1 Naphthyl Acetate - an overview (2023)

Reagents

4.14.13.1 Distinguishing Carboxylesterase from Butyrylcholinesterase

Carboxylesterase and butyrylcholinesterase are often confused because they hydrolyze many of the same esters including p-nitrophenyl acetate, o-nitrophenyl acetate, α-naphthyl acetate, β-naphthyl acetate, irinotecan, and cocaine. They are also inhibited by many of the same inhibitors including diisopropylfluorophosphate, paraoxon, nerve agents, tetraisopropylpyrophosphoramide (iso-OMPA), and cresylbenzodioxaphosphorin oxide (CBDP). A major difference between them is that butyrylcholinesterase reacts rapidly with positively charged compounds, for example, echothiophate, VX, benzoylcholine, and butyrylthiocholine, whereas carboxylesterases prefer substrates and inhibitors that have a neutral charge (Maxwell and Brecht 2001). Confusion in the literature persists to this day, even though distinct protein and gene sequences for butyrylcholinesterase and carboxylesterases are available. The confusion is especially noticeable in studies with rodents and especially in studies with rodent blood. Mouse plasma contains 30 times more carboxylesterase than butyrylcholinesterase and acetylcholinesterase proteins.

Rat plasma contains 800 times more carboxylesterase than butyrylcholinesterase protein (Maxwell et al. 1987b). Interspecies differences in response to soman were eliminated by pretreating animals with 2mgkg⁻¹ CBDP. The CBDP completely inhibited carboxylesterase in plasma and lung, and potentiated the effect of soman so that the LD₅₀ for all species clustered in a narrow range of 11.8–15.6μgkg⁻¹ (Maxwell et al. 1987a).

In contrast to rodents, humans have no carboxylesterase in blood (Li et al. 2005). This huge disparity between the esterase content of human and rodent blood affects the evaluation of toxic substances when rodents are used as models for humans. Effects on butyrylcholinesterase are disregarded because carboxylesterase dominates the outcome. This leads to the mistaken idea that butyrylcholinesterase is not important for detoxication of organophosphorus poisons in humans.

Another significant difference between mouse and human plasma is that mouse plasma has substantial acetylcholinesterase activity, whereas humans have almost no soluble acetylcholinesterase in plasma. In human blood, acetylcholinesterase is mainly in the red blood cells.

Methods to distinguish between carboxylesterase and butyrylcholinesterase in mouse blood as well as in living mice rely on the use of specific inhibitors. CBDP has frequently been used as a specific inhibitor of carboxylesterase. However, we have found that CBDP inhibits both butyrylcholinesterase and carboxylesterase. Living wild-type mice treated with a nontoxic dose of CBDP (2mgkg⁻¹) showed 95% inhibition of plasma carboxylesterase and 80% inhibition of plasma butyrylcholinesterase. Another drawback of CBDP is that it is not commercially available.

Bis(4-nitrophenyl)phosphate is a specific inhibitor of carboxylesterase in rats (Block and Arndt 1978). This conclusion is valid for rat plasma, because rat plasma contains almost no butyrylcholinesterase. However, use of this compound as a specific carboxylesterase inhibitor of mouse carboxylesterase is not valid, because both carboxylesterase and butyrylcholinesterase are inhibited.

A specific inhibitor of human liver and intestine carboxylesterase is the commercially available diketone compound benzil (Wadkins et al. 2005). We found that 4.5μmoll⁻¹ benzil inhibited carboxylesterase in mouse liver and intestine up to 90%, but did not inhibit mouse plasma carboxylesterase. Proteomic analysis has identified the major carboxylesterase in mouse plasma as ES1, accession number gi22135640 in strain 129Sv/J mice (Bhat et al. 2005). ES1 is different from liver and intestine carboxylesterase.

Iso-OMPA is often used as a specific inhibitor of butyrylcholinesterase. However, iso-OMPA is an effective inhibitor of rat plasma carboxylesterase (Yang and Dettbarn 1998).

Eserine inhibits butyrylcholinesterase but not carboxylesterase and is useful for assays of plasma carboxylesterase activity. Eserine cannot be injected into live animals, however, because eserine also inhibits acetylcholinesterase resulting in death of the animal.

Plasma carboxylesterase activity is commonly assayed with p-nitrophenyl acetate. Rodent plasma contains three other esterases that hydrolyze p-nitrophenyl acetate. To specifically measure carboxylesterase activity in mouse blood, one preincubates the plasma with 1mmoll⁻¹ ethylenediamine tetraacetic acid (EDTA) to inhibit paraoxonase, and 0.01mmoll⁻¹ eserine to inhibit acetylcholinesterase and butyrylcholinesterase.

Cytochemical features

Special stains performed on the aspirate can provide information about cell lineage. Myeloperoxidase positivity indicates that a cell is of the granulocytic lineage. Nonspecific esterases (alpha-naphthyl acetate and alpha-naphthyl butyrate) robustly stain cells of the monocytic lineage, and this staining can be blocked in monocytes by the addition of sodium fluoride. The specific esterase, naphthol ASD chloroacetate esterase, is positive in granulocytic lineages, as well as mast cells. Globules of PAS positivity are found in early erythroid precursors (pronormoblasts). An iron stain, which evaluates for the presence of ring sideroblasts as described earlier in the section on MDS, should also be performed.

Cytochemistry

Monocytes stain positively for alpha-naphthyl acetate esterase (nonspecific esterase) and alpha-naphthyl butyrate esterase but are alpha-naphthol AS-D chloroacetate-esterase-negative. The activity of both alpha-naphthyl acetate and butyrate esterase are inhibited by fluoride (in contrast to granulocytes). Monocytes have some granular PAS and Sudan black positivity, slight granular MPO positivity, strong staining for acid phosphatase and lack alkaline phosphatase activity. Monocytes contain lysozyme. Similar proportions of promonocytes, BM monocytes and blood monocytes have IgG-Fc, IgE-Fc and C3b receptors.

7.1.3 Kinetic parameters and specificity

Kinetics and substrate specificity have long been a focus of JHE studies because of the obvious agricultural potential of this information. Elucidation of JHE kinetic constants permits prediction of the rate limits of JH metabolism as well as provides valuable insight into how peripheral JH levels are regulated. A number of reports from the 1980s indicate that among lepidopterans the apparent Km for the naturally occurring JHs ranges from 10⁻⁸ to 10⁻⁶ M (Roe and Venkatesh, 1990). Juvenile hormone titers in the species tested are at least 10–100 times lower than the estimated Km concentration, indicating that the enzyme is very sensitive to changes in JH concentration, but its catalytic potential is wasted. Since the K_ms seemed unduly high compared to substrate concentration, investigators examined the individual rate components of the enzymatic reaction to better explain the observed data (Abdel-Aal and Hammock, 1986; Sparks et al., 1983a).

Several important lessons can be drawn from the kinetic data. First, JHE has a very high affinity for JH. Second, it has a relatively low turnover number or k_cat, where k_cat is the maximum number of substrate JH molecules converted to JH acid per active site per unit time. These factors translate into a very effective enzyme scavenger that can ‘find’ JH at physiological concentrations and convert it to JH acid (Abdel-Aal and Hammock, 1986). The structural elucidation of the protein now provides a rationale for the low turnover (Kamita and Hammock, 2010). The rate-determining event may be release of the lipophilic JH acid, which is buried deep within the enzyme.

Substrate specificity of JHE represents another characteristic that seems counterintuitive. The K_m and V_max that the JHEs display toward the JH homologues is surprisingly low when compared to other substrates, such as α-naphthyl acetate. For example, recombinant M. sexta JHE displays a K_m (410 µM) and V_max (21 µmol/min/mg protein) for α-naphthyl acetate that is considerably higher than for its natural substrate, JH III (K_m=0.052 µM, V_max=1.4 µmol/min/mg protein (Hinton and Hammock, 2003). As noted by Fersht (1985), when discriminating between two competing compounds, specificity should be determined by the k_cat /K_m ratio and not K_m alone. The k_cat/K_m ratios for JH III and α-naphthyl acetate are 27 and 0.04, respectively, underscoring the high degree of specificity of JHE for JH III (Hinton and Hammock, 2003). While these kinetic parameters suggest that the enzyme has a high degree of specificity, it appears that JHEs from several species also hydrolyze methyl and ethyl esters of JH I and III at similar rates (Grieneisen et al., 1997). Moreover, even n-propyl and n-butyl esters of these homologues can serve effectively as substrates albeit at a lower rate of hydrolysis. One of the most unexpected groups of JHE substrates is found in the naphthyl and p-nitrophenyl series (Hanzlik and Hammock, 1987; Kamita et al., 2003; Rudnicka and Kochman, 1984).

Once again, structural analysis provides clues as to how the catalytic site may accommodate certain bulky molecules such as α-naphthyl acetate. If α-naphthyl acetate enters the catalytic site in the acid-first orientation, there is sufficient space to carry out hydrolysis. Computational modeling posits that the catalytic site is flexible enough to accommodate larger molecules such as the ethyl esters of JH I and III or α-naphthyl acetate (Kamita and Hammock, 2010). Another counterintuitive observation regarding JHE activity is that while its major role is the conversion of JH to JH acid, both native and recombinant JHEs from several species can, under the appropriate conditions, transesterify JH to form the higher ester homologues, such as JH ethyl, JH n-propyl, and JH n-butyl esters (Grieneisen et al., 1997). Although JHE-mediated JH transesterification may be a curiosity limited to the test tube, Debernard et al. (1995) demonstrated that when JH III was dissolved in ethanol (10 µl) and injected into L. migratoria, it was converted to JH III acid and also to JH III ethyl ester. Thus, it should be noted that care must be taken to avoid artifacts when alcohols are used as carrier solvents for the hormone in JHE assays. Once again, flexibility of the catalytic site is key. Suggest that low concentrations of detergents and alcohols distort the binding site and allow JH and various substrates better access to the catalytic residues or improve the release of JH acid from the catalytic site. While JHE in a biological milieu clearly serves as an esterase, these new findings imply that the enzyme may have other physiological roles that are as yet undiscovered (Anspaugh et al., 1995).