Multiple molecular forms of soluble esterases in the digestive system of the developing chicken

Soluble esterases in tissues of endodermic origin and of various developmental ages of Gallus gallus were analysed by polyacrylamide slab gel electrophoresis and characterized as carboxylesterases, acetylesterases and cholinesterases. Esterase bands were observed from day 9 in the liver and from day 6 in stomach, intestine and yolk sac. The electrophoretic profiles became more complex after hatching with concomitant increase in the staining intensity. On isoelectric focusing of liver extracts only a major form with pI 5.4 was observed. An eserine sensitive band designated EL-1 was found to be tissue (liver) and age (upon hatching) specific. EL-1-like isozymes were also observed in other species of the Galliformes order.     

Isozyme Polymorphism of Esterases in the Genus Lanistes (Mollusca: Prosobranchiata) and Genetic Analysis of Populations

Esterases from the digestive gland of the snails Lanistes carinatusand Lanistes boltenicollected from four Egyptian governorates were extracted and analyzed using starch gel electrophoresis and five substrates. Twelve esterase bands were detected in both Lanistes species. The esterase bands were distributed in three main zones, which could be classified as acetylesterases, carboxylesterases, and cholinesterases. Depending on the substrate specificity, inhibition properties, and relative mobility of esterase bands, the three zones of esterase activity could be traced to eight genetic loci. Locality-specific loci were found. Inter- and intrapopulation variations are discussed. There is an absence of equilibria at all esterase loci in all populations studied, and a high proportion of genetic diversity in different esterase loci. The absence of interspecific variations proves that Lanistessnails in Egypt belong to one species.     

Genetic variability in esterases and the insecticide resistance in brazilian strains of Oryzaephilus mercator and Oryzaephilus surinamensis (Coleoptera: Silvanidae)

Oryzaephilus mercator and O. surinamensis are stored grains and processed food pests, the latter being responsible for major economical losses. Polyacrylamide gel electrophoresis was used to analyse esterase patterns during insect development. Seven esterases, three cholinesterases, two carboxylesterases and two acetylesterases, were identified in O. mercator, one of which was proper to adults. Five esterases, of which four were cholinesterases, occurred in O. surinamensis. Strains of O. mercator and O. surinamensis were also exposed to malathion and chlorpyrifos-methyl. According to the LC50 estimates, OmLC-M and OmLA strains of O. mercator and OsLB strain of O. surinamensis were the most resistant to both insecticides. However, higher sensitivity to malathion and chlorpyrifos-methyl has also been verified in some of its esterases. Cholinesterases OmEST-1 and OsEST-5 seem to be involved in this resistance. These results suggest that organophosphate tolerance may be related to genetic variability in esterase isoenzymes.     

Purification and characterisation of a carboxylesterase from the latex ofSynadenium grantii Hook, ‘f’

The latex ofSynadenium grantii was found to contain esterolytic activity. Polyacrylamide gel electrophoretic study coupled with substrate and inhibitor specificity studies revealed the presence of multiple forms of carboxylesterases and cholinesterases in the latex. One of the carboxylesterases of the latex was purified by acetone fractionation, carboxymethyl-Sephadex chromatography and Sepharose-6B gel filtration. The homogeneity of the enzyme was established by polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme consists of a single polypeptide chain with a molecular weight of 14,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to basic amino acid residues. The isoelectric pH of the enzyme was found to be 4.0. The enzyme was a glycoprotein as revealed by periodic acid Schiff-staining technique. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was sensitive to organophosphates. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear non-competitive inhibition with 1-naphthol.     

Esterases in quail (Coturnix coturnix). Physicochemical and developmental aspects

• 1.1. Soluble esterases of digestive system organs of various developmental stages in the quail (Coturnix coturnix) were resolved by polyacrylamide gel electrophoresis into several molecular forms which were characterized as carboxylesterases, acetylesterases, cholinesterases and esterases sensitive to eserine.
• 2.2. The pI of the majority of esterasic activity in several quail and chicken tissues was observed in the range of 5.1–5.6, while the apparent molecular weight in liver extracts was 60,000.
• 3.3. The expression of the esterase multiple molecular forms was found to be both tissue- and developmental stage-specific, with electrophoretic patterns becoming more complex in number and/or staining intensity upon hatching and thereafter, especially in liver and intestine.

     

Gene identification and proteomic analysis of the esterases of the cotton bollworm,Helicoverpa armigera

Some of the resistance of Helicoverpa armigera to conventional insecticides such as organophosphates and synthetic pyrethroids appears to be due to metabolic detoxification by carboxylesterases. To investigate the H. armigera carboxyl/cholinesterases, we created a data set of 39 putative paralogous H. armigera carboxyl/cholinesterase sequences from cDNA libraries and other sources. Phylogenetic analysis revealed a close relationship between these sequences and 70 carboxyl/cholinesterases from the recently sequenced genome of the silkworm, Bombyx mori, including several conserved clades of non-catalytic proteins. A juvenile hormone esterase candidate from H. armigera was identified, and B. mori orthologues were proposed for 31% of the sequences examined, however low similarity was found between lepidopteran sequences and esterases previously associated with insecticide resistance from other insect orders. A proteomic analysis of larval esterases then enabled us to match seven of the H. armigera carboxyl/cholinesterase sequences to specific esterase isozymes. All identified sequences were predicted to encode catalytically active carboxylesterases, including six proteins with N-terminal signal peptides and N-glycans, with two also containing C-terminal signals for glycosylphosphatidylinositol anchor attachment. Five of these sequences were matched to zones of activity on native PAGE at relative mobility values previously associated with insecticide resistance in this species.     

Expression of esterases during ontogenesis of the flour beetle Tribolium castaneum (tenebrionidae; coleoptera)

Two electrophoretically fast-migrating, nonspecific esterases were detected in two strains of the flour beetle Tribolium castaneum and designated F (fast) and S (slow) according to their relative migration distances. Both isozymes are associated with the alimentary canal and display ontogenetic changes. Their activity is very low in the egg stage, increases in the larva, and declines dramatically in the pharate pupa and pupa. The overall activity in the pupal stage is low, yet increases gradually throughout this period. In the adult, the activity of the esterases rises sharply. The larval and adult F and S isozymes were found to hydrolyze - and -naphthylacetate and -naphthylpropionate with almost equal capacity. -Naphthyl laurate was cleaved by the F enzyme of both larvae and adults. The F and S were insensitive to inhibitors of arylesterases and cholinesterases and were markedly inhibited by the organophosphate diisopropylphosphorofluoridate (DFP) and could be classified as carboxylesterases. Differential sensitivities of larval and adult esterases to urea and heat treatment as well as to DFP may indicate the expression of different genes during metamorphosis.     

Sensitivity of blood-clotting factors and digestive enzymes to inhibition by organophosphorus pesticides

Organophosphorus pesticide toxicology is normally evaluated in relation to inhibition of cholinesterases (acetyl and butyryl), neuropathy target esterase, and carboxylesterases, with less attention given to other physiologically important hydrolases. This study considers the relative organophosphate sensitivities of the aforementioned serine hydrolases compared with purified blood-clotting factors (thrombin, plasmin, and kallikrein) and digestive enzymes (alpha-chymotrypsin, trypsin, and elastase), assayed under similar conditions. Inhibitors that we examined are organophosphorus insecticides or their activated metabolites (paraoxon, chlorpyrifos oxon, and profenofos) and other toxicants (phenyl saligenin cyclic phosphonate and tribufos) for comparison with values that are found in the literature for the fluorophosphonates (isoflurophate and sarin). Thrombin is the most sensitive blood-clotting factor with IC-50 values of 19 to 160 microM for tribufos, the cyclic phosphonate, isoflurophate, and profenofos; plasmin and kallikrein are less affected (IC-50 >100 microM). Alpha-Chymotrypsin, trypsin, and elastase are most sensitive to the cyclic phosphonate (IC-50 1.3-15 microM) and less so to isoflurophate, sarin, and profenofos (IC-50 values from 3.6 to greater than 100 microM). The cholinesterases, carboxylesterase, and neuropathy target esterase are the most sensitive to inhibition with IC-50 values for the insecticides of less than 0.001 to 0.6, 0.002 to 0.009, and 0.15 to 100 microM, respectively. The generally low potency of these organophosphates for blood-clotting factors and digestive enzymes suggests that associated toxic effects are unlikely at sublethal doses.     

Ox adrenal esterases: their isoenzymes and subcellular localisation

Summary Esterase activity has been shown to exist in multiple forms in the ox adrenal cortical and medullary homogenates and subcellular fractions. Some esterase is adherent to membrane structures. The isoenzymes were resolved into carboxylesterases, arylesterases, acetylesterases and cholinesterases. The distribution of the enzyme groups differed slightly between cortex and medulla.The distribution of specific activity of esterase in ox adrenal medullary and cortical fractions was also investigated. The highest activity was found with the microsomes but there was some activity with the other fractions. Medullary lysosomes were separated by gradient centrifugation and shown to contain esterase activity. The results were discussed in relation to histochemical findings at the light and electron microscopic levels.     

Molecular cloning of cDNAs encoding a range of digestive enzymes from a phytophagous beetle, Phaedon cochleariae

To gain better knowledge of the variety of digestive enzymes in phytophagous coleopteran pests, a sequencing screen of 76 random cDNAs from a gut library from Phaedon cochleariae larvae was performed. The screen yielded 21 cDNAs encoding amino-acid sequences homologous to known digestive enzymes, most of them were cell wall-hydrolysing enzymes. The deduced protein sequences of 7 cDNAs encoding putative α-amylase, cysteine proteinase, trypsin, chymotrypsin, cellulase, pectinase and xylanase display all the structural features that characterize these enzymes in other eukaryotic organisms. Except the α-amylase and chymotrypsin cDNAs, the other cDNAs probably derive from multigene families. The distribution of the corresponding enzymatic activities at various developmental stages of P. cochleariae was examined. α-amylase activity is present in guts of larvae and adults, proteinases are abundant in guts of larvae and adults, but scarce in eggs and larval carcasses, xylanases are present in the guts of larvae and adults, as well as in carcasses of larvae, whereas cellulase and pectinase activities are distributed in larval and adult guts, larval carcasses, and eggs. Only a minor fraction of the cellulases is secreted by microorganisms, suggesting that P. cochleariae synthesizes most of its own cell-wall hydrolysing enzymes. The physiological role of the enzymes is discussed, as well as the significance of these results for pest management strategies involving transgenic plants expressing enzyme inhibitors.     

The Carboxylesterase Gene Family from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Carboxylesterases hydrolyze esters of short-chain fatty acids and have roles in animals ranging from signal transduction to xenobiotic detoxification. In plants, however, little is known of their roles. We have systematically mined the genome from the model plant Arabidopsis thaliana for carboxylesterase genes and studied their distribution in the genome and expression profile across a range of tissues. Twenty carboxylesterase genes (AtCXE) were identified. The AtCXE family shares conserved sequence motifs and secondary structure characteristics with carboxylesterases and other members of the larger / hydrolase fold superfamily of enzymes. Phylogenetic analysis of the AtCXE genes together with other plant carboxylesterases distinguishes seven distinct clades, with an Arabidopsis thaliana gene represented in six of the seven clades. The AtCXE genes are widely distributed across the genome (present in four of five chromosomes), with the exception of three clusters of tandemly duplicated genes. Of the interchromosomal duplication events, two have been mediated through newly identified partial chromosomal duplication events that also include other genes surrounding the AtCXE loci. Eighteen of the 20 AtCXE genes are expressed over a broad range of tissues, while the remaining 2 (unrelated) genes are expressed only in the flowers and siliques. Finally, hypotheses for the functional roles of the AtCXE family members are presented based on the phylogenetic relationships with other plant carboxylesterases of known function, their expression profile, and knowledge of likely esterase substrates found in plants.     

Linkage studies on an esterase locus in the mosquito Aedes togoi

An electrophoretic survey of esterases in 7 wild-type and 10 mutant strains of the mosquito Aedes (Finlaya) togoi was undertaken using thin-layer agar gels. Three esterases (designated the Est-1, Est-2, and Est-3 loci in decreasing order of electrophoretic mobility) could be detected from fourth-instar larvae, pupae, and 2- to 5-day-old adults. Homogenates of the larvae gave the most intensely stained bands in the gels, especially for Est-3. The three esterases were designated carboxylesterases based on their response to the two esterase inhibitors, eserine and paraoxon (diethyl-p-nitrophenyl phosphate). The Est-3 locus was found to have five alleles including at least one null. The linkage results of six backcrosses suggest that Est-3 is located only 5–8 map units from the sex allele (m) and the gene arrangement is Est-3-m-s (straw-colored larva) in linkage group I.This work was supported by National Institutes of Health Grant AI 16983-01.     

The hemocytes of Hyalophora cecropia (Lepidoptera)

The hemocytes of selected stages of Hyalophora cecropia from first instar larvae to four-day-old adults were examined and compared with those of Samia cynthia and Antheraea polyphemus. Five classes and two subclasses of hemocytes are described in these moths: (1) prohemocytes, (2) plasmatocytes (of several morphological types), (3) spherule cells, (4) adipohemocytes (two subclasses), and (5) oenocytoids. All types except oenocytoids and subclass II adipohemocytes, are found in all stages examined. Mitotic figures were common among prohemocytes of most stages, but were seen only rarely among plasmatocytes and adipohemocytes, and were not seen among spherule cells or oenocytoids. Prohemocytes and plasmatocytes often contain lipid but rarely PAS positive material. Spherules of spherule cells are PAS positive, as are occasional cytoplasmic inclusions of oenocytoids. Adipohemocytes of both subclasses contain lipid and PAS positive materials in all stages examined. Adipohemocytes and plasmatocytes proved to be most active in phagocytizing ink. Relationships between hemocytes of these and other insects, and some possible functions of hemocytes are discussed.     

Characterization of a new continuous cell line from the flood water mosquito,Aedes vexans

A new cell line, UM-AVE1, was established from embryos of the mosquito Aedes vexans. Banding patterns for the isozymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), xanthine dehydrogenase (XDH), and esterases were compared with those of larval Aedes vexans tissues as well as those of four other mosquito cell lines and one moth cell line. Karyotype analyses confirmed that the dipteran cell lines were not contaminated with lepidopteran cells, because in all mosquito lines the modal number of chromosomes was 6 (=2n) or 7. Isozyme electrophoresis established a specific profile for each cell line. Two isozymes present in UM-AVE1 (LDH, IDH) were not detected in larvae; this could be a reflection of the different stages used for cell line isolation and enzyme analysis, or lability of sample preparations. It is significant that extracts from UM-AVE1 cells and Aedes vexans larvae had an identical double band for XDH, while all other cell lines examined exhibited only a single band.     

Study of the peptidasic site of cholinesterase: preliminary results

The peptidasic site of highly purified human plasma cholinesterase was investigated using active-site-directed inhibitors. Peptidase activity was assayed taking substance P as substrate. Inhibition by organophosphates indicated that the peptidasic site contained an active serine. The presence of essential histidine residues associated with serine was revealed by histidine modifications. Carboxyl group reagents showed that the active centre contained carboxyl groups in a non-polar environment. The removal of sialic acids did not alter peptidase activity. The peptidasic site of cholinesterase shared many properties with serine proteases sites and esteratic sites of cholinesterases. In addition, with the peptidasic site, as well as the esteratic site, there was always the possibility of 'aging' when inhibited by DFP or soman.     

Tissue specific expression of the acetylcholinesterase gene in Drosophila melanogaster

Summary Acetylcholinesterase (AChE) is mainly membrane bound in the central nervous system (CNS) of larvae and in the head and thorax of adults of Drosophila melanogaster; it is mostly soluble in the larval carcass, the adult abdomen, similar to that of the embryos (Zador et al. 1986). The enzyme shows the same number of isozymes (four or five) in larvae and adults as in the head of the fly or in embryos (Zador et al. 1986). In the Df(3R)GE26/MKRS stock both the membrane bound and the soluble enzyme are at about half normal levels while in the Df(3R)Ace ^HD1/MKRS stock this is true only for the membrane bound AChE. Therefore the effect of the above deficiencies in larvae and adults is consistent with that in embryos (Zador et al. 1986). In heat-sensitive combinations of certain Ace mutant alleles both the membrane bound and the soluble enzyme has reduced activity.Abbreviations AChE acetylcholinesterase (acetylcholine acetyl hydrolase, EC 3.1.1.7) - BAP 1,5-bis(allyldimethylammonium-phenyl)-pentan-3-one dibromide - CNS central nervous system     

A novel lactone-forming carboxylesterase: molecular identification of a tuliposide A-converting enzyme in tulip

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.     

Heterologous Expression and Biochemical Characterisation of Fourteen Esterases from Helicoverpa armigera

Esterases have recurrently been implicated in insecticide resistance in Helicoverpa armigera but little is known about the underlying molecular mechanisms. We used a baculovirus system to express 14 of 30 full-length esterase genes so far identified from midgut cDNA libraries of this species. All 14 produced esterase isozymes after native PAGE and the isozymes for seven of them migrated to two regions of the gel previously associated with both organophosphate and pyrethroid resistance in various strains. Thirteen of the enzymes obtained in sufficient yield for further analysis all showed tight binding to organophosphates and low but measurable organophosphate hydrolase activity. However there was no clear difference in activity between the isozymes from regions associated with resistance and those from elsewhere in the zymogram, or between eight of the isozymes from a phylogenetic clade previously associated with resistance in proteomic and quantitative rtPCR experiments and five others not so associated. By contrast, the enzymes differed markedly in their activities against nine pyrethroid isomers and the enzymes with highest activity for the most insecticidal isomers were from regions of the gel and, in some cases, the phylogeny that had previously been associated with pyrethroid resistance. Phospholipase treatment confirmed predictions from sequence analysis that three of the isozymes were GPI anchored. This unusual feature among carboxylesterases has previously been suggested to underpin an association that some authors have noted between esterases and resistance to the Cry1Ac toxin from Bacillus thuringiensis. However these three isozymes did not migrate to the zymogram region previously associated with Cry1Ac resistance.     

Alcohol dehydrogenase of Biomphalaria glabrata (Mollusca: Pulmonata): Polymorphism,genetic analysis,and interspecific variations

Alcohol dehydrogenase of Biomphalaria glabrata has been characterized by electrophoresis, substrate specificities, and other physicochemical means. It exists as a multiple molecular form possessing a minimum number of three bands in ovotestis, five in digestive gland, and six in albumen gland. Each organ shows characteristic electrophoretic forms which differ in substrate specificities and the response to the organomercurial inhibitor p-hydroxymercuribenzoate. Mercaptoethanol treatment has no effect on any electrophoretic form. Genetic analyses of the electrophoretic variants show that three different loci are responsible for the synthesis of the various electrophoretic forms observed in this species. Different species vary in their electrophoretic patterns. A possible role of alcohol dehydrogenase isozymes in the phylogenetic relationship among three species, B. glabrata, B. tenagophila, and B. straminea, has been discussed.This work was supported by a grant from the Conselho Nacional de Pesquisas, Brazil.