Branching enzyme assay: selective quantitation of the alpha 1,6-linked glucosyl residues involved in the branching points

Methods previously described for glycogen or amylopectin branching enzymatic activity are insufficiently sensitive and not quantitative. A new, more sensitive, specific, and quantitative one was developed. It is based upon the quantitation of the glucose residues joined by alpha 1,6 bonds introduced by varying amounts of branching enzyme. The procedure involved the synthesis of a polysaccharide from Glc-1-P and phosphorylase in the presence of the sample to be tested. The branched polysaccharide was then purified and the glucoses involved in the branching points were quantitated after degradation with phosphorylase and debranching enzymes. This method appeared to be useful, not only in enzymatic activity determinations but also in the study of the structure of alpha-D-glucans when combined with those of total polysaccharide quantitation, such as iodine and phenol-sulfuric acid.     

Initiation of glycogen biosynthesis in Escherichia coli. Studies of the properties of the enzymes involved.

The properties of the enzymes involved in the initiation of glycogen biosynthesis in Escherichia coli were studied. It was found that the enzymic activities which transfer the glycosyl residues from UDPglucose or ADPglucose for the glucoprotein synthesis had differing stabilities upon storage at 4 degrees C. The small amount of glycogen and the saccharide firmly bound to the membrane preparation, were degraded during the storage period. The activity measured in fresh and in stored preparations gave different time dependence curves. The stored preparation had a lag period which could be due to the transfer of the first glucose units to the protein. Both UDPglucose and ADPglucose : protein glucosyltransferases were affected in different ways by detergents. Based on the results presented, it may be concluded that both enzymatic activities are due to different enzymes. Furthermore, both enzymatic activities are different from that which transfers glucose from ADPglucose to glycogen. The following mechanism for the de novo synthesis is suggested. Glycogen in E. coli could be initiated by two different enzymes which transfer glucose to a protein acceptor either from UDPglucose or ADPglucose. Once the saccharide linked to the protein has reached a certain size it is almost exclusively enlarged by another ADPglucose-dependent enzyme. The participation of branching enzyme will produce a polysaccharide with the characteristics of glycogen.     

Regulation of glycogen synthesis and glucose utilization in Escherichia coli during maintenance of the energy charge. Quantitative correlation of changes in the rates of glycogen synthesis and glucose utilization with simultaneous changes in the cellular levels of both glucose 6-phosphate and fructose 1,6-diphosphate.

Treatment of nitrogen-starved cultures of Escherichia coli W4597(K) with sodium azide results in simultaneous changes in both glucose 6-phosphate and fructose 1,6-diphosphate as well as in the rate of glycogen synthesis. Based on these observations, a comprehensive equation was developed which relates the cellular levels of both of these hexose phosphates with the rate of glycogen synthesis. This relationship apparently represents the interaction in vivo between the rate-limiting enzyme of bacterial glycogen synthesis, glucose 1-phosphate adenylyltransferase (adenosine diphosphoglucose synthetase, EC 2.7.7.27), and its substrate glucose 1-phosphate (reflected by glucose 6-phosphate) and its major allosteric activator fructose diphosphate. The form of the equation that describes this relationship was determined from studies presented here of the kinetic properties of the E. coli W4597(K) enzyme in the presence of physiological concentrations of its substrates and modulators. We show here and in subsequent reports of this series that the comprehensive relationship between glycogen synthesis and hexose phosphates can serve as a reference to evaluate the possible participation of new factors in the regulation of glycogen synthesis. Treatment with NaN3 did not change the cellular level of glucose 1-phosphate adenylyltransferase. The value of the adenylate energy charge, (ATP + 1/2 ADP)/(ATP + ADP + AMP), was maintained despite losses of up to 35% in cellular adenylates. The quantitative co-variance between hexose phosphates and the cellular rate of glucose utilization that we previously described for other metabolic conditions was also observed in the azide-treated cultures. We integrate the new information into the system of coordinated regulation of glycogen synthesis, glycolysis, and glucose utilization that we proposed previously.     

荧光发光测定AGPase活力方法初步建立及应用

腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)活力传统上采用32P同位素标记反应底物来测定,但因测定条件的限制而极大地影响了其应用.该研究依据荧光素酶催化荧光素生成发光氧化荧光素的原理,在优化基本反应体系、确立反应体系ATP浓度和荧光强度线性关系等基础上,初步建立了以荧光发光反应测定AGPase活力的新方法,并运用新方法测定了含有不同glgC基因拷贝数菌株的AGPase活力.测定结果显示,不同菌株AGPase活力随glgC拷贝数不同存在显著差异,且其变化趋势与理论预期一致,即新方法可用于AGPase活力的体外测定,且具有更加安全、灵敏、简便和成本低的特点.     

Glycogen phosphorylase, the product of the glgP Gene, catalyzes glycogen breakdown by removing glucose units from the nonreducing ends in Escherichia coli

To understand the biological function of bacterial glycogen phosphorylase (GlgP), we have produced and characterized Escherichia coli cells with null or altered glgP expression. glgP deletion mutants (DeltaglgP) totally lacked glycogen phosphorylase activity, indicating that all the enzymatic activity is dependent upon the glgP product. Moderate increases of glycogen phosphorylase activity were accompanied by marked reductions of the intracellular glycogen levels in cells cultured in the presence of glucose. In turn, both glycogen content and rates of glycogen accumulation in DeltaglgP cells were severalfold higher than those of wild-type cells. These defects correlated with the presence of longer external chains in the polysaccharide accumulated by DeltaglgP cells. The overall results thus show that GlgP catalyzes glycogen breakdown and affects glycogen structure by removing glucose units from the polysaccharide outer chains in E. coli.     

An assay for adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase

Adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5'-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S(0.5) determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.     

Initiation of glycogen biosynthesis in Escherichia coli studies of the properties of the enzymes involved

The properties of the enzymes involved in the initiation of glycogen biosynthesis in Escherichia coli were studied.It was found that the enzymic activities which transfer the glycosyl residues from UDPglucose or ADPglucose for the glucoprotein synthesis had differing stabilities upon storage at 4°C.The small amount of glycogen and the saccharide firmly bound to the membrane preparation, were degraded during the storage period.The activity measured in fresh and in stored preparations gave different time dependence curves. The stored preparation had a lag period which could be due to the transfer of the first glucose units to the protein.Both UDPglucose and ADPglucose: protein glucosyltransferases were affected in different ways by detergents.Based on the results presented, it may be concluded that both enzymatic activities are due to different enzymes. Furthermore, both enzymatic activities are different from that which transfers glucose from ADPglucose to glycogen.The following mechanism for the de novo synthesis is suggested. Glycogen in E. coli could be initiated by two different enzymes which transfer glucose to a protein acceptor either from UDPglucose or ADPglucose. Once the saccharide linked to the protein has reached a certain size it is almost exclusively enlarged by another ADPglucose-dependent enzyme. The participation of branching enzyme will produce a polysaccharide with the characteristics of glycogen.     

GNN is a self-glucosylating protein involved in the initiation step of glycogen biosynthesis in Neurospora crassa

The initiation of glycogen synthesis requires the protein glycogenin, which incorporates glucose residues through a self-glucosylation reaction, and then acts as substrate for chain elongation by glycogen synthase and branching enzyme. Numerous sequences of glycogenin-like proteins are available in the databases but the enzymes from mammalian skeletal muscle and from Saccharomyces cerevisiae are the best characterized. We report the isolation of a cDNA from the fungus Neurospora crassa, which encodes a protein, GNN, which has properties characteristic of glycogenin. The protein is one of the largest glycogenins but shares several conserved domains common to other family members. Recombinant GNN produced in Escherichia coli was able to incorporate glucose in a self-glucosylation reaction, to trans-glucosylate exogenous substrates, and to act as substrate for chain elongation by glycogen synthase. Recombinant protein was sensitive to C-terminal proteolysis, leading to stable species of around 31kDa, which maintained all functional properties. The role of GNN as an initiator of glycogen metabolism was confirmed by its ability to complement the glycogen deficiency of a S. cerevisiae strain (glg1 glg2) lacking glycogenin and unable to accumulate glycogen. Disruption of the gnn gene of N. crassa by repeat induced point mutation (RIP) resulted in a strain that was unable to synthesize glycogen, even though the glycogen synthase activity was unchanged. Northern blot analysis showed that the gnn gene was induced during vegetative growth and was repressed upon carbon starvation.     

植物中的葡糖磷酸变位酶

葡糖磷酸变位酶是催化葡糖1磷酸与葡糖6磷酸之间可逆性转化的酶类,在有机体内的糖原合成及利用中起中枢作用。本文综述了葡糖磷酸变位酶在植物中的分布、重要性、功能及分子特性等,重点介绍了关于植物葡糖磷酸变位酶在遗传及分子生物学方面的研究进展和热点,并讨论了研究葡糖磷酸变位酶的理论和实际意义。     

De novo synthesis of Escherichia coli glycogen is due to primer associated with glycogen synthase and activation by branching enzyme.

Previous reports have demonstrated the incorporation of glucose from ADP-glucose into methanol-insoluble and TCA-insoluble fractions in cell extracts of Escherichia coli in the absence of added primer α-glucan. This activity is reduced 6- to 76-fold in cell extracts of three independently isolated glycogen synthase-deficient mutants of E. coli B. Homogeneous preparations of E. coli B glycogen synthase catalyze incorporation of glucose into both methanol- and TCA-insoluble fractions in the absence of added primer. Since glycogen synthase catalyzes these reactions, it is not necessary to propose a protein acceptor glucose or a unique ADP-glucose-glycosyl transferase to catalyze formation of the glucoprotein in E. coli cell extracts to explain glucose incorporation into TCA-insoluble material (R. Barengo et al. (1975) FEBS Lett.53, 274–278). The incorporation of glucose into methanol-and TCA-insoluble fractions is stimulated by 0.25 m citrate and by branching enzyme. Citrate reduces the K_m for the primer, glycogen, about 11- to 15-fold. Branching enzyme can also reduce the concentration of primer required for incorporation of glucose into methanol-insoluble material. The simultaneous presence of both 0.25 m citrate and branching enzyme enables the glycogen synthase reaction rate to proceed at 30% the maximal velocity at a primer concentration of 1 μg/ml. Incorporation of glucose into methanol- or TCA-insoluble material in the absence of primer is completely inhibited by adding α-amylase. Furthermore, incorporation into methanol- or TCA-insoluble material is reduced 13- to 16-fold relative to the reaction occurring in the presence of primer when glycogen synthase is pretreated with glucoamylase and α-amylase. Previous results show that homogeneous preparations of glycogen synthase contain glucan. Heat-denatured glucogen synthase can act as a primer for the glycogen phosphorylase and glycogen synthase reactions. Both the TCA- and methanol-insoluble products form I₂-glucan complexes with wavelength maxima of about 580–590 nm and 610–615 nm, respectively, suggesting that they are mainly linear chain glucans. The products are completely solubilized with α-amylase. The TCA-insoluble product is not solubilized by pronase treatment. The above results strongly suggest that previous reports on formation of glucoprotein primer for glycogen synthesis or on de novo glycogen synthesis in various similar systems is due to endogenous glucan associated with glycogen synthase rather than formation of glucoprotein which then acts as primer for glycogen synthesis.     

Studies on glycogen synthesis in pigeon liver homogenates. Glycogen synthesis from glucose monophosphates and uridine diphosphate glucose

Comparative time-course studies of glycogen synthesis from glucose 6-phosphate, glucose 1-phosphate and UDP-glucose show that glucose 1-phosphate forms glycogen at an initial rate faster than that obtained with glucose 6-phosphate and UDP-glucose. After 5min. the rates from glucose monophosphates are considerably slower. 2,4-Dinitrophenol decreases glycogen synthesis from both glucose monophosphates, whereas arsenate and EDTA increase glycogen synthesis from glucose 1-phosphate and inhibit the reaction from glucose 6-phosphate, galactose and galactose 1-phosphate. Mitochondria-free pigeon liver cytoplasmic fraction forms less glycogen from glucose monophosphates than does the whole homogenate. 2-Deoxyglucose 6-phosphate inhibits glycogen synthesis from glucose monophosphates. Glycogen formation from UDP-glucose is relatively unaffected by dinitrophenol, by arsenate, by EDTA, by 2-deoxyglucose 6-phosphate and by the removal of mitochondria from the whole homogenate.     

Demonstration of pH control in a commercial immobilized glucose isomerase

The synthesis of a variety of important biochemicals involves multistep enzyme-catalyzed reactions. In many cases, the optimal operating pH is much different for the individual enzymatic steps of such synthesis reactions. Yet, it may be beneficial if such reaction steps are combined or paired, allowing them to occur simultaneously, in proximity to one another, and at their respective optimal pH. This can be achieved by separating the micro-environments of the two steps of a reaction pathway using a thin urease layer that catalyzes an ammonia-forming reaction. In this article, the pH control system in a commercial immobilized glucose (xylose) isomerase pellet, which has an optimal pH of 7.5, is demonstrated. This system allows the glucose isomerase to have near its optimal pH activity when immersed in a bulk solution of pH 4.6. A theoretical analysis is also given for the effective fraction of the immobilized glucose isomerase, which remains active when the bulk pH is at 4.6 in the presence of 20 mM urea versus when the bulk pH is at its optimal pH of 7.5. Both theoretical and experimental results show that this pH control system works well in this case. (c) 1996 John Wiley & Sons, Inc.     

An automated multistep high-throughput screening assay for the identification of lead inhibitors of the inducible enzyme mPGES-1

Prostaglandin E2 synthase (mPGES-1), the enzyme which catalyzes the synthesis of PGE2, is induced during the inflammatory response. For this reason, mPGES-1 could be a potential therapeutic target. A high-throughput screening assay was developed to identify potential inhibitors of mPGES-1. The assay consisted of a 30-s mPGES-1 enzymatic reaction followed by the detection of PGE2 by enzyme immunoassay (EIA). The enzymatic reaction was performed in a batch mode because the instability of the substrate (10 min) limited the number of plates assayed within a working day. The detection of the product by EIA was performed on 3 instruments requiring 14 different steps for complete automation. The authors describe here the optimization and implementation of a 2-part assay on a Thermo CRS robotic system. More than 315,000 compounds were tested, and a hit rate of 0.84% was obtained for this assay. Although the entire assay required multiple steps, the assay was successfully miniaturized and automated for a high-throughput screening campaign.     

A self-glucosylating protein is the primer for rabbit muscle glycogen biosynthesis

In this paper we elucidate part of the mechanism of the early stages of the biosynthesis of glycogen. This macromolecule is constructed by covalent apposition of glucose units to a protein, glycogenin, which remains covalently attached to the mature glycogen molecule. We have now isolated, in a 3500-fold purification, a protein from rabbit muscle that has the same Mr as glycogenin, is immunologically similar, and proves to be a self-glucosylating protein (SGP). When incubated with UDP-[14C]glucose, an average of one molecular proportion of glucose is incorporated into the protein, which we conclude is the same as glycogenin isolated from native glycogen. The native SGP appears to exist as a high-molecular-weight species that contains many identical subunits. Because the glucose that is self-incorporated can be released almost completely from the acceptor by glycogenolytic enzymes, the indication is that it was added to a preformed chain or chains of 1,4-linked alpha-glucose residues. This implies that SGP already carries an existing maltosaccharide chain or chains to which the glucose is added, rather than glucose being added directly to protein. The putative role of SGP in glycogen synthesis is confirmed by the fact that glucosylated SGP acts as a primer for glycogen synthase and branching enzyme to form high-molecular-weight material. SGP itself is completely free from glycogen synthase. The quantity of SGP in muscle is calculated to be about one-half the amount of glycogenin bound in glycogen.     

A continuous coupled polarographic assay for trehalase.

A continuous, coupled polarographic assay, which couples trehalose hydrolysis to O₂ consumption using glucose oxidase (EC 1.1.3.4) and catalase (EC 1.11.1.6) as ancillary enzymes has been developed for the measurement of trehalase (α-α′-trehalose 1-d-glucohydrolase, EC 3.2.1.28) activity. With this procedure, O₂ consumption was a linear function of time and the coupled reaction rate was directly proportional to the amount of protein assayed with both crude and partially purified enzyme preparations. The limits of sensitivity with this assay correspond to the production of 2.5 nmol of glucose/min. The validity of this assay was confirmed by comparative studies with a discontinuous colorimetric assay for the quantitation of glucose. In addition, the applicability of this assay was appraised by determining the K_m of the enzyme for trehalose. The value obtained with the polarographic assay (i.e., 1.3 ± 0.1 mm trehalose) showed excellent agreement with that obtained using a discontinuous colorimetric method (i.e., 1.2 mm trehalose). Thus the equivalence and applicability studies with the polarographic assay demonstrated that this procedure is a valid and sensitive method for the rapid quantitation of trehalase activity.     

Crystal structure of yeast hexokinase PI in complex with glucose: A classical "induced fit" example revised

Hexokinase is the first enzyme in the glycolytic pathway that catalyzes the transfer of a phosphoryl group from ATP to glucose to form glucose-6-phosphate and ADP. Two yeast hexokinase isozymes are known, namely PI and PII. Here we redetermined the crystal structure of yeast hexokinase PI from Saccharomyces cerevisiae as a complex with its substrate, glucose, and refined it at 2.95 A resolution. Comparison of the holo-PI yeast hexokinase and apo-hexokinase structures shows in detail the rigid body domain closure and specific loop movements as glucose binds and sheds more light on structural basis of the "induced fit" mechanism of reaction in the HK enzymatic action. We also performed statistical coupling analysis of the hexokinase family, which reveals two co-evolved continuous clusters of amino acid residues and shows that the evolutionary coupled amino acid residues are mostly confined to the active site and the hinge region, further supporting the importance of these parts of the protein for the enzymatic catalysis.     

Processivity and subcellular localization of glycogen synthase depend on a non-catalytic high affinity glycogen-binding site

Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS.     

Characteristic properties of the complex of glycogen synthase with glycogen

The glycogen synthase I--glycogen complex isolated from rabbit skeletal muscles is stable during precipitation with trichloroacetic acid and Sepharose 2B chromatography. The complex catalyzes the synthesis (lengthening) of the alpha-1.4-glucosyl chains when endogenous or exogenous enzyme-free glycogen is used, the initial rates of this synthesis being identical. Preincubation with glycogen does not cause activation of the complex or formation of additional glycogen synthase I--polysaccharide bonds. The complex is characterized by saturation with respect to glycogen; the molar concentration ratios of the non-reducible chain and protein monomer within the complex does not exceed 100. An increase in the length of the synthesized alpha-1.4-glycosyl chains of glycogen results in a decrease of the rate of the glycogen synthase reaction in time.     

Characterization of the consequence of a novel Glu-380 to Asp mutation by expression of functional P450c21 in Escherichia coli

P450c21 catalyzes an important step in steroid synthesis. Its deficiency leads to symptoms of steroid imbalance. To obtain enough P450c21 for structure and function studies, we developed a method to express P450c21 in Escherichia coli. The 5'-region of the human P450c21 cDNA was modified to ensure efficient translation and the C terminus of the protein was extended with four His residues for easy purification. Mutant proteins with substitutions at residues 172 and 281 exhibited decreased enzymatic activities similar to those found in mammalian cells. One new mutation changing Glu-380 to Asp (D380) caused 3-fold reduction in enzymatic activity. The amount of apoprotein production detected by immunoblotting and the affinity of the mutant protein towards substrate as measured by Km were normal. The defect lies in the decreased ability of the apoprotein to bind heme, which was measured by CO difference and substrate-binding spectra. The D380 mutant protein had 3-fold reduction in peak heights in both spectra. This reduced heme binding resulted in 3-fold lower enzymatic activity.