Substrates methylumbelliferyl

A few acetates of phenols have been used extensively as probes to investigate esterases, e.g., phenyl acetate (7.15), 4-nitrophenyl acetate (7.16), a-naphthyl acetate (7.17) and 7-acetoxy-4-mc(hyl-27/-[l bcnzopyran-2-onc (4-methylumbelliferyl acetate, 7.18). Such substrates are easy to handle and their phenolic metabolite is readily analyzed, allowing convenient monitoring of the reaction. 

Series of homologous esters have been investigated to try to establish structure-metabolism relationships, however partial and limited the latter may be. This aspect will be discussed again in the context of prodrugs (Chapt. 8). Here, we mention a few representative studies in which model substrates were used. Table 7.2 documents the substrate specificity of a rabbit liver carboxylesterase (ES-1A) toward homologous series of methyl, 4-nitrophenyl, a-naphthyl, /1-naphthyl, and 4-methylumbelliferyl esters [41]. In... 

To detect the fate of liposomes, the nonfluorescent 4-methylumbelliferyl-p-D-glycoside [4MU-(3-D-glc)] can be encapsulated. 4MU-(3-o-glc) is a substrate for lysosomal 3-d-glycosidase that shows fluorescence after enzymatic hydrolysis to methylumbelliferone (125). 

The late discovery of acetyl xylan and feruloyl esterases has been partly due to the lack of suitable substrates. Xylans are often isolated by alkaline extraction, in which ester groups are saponified. Treatment of plant materials under mildly acidic conditions, as in steaming or aqueous-phase thermomechanical treatment, leaves most of the ester groups intact. These methods, however, partly hydrolyze xylan to shorter fragments (63,69). Polymeric acetylated xylan can be isolated from delignified materials by dimethyl sulfoxide extraction (70). The choice of substrate is especially important in studies of esterases for deacetylation of xylans. The use of small chromophoric substrates (p-nitrophenyl acetate, a-naphthyl acetate, and methylumbelliferyl acetate) analogously to the assays of disaccharidases may lead to the monitoring of esterases unable to deacetylate xylan (33, 63, 64). 

Figure 2. Specificities of Endoglucanases (EGA, EGB, EGC, EGD) from Clostridium thermocellum cloned in E. coli (10). The substrates (MeUmb-Glc , n = 2-5, MeUmbLac) are depicted (symbols A, (3-1,4 galactopyra-nosyl , / -1,4 glucopyranosyl , 4-methylumbelliferyl) and the arrows indicate scission points as determined by HPLC (1).

Substrate buffer 1.657 mg 4-methylumbelliferyl-a-L-(idopyranosid)-uronic acid-sodium salt (6.6 mM Toronto Research Chemicals) is dissolved in 333 pi demineralized water. The same volume of reaction buffer is added and the solution is vor-texed for 30 s. The substrate buffer is stored at -20°C and protected from light. 

Substrate buffer 0.60 mg 4-methylumbelliferyl-a-iduronate-2-sulfate (Moscer-dam Substrates, Rotterdam, The Netherlands) is dissolved in 1.0 ml acetate buffer to yield a 1.25 mmol/1 solution. 

Substratebuffer 9.10 mg4-methylumbelliferyl-a-D-N-sulfoglucosamide(Moscer-dam Substrates) is dissolved in 1 ml reaction buffer to yield a 20 mM solution. 

Substrate buffer 4.81 mg of the sodium salt of 4-methylumbelliferyl-a-N-acetyl-glucosaminide-6-sulfate (Moscerdam Substrates) is dissolved in 1 ml of reaction buffer. The substrate buffer is stored at -80°C and protected from light. 

Substrate/buffer solution dissolve 1.70 mg 4-methylumbelliferyl-jS-D-galacto-pyranoside (MW 338.3) in 10 ml of reaction buffer to a final concentration of... 

Substrate/buffer solution (prepare fresh) Mix 75 pi oleic acid, 50 pi taurocholate and 250 pi 6-hexadecanoylamino-4-methylumbelliferyl- /3-d-galactopyranosidc stock solution in a 1.5-ml Eppendorf vial and take the mixture to dryness under a gentle stream of nitrogen. Add 500 pi of reaction buffer at room temperature,... 

In practice it is often more convenient to measure the release of a phenol from an aryl phosphomonoester. Standard serum phosphatase methods employ phenyl phosphate (188), p-nitrophenyl phosphate (189), phenolphthalein monophosphate (140), or thymolphthalein monophosphate (141) where the phenol released can be determined spectrophoto-metrically [only the Bodansky method (13) uses a Pi determination]. A number of fluorogenic substrates have been used for phosphatase studies, e.g., jS-naphthyl phosphate (30, 148), 4-methylumbelliferyl phosphate (143), and 3-O-methylfluorescein phosphate (144) The main advantage here is the much greater sensitivity of fluorescence as compared with spectrophotometric assays as little as 1 pmole of 4-methyl-umbelliferone can be detected in continuous assay. 

Coumarins also have a C6-C3 skeleton, but they possess an oxygen heterocycle as part of the C3-unit. There are numerous coumarins, many of which play a role in disease and pest resistance, as well as UV-tolerance. The coumarin umbelliferone (1.21) is popular in enzyme assays. Umbelliferone esters can be used as a substrate for non-specific esterase enzyme assays and in fluorescent immunoassays (Jacks and Kircher, 1967). In order to quantify the enzyme activity of the popular reporter gene P-glucuronidase (GUS), plant extracts can be incubated with 4-methylumbelliferyl P-D-glucuronide (4-MUG 1.22), which upon hydrolysis... 

Hoppe, H.-G. 1983. Significance of exoenzymatic activities in the ecology of brackish water Measurements by means of methylumbelliferyl-substrates. Marine Ecology Progress Series 11 299-308. 

In another report, Michaelis-Menten parameters of ALP have been obtained using a one-shot Lineweaver-Burk (reciprocal) plot. This can be achieved by simultaneously measuring the conversion of 12 independent concentrations of the substrate (4-methylumbelliferyl phosphate) created on-chip. The enzyme was streptavidin-conjugated ALP that was linked to biotinylated phospholipid bilayers coated inside PDMS microchannels. The blue fluorescence of the enzymatic product, 7-hydroxy-4-methyl coumarin, was measured [ 1043]. The surface-bound enzyme was found to have a lower (sixfold) turnover rate than the free enzyme in solutions. After diffusion mixing between two streams (substrate and buffer)... 

Figure 16-4. The P-glucosidase reaction illustrating use of the artificial substrate MUGlc (4-methylumbelliferyl-P-D-glucopyranoside).

Esterases. Acetyl esterase (EC 3.1.1.6) removes acetyl esters from acetylated xylose and short-chain xylo-oligomers. It s polymeracting counterpart, acetyl xylan esterase (EC 3.1.1.72), has a similar activity, but prefers polymeric xylan.244 In addition to acetate-specific enzyme detection kits, HPLC or GC analysis of acetate release from native extracted xylan and chemically acetylated xylan, colorimetric substrates, such as p-nitrophenol acetate and /3-napthyl acetate, or the fluorometric substrate, 4-methylumbelliferyl acetate are also used to assay acetyl esterases.244,253 The third esterase, ferulic acid esterase (EC 3.1.1.73), hydrolyzes the ester bond between ferulic acid or coumaric acid and the arabinose side chain of arabinoxylan. Assays for this activity are usually carried out using starch-free wheat bran or cellulase-treated gramineous biomass as a substrate and monitoring ferulic or coumaric acid released by HPLC or TLC. When preparing enzyme-treated substrates, care must be taken to employ phenolic-acid-esterase-free cellulases.244 Other substrates include methyl and ethyl esters of the phenolic acids, as well as finely ground plant biomass.240,254,255... 

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