Sami Ahmad

Esterases in larval tissues of gypsy moth, Lymantria dispar (L.): optimum assay conditions, quantification and characterization.

Abstract Esterase activity of the gypsy moth, Lymantria dispar (L.), was studied by a spectrophotometric method using 1200 g supernatants of mid-fifth instar larval tissues as the enzyme source and 1-naphthyl acetate as substrate. Using midgut preparation, the K m was determined to be 4.25 × 10 −5 M 1-naphthyl acetate, and the V max 942 nmoles mg −1 min −1 at 30°C. The hydrolysis rate was linear for the first 25–30 min. Enhancement in esterase activity was apparent up to 50°C. The optimum p H was between 7.5–7.7. The specific activities (nmoles mg −1 min −1 at 30°C) of tissues were as follows: midgut (1004), Malpighian tubules (334), nerve cord (125), fat body (111), hindgut (108), foregut (84), gonads (83), muscles (55), brain (43), integument (20), and haemolymph (7). Midgut accounted for 91.5% of total esterase activity of all tissues. With the exception of brain and nerve cord where acetylcholinesterase (EC 3.1.1.7) showed higher activity, all other tissues revealed carboxylesterase (EC 3.1.1.1.) as the predominant enzyme. There was evidence of some arylesterase (EC 3.1.1.2.), particularly in the hindgut, nerve cord, and the brain. Midgut was the tissue richest in esterase actvity, which was largely carboxylesterase activity. Its titre displayed a cyclic pattern that correlated with feeding activity and larval development.

Nonoxidative enzymes in the metabolism of insecticides.

Two major classes of enzymes, i.e., hydrolases and transferases, comprise all the nonoxidative enzymes, and together these enzymes catalyze a wide variety of biotransformations of insecticides. The hydrolytic enzymes involved in insecticide metabolism are carboxylesterase (EC 3.1.1.1), arylesterase alkylamidase, and DFPase (EC 3.8.2.1). Recent experimental evidence suggests that carboxylesterase enzyme(s), formerly known to hydrolyze malathion-type insecticides, can also catalyze hydrolysis of a variety of diversified insecticidal esters such as benzilic acid derivatives, carbanilate compounds, and pyrethroids. These organophosphate-sensitive esterases, with the exception of the enzyme which hydrolyzes malathion, are all present in microsomes. Similarly, the action of amidases now extends to those insecticidal compounds of their intermediates which contain an aminoformyl (N-CHO) moiety. Arylesterase and DFPase catalyze the P-anhydride bond cleavage of the leaving group, a major hydrolytic pathyway for organophosphate insecticides. Transferal enzymes which are presently know to metabolize insecticidal organophosphates are GSH-S-alkyltransferase (EC 2.5.1.12) and GSH-S-aryltransferase (EC 2.5.1.13). These enzymes cleave P-O-R (R = alkyl) or P-0-X (X = aromatic), with subsequent transfer of the R or X group to glutathione. Regarding the other conjugating enzymes, UDP-glucuronyltransferase (EC 2.4.L.17), UDP-glucosyltransferase (EC 2.4.1.35), and arylamine acetyltransferase (EC 2.3.1.5), much work is needed to understand their interactions with insecticidal compounds. There is some evidence that arylsulfotransferase (EC 2.8.2.1) MAY PLAY A PROMINENT ROLE IN THE CONJUGATIVE MECHANISMS OF INSECTS.

Larval and adult housefly carboxylesterase: Isozymic composition and tissue pattern

Abstract Carboxylesterase (EC 3.1.1.1) isozymes of the adult and larval housefly Musca domestica , were separated electrophoretically on polyacrylamide gels. Of the 10 isozymes present in the 17,500 g supernatant of the whole fly homogenate, 9 were detected in the gut and the thoracic muscles. Distribution of isozymes was as follows: isozyme E 1 and E 10 in the foregut as well as in the combined mid-and hindgut preparation, and E 2–8 and E 10 in the thoracic muscles. On the basis of the molecular sieving effect of the gels of different pore sizes, the approximate mol. wt of the isozymes E 1–9 were found to be in the range of 67,000 (±3000)–300,000. Isozyme E 10 had the highest molecular weight of ca. 1 × 10 6 . Ten isozymes were resolved from larval tissues, and these corresponded to E 1 and E 3–10 of the adult, and another isozyme E x with no correspondence to any of the adult isozymes. Isozyme E 2 was absent. Distribution pattern of larval isozymes was as follows: E 1 , E 3–7 and E 9–10 in the gut; E 1 , E 6–8 and E 10 in the muscles; E 1 , E 3–8 , E 10 and E x in the fat body. Densitometric scans showed that the activity detected histochemically and by pH-titrimetric method ( Ahmad , 1970a) was due primarily to isozyme E 1 . Although nothing further is known concerning this isozyme, its wide distribution in larval tissues and restriction to gut in the adult stage may be biologically significant. The molecular diversity and varied tissue pattern indicate several roles for carboxylesterase in the housefly. The physiological roles discussed are: (1) regulation of JH titre, (2) mobilization of fat in fat body, (3) energy-related catabolism of fatty acid esters in flight muscles, (4) cuticular wax synthesis and transport, and (5) degradation of undesirable, metabolically inert esters.

Nadph oxidation by microsomal preparations of gypsy moth larval tissues

Abstract NADPH oxidation by microsomal preparations from several tissues of fifth-instar larvae of gypsy moth, Porthetria dispar Linn. was studied. The distribution of this activity was as follows: 53.9, 27.8, 9.6, 4.4, and 4.3 per cent in midgut, fat body, hindgut, Malpighian tubules, and foregut respectively. Relatively specific activities of these microsomal preparations were 100, 81, 77, 61, and 55 for hindgut, Malpighian tubules, midgut, foregut, and fat body. NADPH oxidation was rapid for the first 20 minutes of the reaction and exhibited a K m of 1.75 × 10 −5 M NADPH. Cytochrome C greatly enhanced NADPH oxidation without affecting the K m . Carbon monoxide inhibited microsomal oxidation of NADPH and the inhibition was partially reversed by cytochrome C. This suggested the possibility of the presence of cytochrome P 450 . SKF 525-A inhibited NADPH oxidation and the locus of inhibition appeared to be cytochrome P 450 . Sulphhydryl inhibitors, p -chloromercuri-benzoate, p -chloromercuriphenyl sulphonate and Cu 2+ , inhibited NADPH oxidation and the inhibition was not reversed by cytochrome C. This indicated the involvement of NADPH-cytochrome C-reductase in the electron transport chain in gypsy moth.

NADPH-cytochrome-c-reductase: changes in specific activity in gypsy moth larvae

Abstract NADPH-cytochrome- c -reductase from guts of gypsy moth, Porthetria dispar , oxidized NADPH at the following rates: 11·6, 41·9, and 57·6 nmol/30 min per 0·6 mg protein, in the third, fourth and fifth instar, respectively. The increase in specific activity may be due to a natural induction process controlled by materials in the food. This could also explain the observed decreases just prior to ecdysis at which time the larvae have ceased feeding.

Hydroxylation and demethylation by gut microsomes of gypsy moth larvae

Abstract 1. 1. Biotransformations of certain aromatic xenobiotics by microsomes from the gut of gypsy moth Porthctria dispar L. larvae were studied. 2. 2. The specific activities for hydroxylation (aniline), N-demethylation (N,N-dimethyl-p-nitrophenyl carbamate) and O-demethylation (p-nitroanisole) were 11.3, 8.0, and 7.0 nmoles product per hour, respectively. Bovine serum albumin enhanced these rates by 11.9, 50.9, and 24.5 per cent, indicating the presence of some inhibitory material in the microsomal preparation. 3. 3. Maximal N-demethylase activity was observed at 37°C., and at higher temperatures enzymic activity declined rapidly, probably through thermal deactivation. Highest activity was observed between pH 7.0 and 7.8. 4. 4. All three reactions were inhibited by mixed-function oxidase inhibitor, SKF525-A. The addition of Mg3+ did not improve enzymic activity.

Metabolism of Radiolabeled Insecticides in Insects and Related Arthropods: A Critical Study of Various Techniques

The metabolism of radiolabeled insecticides in insects and acarina is studied largely by coupling radiotracer techniques with analytical methods, such as TLC, paper and column chromatography, gel-permeation chromatography, and enzymatic assays. These techniques in various combinations yield both the identification and quantification of the metabolites. Other analytical methods such as gas chromatography or IR spectrometry may also be used to obtain additional support for identification of metabolites. In the absence of authentic chromatographic standards, however, NMR and mass spectrometry are necessary in the identification of the unknown compound. The quantity of the radiolabeled insecticide administered should be within the toxicological range of the insect. Therefore, the dosage-mortality response of the insect using unlabeled material should be determined. A dose should be selected that keeps insect mortality to a minimum in order to avoid complications in the computation of the balance data. The radiolabeled insecticide is usually applied topically to the insect. Alternately, the material may be administered by dipping in a solution containing the radiolabeled compound or by exposure to filter paper impregnated with radiolabeled material. Administration of the radiolabeled material by the oral route presents several problems. Sterile rearing conditions are mandatory to avoid contamination of treated diet with microorganisms. Some knowledge of the insects feeding rhythm is desirable so that the labeled diet is given at peak feeding time. Synthetic diets should be adjusted to pH 7.0. These precautions minimize degradation of the insecticide in the diet prior to consumption by the insect. Precise doses of radiolabeled materials may be administered by injection. The technique is mainly useful in metabolism studies of intermediate materials resulting from the biotransformation of the parent compound.