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 mutant strain of Escherichia coli K-12, designated 618, accumulates glycogen at a faster rate than wild-type strain 356. The mutation affects the ADPglucose pyrophosphorylase regulatory properties (N. Creuzat-Sigal, M. Latil-Damotte, J. Cattaneo, and J. Puig, p. 647-680, in R. Piras and H. G. Pontis, ed., Biochemistry of the Glycocide Linkage, 1972). The enzyme is less dependent on the activator, fructose 1,6 bis-phosphate for activity and is less sensitive to inhibition by the inhibitor, 5'-AMP. The structural gene, glgC, for this allosteric mutant enzyme was cloned into the bacterial plasmid pBR322 by inserting the chromosomal DNA at the PstI site. The glycogen biosynthetic genes were selected by cotransformation of the neighboring asd gene into an E. coli mutant also defective in branching enzyme (glgB) activity. Two recombinant plasmids, pEBL1 and pEBL3, that had PstI chromosomal DNA inserts containing glgC and glgB were isolated. Branching enzyme and ADPglucose pyrophosphorylase activities were increased 240- and 40-fold, respectively, in the asd glgB mutant, E. coli K-12 6281. The E. coli K-12 618 mutant glgC gene product was characterized after transformation of an E. coli B ADPglucose pyrophosphorylase mutant with the recombinant plasmid pEBL3. The kinetic properties of the cloned ADPglucose pyrophosphorylase were similar to those of the E. coli K-12 618 enzyme. The inserted DNA in pEBL1 was arranged in opposite orientation to that in pEBL3.
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PMID:Cloning and expression of the Escherichia coli glgC gene from a mutant containing an ADPglucose pyrophosphorylase with altered allosteric properties. 301 41

A technique is presented by which mutations can be introduced into the Escherichia coli chromosome by gene replacement between the chromosome and a plasmid carrying the mutant gene. The segregational instability of plasmids in E. coli is used with high efficiency to isolate E. coli mutants. The method should be applicable to construction of mutants for any E. coli chromosomal gene provided it is dispensable, and for any E. coli strain provided it is capable of homologous recombination. The use of the method was demonstrated by constructing E. coli mutants for the glycogen branching enzyme gene (glgB) and the beta-galactosidase gene (lacZ). The results show that recombination occurs via a reciprocal mechanism indicating that the method should, in a slightly modified form, also be useful in transferring chromosomal mutations onto multicopy plasmids in vivo.
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PMID:A general method for the construction of Escherichia coli mutants by homologous recombination and plasmid segregation. 311 16

A 7 1/2-year-old girl had exercise intolerance and exertional dyspnea. Four months later, congestive heart failure developed, with recurrent chylous pleural effusions, and she died at age 8 1/2 years. Endomyocardial biopsy tissue showed abundant PAS-positive, diastase-resistant cytoplasmic deposits. Similar inclusions were seen in muscle, skin, and liver specimens. Postmortem studies showed that the abnormal polysaccharide was especially abundant in heart and muscle, but was also present in all other tissues, including the central nervous system. Glycogen isolated from heart, muscle, and spinal cord showed a shift of the iodine spectrum toward higher than normal wavelengths. Branching enzyme activity was lacking in the muscle biopsy specimen and in all postmortem tissues; glycogenolytic enzymes had normal activities. These studies show that cardiomyopathy can be the first symptom of generalized branching enzyme deficiency and that the degree of accumulation of the abnormal polysaccharide may vary in different tissues.
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PMID:Severe cardiopathy in branching enzyme deficiency. 347 93

Two molecular forms (BE-I and -II) of 1,4-alpha-glucan branching enzyme were purified from rat liver by affinity chromatography on glycogen-adipoyldihydrazide-Sepharose 4B and glycogen-ethylenediamine-Sepharose 4B, respectively, after hydrophobic chromatography on hexylamine-Sepharose 4B, as single proteins with the same molecular weight of 82,000 on SDS-polyacrylamide gel electrophoresis. By comparing their UV spectra and by RNA detection on polyacrylamide gels after electrophoresis, BE-I and -II were identified to be RNA associated and unassociated forms, respectively. The molecular weights of BE-I and -II estimated by Sephadex G-200 gel filtration were 91,000 and 98,000, respectively, indicating that both forms consist of a monomer. As Be-II had about half the specific activity of BE-I, the RNA component was not essential for the activity of the rat liver branching enzyme, and it was also dissociable from the protein component (BE-II) on polyacrylamide gel electrophoresis at pH 7.3, whereby BE-II showed a heterogeneity of at least three microspecies as revealed by protein staining as well as by activity staining of the gels. Using the antibody against BE-I, BE-I and -II were indistinguishable on immunodiffusion, and the activity inhibition rate of BE-II by the antibody was almost the same as that of BE-I if their specific activities are considered, indicating that the RNA component may have no effect on the enzyme-antibody reaction. Immunochemically no isoenzyme was detected for this enzyme in rat tissues.
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PMID:Purification of RNA associated and unassociated forms of 1,4-alpha-glucan branching enzyme from rat liver. 617 27

The fluorescence yield and lifetime of ethidium bromide complexes with 1,4-alpha-glucan branching enzyme and its free nucleic acid component 2.5S RNA were measured. Both fluorescence parameters showed a 10-fold increase in comparison with those characteristics for the free dye. This increase allows to suggest the existence of double-stranded regions in 2.5S RNA both in the free as well as in the protein bound state. The coefficients of fluorescence polarization were also determined for ethidium bromide complexed with free and protein bound 2.5S RNA. They proved to be 13 and 18% respectively. No concentration depolarization was observed in both types of ethidium bromide and ethidium bromide--enzyme--RNA complexes. This proves that the double-stranded regions are rather short and that two ethidium bromide molecules can't be bound to each of them. The binding isotherms were measured for ethidium bromide absorbed on 2.5S RNA and on the holoenzyme. Their parameters napp and rmax are identical in the cases of free and protein bound 2,5S RNA (rmax = 0.046 +/- 0.001). However the binding constants of ethidium bromide complexes with free and protein bound 2.5S RNA differ significantly (Kapp = 2.2 X 10(6) M-1 for free 2.5S RNA and Kapp = 1.6 X 10(6) M-1 for the holoenzyme). The quantity of nucleotides involved in the two double-stranded regions accessible for ethidium binding is estimated to be about 28%. Increasing of Mg2+ ion concentration up to 10(-3) results in a decrease of ethidium bromide binding with double stranded regions. It may be due to a more compact tertiary structure of 2.5S RNA in the presence of Mg2+ in the free as well as in protein bound state.
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PMID:[Study of 2.5S RNA of 1,4-alpha-glucan branching enzyme by fluorescent methods using ethidium bromide]. 619 23

1.4-alpha-glucan branching enzyme (EC 2.4.1.18) from rabbit muscles with an essential 2.5S RNA component has been studied by limited trypsin treatment. Under a great variety of hydrolysis conditions the product resistant to subsequent action of trypsin was obtained. This product contains about 70% of protein and all 2.5S RNA of the original nucleoprotein and retains about 50% of original activity. Amino acids Composition showed, that the protein is of alkaline nature and is rich in lysine. The alkaline nature of protein remains unchanged after trypsinolysis. On the basis of these studies it was assumed that the presence of firmly attached to the protein 2.5S RNA protects the branching enzyme against more powerful trypsinolysis and hinders loss of activity of the branching enzyme.
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PMID:[Structure-function study of 1,4-alpha-glucan branching enzyme by limited trypsin treatment]. 621 6

Human skin fibroblasts from patients with Type IV glycogen storage disease, in which there is a demonstrable deficiency of glycogen branching enzyme, were shown to be able to synthesize [14C]glycogen containing [14C]glucose at branch points when sonicates containing endogenous glycogen synthase a were incubated with UDP[14C]glucose. The branch point content of the glycogen synthesized by the Type IV cells was essentially the same as that formed by normal cells, but the total synthetic capacity of the Type IV cells was lower. A new assay for the branching enzyme using glycogen synthase as the indicator enzyme has been developed. Using this assay it has been shown that the residual branching enzyme of affected children and of their heterozygote parents is less easily inhibited by an IgG antibody raised in rabbits against the normal human liver enzyme than is the branching enzyme of normal fibroblasts.
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PMID:Studies of the residual glycogen branching enzyme activity present in human skin fibroblasts from patients with type IV glycogen storage disease. 622 Jul 6

Glycogen branching enzyme was isolated from rabbit liver. The highly purified enzyme shows a monomer molecular weight of 71 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and apparent molecular weights of 93 000 by sucrose density gradient sedimentation and 52 000 by gel-exclusion chromatography on Sephacryl S-300. No glucosamine, mannosamine, galactosamine, or sialic acid was detected in the protein. An amino acid analysis is reported. The spectrum of branching enzyme is that of a simple polypeptide, with A1%280nm = 24.6. Highly purified branching enzyme consists of several closely related active enzyme forms that can be resolved by isoelectric focusing in polyacrylamide gel. The major species of pI 5.7 is flanked by less abundant forms of pI 5.6 and 5.8. Seemingly identical enzyme forms are observed in crude extracts of rabbit liver, skeletal muscle, brain, and heart, although the absolute and relative concentrations vary among the tissues. Branching enzyme apparently does not exhibit tissue-specific isoenzymes.
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PMID:Isolation and characterization of glycogen branching enzyme from rabbit liver. 622 54

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

1,4-alpha-glucan:1,4-alpha-glucan 6-alpha-D-(1,4-alpha-D-glucano) transferase (branching enzyme) was purified by ammonium sulphate precipitation, chromatography on DEAE-cellulose, fractionation with poly(ethyleneglycol) 6000, chromatography on DEAE-Sepharose and gel filtration on Sephadex G150. The final specific activity was 3000 U/mg corresponding to a purification of approximately 10000-fold over the muscle extracts. 0.6 mg of enzyme was isolated from 4000 g muscle within eight days corresponding to an overall yield of 7%. The purified protein was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, and this technique yielded a molecular weight of 77000 for the subunit molecular weight of branching enzyme. The apparent molecular weight of the native enzyme determined by gel filtration on Sephadex G150 was 60000, demonstrating that branching enzyme is a monomeric protein. Only a very small proportion of the branching enzyme activity in muscle extracts (2%) precipitated with the protein-glycogen complex. This finding, and its low concentration in muscle, explain why a protein-staining band corresponding to branching enzyme cannot be detected by polyacrylamide gel electrophoresis of the protein-glycogen complex.
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PMID:Purification and subunit structure of glycogen-branching enzyme from rabbit skeletal muscle. 644 99


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