Krishnappa Venkatesh

Haemolymph juvenile hormone esterase during the life cycle of the tobacco hornworm, Manduca sexta (L.)

Abstract Juvenile hormone (JH) esterase activity was found in the plasma of larvae, pupae and adults of wild-type tobacco hornworms, Manduca sexta . There was a single peak of plasma JH esterase activity approx. 28 h prior to ecdysis in each instar from the second through the fourth instar and a peak of activity prior to both wandering and pupation in the fifth (last) instar. JH esterase activity was high in newly formed male and female pupae but declined to minimal levels by day 1 of the pupal stage. For the remainder of the pupal period, activity was at background levels. JH esterase activity increased again in newly emerged, virgin male and female adults but declined and remained at a low level 1 day after emergence through death. Gel filtration analysis of larval, pupal and adult plasma resolved a single peak of JH esterase activity with an apparent molecular weight of 66,000. However, isoelectric focusing revealed three forms with isoelectric points of 5.5, 5.8 and 6.1. These isoelectric forms were also found in black and white mutants of last instar M. sexta and in purified JH esterase from wild-type larvae. The plasma JH esterase activity metabolized JH I 2–3 times faster than JH III and was sensitive to inhibition by octylthio-1,1,1-trifluoro-2-propanone and insensitive to O , O -diisopropyl phosphorofluoridate. Gel filtration, isoelectric focusing, substrate specificity and developmental studies suggest that the same JH esterases are found in the plasma of larvae, pupae and adults and appear to be different from general (α-NA) esterase.

Characterization and the developmental role of plasma juvenile hormone esterase in the adult cabbage looper, Trichoplusia ni

Abstract Juvenile hormone (JH) esterase was characterized from the plasma of adult females of the cabbage looper, Trichoplusia ni , and compared with that present in 4th and 5th instar larvae. Ester hydrolysis was the principal route of JH metabolism. Gel filtration of plasma resolved a single peak of JH esterase which was distinct from that of the α-naphthyl acetate (α-NA) esterase activity. The JH esterase apparent molecular weight was 62,000 in prepupae and virgin, female adults and 69,000 in 2-day-old 4th instar larvae. Broad range isoelectric focusing of plasma of prepupae and adults resolved a major peak of activity at pH 5.5 with a minor peak of activity at pH 6.1 and in 4th instar larvae at pH 5.45 and 5.8, respectively. By this method JH esterase was resolved from the α-NA esterase activity. The plasma of prepupae and adults metabolized JH I at about twice the rate of JH III. JH esterase activity from adult plasma was more stable than the α-NA esterase activity. Adult JH esterase activity was insensitive to inhibition by O , O -diisopropyl phosphorofluoridate in contrast to that of the α-NA esterase activity. Mated females oviposited 8 times more eggs than virgin females to 10 days after emergence. The total haemolymph protein content of virgin females remained high throughout the period of study whereas mated females showed a significant decline beginning on day 4. JH esterase activity remained unchanged in virgins whereas it declined drastically in mated females. The α-NA esterase activity declined to low levels shortly after emergence in both groups. JH and α-NA esterase activity was not affected by the application of the juvenoid, ( RS )-methoprene. The present study provides evidence of a functional role for JH esterase in JH metabolism and reproduction in adult T. ni . JH esterases in the adult were identical to that of prepupae by the methods described above.

19F-NMR spectral evidence that 3-octylthio-1,1,1-trifluoropropan-2-one, a potent inhibitor of insect juvenile hormone esterase, functions as a transition state analog inhibitor of acetylcholinesterase

Abstract Fluoroketones have been developed as highly potent, specific inhibitors of insect juvenile hormone esterase. These compounds are believed to function as transition state analog inhibitors of the enzyme. Of the fluoroketones prepared, 3-octylthio-1,1,1-trifluoropropan-2-one has been extensively studied, yet no analysis of the actual mode of enzyme inhibition has been reported. A 19 F-NMR analysis of the inhibition of a hydrolytic enzyme, acetylcholinesterase, by 3-octylthio-1,1,1-trifluoropropan-2-one was carried out. Experiments with phosphorylated acetyl-cholinesterase determined that interaction of the fluoroketone with the enzyme is specific for the esteratic active site serine residue. The carbonyl moiety of the fluoroketone is required for binding and inhibition. The fluoroketone binds to the enzyme active site in a tetrahedral form, indicating that 3-octylthio-1,1,1-trifluoropropan-2-one does function as a transition state analog inhibitor of a hydrolytic enzyme.

The inhibition of insect juvenile hormone esterase by trifluoromethylketones: Steric parameters at the active site

Abstract A rationally designed structure-activity relationship study has been accomplished using trifluoromethylketone inhibitors of insect juvenile hormone esterase from the cabbage looper, Trichoplusia ni (Hubner) (Lepidoptera: Noctuidae). Several α- and α′-substituted derivatives of 3-octylthio-1,1,1-trifluoropropan-2-one have been prepared and assayed for inhibitory potency against juvenile hormone esterase. The results indicate that the sulfur/protein interaction does not occur in a sterically constrained environment. Substitution adjacent to sulfur did not dramatically effect activity. However, substitution adjacent to the carbonyl of the trifluoromethylketone moiety reduced inhibitory potency substantially, indicating that the active site region of juvenile hormone esterase which interacts with the carbonyl is restricted to rather small substrates. A small hydrophobic pocket near the active site has been identified and can serve to increase inhibitory potency by secondary binding of appropriate substituents. The present study has resulted in the preparation of two more effective in vitro inhibitors of juvenile hormone esterase than those previously reported. Evidence that there are two naturally occurring forms of juvenile hormone esterase has also been provided.

Enzymatic and immunohistochemical studies on the role of cytochrome P450 and the flavin-containing monooxygenase of mouse skin in the metabolism of pesticides and other xenobiotics

Abstract The cytochrome P450 (P450) content, the cytochrome c reductase activity, the metabolism of a variety of P450 substrates, and the presence and role of flavin-containing monooxygenase (FMO) in xenobiotic metabolism were studied in skin microsomes and compared to those of liver. The cytochrome P450 content of skin as determined by CO-dithionite-reduced minus CO-oxidized spectra was approximately 6.8% of the liver P450 content. By comparison, cytochrome c reductase activity in skin microsomes was high, being equivalent to approximately one-third of the liver microsomal enzyme activity. Skin microsomes metabolized several known P450 substrates and, depending upon the substrate used, the specific activity ranged from 2.5 to 13.4% of the corresponding rates seen in liver microsomes. Skin microsomes exhibited the highest enzymatic activity with benzo[ a ]pyrene and ethoxyresorufin, moderate activity with parathion and aldrin, and low activity with benzphetamine and ethoxycoumarin. Skin microsomes also metabolized the triazine herbicides atrazine, simazine, and terbutryn, with the activity being 2 to 5% of the liver microsomal activity. FMO activity in skin microsomes with thiobenzamide and methimazole as substrates ranged from 10 to 20% of the liver FMO activity. Immunohistochemical studies using antibodies to mouse liver FMO showed localization primarily in the epidermis. Additional studies using pig skin showed a similar distribution pattern. Antibodies developed to mouse liver FMO and the constitutive liver P450 isozyme, 1A2, showed cross-reactivity on Western blots with proteins in skin microsomes that appeared identical to the cross-reacting proteins present in liver microsomes. The relative contribution of P450 and FMO in mouse skin to the sulfoxidation of phorate was investigated and compared to that of liver microsomes. Several procedures were employed to selectively inhibit either P450 or FMO to determine the role of each monooxygenase system in the absence of the other system. In liver microsomes, P450 was responsible for 68 to 85% of the phorate sulfoxidation activity. In contrast, in skin microsomes 66 to 69% of the phorate sulfoxidation activity was due to FMO, while P450 was responsible for the remainder of the activity. Thus, although the overall phorate sulfoxidation rate in mouse skin microsomes was only 3 to 4% of the rate seen in liver, FMO appears to assume a greater relative role to P450 in the metabolic processes in skin.

The flavin-containing monooxygenase of mouse kidney: A comparison with the liver enzyme

Flavin-containing monooxygenase (FMO; EC 1.14.13.8) was purified from mouse kidney microsomes and compared to that isolated from mouse liver microsomes. The purified enzymes from kidney and liver appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 58,000 daltons. On wide range (pH 3.5 to 9.0) isoelectric focusing, FMOs from kidney and liver resolved as a single band with an isoelectric point of 8.2. The enzymes from both kidney and liver have a pH optimum of 9.2. Thiobenzamide-S-oxidation catalyzed by both enzymes was sensitive to inhibition by the competitive inhibitors thiourea and methimazole. At an n-octylamine concentration of 3 mM, thiobenzamide-S-oxidation by the kidney FMO was increased by 122% and that by the liver FMO by 148%. Km and Vmax values were determined and compared between the two tissue enzymes for xenobiotic substrates containing nucleophilic nitrogen, sulfur or phosphorus atoms. In general, for most FMO substrates, Km and Vmax values were similar between kidney and liver FMO with only a few exceptions. The Km and Vmax values for fenthion for kidney were only half of those observed for liver FMO. Fonofos was unusual in having a low Km as well as a low Vmax for both tissue enzymes. Anti-sera developed to the FMO purified from kidney and liver showed cross-reactivity with each purified enzyme as well as with a protein with the same molecular weight as the purified FMO present in both kidney and liver microsomes. These bands showed equal intensity based on an equivalent amount of protein. Analysis of kidney and liver FMO by proteolytic digestion followed by visualization of peptides by silver staining or immunoblotting showed only minor differences between the enzymes of the two tissues. The amino acid composition of both mouse kidney and liver FMO was low in methionine and histidine and rich in aspartate/asparagine, glutamate/glutamine, leucine, valine and glycine. Edman degradation of the purified mouse kidney and liver FMO provided a single amino acid sequence of the NH2-terminus. This sequence matched exactly with the cDNA-deduced sequence reported for the pig and rabbit liver beginning with the fifth amino acid and contained the highly conserved FAD-binding domain Gly-X-Gly-X-X-Gly, commonly found in a number of other FAD-binding proteins. These studies indicate that the renal and hepatic forms of FMO from mouse are similar enzymes that are immunologically related and show only a few minor differences.

A novel geminal diol as a highly specific and stable in vivo inhibitor of insect juvenile hormone esterase

Thio-containing and acetylenic trifluoromethyl ketones were potent inhibitors of insect juvenile hormone (JH) esterase with greater inhibitory activity than aliphatic and α,β-unsaturated homologs. Octylthio-1,1,1-trifluoropropan-2-one was the most potent inhibitor with the greatest equilibrium hydration constant in pure water. However, a keto/hydrate equilibrium was not necessary for JH esterase inhibition. The carbonyl tautomer of 1-octyl [1-(3,3,3-trifluoropropan-2,2- dihydroxy)] sulfone (OTPdOH-sulfone) was not detectable, and yet OTPdOH-sulfone was a potent in vitro inhibitor of JH esterase with an I50 of 1.2 nM. The mechanism of JH esterase inhibition by these compounds is discussed. OTPdOH-sulfone inhibited JH esterase with minimal activity toward insect 1-naphthyl acetate esterase and electric eel acetylcholinesterase. The inhibitor was also active in vivo, selective for JH esterase, and persistent for over 32 h. OTPdOH-sulfone when topically applied to larval and adult cabbage loopers, Trichoplusia ni, elicited juvenoid activity apparently because of the specific in vivo inhibition of JH metabolism. Arch. Insect Biochem. Physiol. 36:165–179, 1997.

The role of juvenile hormone and brain factor(s) in the regulation of plasma juvenile hormone esterase activity during the last larval stadium of the tobacco hornworm, Manduca sexta

Abstract During the last (fifth) larval stadium of gate-1 Manduca sexta there were two peaks of juvenile hormone esterase activity in the plasma. The first peak occurred on day 3 preceding wandering and the second of a similar size on day 8 preceding pupation. The topical application of juvenoids (juvenile hormone I, epofenonane, and ( RS )-methoprene) to the abdomens of prewandering (day 0 to 3) and postwandering (day 5 and 7) larvae increased the plasma juvenile hormone esterase activity. Head ligation and starvation of prewandering larvae blocked the first peak and ligation of postwandering larvae blocked the second peak. Application of juvenoids could not rescue the effects of ligation and starvation in prewandering day 0 to 2 larvae, but ligation of day 1 and 2 larvae for 24 h followed by juvenoid treatment restored normal juvenile hormone esterase activity levels in ligated but not starved larvae. Juvenile hormone acted directly on the abdomen presumably on the fat body to increase juvenile hormone esterase activity throughout the last stadium but day-3 and -7 larvae were most responsive. Injection of head extracts of day-1, -2 and -3 fifth-instar larvae into the abdomens of head-ligated day-1 larvae did not increase the plasma juvenile hormone esterase activity, whereas it increased the α-naphthyl acetate esterase activity. Day-1 head extract decreased the plasma juvenile hormone esterase activity in unligated day-0, -1 and -6 larvae and in 24 h head-ligated, day-2 larvae that were treated with epofenonane. In day-1 unligated larvae treated with epofenonane one head equivalent reduced the juvenile hormone esterase activity by 48%. Injection of 4 head equivalents from day-1 larvae into larvae of the same age not treated with juvenoids completely inhibited the normal increase in juvenile hormone esterase activity seen between day 1 and 2. The inhibitory factor was specific in action not affecting the α-naphthyl acetate esterase activity, was dose-dependent, and found both in head and brain but not in whole body extracts. The inhibitory effect was present in head extracts prepared from day-1, -2 and -3 larvae. Head extracts also significantly decreased the juvenile hormone esterase activity in the whole body of normal as well as epofenonane-treated larvae without affecting the α-naphthyl acetate esterase activity.

Inhibition of insect juvenile hormone esterase by α,β-unsaturated and α-acetylenic trifluoromethyl ketones

Abstract A series of β,β-disubstituted-α,β-unsaturated and β-substituted-α-acetylenic fluoroketones have been prepared and assayed as inhibitors of insect juvenile hormone esterase from the cabbage looper, Trichoplusia ni (Hubner) (Lepidoptera: Noctuidae). The most potent inhibitors of each series, 4-methyl-1,1,1-trifluorododec-3-en-2-one 3 and 1,1,1-trifluorododec-3-yn-2-one 15, were also assayed as in vivo inhibitors of juvenile hormone esterase. None of the new compounds assayed were more effective inhibitors than structurally analogous α-thioalkyl substituted trifluoropropanones. The acetylenic series were uniformly more potent inhibitors than the unsaturated series of fluoroketones. The difluoroketone derivatives of the most potent inhibitors were markedly less active. This study clearly reveals that the S atom of α-thioalkyl substituted fluoroketone inhibitors has a more complex function than simply mimicking the site of unsaturation in the natural substrate.

The effect of detergents on the purified flavin-containing monooxygenase of mouse liver, kidney and lungs

1. The effect of various commonly used membrane solubilizing detergents on the activity of the microsomal xenobiotic metabolizing enzyme, the flavin-containing monooxygenase (FMO) purified from mouse liver, kidney and lungs was determined. 2. Regardless of the type of detergent used, the effect on the enzyme activity was variable depending on the type of substrate used. 3. Emulgen 911 concentrations of up to 10% had very little effect on thiobenzamide-S-oxidation by liver, kidney or lung FMO. 4. While Emulgen 911 increased substrate dependent NADPH oxidation rate by thiourea and thioacetamide, it drastically reduced the activity toward the organophosphorous compounds, disulfoton, fenthion, fonofos and phorate at low concentrations. 5. Activities of fenthion, phorate and fonofos were decreased by 80, 65 and 55% by the inclusion of 0.25% Emulgen 911 in the assay mixture. 6. This decline in FMO activity for phorate was evident regardless of the type of detergent used. In contrast, thiourea dependent NADPH oxidation rate in the presence of various detergents was variable. 7. Thiourea oxidation rate was decreased by cholate and Zwittergent 3-12, whereas it was increased in the presence of Emulgen 911, Triton X-100 and Tween 20. 8. This study shows that before FMO activity is determined in the presence of detergents their effects should be carefully evaluated.