Enzyme assays for

Figure 7-10. Coupled enzyme assay for hexokinase activity. The production of glucose 6-phosphate by hexokinase is coupled to the oxidation of this product by glucose-6-phosphate dehydrogenase in the presence of added enzyme and NADP". When an excess of glucose-6-phosphate dehydrogenase is present, the rate of formation of NADPH, which can be measured at 340 nm, is governed by the rate of formation of glucose 6-phosphate by hexokinase.

Finally, one must take into account that in using a coupled enzyme assay one must produce, or purchase, not only the target enzyme of interest but also the coupling enzymes and any co-substrates required for these additional protein reagents. Hence a coupled enzyme assay can be quite expensive to implement, especially for large library screening. In some cases the cost may be prohibitive, precluding the use of a particular coupled enzyme assay for HTS purposes. 

Figure 7.1 Concentration-response plots for a series of compounds displaying Kf9p values ranging from 100 to 0.01 nM, when studied in an enzyme assay for which the enzyme concentration is 50nM. The lines through the data sets represent the best fits to the standard isotherm equation that includes a non-unity Hill coefficient (Equation 5.4). Note that for the more potent inhibitors (where Kf" < [E]T), the data are not well fit by the isotherm equation.

Enzyme assays For enzyme activities, total protein is extracted from seedlings of controls and after 72 h-treatment under native conditions. Primary roots are homogenized in extraction buffer (for formulation see... 

Goven, A.J., S.C. Chen, L.C. Fitzpatrick, and B.J. Venables. 1994. Lysozyme activity in earthworm (Lumbricus terrestris) coelomic fluid and coelomocytes enzyme assay for immunotoxicity of xenobiotics. Environ. Toxicol. Chem. 13 607-613. 

McClure was among the first to treat the kinetics of coupled enzyme assays, and others have discussed essential aspects of experimental design. Rudolph et al provided an extensive list of coupled-enzyme assays for selected enzyme reaction products, and several examples are shown in Table I. 

He W, Voznyi YV, Boer AM, Kleijer WJ, van Diggelen OP (1993) A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease type D (MPS HID). J Inherit Metab Dis 16 935-941... 

Van Diggelen OP, Zhao H, Kleijer WJ, Janse HC, Poorthuis BJHM, van Pelt J, Kamerling JP, Galjaard H (1990) A fluorimetric enzyme assay for the diagnosis of Morquio disease type A (MPS IV A) Clin Chim Acta 187 131-140... 

A.l. 4 Enzyme Assay for CDG-lld (Golgi UDP-Galactose N-Acetylglucosamine /3-1,4-Galactosyltransferase I Deficiency [11])... 

Verhoeven NM, Roos B, Struys EA, Salomons GS, van der Knaap MS, Jakobs C (2004) Enzyme assay for diagnosis of guanidinoacetate methyltransferase deficiency. Clin Chem 50 441-443... 

Stauffer, C.E. 1989. Peptide hydrolases. In Enzyme Assays for Food Scientists, pp. 133-161. VanNos-trand Reinhold, New York. 

Mary Ellen Jones and her colleagues set out to purify the enzyme or enzymes involved in these two reactions. Their main goal was to determine whether the two reactions are carried out by one protein or more than one. Their findings indicated that the two reactions were both catalyzed by the same enzyme, consisting of a single polypeptide chain. To demonstrate this fact, it was necessary to monitor both enzyme activities at each step in the purification and show that both activities copurified. For this purpose, Jones used specific enzyme assays for both enzyme activities. All fractions were assayed for both enzymatic activities at each stage of the purification. 

Bitton, G. and Morel, J.L. (1998) Microbial enzyme assays for the detection of heavy metal toxicity, in P.G. Wells, K. Lee and C. Blaise (eds.), Microscale Testing in Aquatic Toxicology Advances, Techniques, and Practice, CRC Press, Boca Raton, FL, pp. 143-152. 

Bitton, G. and Morel, J.L. (1998) Enzyme assays for the detection of heavy metal toxicity, in P.G. Wells,... 

Cohen, C.B., Chin-Dixon, E., Jeong, S., Nikiforov, T.T., A microchip-based enzyme assay for protein kinase A. Anal. Biochem. 1999, 273, 89-97. 

L(+)-Lactate dehydrogenase is specific for l(+)-lactate and does not react with d(—)-lactate. LDH is used in coupled enzyme assays, for example in the determination of ATPase (Penefsky and Bruist 1984), myokinase (Brolin 1983), and pyruvate kinase (Beutler 1971). It may also be used in the determination of lactate (Noll 1984), pyruvate (Lamprecht and Heinz 1984), and various other metabolites. 

Two bacterial enzymes were used in a linked-enzyme assay for heroin plus metabolites. 

I. LCAT deficiency. Cholesterol associated with HDL cannot be esterified. There is a buildup of unesterified cholesterol, with comeal opacities, renal insufficiency, hemolytic anemia, and premature atherosclerosis. The diagnosis may be made on enzyme assay for plasma LCAT. 

ECE inhibitors can be evaluated in enzyme assays, isolated tissues and whole animals. A range of tissues (for example, vascular endothelium, vascular smooth muscle, lung) demonstrate phosphoramidon-sensitive ECE activity and provide a choice of starting points for an enzyme assay for ECE inhibitors. Partially purified ECE, such as that from rabbit lung, is... 

T6. Tiffany, T. 0., Johnson, G. F., and Chilcote, M. E., Feasibility of multiple simultaneous enzyme assays, for diagnostic purposes, with the GeMSAEC fast analyzer. Clin. Chem. 17, 715-720 (1971). 

El55 Gerard, S. and Khayam-Bashi, H. (1984). Negative interference with the Ektachem (Kodak) enzymic assay for creatinine by high serum glucose. Clin. Chem. 30, 1884. 

Anhydro-D-glucitol (AG, 164 g/mol) is one of the main sugar alcohols in human cerebrospinal fluid and blood. In plasma, the normal AG level is 24.6 7.2 mg/L (539 patients tested), while patients with diabetes mellitus show reduced AG levels of 7.3 7.1 mg/L (808 patients tested). Because reduced AG levels are specific indicators of this type of diabetes, a diagnostic enzyme assay for AG has been developed. The assay employs the enzymes... 

Product inhibition is a cause of nonlinearity of reaction progress curves during fixed-time methods of enzyme assay. For example, oxaloacetate produced by the action of aspartate aminotransferase inhibits the enzyme, particularly the mitochondrial isoenzyme. The inhibitory product may be removed as it is formed by a coupled enzymatic reaction malate dehydrogenase converts the oxaloacetate to malate and at the same time oxidizes NADH to NADL... 

Ludvigsen CW, Thurn JR, Pierpont GL, Eckfeldt JH. Kinetic enzymic assay for D(-)-lactate, with use of a centrifugal analyzer. Clin Chem 1983 29 1823-5. 

Morris HC, Overton PD, Ramsay JR, Campbell RS, Hammond PM, Atkinson T, et al. Development and validation of an automated enzyme assay for paracetamol (acetaminophen). Clin Chem Acta 1990 187 95-104. 

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