Total Esterase Activity Research Articles

Comparison of chromaffin cells from several animal sources for their use as an in vitro model to study the mechanism of organophosphorous toxicity

It had been observed that the chromaffin cells of bovine adrenal medulla contain high levels of neuropathy target esterase (NTE), the esterase whose inhibition and aging is associated with induction of the organophosphorous induced delayed neuropathy. In this study, total esterase and NTE activities, and their inhibition kinetics by OPs are characterized in adrenal medulla of several species in order to find the best source for chromaffin cells. Total esterase activity in membrane fraction of bovine, equine, porcine, ovine and caprine were 6100 ± 840, 4200 ± 270, 5000 ± 120, 28800 ± 3000, and 10800 ± 2400 mU/g tissue, respectively (mean ± S.D., n = 3–4). NTE represented around 70%, 24%, 58%, 10% and 24% of the total esterases in the same tissues, respectively. It was deduced that NTE represents between 69% and 89% of the “B-activity” (activity resistant to 40 μM paraoxon) in the membrane fraction of all species. The mipafox I 50 calculated for 30-min inhibition of NTE at 37 °C ranged between 7.4 and 12 μM. These values are in the range of that for brain NTE in hen (the usual model for testing OP delayed neurotoxicity). Considering that bovine adrenal medulla contains high NTE activity, that it represents a high proportion of total activity, it is easier to dissect than adrenal medulla from equine, caprine or ovine, and is more readily available than species cited previously, and that its inhibitory properties are similar to the classical hen brain model, it is deduced that bovine adrenal medulla is the most appropriate source of chromaffin cells to study OP toxicity, with porcine as the second alternative. The kinetic properties of chromaffin cell cultures from bovine and porcine were in accordance with their properties in homogenate and subcellular fractions, and they displayed an appropriate stability and viability of the primary culture to be used in in vitro toxicological studies for both mechanistic and testing purposes.

Stability of Oxymethyl-Modified Coumarinic Acid Cyclic Prodrugs of Diastereomeric Opioid Peptides in Biological Media from Various Animal Species Including Human

In vitro stability studies of oxymethyl-modified coumarinic acid (OMCA) cyclic prodrugs of the diastereomeric opioid peptides DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), [Ala2,D-Leu5]-Enk (H-Tyr-Ala-Gly-Phe-D-Leu-OH), [D-Ala2,Leu5]-Enk (H-Tyr-D-Ala-Gly-Phe-Leu-OH), and [Ala2,Leu5]-Enk (H-Tyr-Ala-Gly-Phe-Leu-OH) were conducted to evaluate how the chirality of specific amino acid residues (Ala2 and Leu5) in the peptide portion affects their bioconversion by esterases. The stability studies were conducted at 37 degrees C in plasma and tissue homogenates (liver and brain) from five animal species (rat, mouse, canine, guinea pig, and hamster) and human in an attempt to identify an animal species that had a "prodrug bioconversion profile" comparable to that of humans. Initially, the total esterase activity in these biological media was measured using p-nitrophenyl butyrate (PNPB) as a substrate. By repeating this activity assay in the presence of paraoxon, a potent esterase B inhibitor, it was possible to estimate the relative amounts of esterases B and esterases A/C in a biological sample. Stability studies of the cyclic prodrugs were carried out under identical conditions, that is, in the presence and absence of paraoxon. Significant differences in the rates of hydrolysis of the cyclic prodrugs were observed, particularly between cyclic prodrugs with differences in the chirality of the amino acid on the C-terminus of the peptide portion, for example, L-amino acids at the C-terminus hydrolyzed more rapidly than D-amino acids. This stereoselective hydrolysis was independent of the animal species but tended to be more pronounced in brain and liver homogenates compared to plasma. Increased esterase specific activity, as measured by PNPB, in the biological media did not necessarily correlate with increased bioconversion rates of the cyclic prodrugs. The enzymatic stability profiles of the cyclic prodrugs in biological media from canine and guinea pig most closely resembled the profiles from human biological media. Therefore, canine and guinea pig appear to be the most relevant animal models for conducting pharmacokinetic studies on these cyclic prodrugs of opioid peptides.

Relationship among expression, amplification, and methylation of FE4 esterase genes in Italian populations of Myzus persicae (Sulzer) (Homoptera: Aphididae)

The wide use of insecticides containing an esteric group selected resistant Myzus persicae populations characterised by the overproduction of one of two closely related carboxylesterases (E4 and FE4). In this paper, we present data collected from Italian population indicating that all the 22 populations analysed possess amplified FE4 gene only. The estimation of FE4 copy number, carried out by densitometric scanning of dot and Southern blots, puts in evidence that the different populations possess a gene copy number ranging from 6 to 104. Statistical analysis shows the existence of a high positive correlation between gene copy number and total esterase activity. In aphid strain with low FE4 copy number, these genes are almost totally methylated. On the contrary, aphid strains with high FE4 gene number evidenced highly variable methylation levels and absence of correlation between the number of genes and their methylation state. The same result has been observed when comparing FE4 methylation levels and esterase activity.

Separation and quantitative recovery of mouse serum arylesterase and carboxylesterase activity

Paraoxonase-1 (PON1) is known to be associated with high density lipoproteins. We optimized buffer conditions to obtain quantitative recovery of PON1 (arylesterase) activity and analyzed the distribution of PON1 in mice using a combination of size-exclusion chromatography and ultracentrifugation. Size-exclusion chromatography of mouse serum separated the esterase activity into two peaks, one overlapping the high density lipoproteins and a second peak of lower molecular weight, consistent with serum carboxylesterase, which accounted for approximately 20% of the total esterase activity of normal mouse serum. Using conditions for the quantitative recovery of arylesterase activity, we fractionated serum by ultracentrifugation into d < 1.21 g/ml, d < 1.25 g/ml, d > 1.21 g/ml, and d > 1.25 g/ml fractions. We observed that PON1 arylesterase activity and mass were isolated in the d < 1.21 g/ml fraction and that serum carboxylesterase was recovered in the d > 1.25 g/ml fraction. The significance of the confounding of PON1 arylesterase activity by serum carboxylesterase was demonstrated by studying mice challenged with a high-fat, high-cholate diet for 14 days. It was shown that all of the decrease in arylesterase activity in response to this diet is attributable to the HDL-associated arylesterase activity (PON1). We conclude that mouse PON1 is quantitatively associated with high density lipoproteins. The contribution of serum carboxylesterase to the total esterase activity significantly confounds the interpretation of total arylesterase activity in mouse serum.

Characterizations of general esterases in relation to malathion susceptibility in two field populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen)

General esterases were characterized and compared from two populations of the oriental migratory locust, Locusta migratoria manilensis, collected from Huanghua and Pingshan Counties, Hebei Province, China. General esterases were most concentrated in the thorax and abdomen, which contained 46.1 and 36.1% of total esterase activity in females, and 42.7 and 36.0% in males, respectively, when α-naphthyl acetate was used as a substrate. There was no distinct difference in esterase banding patterns in different body regions for the substrates α-naphthyl acetate and β-naphthyl acetate on non-denaturing polyacrylamide gel electrophoresis (PAGE). However, the general esterase activities in the Huanghua population were 1.8-fold higher than those in the Pingshan population in both females and males. Increased esterase activity in the Huanghua population appeared to be mainly due to several additional esterase bands detected on non-denaturing PAGE. Inhibition studies of general esterases using four inhibitors, including paraoxon, malaoxon, eserine, and carbaryl, indicated that most general esterases in the two populations were B-type. The increased esterase activity in the Huanghua population appeared to be associated with a 1.8-fold decreased susceptibility to malathion. Such differences may attribute to the difference in control practices for the locust between Huanghua and Pingshan Counties.

The Use of Polyacrylamide Gel Electrophoresis for the Investigation and Detection of Fenitrothion and Chlorpyrifos-Methyl Resistance in Oryzaephilus surinamensis (Coleoptera: Silvanidae)

Esterase activity in 16 strains of Oryzaephilus surinamensis (L.) with different degrees of resistance to chlorpyrifos-methyl and fenitrothion were investigated quantitatively and qualitatively. Chlorpyrifos-methyl resistance strongly and positively correlated with total esterase activity, whereas there was a moderate relationship between fenitrothion resistance and esterase activity. Polyacrylamide gel electrophoresis (PAGE) provided visual confirmation of these relationships and identified qualitative differences in esterase activity between the two highly chlorpyrifos-methyl strains. Both methods revealed that the elevated esterase activity was not acetylcholinesterase based. The involvement of elevated esterase activity in resistance was confirmed through its inhibition by chlorpyrifos and chlorpyrifos oxon. PAGE was identified as an effective tool for the simple and rapid detection of resistance due to the activity of esterases and is more powerful than quantitative total esterase assays due to its ability to identify both quantitative and qualitative differences.

Purification and properties of intracellular esterases from Streptococcus thermophilus

Two of the three intracellular esterases identified in Streptococcus thermophilus were purified to homogeneity using ammonium sulphate fractionation and three chromatographic steps: anion exchange, hydrophobic interaction and gel filtration. The subunit molecular masses of esterases I and II were ∼34 and ∼60 kDa, respectively. The holoenzyme molecular masses of esterases I and II were ∼50 and ∼60 kDa, respectively, indicating that esterase I could be a dimer and that esterase II was a monomer. Phenylmethylsulphonyl fluoride inhibited the activity of both esterases, but to different degrees. Dithiothreitol, N-ethylmaleimide and EDTA strongly inhibited the activity of esterase I but significantly enhanced the activity of esterase II. Esterase I was active on p-nitrophenyl esters of the short-chain fatty acids from C2 to C10 and esterase II was active on p-nitrophenyl esters of the C2–C6 fatty acids. For both enzymes, maximum activity was obtained with p-nitrophenyl butyrate (C4). The Km values of esterase I on p-nitrophenyl esters of C2–C8 fatty acids ranged from 6.7 to 0.004 mm and the corresponding Vmax values ranged from 8.12 to 1.12 μmol min−1 mg−1 protein. The N-terminal amino acid sequences of the two esterases also differed. The major esterase (I), accounting for ∼95% of the total esterase activity, was further characterized. Esterase I was also active against tributyrin (C4), dicaproin (C6) and monoglycerides of up to C14 with maximum activity on monocaprylin (C8). Decreasing pH (from 8.0 to 5.5), temperature (from 37° to 25°C) or water activity (from 0.99 to 0.80) considerably reduced the activity of esterase I, whereas increasing NaCl concentration up to 7.5% (w/v) markedly enhanced the activity of this enzyme. Esterase I may play a role in the development of cheese flavour with respect to lipolysis.

Changes in the activity and pattern of hemolymph esterases in the larvae of wax moth Galleria mellonella L. (Lepidoptera, Pyralidae) during mycosis

The extended pattern of multiple esterase forms has been revealed in the hemolymph of wax moth Galleria mellonella larvae infected by the fungi Metarhizium anisopliae, Beauveria bassiana or Paecilomyces fumoso-roseus. The total esterase activity of the hemolymph also increases during mycosis. Mechanical damage of the cuticle, treatment with deltamethrin, and chilling of the caterpillars induced similar changes in the hemolymph pattern of esterase activity. Presumably, the changed spectrum and activity of the hemolymph esterases during mycosis is due to the damaged cuticle and epidermis cells.

Effect of pirimicarb application on field ppulations of Myzus persicae (Sulz.) (Homoptera: Aphididae) with different esterase levels

The frequency and intensity of Myzus persicae (Sulz.) resistance were studied using bioassays and biochemical tests in two potato crops in Ibicoara, Bahia. Both areas received several insecticide applications which failed to control M. persicae. By the end of the crop season, one of the areas received two applications of pirimicarb and the other was not sprayed. Six samples of 700 aphids each were randomly collected before and after the pirimicarb applications, with an interval of four days. Specimens of M. persicae were also collected from isolated potato plants. At the laboratory, the samples were characterized by the CL50 based on insecticide bioassays and by total esterase activity using colorimetric assays. After the pirimicarb applications, the susceptible (S), the moderate resistant (R1) and mixed S/R1populations presented decreasing resistance frequencies (from 36.6 to 9.9; 12,0 to 7.5 and 11.4 to 5.9%, respectively). On the other hand, high resistant individuals (R2), extremely resistant (R3), and mixed populations of R1/R2 had increasing frequencies (from 17.7 to 36.7%; 2.3 to 9.1%, and 20.0 to 30.8%, respectively). The survival of S individuals was probably due to their wandering behavior on the plants to avoid sprayed areas. All changes in frequency were reflected in the LC50 and in the resistance ratio (rr). In the pirimicarb untreated area, high frequencies of R2, R3 and R1/R2 were observed. High frequency and resistance intensity of M. persicae in areas under intensive insecticide applications can be related to the selection of resistant populations and due to the entrance of winged migrants from spontaneous plants, where the frequency of R2+R3 was 81.4%.

Chloramphenicol and ampicillin-induced changes in rat hepatic esterase and amidase activities.

The influence of ampicillin and chloramphenicol administered intraperitoneally singly or in combination on the protein content and the activities of hepatic esterase and amidase have been investigated in rats. The results have been compared to the effects of phenobarbitone (inducer) and p-nitrophenyl-phosphate (inhibitor) of hepatic hydrolases. Ampicillin pretreatment reduced protein level and amidase activity by 3.5% each but caused a significant increase (8.1%) in total esterase activity compared to controls. Chloramphenicol treatment caused an overall decrease in protein level, esterase and amidase activities respectively by 11%, 11%, and 35% over controls. Combined administration of both drugs resulted in a decrease in protein, esterase and amidase activities by 11.5%, 12.5%, and 41.2% respectively, thus mimicking the effects obtained with chloramphenicol alone. The changes induced by administration of the drugs particularly in combination on the constituent enzymes of rat hepatic hydrolases may affect the ability of the body to deal with exposure to environmental chemicals if extrapolated to man.

Toxicity of Cytisine against the Mustard Aphid Lipaphis erysimi Kaltenbach (Homoptera: Aphididae) and Its Effect on Esterases

The toxicity of cytisine from Sophora alopecuroides (L.) (Papilionaceae) against the mustard aphid Lipaphis erysimi (Kaltenbach) and its effect on esterase activities were studied. The efficacy of cytisine against aphids was higher than that of anabasine and nicotine, two botanical aphidicides from Nicotiana tabacum and Anabasis aphylla. The median lethal concentrations of the three alkaloids against the apterous adult were 432.59, 648.70, and 1090.65 mg/L, respectively, at 48 h of pest treatment using the cage-dip method. A second set of experiments utilized colorimetry to measure total esterase and carboxylesterase activity from 4th-instar larvae of the diamondback moth, Plutella xylostella (L.), which were pretreated with the alkaloids. Cytisine inhibited esterase and carboxylesterase activities in this species; moreover, there was an inverse relationship between alkaloid concentration and esterase activity.

The influence of individual susceptibility in pyrethroid exposure

The aim of this study was to find a suitable biomarker for pyrethroid adverse effects. It was shown that there is a correlation between the half-life time ( t 1/2) of pyrethroids in plasma and the clinical findings. We hypothized that this finding indicates an interindividual different amount of total esterase activity or even a polymorphism. By in vitro experiments it was demonstrated that pyrethroids are cleaved by carboxylesterases. After it turned out that carboxylesterase activity in human plasma is too low for detection, a method for specific determination of carboxylesterase activity in human isolated lymphocytes was developed. As a substrate for carboxylesterase activity, cyfluthrin was added to the lymphocyte suspension. As a proof for cyfluthrin degradation by carboxylesterases the produced hydrocyanic acid was determined by GC/MS. First hints for interindividual differences in carboxylesterase activity in lymphocytes were found.

Molecular Analysis of a Gene Encoding a Cell-Bound Esterase from Streptomyces chrysomallus

A gene (estA) encoding a 42-kDa cell-bound esterase, EstA, was found to be located 75 bp upstream of the cyclophilin A gene (cypA) of Streptomyces chrysomallus. Western blot analysis revealed the presence of EstA (42 kDa) in cell extracts of S. chrysomallus X2 and Streptomyces lividans. EstA specifically hydrolyzes short-chain p-nitrophenyl esters. EstA formation starts at the end of growth phase, and its activity level remains constant throughout stationary phase. Expression of estA from the melanin (mel) promoter in plasmid pIJ702 led to a substantial increase of total esterase activity in streptomycetes.

Control of malaria vectors with the insect growth regulator pyriproxyfen in a gem mining area of Sri Lanka

The general esterase activity in larvae, pupae and adult mosquitoes of Aedes togoi, Aedes aegypti (ROCK), Aedes albopictus, Culex quinquefasciatus (Kembangan) and Armigeres subalbatus was determined by a continuous fluorimetric assay which employs 4-methylumbelliferylacetate (4-MUA) as the substrate. The specific activities of the fourth-instar larva and adult of C. quinquefasciatus (Kembangan) were significantly higher than the corresponding activities of the other species of mosquitoes. When measured in the fourth-instar larvae, the total esterase activity per larva was significantly higher in C. quinquefasciatus (Kembangan) and Armigeres subalbatus compared to the other three species examined. This was correlated with correspondingly higher LD50 values, measured with malathion, for these two species. 1-Naphthylacetate (1-NA) showed inhibition of 4-MUA-esterase activity. A comparison of the total esterase activity measured in parallel with 4-MUA and 1-NA as substrates in 11 extracts of adults and pupae of different species of mosquitoes showed a good correlation (r = 0.96). The procedure was also modified as a quantitive end-point assay suitable for rapid screening of multiple samples. For investigation of field populations, the fluorescence of the released 4-MU could be observed under illumination of a 96-well plate with a hand-held u.v. lamp.

Influence of phosphorus and formononetin on isozyme expression in the Zea mays‐Glomus intraradices symbiosis

Maize (Zea mays L. cv. Great Lakes 586) plants were either inoculated with the vesicular‐arbuscular mycorrhizal (VAM) fungus Glomus intraradices Schenck and Smith, or grown in the presence of the isoflavone formononetin or were provided with both G. intraradices and formononetin. All plants were grown in soil containing one of five levels of inorganic P (between 8 and 110 µg g−1 soil). By 3 weeks there were significant differences in a number of enzyme activities and in the pattern of isoenzymes in roots colonized by the VAM fungus or treated with formononetin. One NAD‐malate dehydrogenase (MDH) isozyme was expressed only in mycorrhizal roots, whether treated or not with formononetin. Despite differences in the soil P level, the expression of this isozyme was not observed in non‐mycorrhizal roots, indicating specific expression in the mycorrhizae. We suggest that MDH isozyme could serve as a specific, early indicator of the Zea‐Glomus symbiosis. Differences in the esterase (EST) isozyme pattern were not detectable between VAM and non‐VAM roots, suggesting that this enzyme system is not a good parameter for the evaluation of mycorrhizal colonization. As available P in the soil increased, total EST activity appeared to increase as well. Interestingly, total peroxidase (POX) activity increased along with P suggesting that as plant P nutrition improved, both cell wall ramification and the quantity of defense peroxidases increased as well. Total POX activity from mycorrhizal roots was inversely correlated with root colonization, indicating that there was suppression of POX activity by the host under low soil P. Most interestingly, formononetin further decreased POX activity regardless of the level of P or mycorrhizal status. This may suggest one mechanism by which formononetin enhances root VAM colonization. The presence of this isoflavone suppressed POX activity in mycorrhizal roots allowing a rapid penetration and spread of the fungus in the root cortex. The interplay between host root, soil P levels, secondary metabolites and endogenous host enzyme activities and a particular VAM fungus has a profound effect on the efficiency, duration and functioning of an endomycorrhizal symbiosis.

Changes of Insecticide Resistance Levels and Detoxication Enzymes Following Insecticide Selection in the German Cockroach,Blattella germanica(L.)

Insecticide resistance in the German cockroach can be mediated by a number of mechanisms, the most common being enhanced enzymatic metabolism. Changes in metabolic enzymes following insecticide selection of a hybrid German cockroach strain (JM) were investigated. The JM strain originated from matings of the Munsyana (multiple resistance) strain and Johnson Wax (susceptible) strain. Two subpopulations of the JM strain were selected over three generations: one with a pyrethroid insecticide (cypermethrin) and one with an organophosphate insecticide (chlorpyrifos). Significant increases in resistance levels were observed in the first generation following selection. These trends continued for three generations and resistance levels to each insecticide approached those of the original Munsyana strain by the third generation of selection. Cypermethrin selected for cross-resistance to chlorpyrifos; however, chlorpyrifos selected for negative cross-resistance to cypermethrin. Increased body weight was weakly but positively correlated with increases in insecticide resistance to each insecticide. Examinations of metabolic enzymes indicated no change in total esterase (1-NA, 2-NA, and PNPA hydrolysis) or glutathione S-transferase (DCNB and CDNB conjugation) activity. Native PAGE of esterases identified one electromorph (E2) which stained more intensely following chlorpyrifos selection and which was inhibited by both chlorpyrifos and chlorpyrifosoxon. Examination of cytochromes P450 by carbon monoxide difference spectra indicated that each insecticide selected for increases in total cytochrome P450 content. However,in vitrodemethylation and Western analysis results suggest that different cytochrome P450 isoforms were selected by each insecticide.

Assessment of fluorescein diacetate hydrolysis as a measure of total esterase activity in natural stream sediment biofilms

I applied the fluorescein diacetate (FDA) hydrolysis technique as a rapid and sensitive estimator of total esterase activity in stream sediment biofilms. I investigated the effects of temperature, pH and incubation time and optimized the assay for low blanks and high fluorescein extraction. The FDA procedure was precise (c.v. = 4.15%) and could detect 10 nM fluorescein. Spatial patterns of esterase activity within stream sediment biofilms correlated with electron transport system activity, bacterial thymidine incorporation, glucosidase activity and chlorophyll a. As such, I suggest the modified and optimized technique as applicable to the investigation of total stream bioflim esterase activity.

Characterisation of Insensitive Acetylcholinesterase in Insecticide-Resistant Cotton Aphids,Aphis gossypiiGlover (Homoptera: Aphididae)

Levels of insecticide resistance, total esterase activity, and acetylcholinesterase (AChE) sensitivity to inhibition by insecticides were investigated in three clones ofAphis gossypiiGlover. Compared with LC50values for a susceptible clone (171B), clone 968E showed high (>20-fold) resistance to most of the carbamate and organophosphorus insecticides tested, whereas clone 1081K only exhibited strong resistance to some carbamate insecticides.171B aphids had low esterase activity combined with an AChE variant showing baseline sensitivity to organophosphorus and carbamate insecticides. 968E combined high esterase activity with broad-spectrum AChE insensitivity to these chemicals. 1081K had low esterase activity and an AChE variant that was insensitive to pirimicarb and a smaller range of OP insecticides. These findings demonstrate the occurrence of at least two insensitive AChE variants inA. gossypii,whose contrasting insensitivity profiles permit rapid diagnosis using a kinetic microplate assay. Altered AChE appeared to be the main mechanism of resistance to both organophosphorus and carbamate insecticides in these clones.

Evidence for the existence of juvenile hormones in ticks.

Juvenile hormones (JHs) comprise a family of sesquiterpenoid compounds identified primarily from insects. JHs are involved in preventing metamorphosis during moults in immature stages and regulating aspects of reproduction in adults[l]. The JH titre is strictly regulated by a number of different mechanisms at specific times during development. JHs are synthesized and released into the haemolymph, where they are bound to haemolymph binding-proteins which protect them from esterases[2]. When JHs are no longer needed, they are degraded by specific JH-esterases and JH-epoxidases to the inactive products, JH-acids and JH-diols[3]. Ticks (Acarina:lxodidea), like insects, pass through several immature stages before becoming reproductively competent. JHs have not been identified in ticks, but several reports suggest that JHs are present and have roles similar to those in insects[4,5]. This study presents evidence for the occurrence of haemolymph JH-binding proteins and of specific JH-esterase and epoxidase activities, thus, indirectly supporting the presence of JH-like compounds in the adult tick, Amblyomma hebraeum. Ticks were raised as previously described[6] and taken for assay at various times during the feeding cycle. For JHesterase and epoxidase studies, tick tissues were divided into three categories: haemolymph, gut tissue and 'remaining tissues' (included fat body, muscle, ovary, salivary glands, and synganglion). Total esterase and epoxidase activities were determined by homogenizing the tissue in phosphate buffered saline (0.1M phosphate buffer, 100 mM NaCI) and incubating an aliquot with 0.12 HCi [3H]JHIII (0.58 Ci/mol) for 4h at 26'C. The sample was then extracted into acetonitrile and radioactivity analyzed by HPLC connected to an on-line radioactivity detector. Separation was performed on a C I S column using a linear gradient of acetonitri1e:water (35:65. v/v) to acetonitrile over 45 min at a flow rate of 1 ml/min. Non-specific esterase activity was assessed according to Rotin et a1.[7]. Haemolymph binding studies using the photoaffinity label, [3H]epoxy-bis-farnesyI diazoacetate ([3H]EBDA), followed the procedure of King ef al.[8]. For this. diluted haemolymph was incubated with 1 pCi [3H]EBDH or I 1.11 [3H]EBDA + 10 nmol JHI in polyethylene glycol-treated quartz cuvettes and exposed to 254 nm light for 5 min in a Rayonete Photoreactor. Following SDS-PAGE, gels were prepared for fluorography according to Prestwich et al. [9]. Incubation of tissues with [3H]JHIII suggests that only the 'remaining tissues' group showed specific esterase activity (total esterase activity non-specific esterase activity). Epoxidase and JH-specific esterase activities in this group of tissues were low in small, partially fed females, peaked just prior to engorgement. then declined to initial levels at the time of engorgement. Of the two enzymes examined, the JHepoxidase showed the greatest activity. Gut tissue apparently could only non-specifically metabolize JHIIl to the inactive -acid, -diol, and -acid-diol derivatives. Haemolymph tissue showed no detectable esterase or hydrolase activity but this could be due to difficulties in obtaining a sufficient volume of haemolymph for the assay. Photoaffinity labelling experiments using the JHI photoaffinity analogue, [3H]EBDA, with haemolymph from partially fed females indicated the presence of two regions of [3H]EBDA binding on SDS-PAGE gels that could be displaced by excess JHI. These corresponded to a high molecular weight region (75-100 kDa) and a single, low molecular weight band (approximate molecular weight 44 kDa). Kulcsar et al.[10] identified a similar, high molecular weight binding region (66-97 kDa) using the JHIII-specific photoaffinity label [3H]epoxy-farnesyl diazoacetate but suggested that this labelling was due to non-specific binding of [3H]EBDA to lipoproteins. It is likely that the high molecular weight region we observe is also due to nonspecific binding of lipoproteins. The 44 kDa labelled protein observed on SDS-PAGE gels is more suggestive of a JHIdisplaceable binding protein, however, at present i t is not known whether this protein is specific for a JH-like compound or is developmentally regulated throughout the feeding cycle. Although direct identification of JHs in tick haemolymph/tissue is ultimately needed, a combination of indirect evidence using complementary techniques can be strongly suggestive of the presence of JH. The identification of a haemolymph JHI-displaceable binding protein using the photoaffinity label EBDA indicates that a mechanism for JH protection and distribution exists in ticks. However, binding displacement by JHI does not imply that endogenous JHI is present in ticks and may merely reflect cross-reactivity to another related compound. Furthermore. the enzymes responsible for JH degradation in insects are also present in tick tissues and these enzymes appear to be developmentally regulated. Given that ticks possess these basic mechanisms for strict JH titre regulation, it is compelling to suggest that JHs do have important functions in tick development.

Soybean seed deterioration and response to osmotic priming: changes in specific enzyme activities in extracts from dry and germinating seeds

The effect of accelerated aging (AA) and seed priming on specific seed enzymes was studied in soybeans. Aging seeds for 48 and 96 h at 41°C and 100% humidity reduced germination from 92% to 68% and 0%, respectively. Malate dehydrogenase (MDH) glutamate dehydrogenase (GDH), and esterase were assayed in extracts from dry and imbibing seeds. Malate dehydrogenase activity was the least affected by AA treatments. Certain GDH isozyme activity was reduced by 17% after 48 h of AA and almost completely lost after 96 h. Imbibing for 24 h increased GDH activity in 48 h-accelerated-aged seeds but not the 96 h-aged seeds. Two esterase isozymes in dry seeds showed a 77% loss of activity after 48 h of AA. This treatment increased the total esterase activity, with the greatest activity observed after 96 h of AA. This was due to the appearance of a diffuse high molecular weight (HMW) region of activity observed on native polyacrylamide gels and specifically induced in the embryonic axes. Imbibing 48-h-accelerated-aged seeds for 48 h caused a reappearance of the two esterase enzymes lost during aging. The HMW esterase staining region also appeared after imbibing unaged seeds for 24 h. Seed priming, caused a response similar to imbibition for each enzyme except the HMW esterase activity. Priming 48-h-accelerated-aged seeds reduced the HMW esterase activity. This was not observed during imbibition of aged seeds and indicates the first identification of a specific enzymatic response unique to a seed priming treatment.