4-Nitrophenyl butyrate

FIGURE 68.1. CarbE and ChE activities in liver versus blood plasma of thiouracil fed rats. The animals received rat chow supplemented with 0.05% thiouracil for 22, 30, or 38 days. Methyl butyrate hydrolysis (A A) and 4-nitrophenyl butyrate hydrolysis ( O) were measured in liver homogenate and plasma in accordance with Sterri et al. (1985b), and acetylcholine hydrolysis ( ) by the method of Sterri and Fonnum (1978). The activities are percent of corresponding control (=100%) in male (closed symbols) and female (open symbols) animals fed standard rat chow. 

The various isoenzymes of plasma CarbE might have different importance with respect to their scavenger function. Results show a 2.5-fold higher plasma CarbE activity with methyl butyrate in adult female rats compared to males, whereas the soman toxicity does not differ between them (Sterri et al., 1985a). The same authors also report adult values of soman toxicity in 31 day old rats of both sexes, whereas at that time the methyl butyrate hydolyzing capacity of female plasma is far from fully developed. Thus, the isoenzyme with the highest preference for 4-nitrophenyl butyrate as substrate (Sterri and Foimum, 1989) may be the most effective soman scavenger of plasma, at least in rats. 

Sterri, S.H., Johnsen, B.A., Fonnum, F. (1985b). A radiochemical assay method for carboxylesterase, and comparison of enzyme activity towards the substrates methyl[l- C]butyrate and 4-nitrophenyl butyrate. Biochem. Pharmacol. 34 2779-85. 

Lipase-Catalyzed Hydrolysis of 4-Nitrophenyl Butyrate Huang... 

Several different carboxylic esters may be hydrolyzed by CarbEs among them, several may also be hydrolyzed by BuChE and A-esterases. For CarbEs, the aromatic but5uyl esters are better substrates than the acetyl esters, whereas the opposite holds for A-esterase (Augustinsson, 1959 Aldridge 1953 Ecobichon, 1970). The hydrolysis of two simple aliphatic and aromatic but57ryl esters (namely, methyl but5n-ate and 4-nitrophenyl butyrate) is catalyzed by CarbE, with only minimal contributions by ChE and A-esterase in a series of rat tissues, except... 

The results presented in Table XVIII imply that rate enhancements are derived from association of the catalyst and substrate prior to reaction. In accord with this idea, the release of p-nitrophenol from p-nitrophenyl butyrate follows saturation kinetics in the presence of 16. The dissociation constant of the complex is reported to be 9.9 X 10-4 M, indicating that binding is very strong. Unfortunately, dissociation constants of the complexes of 16 with the other substrates have not yet been obtained. 

Remarkably, Brassica napus pollen was reported to have a 22 kDa cutinase that cross-reacted with antibodies prepared against F. solani f. pisi cutinase [134]. Although a 22 kDa and a 42 kDa protein that catalyzed hydrolysis of p-nitrophenyl butyrate were found in this pollen, only the former catalyzed cutin hydrolysis. Immunofluorescence microscopic examination suggested that the 22 kDa protein was located in the intine. Since the nature of the catalytic mechanism of this enzyme has not been elucidated, it is not clear whether this represents a serine hydrolase indicating that plants may have serine and thiol cutinases. The role of the pollen enzyme in controlling compatibility remains to be established. 

Fontes et al. (1998b) Batch Hydrolysis of p-nitrophenyl butyrate Cutinase... 

Shirai, K., Jackson, R.L. 1982. Lipoprotein lipase-catalyzed hydrolysis of />-nitrophenyl butyrate. J. Biol. Chem. 257, 1253-1258. 

These events all occur prior to the time of conidial germination. Concurrent with the release of the liquid film (Afi.) it was shown that there is a release of esterase with activity against p-nitrophenyl butyrate (A5). Activity was released in two stages, the first occurring within 2 min of contact and the second between 10 and 15 min of contact (Figure 2). 

Geldmacher von Mallinckrodt (Gil) has shown that the choice of substrate is of paramount importance when measuring the activation by oximes of plasma cholinesterase which has been inhibited by alkyl phosphates. This is because oximes have been found to cause spontaneous hydrolysis of o-nitrophenyl butyrate and the thiocholines, but not of butyrylcholine or benzoylcholine. 

Yarrowia lipolytica lipase (YLL) activity was performed by continuously measuring the increase in the absoibance in 348 nm produced by the release of p-nitro-phenol in the hydrolysis 0.4 mM p-nitrophenyl-butyrate (pNPB) in 25 mM sodium phosphate buffer pH 7 and 28°C. The reaction was initialized by addition of 0.2 ml of lipase suspension to 2.5 ml of substrate solution. One international unit (lU) of pNPB was defined as the amount of immobilized YLL necessary to hydrolyze 1 pmol ofpNPB per minute in assay conditions [21]. 

This material is not very surface-active, but it is very efficient at solubilizing organic solutes. The electronic spectra of absorbed dyes is shifted and hexapus will bind /)-nitrophenyl butyrate and inhibit its reaction with OH . Hexapus apparently forms a hydrophobic cavity which can incorporate solutes. 

Figure 8. Time course of appearance of cutinase as measured by immunochemical techniques (left) and cutinase activity as measured by -nitrophenyl butyrate hydrolysis (right) in the extracellular fluid of spore suspensions of F. solani f. sp. pisi induced with either cutin hydrolysate or purified Cie dihydroxy acid. (Reproduced with permission from Ref. 35. Copyright 1986 The National Academy of Sciences.)...

Now that it has been clearly established that Upase hydrolyzes only water-insoluble substrates in a heterogeneous system (6), any method proposing the use of a water-soluble substrate must be rejected. Examples of water-soluble substrates that have been used are the Tweens (189,190) and p-nitrophenyl aeetate and p-nitrophenyl butyrate (191). In fact an entirely different enzyme appears to hydrolyze the Tweens it has been purified from pancreas (192) and been termed a Tweenase (193). An enzyme catalyzing a rapid rate of hydrolysis of Tweens has also been purified from rat adipose tissue (194). 

Several methods have been described that employ a special substrate designed to give a colored end product after hydrolysis, or one that can be easily converted to a colored product. The p-nitrophenol liberated from the hydrolysis of p-nitrophenyl butyrate is readily measured directly (191). y8-Naphthyl laurate has been used as a substrate for serum lipase. The )8-naphfhol hberated is combined with tetrazotized di-o-anisidine to give a pigment (211, 212). Several esters of 2-naphthol-6-sulfonic acid have been used as substrates for milk lipase. The naphthol-sulfonic acid liberated is determined by the Folin-Ciocalteu reagent (213). 

The addition of alcohol, as cosurfactant, to the [Cgmim][TfjN]/AOT/water system leads to stable w/IL microemulsions. DLS and protein solubilization experiments confirm the existence of an aqueous nanoenvironment in the IL phase of [C mirnTf N]/ AOT/l-hexanol/water microemulsions [67]. The kinetics of the enzymatic reactions were performed in this quaternary system. Specifically, lipase-catalyzed hydrolysis of p-nitrophenyl butyrate (p-NPB) was used as a model reaction [68]. In a similar way, the hpase-catalyzed hydrolysis of p-NPB was investigated to evaluate the catalytic efficiency in water/AOT/Triton X-100/[C mim][PFJ [69]. A large single-phase microemulsion region can be obtained from the combination of two surfactants in IL. 

The lipase-catalyzed hydrolysis of p-nitrophenyl butyrate (p-NPB) was used as a model reaction. It was found that the hydrolysis rate was faster in the water-in-IL microemnlsions than in the water-in-isooctane microemulsions. Hie intrinsic activity of lipase in the IL microemulsion was about three times higher than that of water/ AOT/isooctane microemulsions of AOT under the given experimental conditions. The enhanced catalytic activity of lipase in water-in-IL microemulsions may be due to (i) aqueous microenvironmental changes, (ii) the partition of the substrate or other molecules involved in the reaction between water and IL phases, and (iii) the existence of 1-hexanol as a cosurfactant. 

Lipase immobilized on SMOF by glutaraldehyde method Lipase [166] activity tested by determination of p-nitrophenyl butyrate hydrolysis products SMOF evaluated for synthesis of butyl laurate from lauric acid and n-butanol in n-hexane and n-heptane percentage yield up to 99% with 38 s residence time at a flow rate of 1 pl/min... 

A soln. of y-(p-nitrophenyl)butyric acid and anisole in polyphosphoric acid heated 0.5 hr. at 120-125° in an oil bath p -methoxy-y-(p-nitrophenyl)butyrophenone. 

Figure 11.5 First-order rate constants plotted against CTAB concentrations for the three p-nitrophenyl esters in the AMP buffer (pH 9.59) at 25° C. The solid lines are calculated from Equation 11.26. The dashed line represents the rate-CTAB concentration profile for p-nitrophenyl butyrate in a 0.01 m carbonate buffer (pH 9.87) at 25° C. From Funasaki [29].

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