Kinetic analysis of glucose-6-phosphatase: an investigative approach to carbohydrate metabolism and kinetics

The enzyme glucose-6-phosphatase (G-6-Pase) catalyzes the hydrolysis of glucose-6-phosphate (G-6-P) to glucose. This is one of the key steps in gluconeogenesis and is critically important in maintaining stable blood glucose levels in most mammals. G-6-Pase is primarily found in the endoplasmic reticulum (ER) of hepatocytes and can easily be studied using isolated microsomes prepared from liver ER. A three-part undergraduate laboratory exercise uses rat liver microsomes to focus on the enzymatic analysis of G-6-Pase. The assessment of G-6-Pase activity is conducted using a stopped assay protocol combined with a colorimetric determination of inorganic phosphate (P_i) levels. The laboratory exercise was designed to carry out an independent inhibition investigation using orthovanadate, a competitive inhibitor of G-6-Pase with potential clinical importance. The format of the three-part investigation provides a useful mechanism for demonstrating enzyme kinetics and competitive inhibition using an enzyme that is important for carbohydrate metabolism and glycogen storage disease.     

Synthesis of methyl 6-(ammonium 2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate)-alpha-D-mannopyranoside and use of this compound for the determination of N-acetylglucosamine-1-phosphotransferase

Methyl 6-(ammonium 2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate)-alpha-D-mannopyranoside was synthesized and identified by 1H-n.m.r. and 13C-n.m.r. data, acid hydrolysis, and elemental analysis. It was utilized for the determination of UDP-N-acetylglucosamine-1-phosphotransferase in an assay procedure that employed methyl alpha-D-mannopyranoside as an acceptor. The assay product was identified and characterized by thin-layer chromatography with the title reference compound. The present technique does not require [32P]UDP-N-acetylglucosamine, but effectively uses commercially available UDP-[14C]GlcNAc.     

O~6-甲基鸟嘌呤受体蛋白的定量测定

本文介绍了一种细胞提取液O~6—甲基鸟嘌呤(O~6—MeGua)受体蛋白测定及其底物O~6-[~8H-Me]Gua DNA的制备方法。废物与受体蛋白反应后,甲基从O~6-[~3H-Me]Gua DNA转移到受体蛋白,生成甲基-S-半胱氨酸(Me-S-Cys)。经盐酸水解后,直接测定酸不溶部分的受体蛋白沉淀。方法简便、快速、准确。     

Specific assay of alpha-dextrin 6-glucanohydrolase using labeled pullulan

A new method was developed for the assay of alpha-dextrin 6-glucanohydrolase (EC 3.2.1.41), which hydrolyzes the alpha-D (1----6) glucosidic bonds occurring in polyglucans such as pullulan and amylopectin limit dextrins. After enzymatic hydrolysis of a pullulan-dye conjugate, the remaining substrate is precipitated by adding ethanol. Colored reaction products are determined by measuring the supernatant absorbance at 534 nm. The assay is simple, specific, and suitable for both plant and bacterial enzymes.     

An ultraviolet-spectrophotometric method with 2-cyanoacetamide for the determination of the enzymatic degradation of reducing polysaccharides.

A rapid, facile, and sensitive uv-spectrophotometric assay has been developed for the determination of the enzymatic degradation of polysaccharides that generates reducing sugars. The assay was carried out with 2-cyanoacetamide in a single test tube. The solution was left at pH 9 by the addition of borate buffer within 5 min. Measurement of the reaction mixture at 274 nm allows a simple determination up to 600 mumol/liter of reducing sugars. The coefficient of variation was less than 2% on all measurements. The assay was developed with pectin and polygalacturonic acid from apples and has been compared with the Somogyi-Nelson method. The new assay was then exemplarily used for the determination of the enzymatic hydrolysis products of pectin from cotton.     

Thermostable continuous coupled assay for measuring glucose using glucokinase and glucose-6-phosphate dehydrogenase from the marine hyperthermophile Thermotoga maritima

A novel, thermostable adaptation of the coupled-enzyme assay for monitoring glucose concentrations was developed for an optimal temperature of 85 degrees C. This is the first report of a thermostable glucostat from a marine hyperthermophile. The continuous assay, using glucokinase (Glk) and glucose-6-phosphate dehydrogenase (Gpd) from Thermotoga maritima, demonstrated robust activity over a range of temperatures (75-90 degrees C) and pH values (6.8- 8.5). Purified glucokinase had a monomeric molecular mass of 33.8kDa while that of glucose-6-phosphate dehydrogenase (D-glucose 6-phosphate:NADP oxidoreductase) was 57.5kDa. The high-temperature assay provided a method for directly assaying the activity of another hyperthermophilic enzyme, 1,4-beta-D-glucan glucohydrolase (GghA) from Thermotoga neapolitana. To provide a benchmark for protein-engineering experiments involving GghA, a three-enzyme continuous assay (performed at 85 degrees C), linking wild-type GghA, Glk, and Gpd, measured glucose produced from GghA's hydrolysis of cellobiose, one of GghA's secondary substrates. The assay established the kinetic behavior of wild-type GghA toward cellobiose and was used to screen for changes in the catalytic efficiency of variant GghA(s) induced by random mutagenesis. The assay's development will allow high-throughput screening of other thermostable glucose-producing enzymes, including those applicable to commercial biomass conversion.     

A sensitive fluorimetric rate assay for biotinidase using a new derivative of biotin, biotinyl-6-aminoquinoline

The previously reported method for the estimation of biotinidase (EC 3.5.1.12) is an endpoint colorimetric assay based on the hydrolysis of biotinyl-4-aminobenzoate, followed by diazotization, and is not suitable for our studies of biotinidase. A fluorimetric rate assay of biotinidase which uses a newly synthesized derivative biotinyl-6-aminoquinoline is described here.     

An assay for bacterial and eukaryotic chondroitinases using a chondroitin sulfate-binding protein

A simple, rapid, and reproducible microtiter-based chondroitinase (CSase) assay is reported here, based on the competition of chondroitin sulfate (CS) with immobilized hyaluronan (HA) for the binding of TSG-6 protein, the product of TNF-inducible gene 6. Although the catabolic reaction of bacterial and other prokaryotic CSase enzymes, often referred to as the chondroitin lyases, can be followed by tracking the generation of unsaturated bonds by the spectrophotometrical determination of the absorbance at 232 nm, no rapid, sensitive, and simple assay has been devised to date for measuring the activity of the vertebrate enzymes that cleave their substrate exclusively by hydrolysis. We provide data demonstrating that the CSase assay described here is suitable for the determination of the activities of both classes of enzymes. For the bacterial enzyme CSase ABC, both the determination of the absorbance at 232 nm and the assay based on TSG-6 binding are suitable using the same range of enzyme activities. However, for testicular hyaluronidase, considerably higher enzyme activities were needed to cleave CS than to cleave HA. Using the HA-binding domain of aggrecan for a comparison, we determined that the interaction between TSG-6 and chondroitin sulfate is uniquely suited for this CSase assay.     

Use of Yarrowia lipolytica hexokinase for the quantitative determination of trehalose 6-phosphate

Abstract This paper describes a procedure for the quantitative determination of trehalose 6-phosphate (T6P) based on its ability to inhibit hexokinase from Yarrowia lipolytica . The assay is linear between 1 nmol and at least 8 nmol. The concentration of T6P in wild-type Saccharomyces cerevisiae (0.15 mM) and in ras2 mutants (0.25 mM) remained unchanged in the exponential or stationary phase of growth or after heat shock. A tps1 mutant affected in T6P synthase did not show detectable T6P. Heat shock increased the concentration of T6P in Schizosacharomyces pombe from 0.43 to 0.75 mM.     

Recruiting mechanism of the AAA peroxins, Pex1p and Pex6p, to Pex26p on the peroxisomal membrane

A peroxisomal C-tail-anchored type-II membrane protein, Pex26p, recruits AAA ATPase Pex1p-Pex6p complexes to peroxisomes. We herein attempted to gain mechanistic insight into Pex26p function. Pex26pΔ33-40 truncated in amino-acid residues at 33-40 abolishes the recruiting of Pex1p-Pex6p complex to peroxisomes and fails to complement the impaired phenotype of pex26 CHO cell mutant ZP167, thereby suggesting that peroxisomal localization of Pex1p and Pex6p is indispensable for the transport of matrix proteins. In in vitro transport assay using semipermeabilized CHO cells, Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. This finding is confirmed by the assay using Walker-motif mutants. Transport of Pex1p and Pex6p is temperature-dependent. In vitro binding assays with glutathione-S-transferase-fused Pex26p, Pex1p and Pex6p bind to Pex26p in a manner dependent on ATP binding but not ATP hydrolysis. These results suggest that ATP hydrolysis is required for stable localization of Pex1p to peroxisomes, but not for binding to Pex26p. Moreover, Pex1p and Pex6p are altered to a more compact conformation upon binding to ATP, as verified by limited proteolysis. Taken together, Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by the ATPase cycle.     

A novel fluorometric assay for ADP-ribose pyrophosphatase activity

ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose to ribose-5-phosphate and AMP. The ADPRase activity have been assessed by coupling the reaction to alkaline phosphatase and colorimetrically measuring the amount of inorganic phosphate released from AMP that is one of the products of ADPRase. Another but less sensitive colorimetric method has been employed: the reaction mixture was treated with charcoal to adsorb the adenine-containing compounds such as AMP and ADPR and subsequently remaining ribose-5-phosphate was measured colorimetrically. However, the measurement of inorganic phosphate cannot be feasible to assay ADPRase in phosphate-containing samples and the determination of ribose-5-phosphate also is less sensitive. Here we develop a fluorescent assay for ADPRase that utilizes 1, N(6)-etheno ADP-ribose, a fluorescent analogue of ADP-ribose. This method measures fluorescent 1, N(6)-etheno adenosine that is produced by coupling the hydrolysis of 1, N(6)-etheno ADP-ribose to dephosphorylation with alkaline phosphatase. The fluorometric assay is comparable in sensitivity and useful for ADPRase assay in phosphate-containing samples.     

An enzymatic colorimetric assay for glucose-6-phosphate

A specific colorimetric assay for the determination of glucose-6-phosphate (G6P) was developed. This assay is based on the oxidation of G6P in the presence of glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP⁺); the NADPH thereby generated reduces the tetrazolium salt WST-1 [2-(4-indophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt] to water-soluble yellow-colored formazan with 1-methoxy-5-methylphenazium methylsulfate (1-mPMS) as an electron carrier. The assay is optimized for reaction buffer pH, enzyme/dye concentration, and reaction time course. The limit of detection of the assay is 0.15 μM (15 pmol/well). The usefulness of the assay is demonstrated by the accurate measurement of the G6P concentration in fetal bovine serum (FBS).     

An enzyme-linked immunosorbent assay for 6-keto PGF1 alpha

An enzyme-linked immunosorbent assay for 6-keto prostaglandin F1 alpha, a stable metabolite of prostacyclin, has been developed. The assay allows quantitation of 6-keto PGF1 alpha in the range 1-200 pg/0.1 ml and shows very low cross reactivity to nine other prostaglandins. Dose dependent stimulation by thrombin of 6-keto PGF1 alpha formation in human endothelial cells in culture has been used to verify the assay. Quantitation by the enzyme linked immunosorbent assay agrees closely with determination by radioimmunoassay.     

Determination of the substrate specificity of the phospholipase D from Streptomyces chromofuscus via an inorganic phosphate quantitation assay

The substrate specificity for phospholipase D from Streptomyces chromofuscus (PLD(Sc)) has been determined utilizing an assay based on the quantitation of inorganic phosphate. 1,2-Di-n-hexanoyl phosphatidylcholine (C6PC), phosphatidylethanolamine (C6PE), phosphatidylserine (C6PS), phosphatidylglycerol (C6PG), and an unnatural phospholipid bearing a neohexyl headgroup (C6PDB) were examined as substrates. The assay relies on the quenching of the PLD(Sc)-catalyzed hydrolysis of the phospholipid substrates with EDTA followed by the hydrolysis of the phosphatidic acid product with alkaline phosphatase. The inorganic phosphate thus released is quantitated through the formation of a complex with ammonium molybdate, which has an absorbance maximum at 700 nm. To minimize the time involved and the reagents consumed, the assay is conducted in 96-well plates. The results of this study indicate that the catalytic efficiency for PLD(Sc) on the substrates is C6PC > C6PS approximately C6PE > C6PG > C6PDB.     

Determination of CoASH by high-performance liquid chromatography and its application in the assay of long-chain acyl-CoA derivatives.

A high-performance liquid chromatographic procedure is described for the determination of picomole amounts of CoASH, using a microparticulate, strong anion-exchange resin. The method was applied in a systematic study to optimize the conditions for alkaline hydrolysis of palmitoyl-CoA. The procedure, which ensures 100% recovery of CoASH by hydrolysis of paimitoyl-CoA, was found to be convenient also for the assay of the endogenous content of long-chain acyl-CoA derivatives in biological material.     

Oxidation in H2O and D2O of 6-ethyl-5H-dibenz(c,e)azepine and 1-methylnicotinamide by aldehyde oxidase from rabbit liver

We report the solvent hydrogen isotope effects associated with the oxidation of 6-ethyl-5H-dibenz(c,e)azepine (6-ED) and 1-methylnicotinamide (1-MN) catalyzed by aldehyde oxidase from rabbit liver using two assay methods. The first uses 2,6-dichlorophenolindophenol (DCI) as electron acceptor in an indirect assay in which the bleaching of DCI is measured as the substrate is oxidized. The second uses molecular oxygen as electron acceptor in a direct assay in which the oxidation of 1-MN to its pyridones is accompanied by an increase in absorbance at 300 nm and the oxidation of 6-ED to its lactam product is accompanied by a decrease in absorbance at 335 nm. We have found a solvent hydrogen isotope effect close to unity in the turnover number for each substrate and for each assay method. The solvent hydrogen isotope effects on kcat/Km ranged from 0.4 to 1.1. We conclude that changes in bonding of hydrogen in solvent water, including hydrolysis of or general base attack on an enzyme-intermediate complex, do not play a rate-contributing role in the maximal velocity of oxidation of 1-MN and 6-ED catalyzed by aldehyde oxidase from rabbit liver.     

A sensitive fluorometric assay for protein-bound DOPA and related products of radical-mediated protein oxidation

Oxidative attack on proteins results in the hydroxylation of tyrosyl residues to protein-bound DOPA (3,4-dihydroxyphenylalanine). Existing methods for assaying protein-bound DOPA have poor sensitivity and numerous possible interferences, such that accurate determination (especially of very low DOPA concentrations) has required time-consuming acid hydrolysis and HPLC analysis with fluorometric detection. This work presents a sensitive and selective assay for peptide or protein-bound o-benzoquinones derived from DOPA based on fluorometric detection of ethylenediamine derivatives. Detection limits for protein-bound DOPA are in tbe range 0.53–4.70 ng/mL for the assay mixture, corresponding to sample DOPA concentrations of 0.59–5.30 ng/mL (representing a minimum of 6–54 pmole detected), depending on the particular protein/peptide under study. The assay response increases linearly with DOPA concentration, and also with the extent of radical exposure of the protein. The assay is a simple and fast way to assess DOPA formation and thus oxidative damage in a protein.     

An enzyme-linked immunosorbent assay for 6-keto PGF

An enzyme-linked immunosorbent assay for 6-keto prostaglandin F_1α, a stable metabolite of prostacyclin, has been developed. The assay allows quantitation of 6-keto PGF_1α in the range 1–200 pg/0.1 ml and shows very low cross reactivity to nine other prostaglandins. Dose dependent stimulation by thrombin of 6-keto PGF_1α formation in human endothelial cells in culture has been used to verify the assay. Quantitation by the enzyme linked immunosorbent assay agrees closely with determination by radioimmunoassay.     

Improved viscometric assay for cellulase

A viscometric assay for the determination of carboxymethylcellulase is described. The method, which is developed from a direct theoretical approach, takes into account a kinetic energy correction and the non-Newtonian behavior of carboxymethylcellulose solutions. The enzymic hydrolysis of carboxymethylcellulose shows strong competitive inhibition by the products of the reaction and a method is presented for estimating the initial velocity of such reactions. A direct chemical determination of reducing end groups was used to confirm the validity of this approach.     

Determination of glucose-6-phosphatase activity using the glucose dehydrogenase-coupled reaction

We present a method to determine glucose 6-phosphate activity. This assay measures the rate of glucose released in the glucose-6-phosphatase reaction. The glucose is oxidized to beta-D-gluconolactone by glucose dehydrogenase in a coupled reaction that uses NAD(P)+. The determination is rapid, reproducible, and does not require withdrawal, precipitation, centrifugation, or neutralization steps. This method provides a simple resolution to the problem of the nonspecific appearance of Pi, which is especially important in studies of regulation of glucose-6-phosphatase performed in the presence of ATP.