Carboxylic ester hydrolases in the thyroid gland of the guinea-pig. A light microscopic study

Synopsis The location of cholinesterase and non-specific esterase in the thyroid gland of the guinea-pig was studied with the light microscope. It was found that the inxodyl method for non-specific esterase activity under special conditions is superior to the cholinesterase method in a number of respects for the demonstration of the intra-, inter-and parafollicular cells. When using the indoxyl method the incubation period can be reduced from 2.5–3 hr to 40–50 min. Further, the reaction can be followed during the incubation. False localization of the reaction products is avoided, and nerves and erythrocytes are not stained.By varying the fixation time and the time of storage in gum arabic-sucrose, it was found that the miscellaneous activity of non-specific esterase in APUD cells (C-cells) and follicle cells may be due to both factors. In fresh tissue the activity of the enzyme was equal in follicle and C cells.Special cyst-like structures containing an esterase which is NaF-resistant whenn-naphthyl acetate is employed as a substrate and which gives a strong reaction at low pH values when 5-bromo indoxyl acetate is the substrate, are described, and their nature and possible origin are discussed.     

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections

Summary The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study whow that the enzyme histochemical technique — except for some limitations — is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.     

Esterase Autodisplay: Enzyme Engineering and Whole-Cell Activity Determination in Microplates with pH Sensors

Among the GDSL family of serine esterases/lipases is a group of bacterial enzymes that posses C-terminal extensions involved in outer membrane anchoring or translocation. ApeE from Salmonella enterica serovar Typhimurium, a member of this group, has been expressed in Escherichia coli and was resistant to protease digestion when the protease was added to whole cells, indicating a periplasmic localization. The five consensus blocks conserved within all GDSL esterases were identified in ApeE by multiple sequence alignment and separated from the C-terminal extension. The DNA sequence spanning the four invariant residues Ser, Gly, Asn, and His, and hence representing the catalytic domains of ApeE, was amplified by PCR and fused in frame to the transport domains of the autodisplay system. The resulting artificial esterase, called EsjA, was overexpressed in the cell envelope of E. coli and was shown to be active by the use of α-naphthyl acetate (α-NA) as a substrate in an in-gel activity stain after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface exposure of EsjA was indicated by its accessibility to protease added to whole cells. The esterase activity of whole cells displaying EsjA was determined by a pH agar assay and by the use of microplates with integrated pH-dependent optical sensors. α-NA, α-naphthyl butyrate, and α-naphthyl caproate were used as substrates, and it turned out that the substrate preferences of artificial EsjA were altered in comparison to original ApeE. Our results indicate that autodisplay of esterase in combination with pH sensor microplates can provide a new platform technology for the screening of tailor-made hydrolase activities.     

Rat small-intestinal β-galactosidases: Influence of pH on the hydrolysis of different substrates

1. The influence of pH and the kind of buffer on the hydrolysis of lactose and four hetero-β-galactosides (phenyl β-galactoside, o-nitrophenyl β-galactoside, p-nitrophenyl β-galactoside and 6-bromo-2-naphthyl β-galactoside) by homogenates of rat small-intestinal mucosa has been studied. 2. There are at least two β-galactosidases present in the homogenates, one with optimum pH3–4 and another with optimum pH5–6. 3. The enzyme with the lower pH optimum is mainly a heterogalactosidase. It hydrolyses lactose slowly. The other enzyme is mainly a disaccharidase, since it hydrolyses lactose much more rapidly than the heterogalactosides. 4. Under the conditions used, citrate had an inhibitory effect on the 6-bromo-2-naphthyl β-galactosidase activity at pH3–4, but did not influence the 6-bromo-2-naphthyl β-galactosidase activity at pH5–6 or the hydrolysis of the other substrates at any pH.     

Substrate antagonism in the kinetic mechanism of E. coli phosphofructokinase-1

In the presence of its allosteric activator GDP, the major phosphofructokinase-1 from Escherichia coli K12 follows Michaelis—Menten kinetics. The kinetic behavior observed at steady-state using different concentrations of the substrates ATP and fructose-6-phosphate and the pattern of inhibition by the substrate analogs adenylyl-(β,γ-methylene)-diphosphonate and D-arabinose-5-phosphate are consistent with a random sequential mechanism in rapid equilibrium, rather than with an ordered binding as was suggested earlier. However, ATP and fructose-6-phosphate do not bind independently to the same active site, since the apparent affinity for one substrate is decreased about 20-fold when the other substrate is already bound. The antagonism between ATP and fructose-6-phosphate shows that a negative interaction occurs during the reaction with E. coli phosphofructokinase-1 which must be considered in addition to its allosteric properties.     

A rapid spectrophotometric method for the determination of esterase activity

We have developed a spectrophotometric assay method which continuously records esterase activity at 510 nm by monitoring absorbance changes due to the formation of a diazo dye complex. In our method, α-naphthyl ester substrates are hydrolyzed by enzymatic action to α-naphthol which couples to Fast Blue RR salt (a diazonium salt) forming a diazo dye complex. Our method is unique in directly monitoring the formation of the diazo dye complex without extracting the color of the complex as in other methods that use naphthyl esters and diazo coupling of reaction products. The method appears to be limited to α-naphthyl ester substrates, however, since β-naphthyl esters did not give a linear change in absorbance in the enzymatic reactions tested. With this assay method, one can use a single substrate both to determine esterase units quantitatively in solution and to detect esterase staining activity on gel electrophoresis.     

Genetics of a tissue esterase polymorphism (Est-6) in the rabbit (Oryctolagus cuniculus)

Genetic analysis of a polymorphic tissue esterase revealed a new locus (Est-6) with two alleles (Est-6 ^a andEst-6 ^b) on linkage group VI of the rabbit.Est-6 is closely linked to theEst-1,2,4 cluster. Esterase ofEst-6 is found in many organs, particularly in liver and small intestine, but not in erythrocytes and serum.Est-6 esterase hydrolyzes -naphthyl acetate and butyrate, naphthol AS-D acetate, indoxyl acetate, and butyrate as well as 5-bromoindoxyl acetate,N-acetyl-l-alanine--naphthyl ester but not 4-methylumbelliferyl acetate and fluorescein diacetate. The enzyme is inhibited by bis-p-nitrophenyl phosphate and eserine but not byp-chloromercuribenzoate. It was classified as a carboxylesterase (EC 3.1.1.1). Based on chromosomal localization, tissue distribution, substrate specificity, inhibitor sensitivity, and range ofpI's, rabbitEst-6 is assumed to be homologous with mouseEs-7.The contribution of Dr. O. von Deimling (No. 59) was supported by the Deutsche Forschungsgemeinschaft (De 315/2-2).     

Lipase-catalyzed optical resolution of trifluoro(aryl)ethanols

Optical resolutions of racemic 2,2,2-trifluoro-1-(aryl)ethanols — (1-naphthyl), (2-naphthyl), (4-methylnaphthyl), (phenyl), (1-pyrenyl) — were achieved by lipase-catalyzed enantioselective acetylations with vinyl acetate as an acetyl donor in octane, and (S)-acetates and (R)-alcohols were obtained. Among the lipases tested, lipase from Pseudomonas aeruginosa (lipase LIP, Toyobo) showed good enantioselectivity for above ethanols. However, no acetylation occurred with sterically hindered alcohols — (9-phenanthryl), (9-anthryl), (2-methylnaphthyl), (2, 4, 6-trimethylphenyl) — by various lipases. The resolutions of the three alcohols were carried out by the enantioselective alcoholysis or hydrolysis of their chloroacetates by lipase LIP.     

Acid phosphatases of rabbit spermatozoa: II. Partial purification and biochemical characterization of the multiple forms of rabbit spermatozoan acid phosphatase

Five acid phosphatases, S4, S3, S2, Szn and S1 (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) of ejaculated rabbit spermatozoa were either partially purified by DEAE-Sephadex column chromatography or prepared by specific extraction methods.The pH optimum of S4 was 6.0–6.5 in acetate buffer and 7.0 in Tris-HCl buffer; the pH optima of S3, S2, Szn, and S1 were 4.5, 5.5., 6.0 and 5.2, respectively, in acetate buffer. The apparent molecular weights of S3, S, Szn and S1, determined by disc gel electrophoresis, were 123 000, 86 000, 64 000 and 45 000–49 000, respectively. Incubation with neuraminidase did not alter the electrophoretic mobilities of any of the enzymes.Ten natural phosphoric esters were tested as substrates. S4 preferentially hydrolyzed ATP, ADP, PP_i and 3′-AMP. S3 hydrolyzed only β-glycerophosphate and glucose 6-phosphate to a significant extent. S2 hydrolyzed β-glycerophosphate, glucose 1-phosphate, the phosphoproteins, casein and phosvitin. S1 hydrolyzed ADP and β-glycerophosphate most readily. Szn may be an ATPase since it exhibits very high Zn²⁺-stimulated against ATP.These characteristics combined with the effects of NaF, ZnCl₂, l-(+)-tartaric acid, and formaldehyde on the activity of each partially purified enzyme with α-naphthyl phosphate as substrate indicate that these phosphatases are structurally and functionally different.     

Esterases in histochemistry and ultrahistochemistry

Synopsis The histochemical identification of individual esterases is a problem that has not yet been overcome. Inhibitors and different substrates reveal different patterns of distribution. 8-hydroxyquinoline acetate is a useful substrate in ultrahistochemistry. There is evidence of a relationship between esterase distribution and function.ACTH adrenocorticotropic hormone - 5Bri–O-2 5-bromoindoxyl acetate - 5Br–4ClI–O-2 5-bromo-4-chloro indoxyl acetate - cAMP cyclic adenosine monophosphate - DFP di-isopropyl-fluorophosphate - hCG human chorion gonadotropin - HS-2/4 thiol acetate/butyrate - I-O-2/4 indoxyl acetate/butyrate - N-O-2/3/4 -naphthyl acetate/propionate/butyrate - N-O-2 -naphthyl acetate - N-S-2/9 -naphthyl thiolacetate/nonanoate - NAS-O-2 naphthol AS acetate - NASD-O-2 naphthol AS-D acetate - 4NP-O-2/3 p-nitrophenyl acetate/propionate - 4NP-S-2 p-nitrophenyl thiol acetate - P-O-2 phenyl acetate - Q-O-2/4 8-hydroxyquinoline acetate/butyrate - Q-S-2/4 8-mercaptoquinoline acetate/butyrate - TBA-S-2/9 -thiolbenzanilide acetate/nonanoate - TSH thyroid-stimulating hormone     

Microchemical investigation of alpha D-glucosidases using 4-methylumnelliferyl- and 2-naphthyl-alpha-d-glucoside (author's transl)]

In crude homogenates prepared from freeze-dried cryostate sections of various rat organs the Km and Vmax of acid and neutral alpha-glucosidase as well as the effect of the pH, substrate and enzyme concentration and the incubation time on the activity were determined fluorometrically with 4-methylumbelliferyl- and 2-naphthyl alpha-d-glucoside as substrates. On the basis of the biochemical data 2 assays were developed for the microchemical measurement of both alpha-glucosidases in groups of epithelial cells isolated from freeze dried cryostate sections of the epididymis, jejunum, ilium, liver and kidney of suckling and adult rats. The rate of hydrolysis of 2-naphthyl and 4-methylumbelliferyl alpha-d-glucoside differs moderately. However, due to the higher sensitivity of 4-methylumbelliferone the methylumbelliferyl derivative is preferable especially for the evaluation of alpha-d-glucosidases in cells with low enzyme activity.     

A Diazo Coupling Method for the Electron Microscopic Localization of Cholinesterase

The presence of cholinesterase at the myoneural junction of intercostal muscle has been demonstrated in both light and electron microscopic preparations. A new simultaneous diazo coupling technique using α-naphthyl acetate as substrate and "hexazonium pararosanilin" as coupler has been applied to cold formalin-fixed tissues. After postfixation in buffered osmium tetroxide the sites of esterase activity are faithfully demonstrated at a high level of resolution. The details of cholin-esterase distribution and some technical aspects of the procedure are discussed.     

Characterization and affinity purification of juvenile hormone esterase from Bombyx mori

Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.     

Characterization of esterases in malathion-resistant and susceptible strains of the pteromalid parasitoid Anisopteromalus calandrae

General esterase, malathion-specific carboxylesterase, phosphotriesterase, glutathione S-transferase, cytochrome P-450-dependent monooxygenase activity, and target site sensitivity were compared in malathion-resistant (R) and malathion-susceptible (S) strains of the parasitoid Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae). Activity against -naphthyl acetate was not significantly different in male and female wasps for either strain. General esterase activity ranged from 1.2-fold to 2.5-fold higher in the R strain compared with the S strain, but these differences between strains were not consistent. Based on V_max/K_m ratios estimated for a number of analogs of four substrates (-naphthyl acetate, β-naphthyl acetate, 4-methylumbelliferyl acetate, and p-nitrophenyl acetate) there was no evidence that general esterase activity was elevated or reduced in the R strain. Malathion-specific carboxylesterase (MCE) activity, determined by using 2,3-¹⁴C-malathion as substrate, was 10- to 30-fold higher in the R strain compared with that in the S strain. The MCE has a pH optima at about pH 7, is cytosolic, and is labile upon storage at −80°C. MCE activity could be recovered from native 10% PAGE gels and IEF–PAGE gels (pI=5.2), but the peak of MCE activity also contained the major peak of activity against -naphthyl acetate. There was no evidence for major involvement of phosphotriesterase, glutathione S-transferase, monooxygenase, or altered acetylcholinesterase in the resistance. These data suggest that an increased activity of a MCE in the R strain is the probable major mechanism conferring resistance to malathion in A. calandrae. This study provides the first characterization of a biochemical resistance mechanism in a parasitoid with a high level of resistance to an organophosphate insecticide.     

Purification and Characterization of Carboxylesterase from the Seeds of Jatropha curcas

Carboxylesterases are hydrolases which catalyze the hydrolysis of various types of esters. Carboxylesterase from the seeds of Jatropha curcas has been purified to homogeneity using ammonium sulfate fractionation, CM-cellulose chromatography, Sephadex G-100 chromatography and preparative polyacrylamide gel electrophoresis (PAGE). The homogeneity of the purified enzyme was confirmed by PAGE, iso-electrofocusing and SDS-PAGE. The molecular weight of the purified enzyme was determined by both gel-permeation chromatography on Sephadex G-150 and SDS-PAGE. The molecular weight determined by Sephadex G-150 chromatography and SDS-PAGE both in the presence and absence of 2-mercaptoethanol was 31 kDa. The isoelectric point of the purified enzyme was found to be 8.9. JCSE-I (J. curcas seed esterase-I) was classified as carboxylesterase on the basis of substrate and inhibitor specificity. The K_m of JCSE-I with 1-naphthyl acetate, 1-naphthyl propionate, 1-naphthyl butyrate and 2-naphthyl acetate as substrates were found to be 0.0,794, 0.0,658, 0.0,567 and 0.1 mM, respectively. The enzyme exhibited an optimum temperature of 45 °C and an optimum pH of 6.5. The enzyme was stable up to 15 min at 65 °C. The enzyme was resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB) and inhibited by organophosphates (dichlorvos, parathion and phosphamidon).     

The use of 2-thionaphthyl acetate as a substrate for the localization and characterization of nonspecific esterase activity in rat alveolar and peritoneal macrophages

Summary A 2-thionaphthyl acetate substrate was utilized to assess the subcellular distribution of nonspecific esterases in rat pulmonary alveolar and peritoneal macrophages. The enzymatically liberated 2-thionaphthol was visualized at pH 7.1 by utilizing gold as a capture agent. Glutaraldehyde-fixed macrophages derived from healthy animals using standard lavage techniques exhibited a high affinity for the substrate and reaction times were thus relatively short (30–60 min). Alveolar macrophages had heavy reaction product on the external surface of the plasma membrane and membranes limiting cisternae of rough endoplasmic reticulum, Golgi complex and mitochondria. Only a thin layer of reaction density was observed associated with the limiting membranes of lysosomes and phagosomes. Peritoneal macrophages were similarly but much less intensely reactive, although they generally lacked or had very little plasma membrane-associated staining. The 2-thionaphthyl acetate esterase activities in both alveolar and peritoneal macrophages were sensitive to diisopropylfluorophosphate (DFP), while only the latter was inhibited by sodium fluoride. Polyacrylamide gel isoelectric focusing of whole cell homogenates indicated that the 2-thionaphthyl acetate esterase activity was the same as that for -naphthyl acetate in these cells. The data indicate that a significantly different distribution of nonspecific esterase activity results with use of a 2-thionaphthyl acetate substrate in the presence of gold ions than that previously reported with other methods. The rapid penetrability and sensitivity of this substrate make it a potentially useful tool for evaluating subcellular localization of esterase activity and probing characteristics of cellular organelles.     

Isoenzymes and histochemistry of non-specific esterases in normal and cancerous skin

Synopsis Non-specific esterases in normal and carcinomatous skin of the mouse have been investigated electrophoretically and histochemically. Three esterase bands were obtained on electrophoresis from homogenates of normal skin; homogenates of carcinomas showed an accumulation of esterase-Ia and esterase-Ib.^* However, using several ester substrates, substrate-specific patterns were demonstrated in the electrophoresis separations and histochemically in tissue sections. On the electrophoresis separations, -naphthyl acetate, -naphthyl acetate, 6-bromo-2-naphthyl acetate, naphthol AS acetate, naphthol AS-D acetate and naphthol AS-LC acetate gave rise to similar patterns, but with -naphthyl propionate as subsmate, more esterase-Ib was indicated and with 5-bromo-indoxyl acetate a distinctive preponderance. Peripheral or uniformly distributed staining was found histochemically in tumour epithelium using -naphthyl acetate, -naphthyl propionate and -naphthyl acetate, whereas with the substrates of naphthol AS acetate, naphthol AS-D acetate and indoxyl acetate an intermediate pattern of staining related to keratinization was obtained.     

Distinct effect of a cationic surfactant on the transient and steady state phases of 2-naphthyl acetate hydrolysis catalyzed by α-chymotrypsin

Results obtained on the effect of addition of dodecyltrimethylammonium bromide (DTAB) upon the α-chymotrypsin (α-CT) catalyzed hydrolysis of 2-naphthyl acetate (2-NA) under steady state conditions for the acyl–enzyme intermediate are compared with those previously obtained in the transient (pre-steady state or “burst”) phase. It is found that, while in the transient phase there is no effect of DTAB addition on the kinetic parameters at concentrations below the critical micelle concentration (CMC) of the surfactant, super-activity is observed when the acyl–enzyme intermediate reaches the steady state condition. This difference implies that the surfactant does not modify either the formation or the decomposition of the enzyme–substrate complex (transient phase) but notably increases the rate of disruption of the acyl–enzyme intermediate.     

The genetic control of arylesterase activity in the serum of Gotland Sheep

Esterase activity was determined in 955 serum samples of Gotland sheep. The activities showed a wide range and could be distinguished in six types. Family data were in agreement with the theory of three multiple alleles controlling the esterase activities (Lee, 1966). The following gene frequencies were estimated: Es ^a= 0.15, Es ^b= 0.75 and Es ^c= 0.10. The Es ^a allele controlled an enzym with high hydrolysis rate of α-naphthyl acetate, medium high of β-naphthyl acetate and low of indophenyl acetate. The enzym controlled by the Es ^b allele had medium high hydrolysis rate of α- and β-naphthyl acetate and relatively high of indophenyl acetate. The enzym of the Es ^c allele had low activities for all three substrates. The hydrolysis rate was increased by the addition of calcium. In starch-gel electrophoresis the esterase types could be divided in four groups due to the staining intensity of the arylesterase zone but no difference in migration rates was observed.     

Substrate antagonism in the kinetic mechanism of E. coli phosphofructokinase-1.

In the presence of its allosteric activator GDP, the major phosphofructokinase-1 from Escherichia coli K12 follows Michaelis—Menten kinetics. The kinetic behavior observed at steady-state using different concentrations of the substrates ATP and fructose-6-phosphate and the pattern of inhibition by the substrate analogs adenylyl-(β,γ-methylene)-diphosphonate and D-arabinose-5-phosphate are consistent with a random sequential mechanism in rapid equilibrium, rather than with an ordered binding as was suggested earlier. However, ATP and fructose-6-phosphate do not bind independently to the same active site, since the apparent affinity for one substrate is decreased about 20-fold when the other substrate is already bound. The antagonism between ATP and fructose-6-phosphate shows that a negative interaction occurs during the reaction with E. coli phosphofructokinase-1 which must be considered in addition to its allosteric properties.