearance of glycogenin with a lower molecular weight suggestive of reduced glucosylation. Increased glycogen synthesis may lead to competition between glycogenin and glycogen synthase for their common substrate UDP-glucose. In summary, we conclude that (i) glycogen synthase is a primary rate-limiting enzyme of glycogen biosynthesis in COS cells, (ii) that phosphorylation of glycogen synthase is regulatory for glycogen accumulation, and (iii) once glycogen synthase is activated, the reaction mediated by UDP-glucose pyrophosphorylase can become rate-determining.
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PMID:Rate-determining steps in the biosynthesis of glycogen in COS cells. 864 5

A chromosomal region of Bacillus stearothermophilus TRBE14 which contains genes for glycogen synthesis was cloned and sequenced. This region includes five open reading frames (glgBCDAP). It has already been demonstrated that glgB encodes branching enzyme (EC 2.4.1.18 [H. Takata et al., Appl. Environ. Microbiol. 60:3096-3104, 1994]). The putative GlgC (387 amino acids [aa]) and GlgD (343 aa) proteins are homologous to bacterial ADP-glucose pyrophosphorylase (AGP [EC 2.7.7.27]): the sequences share 42 to 70% and 20 to 30% identities with AGP, respectively. Purification of GlgC and GlgD indicated that AGP is an alpha2beta2-type heterotetrameric enzyme consisting of these two proteins. AGP did not seem to be an allosteric enzyme, although the activities of most bacterial AGPs are known to be allosterically controlled. GlgC protein had AGP activity without GlgD protein, but its activity was lower than that of the heterotetrameric enzyme. The GlgA (485 aa) and GlgP (798 aa) proteins were shown to be glycogen synthase (EC 2.4.1.21) and glycogen phosphorylase (EC 2.4.1.1), respectively. We constructed plasmids harboring these five genes (glgBCDAP) and assayed glycogen production by a strain carrying each of the derivative plasmids on which the genes were mutated one by one. Glycogen metabolism in B. stearothermophilus is discussed on the basis of these results.
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PMID:Characterization of a gene cluster for glycogen biosynthesis and a heterotetrameric ADP-glucose pyrophosphorylase from Bacillus stearothermophilus. 924 54

Rat brain glycogen branching enzyme was partially purified in order to elucidate its mechanism of action. The alpha1,4-alpha1,6-glucan polysaccharide was synthesized using rat brain branching enzyme under two different elongation conditions: Glc-1-P and phosphorylase or UDP-Glc and glycogen synthase. The products obtained demonstrated that the cpolysaccharides synthesized (pattern of the spectra obtained in the presence of Krisman's reagent, lambda max, parameter A and R, % beta-amylolysis and degree of branching) under different incubation times are nearly constant. These results imply that the degree of branching of a polysaccharide depends only on the enzyme specificity.
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PMID:Glycogen brain branching enzyme. 962 Apr 41

Glycogen synthase of bovine retina was found associated with the acid-insoluble and acid-soluble proteoglycogen fractions. The synthase associated with the acid-insoluble proteoglycogen precursor showed an 8-fold lower Km for UDP-glucose than the synthase associated with the acid-soluble fraction, and was inhibited by detergent. A short digestion with pronase resulted in conversion of the acid insoluble fraction into acid-soluble. The results lead us to postulate that the acid-insolubility of the proteoglycogen fraction and the association with retina membrane proposed before, is caused by glycogen synthase strongly associated to its polysaccharide moiety. The enlargement of the polysaccharide moiety during proteoglycogen biosynthesis, from glycogenin linked to a few 11 to 12 glucose units to the acid-insoluble proteoglycogen precursor (Mr 470,000) would be carried out, together with the branching enzyme, by the glycogen synthase showing a low Km for UDP-glucose. The glycogen synthase with the highest Km for UDP-glucose would participate in conversion of the precursor into mature acid-soluble proteoglycogen.
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PMID:Two glycogen synthase activities associated with proteoglycogen in retina. 1068 12

A 6-kb DNA fragment of the Rhodobacter sphaeroides 2.4.1 glg operon was cloned from a genomic library using a polymerase chain reaction probe coding for part of the ADP-glucose pyrophosphorylase (glgC) gene. The DNA fragment was sequenced and found to harbor complete open reading frames for the glgC and glgA (glycogen synthase) genes and partial sequences corresponding to glgP (glycogen phosphorylase) and glgX (glucan hydrolase/transferase) genes. The genomic fragment also contained an apparent truncated sequence corresponding to the C-terminus of the glgB gene (branching enzyme). The presence of active branching enzyme activity in crude sonicates of Rb. sphaeroides cells indicates that the genome contains a full-length glgB at another location. The structure of this operon in relation to other glg operons is further discussed. The deduced sequence of the ADP-glucose pyrophosphorylase enzyme is compared to other known ADP-glucose pyrophosphorylase sequences and discussed in relation to the allosteric regulation of this enzyme family. The glgC gene was subcloned in the vector pSE420 (Invitrogen) for high-level expression in E. coli. The successful overexpression of the recombinant enzyme allowed for the purification of over 35 mg of protein from 10 g of cells, representing a dramatic improvement over enzyme isolation from the native strain. The recombinant enzyme was purified to near homogeneity and found to be physically, immunologically, and kinetically identical to the native enzyme, verifying the fidelity of the cloning step.
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PMID:Cloning and sequencing of glycogen metabolism genes from Rhodobacter sphaeroides 2.4.1. Expression and characterization of recombinant ADP-glucose pyrophosphorylase. 1072 89

We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c(1) and CycM and a small increase in the amount of cytochrome aa(3). In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene.
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PMID:Enhanced symbiotic performance by Rhizobium tropici glycogen synthase mutants. 1120 82


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