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Paraoxon inhibition of an insect egg lipase
Abstract
A lipase present in an insect egg (Diabrotica undecimpunctata howardi Barber) is sensitive to inhibition by low concentrations (approx. 10−6M) of diethyl-p-nitrophenyl phosphate. The inhibition occurs only in the presence of substrate; i.e., exposure of the enzyme to inhibitor in the absence of substrate does not result in inhibition. The high sensitivity of this enzyme to the inhibitor makes it a likely target of organophosphate poisoning in those insect embryos killed before the appearance of the generally accepted target, cholinesterase.
... Although lipases preferentially cleave long-chain acylglycerols, they can also hydrolyze naphthyl esters and 4-nitrophenyl esters (Gilham and Lehner, 2005). Moreover, lipases can be inhibited by OP pesticides (Krysan and Guss, 1971; Casida et al., 2008). For example, four of the six esterase isozymes described by Cook et al. (1969) in the digestive secretion of the cockroach could be lipases because triglyceride hydrolysis decreased after full inhibition of these isozymes with 10 − 4 M dichlorvos. ...
Carboxylesterases (CbEs) are key enzymes in pesticide detoxification. These esterases are involved in the biochemical mechanism for pesticide resistance in some pest species, and further they are considered an efficient protective mechanism against acute toxicity by organophosphate (OP) pesticides in mammals. To gain knowledge on the role of CbEs in pesticide toxicity and natural tolerance in earthworms, we performed an enzyme kinetic analysis to investigate whether these annelids are able to secrete them into their gut lumen. We determined levels of CbE activity and isozyme abundance in the gut wall and ingested soil collected from different portions of the gastrointestinal tract of Lumbricus terrestris. Moreover, modulation of enzyme activity by selected substrates (alpha-naphthyl acetate [alpha-NA], 4-nitrophenyl valerate [4-NPV] and 4-nitrophenyl acetate [4-NPA]) and OP pesticides was examined to compare the response between tissue and soil CbEs. We found a high CbE activity in the ingested soil extracts from the crop/gizzard (alpha-NA-CbE=8.43+/-2.76U mg(-1) protein and 4-NPA-CbE=5.98+/-2.11U mg(-1) protein) compared to the gut wall. Three lines of evidences suggest that the gut epithelium is the main source of this luminal CbE activity. First, the effect of substrate concentrations on CbE activity from both the ingested soil extracts and gut tissues resulted in similar apparent K(m) and V(max) values. Second, native PAGE gels revealed that some of the CbE isozymes in the gut tissue were also present in the soil extracts. Third, tissue and soil CbEs showed the same sensitivity to inhibition by OPs. The concentrations of insecticide causing 50% of esterase inhibition (IC(50)) was comparable between tissue (IC(50)s range=4.01-9.67nM dichlorvos and 8480-6880nM paraoxon) and soil (IC(50)s range=6.01-11.5nM dichlorvos and 8400-7260nM paraoxon). Our results suggest a set of (eco)toxicological implications and environmental applications derived from the ability of earthworms to secrete these pesticide-detoxifying enzymes.
On effects of various organophosphorus insecticides such as chlorfenvinphos (phosphate type), EPN (phosphonothionate type), fenitrothion (phosphorothionate type) and mecarbam (phosphorothiolothionate type), and carbaryl (carbamate) on serum pseudocholinesterase, serum nonspecific esterase, serum lipoprotein lipase, adipose tissue lipase and epinephrineinduced lipolysis, this investigation was caried out in vitro. The results were as follows;1. These insecticides caused a dose-dependent inhibition of serum nonspecific esterase activity as well as serum pseudocholinesterase. Chlorfenvinphos exhibited the strongest inhibition of serum pseudocholinesterase, but severe inhibition of serum nonspecific esterase was recognized carbaryl rather than chlorfenvinphos.2. Carbaryl and fenitrothion caused slight stimulation of the serum lipoprotein lipase activity with addition of 10-5 M, but inhibited this enzyme with 10-3 M of these insecticides3. Addition of 10-3 M of these insecticides caused inhibition of adipose tissue lipase in lipolytic system using ediol as substrate. These insecticides also inhibited epinephrineinduced lipolysis as well as inhibition of the adipose tissue lipase.4. On the base of these findings, it was suggested that these insecticides caused the inhibition of lipolysis by direct suppression of adipose tissue lipase without reference to cAMP dependent proteinkinase.
Soluble esterases from eggs of Diabrotica undecimpunctata howardi and D. virgifera were examined by polyacrylamide gel electrophoresis. Eggs from D.undecimpunctata howardi exhibited from 10 to 16 electrophoreticallyseparable esterases depending on age; prediapause eggs from D. virgifera contained 6 or 7 esterases depending on whether α- or β-naphthol substrates were used.Zymograms of eggs of both species contained a single zone of activity that was inhibited by 5 × 10−5 M eserine. Organophosphorus compounds produced inhibitory effects depending on which compound was used.Two esterase bands from eggs of D. undecimpunctata howardi were identified as lipases by direct comparisons of estrolytic and lipolytic activities in eluates from gel sections. The lipases could not be distinguished from other esterases on polyacrylamide gel zymograms with α-naphthyl caprylate or longer-chain naphthol esters.
The activity of tefluthrin, carbofuran, terbufos and dieldrin in soil against eggs of different ages of the southern corn rootworm, Diabrotica undecimpunctata howardi (Barber) (Coleoptera: Chrysomelidae) was determined. Embryological development following treatment with these insecticides was also investigated to determine the stage of developmental arrest. Generally, younger eggs were found to be more susceptible, dieldrin being the least and tefluthrin the most potent ovicide of combined egg age mortalities. Terbufos and carbofuran were relatively inactive against older eggs compared with tefluthrin. Terbufos stopped embryonic development at the time when acetylcholine esterase activity has been shown to commence. Carbofuran, dieldrin and tefluthrin slowed but did not arrest embryonic development, although the two former compounds prevented eclosion to a greater degree than tefluthrin, suggesting death during absorption of serosal fluids at eclosion. Neonate emergence following treatment of eggs with tefluthrin resulted in death within a few hours. The results are discussed in relation to insecticidal action and permeability changes of egg membrane structure with age. © 1997 SCI.
The exposure of newly fertilized amphibian embryos to malathion (44 mg/L) produces 67% mortality on the fifth day of exposure. Clinical signs of intoxication are evident on the fourth day. A variety of biochemical parameters, such as enzyme activity, lipid and protein content, was examined in control and malathion-treated embryos. The activity of acetyl, butyrylcholinesterase and aliesterase are quickly inhibited. An interference with cellular protein synthesis and redistribution is possible. A slight increase in total phospholipids is evident after 24 hr of exposure.
An extracellular lipase from Corynebacterium acnes has been partially purified and characterized. The enzyme was obtained from the crude culture broth after ultrafiltration, gel filtration through Sephadex G-100, and chromatography on CM-cellulose. This preparation demonstrates broad specificity and its activity against tri-, di-, and monoglycerides and also p-nitrophenyl acetate is completely inhibited after reaction with diisopropyl phosphorofluoridate (Dip-F). These substrates apparently react at the same catalytic site of the enzyme since: (1) a constant ratio of specific activities for the substrates p-nitrophenyl acetate and tributyrin is obtained during purification; (2) competitive inhibition can be demonstrated between these two substrates; (3) similar rates of decay of hydrolytic activity are observed with p-nitrophenyl acetate and triglycerides during either reaction with Dip-F or heat denaturation; and (4) reaction with [32P]Dip-F yields only one [32P]diisopropylphosphoryl (Dip) protein derivative with a molecular weight of approximately 54,000. This lipase is stable from pH 4-8 at 23°. It is relatively stable to heat at pH 5.5 but is heat labile at pH 7.8. [32P]Dip-F was used to identify a reactive serine residue at the catalytic site. Presence of this serine residue suggests that the lipase contains a charge-transfer system similar to that described in such serine proteases as chymotrypsin, subtilisin, and elastase. Micellar particles of lipid substrates were analyzed for number, size, and stability with the Coulter counter, Model T. Following dilution of these particles into the concentration range required for KM measurements, the micelles appear to dissolve. This physical instability of the substrate may explain the non-linear enzyme kinetics often observed with the glycerol ester hydrolases.
The effects of the pesticide, Parathion (0,0-diethyl 0-p-nitrophenyl thiophosphate) and various lipolytic and antilipolytic agents on lipolysis in adipose tissue were studied with
isolated fat cells from rat epididymal fat pads. Lipolysis was measured as the release of free fatty acids into an albumin-bicarbonate
medium. Parathion depressed lipolysis in a linear manner at concentrations ranging from 10−9 to 10−3M. At a concentration of 10−5M, Parathion depressed the lipolytic response to epinephrine (0.15 μg/ml) and slightly to cyclic 3′,5′-adenosine monophosphate
(10−3 M) and enhanced the antilipolytic response to nicotinic acid (33 μM). It is possible that Parathion may interfere with adenyl
cyclase activity.
Paraoxon exerts its antilipolytic effect on the lipolytic system of fat cells by a direct interaction with the triglyceride
lipase. It does not affect other components of the lipolytic system such as adenyl cyclase, phosphodiesterase, ATP-levels
or binding of cyclic 3′,5′-AMP-dependent protein kinase. Phosphorylation of protein mediated by cyclic 3′,5′-AMP-dependent
protein kinase is not impaired by paraoxon.
In experiments with isolated epididymal fat cells, paraoxone caused a dose-dependent inhibition of lipolysis in the LD50dose range (10−7- 10−5M). The inhibition occurred in unstimulated as well as stimulated lipolysis elicited by 10−6M norepinephrine or 3 10−3M dibutyryl-A-3′, 5′-MP. The type of inhibition showed a noncompetitive pattern at an apparent Ki= 1.1 10−6M. Obidoxime, HS 3 and HS 6 did not abolish the 50% inhibition of maximum lipolysis elicited by 10−6M paraoxone. Neither a reactivation nor a direct interaction between paraoxone and oximes was observed in this test system.
The triglycerides of eggs of the western corn rootworm (Diabrotica virgifera LeConte, Coleoptera: Chrysomelidae) are unavailable to an apparently freely active triglyceride lipase (glycerol ester hydrolase, EC 3.1.1.3) in isotonic homogenates. Homogenization of the eggs in a hypotonic medium or treatment of isotonic homogenates with freeze-thaw or sonication will release the triglycerides permitting hydrolysis by endogenous lipase. The observations suggest that a case of structure-linked substrate latency has been observed.
The lipolytic activity in homogenates and aqueous extracts of acetone powders of eggs of the southern corn rootworm (Diabrotica undecimpunctata howardi Barber) was studied. The general properties were determined using as substrate olive oil in an emulsion stabilized by gum
arabic. Bovine serum albumin or Triton X-100 were required in the assay system; they protected the enzyme from spontaneous
denaturation. The lipase had activity optima at pH 7 and at 45 C. The preparation was inactive towards triacetin, and activity
increased in the series tripropionin<tributyrin<trihexanoin< trioctanoin<tridecanoin. Activity towards tridecanoin. Triolein
was hydrolyzed to oleic acid and glycerol with no marked accumulation, even transient, of partial glycerides.
Four different lipolytic preparations have been isolated from rat skeletal muscle. Two of these, provisionally designated monopalmitin lipase (MPL) and monomyristin lipase (MML), are associated with insoluble cellular particulate fractions. The other two enzymes, provisionally designated tricaproin lipase (TCL) and monolaurin lipase (MLL), are found in the high-speed supernatant fraction. Taken as a group, these enzymes are capable of hydrolyzing short-chain triglycerides (acyl moieties of C3 to C8) and monoglycerides of lauric, myristic, palmitic, stearic, and oleic acids. All of these enzymes have a serine residue at or near the catalytic site as they are strongly inhibited by diisopropyl fluorophosphate. The two particulate preparations contain a sulfhydryl group and are sensitive to p-chloromercuribenzoate and N-ethylmaleimide, while the soluble preparations are not.
The MLL, MML, and MPL preparations all have alkaline pH optima, while the TCL preparation has an acidic optimum. Buffer type is important: some buffer compounds completely inhibited one or more preparations.
Of the soluble enzymes, MLL withstood heating to 60°C, while TCL is completely inactivated at this temperature. Of the particulate preparations, only MML was stable to lyophilization. It is concluded that there are at least four lipolytic enzymes in rat skeletal muscle. The possible significance of the presence of these enzymes in muscle is discussed.
Reports in the late 1940’s of work done earlier with various synthetic organophosphorus chemicals as potential insecticides (Schrader 1947) have stimulated enormous industrial activity in this field. By way of illustration, Moorefield and Lanham (1959) estimated that over 50,000 such compounds had already been made and tested against insects. Thirty-six of these were said to be in commercial production: 33 dialkyl phosphates, 2 aminophosphates, and 1 phosphonate. Fukuto et al. (1959, p. 1121) found the number in use to be “30 or more”, of which 2 were esters of phosphonic acid.
Zone electrophoretic analysis of the gastric secretion of the cockroach, Periplaneta americana, revealed the presence of six carboxylic esterases. Two of these enzymes are resistant to 10−4 M dichlorvos (DDVP) and one, E6, is localized in the epithelium of the gastric caecum and the midgut. A purification is described for esterase E6 which yields a single zone after starch-gel electrophoresis. The purified enzyme readily hydrolysed 14C-labelled tripalmitin to free fatty acid, diglyceride, and monoglyceride, and 1-naphthyl acetate to 1-naphthol and acetic acid, and 86 per cent of both the esterolytic and lipolytic activity was inhibited by 10−3 M dichlorvos. Both the calcium and manganous ions served as activators in triglyceride hydrolysis.Three different phosphatases were isolated from the gastric fluid, one having an alkaline pH optimum and the second having an acid pH optimum (5·0) and a third having a pH optimum near 7. The acid phosphatase was inhibited by 10−3 M manganous ion; the neutral phosphatase showed a marked increase in activity in the presence of the same cation.The rôle of these hydrolases in the normal function of the insect and in the metabolism and mode of action of insecticides is discussed.
The purposes of this study were to determine the accordance or disagreement of the milkweed bug egg (Oncopeltus fasciatus) with generalizations set forth for eggs of terrestrial oviparous animals, i.e., that a high degree of lipid catabolism is characteristic, that lipids constitute the major source of energy for development, and that the greatest use of lipids occurs in the later stages of development. Eggs were found to contain an average of 32.2 μg. of lipid, or 11.6% of the wet weight. Shortly before hatching, the content was 28.5 μg., or 10.7% of the wet weight. The over-all decrease represented utilization of 11.5% of the initial lipid store, with the decrease occurring mostly in the latter half of development. Lipids therefore do not seem to play as dominant a role in development as expressed by other insect eggs. The apparent increase in pterine pigments suggests that protein catabolism may be more important than lipids in supplying metabolic energy.
Mittels der Warburg-Methode wurde die Cholinesteraseaktivitt der Eier von Bombyx mori vom Ende der Diapause bis zum Schlpfen fortlaufend untersucht.Fnf Tage vor dem Schlpfen konnte erstmals eine Fermentaktivitt nachgewiesen werden. Von diesem Zeitpunkt ab stieg die Aktivitt bis zum Schlpfen gleichmig stark an.Auf Grund der Untersuchungen der Abhngigkeit der Fermentaktivitt von Substratkonzentration liegt eine echte Cholinesterase (Cholinesterase I nach Augustinsson) vor.50%-Hemmung ergab eine 0,6 10–6 m Eserinlsung.Ein Zusammenhang zwischen den zeitlichen Eintritt der Giftwirkung von E 605 wie auch von E 600 und der Entwicklung der Cholinesteraseaktivitt konnte nicht gefunden werden.
Nachtrag bei der Korrektur: Whrend der Drucklegung dieser Arbeit erhielt ich Kenntnis von einer Arbeit von K. A. Lord und C. Potter (Organo-phosphorus insecticides-insecticidal and anti-esterase activity of organophosphorus compounds, Chemistry a. Industry 1954, 1214–1217). Die Autoren fanden bei Diataraxia oleracea (= Mamestra) ebenfalls eine Cholinesterase-Aktivitt der Eier erst kurz (etwa 3 Tage) vor dem Schlpfen derselben. Auch konnten sie keinen Zusammenhang zwischen der Fermentaktivitt und der Giftwirkung von Tepp feststellen.
A modification of the Duncombe method for measurement of free fatty acids is presented. Substitution of diphenylcarbohydrazide for diethyldithiocarbamate color complexing agent increases the sensitivity of the method 4- to 6-fold. The absorbancy of the color produced is measured at 540 mμ. Applications of the method for the assay of pancreatic lipase, and rat liver lysosomal lipase and phospholipase are described.
Lipase of Mucor pusillus NRRL 2543 was recovered with ammonium sulfate precipitation, gel filtration on Sephadex G-75, and anion-exchange chromatography on diethylaminoethyl-Sephadex A-50. Maximal glycerol ester hydrolase (lipase) activity was observed at pH 5.0 to 5.5 and 50 C when trioctanoin and olive oil were used as substrates. The enzyme also showed esterase activity; it hydrolyzed, with the exception of methyl butyrate, all methyl esters tested. A minimum chain length of six carbons appeared to be a requirement for esterase activity, which was maximal at about pH 5.5 with methyl dodecanoate (C(12)) as the substrate. Neither the glycerol ester hydrolase (lipase) nor the esterase activity of the enzyme appeared to be affected by thiol group inhibitors, chelating agents, and reducing compounds. On the other hand, hydrolysis of triolein and methyl dodecanoate was arrested to the same extent in the presence of diisopropyl fluorophosphate, which suggested the involvement of serine in the active center of the enzyme. The enzyme remained stable during a 30-day storage at - 10 C.
Eggs of the large milkweed bug, Oncopeltus fasciatus (Dallas), are totally insensitive to parathion vapor, but freshly emerged larvae are sensitive. Work with H3- parathion showed that large quantities are taken up by the eggs, but very little reaches the embryo. However, on emergence from poisoned eggs, larvae rapidly take up parathion from the egg and die soon thereafter. Ten other organophosphates and 2 carbamates are, like parathion, without ovicidal action on this species; but dichlorvos and trichlorfon are lethal to the late stages of developing eggs. Studies with C14-dichlorvos showed that extremely large quantities are taken up by the eggs, and a large fraction of it reaches the embryo. It is concluded that the nontoxicity of parathion is a penetration phenomenon, involving either a barrier or very poor partitioning from external lipid into the egg interior; but the ineffectiveness of dichlorvos to early stages may be due to their intrinsic insensitivity.