Continuous assay method for glycogen synthase activity

An assay method for glycogen synthase (EC 2.4.1.11) has been developed based on the continuous measurement of the change of pH accompanying the glycogen synthesis reaction. The use of low buffer concentrations and an amplifier with variable gain and offset voltage allow us to register changes in the pH of the system small enough to ignore the significant pH dependence of the enzyme activity. A theoretical approach has been used to correlate the pH measurements with the progres of the reaction in terms of glucose incorporated into glycogen. The method offers the advantages of being continuous and of low cost.     

The family of glycogen phosphorylases: structure and function

Glycogen phosphorylase plays a central role in the mobilization of carbohydrate reserves in a wide variety of organisms and tissues. While rabbit muscle phosphorylase remains the most studied and best characterized of phosphorylases, recombinant DNA techniques have led to the recent appearance of primary sequence data for a wide variety of phosphorylase enzymes. The functional properties of rabbit muscle phosphorylases are reviewed and then compared to properties of phosphorylases from other tissues and organisms. Tissue expression patterns and the chromosomal localization of mammalian phosphorylases are described. Differences in functional properties among phosphorylases are related to new structural information. Evolutionary relationships among phosphorylases as afforded by comparative analysis of proteins and gene sequences are discussed.     

Development and application of a screening assay for glycoside phosphorylases

Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They require only a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzyme’s substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen a residue E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity toward alkyl β-glucosides and phenyl β-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity.     

Radioactive assay for aryl hydrocarbon hydroxylase. Improved method and biological importance

By addition of two volumes of a 1M aqueous KOH/dimethylsulfoxide ($\text{15}\text{85}$; v/v) mixture to the enzymatic incubation medium, it is possible to selectively extract the unmetabolized benzo(a)pyrene in hexane. Therefore, the radio-activity remaining in the water phase corresponds to all the in vitro synthesized metabolites. This isotopic method is very sensitive (2 × 10^?11 moles) and is almost insensitive to the room lighting. The aryl hydrocarbon hydroxylase activities found with this method are 2,3 and 10 times higher in the liver, lung and kidney respectively compared to those obtained with the fluorimetric method.     

Kinetic analysis and modelling of the allosteric behaviour of liver and muscle glycogen phosphorylases

Allosteric enzymes have very complex kinetic behaviours which are primarily interpreted through simplified models. To describe the functional properties of liver and muscle glycogen phosphorylase isozymes we have developed an experimental strategy based on the measurements of initial reaction rates in the presence of different concentrations of the effectors glucose-1-phosphate and methyl-xanthines. Using the extensive structural information available for the two glycogen phosphorylase conformers T (inactive) and R (active) with different ligands, we have applied the Monod-Wyman-Changeux model and analysed the results in the context of the exclusive binding of the inhibitors to the T state, meanwhile the substrate glucose-1-phosphate binds to both, the R and T states. The kinetic analysis shows a good agreement between our model and the results obtained from the glycogen phosphorylases and inhibitors included in this study, which demonstrates the validity of the approach described here.     

A semicontinuous assay for glycogen phosphorylase

An assay for phosphorylase a or b is described in which the rate of release of phosphate from glucose 1-phosphate is followed by means of an “instantaneous” method for determination of phosphate.     

A titrimetic assay for glycogen phosphorylase

A titrimetric method for the assay of glycogen phosphorylase is presented in which a direct and continuous course of reaction is obtained over a wide range of enzyme concentrations (7.2–378.3 μg/ml). The method resulted in rates which were in agreement with those obtained using the inorganic phosphate method, and the expected value of the equilibrium concentration ratio of inorganic phosphate to glucose-1-phosphate was obtained. The method can be extended to higher concentrations, and it can be used to measure the rate in either direction. The K_m and V_max values of each substrate, glucose-1-phosphate and inorganic phosphate, were determined.     

Efficient inhibition of muscle and liver glycogen phosphorylases by a new glucopyranosylidene-spiro-thiohydantoin.

Reaction of C-(1-bromo-1-deoxy-beta-glucopyranosyl)formamide 2 with thiocyanate ions was the key step of a short synthesis of D-glucopyanosylidene-spiro-thiohydantoin 7 which proved to be a potent inhibitor of muscle and liver glycogen phosphorylases.     

An enzyme-coupled continuous spectrophotometric assay for glycogen synthases

The metabolic pathways leading to the synthesis of bacterial glycogen involve the action of several enzymes, among which glycogen synthase (GS) catalyzes the elongation of the α-1,4-glucan. GS from Agrobacterium tumefaciens uses preferentially ADPGlc, although UDPGlc can also be used as glycosyl donor with less efficiency. We present here a continuous spectrophotometric assay for the determination of GS activity using ADP- or UDPGlc. When ADPGlc was used as the substrate, the production of ADP is coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH). With UDPGlc as substrate, UDP was converted to ADP via adenylate kinase and subsequent coupling to PK and LDH reactions. Using this assay, we determined the kinetic parameters of GS and compared them with those obtained with the classical radiochemical method. For this purpose, we improved the expression procedure of A. tumefaciens GS using Escherichia coli BL21(DE3)-RIL cells. This assay allows the continuous monitoring of glycosyltransferase activity using ADPGlc or UDPGlc as sugar-nucleotide donors.     

A bioluminescent assay for glycogen phosphorylase in cultured cells

A new method for the determination of glycogen phosphorylase (1,4 alpha-D-glucose:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) in cultured cells is described. The assay utilizes bacterial luciferase (EC 2.7) in a liquid scintillation spectrometer to measure NAD(P)H formed in a coupled enzyme reaction comprising glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and phosphoglucomutase (EC 2.7.5.1). This assay is highly sensitive, easily detecting as little as 10 microU phosphorylase, fast and simple to perform. With modifications this procedure can be extended to measure other glycogenolytic enzymes and intermediates.     

Complementation of subunits from glycogen phosphorylases of frog and rabbit skeletal muscle and rabbit liver.

Activity can be induced in potentially active rabbit skeletal muscle phosphorylase monomers covalently bound to Sepharose by noncovalent interaction with soluble subunits carrying inactive pyridoxal 5'-phosphate analogs or even salicyladlehyde. These analogs are themselves incapable of reconstituting active holophorphorylase from apophosphorylase. Phosphorylases with one intrinsically inactive and one potentially active subunit have about one half of the activity of the native phosphorylase dimer. The usefulness of this technique for subunit complementation was demonstrated by forming hybrid phosphorylases with inactive Sepharose-bound rabbit skeletal muscle subunits containing pyridoxal 5'-phosphate monomethylester and soluble activatable frog muscle and rabbit liver phosphorylase monomers. The inactive Sepharose-bound subunit induced in each case activity in the soluble subunit. But whereas the inactive rabbit muscle phosphorylase subunit even transmitted its characteristic temperature dependence of the rate of the reaction to the frog muscle subunit, it could not propagate its control properties to the liver enzyme. Differences of hybrid phosphorylases are related to immunological and amino acid divergencies among the component enzymes.