RESUMEN

The flavoprotein DT-diaphorase (EC 1.6.99.2) is believed to play an important role in the body's defense system. This enzyme has been purified 13,000-fold with a recovery of 58% from a cytosolic fraction of abdominal fat obtained from an obese patient undergoing elective surgery. Purification of the enzyme to electrophoretic homogeneity was achieved after two chromatographic steps: (1) affinity chromatography on azodicumarol Sepharose 6B; (2) anion exchange chromatography on DEAE Sephacel. The enzyme exhibits a monomer molecular mass of 32 kDa in SDS-PAGE and has 1 FAD prosthetic group per 32 kDa monomer. The FAD prosthetic group appears to be firmly attached to the apoproprotein. The enzyme reduces azodyes and quinones and demonstrates a broad substrate specificity. The enzyme has characteristics that are similar to DT-diaphorase purified from rodent liver, especially the rat liver enzyme. Estimated Km values for NADH, NADPH and menadione are 200, 140 and 3.3 microM, respectively. Vmax values for these substrates in the same order are 762, 667 and 294 mumol/mg.min. Dicumarol and warfarin exhibited competitive inhibition with pyridine nucleotides. The inhibition constants (Ki) for the drugs were estimated to be 10 nM and 2.2 microM, respectively. When compared to several other tissues, abdominal fat has one of the highest DT-diaphorase activities (Martin, L.F., Patrick, S.D. and Wallin, R. (1987) DT-diaphorase in morbidly obese patients. Cancer Lett., 36, 341-347), but the specific role of the enzyme in human fat is unknown.

Asunto(s)

RESUMEN

We have investigated the form I cyclic nucleotide phosphodiesterase (PDE) from Drosophila melanogaster and shown that whereas heads and male thoraces and abdomens contain high levels of Ca2+-stimulated enzyme, female thoraces and abdomens contain little Ca2+-stimulated activity. The electrophoretic patterns of form I PDE from these 3 sources have also been studied and reveal that heads, and male thoraces and abdomens, produce two bands of form I PDE both of which are stimulated by Ca2+. Extracts of female thoraces and abdomens, on the other hand, show only a single, faster running band of PDE activity which is only marginally stimulated by Ca2+, if at all. Surveying wild-type strains of Drosophila has revealed that one strain, Swedish, shows altered electrophoretic mobility of the PDE band from female thoraces and abdomens. The alteration is such that the Swedish PDE band runs more anodally than the Oregon-R and Canton-S PDE activities. Mixing experiments, using co-homogenization of heads with female thoraces and abdomens, yield a single faster running band on electrophoresis. This band contains only Ca2+-insensitive PDE. Attempts to reconstruct this loss of Ca2+-sensitive PDE without electrophoresis have failed. The Swedish electrophoretic variation of the PDE from female thoraces and abdomens has been found to be recessive with respect to the Canton-S phenotype, but the variation is observed to re-emerge and segregate with the third chromosome in the F2 generation. The results indicate that electrophoretic variation in the form I PDE is, by itself, insufficient to allow the location of the structural gene for this enzyme.

Asunto(s)

RESUMEN

Adenylate cyclase in homogenates of Drosophila melanogaster is heterogeneous with respect to its affinity toward MgATP and its subcellular distribution. Km values for MgATP range, under similar assay conditions, from approximately 10(-5) M to approximately 10(-3) M, depending on the body region and on the subcellular localization of the enzyme. The majority of the enzyme in whole-body preparations is particulate, but various body regions differ in the relative proportion of the soluble enzyme. The memory mutant rutabaga lacks up to 35% of the total particulate activity. Even ligands that stimulate directly the catalytic subunit are incapable of bringing the activity of the mutant's enzyme to normal levels. The defect is differentially pronounced in various body parts and is associated with an altered responsiveness of the enzyme to Mg2+, to Ca2+, and to forskolin. It is suggested that rutabaga is lesioned in a subpopulation, or a functional state, of adenylate cyclase, which may play a role in memory formation.

Asunto(s)

RESUMEN

The activity of adenylate cyclase in homogenates prepared from the Drosophila memory mutant rutabaga is lower than normal. The effect is most pronounced in washed membranes prepared from the abdomen, in which the enzyme displays both a lower Vmax and a higher Km. Analysis of the effect of divalent cations suggests a lesion in the responsiveness of the enzyme to Mg2+. Studies of adenylate cyclase in rutabaga may potentially pinpoint a subpopulation of the enzyme which may prove relevant to molecular mechanisms underlying conditioning.

Asunto(s)

RESUMEN

1. Phenolphthalein, halogenated fluoresceins, and other triphenylmethane and diphenylmethane derivatives inhibited biphenyl hydroxylation, aldrin epoxidation and several O-dealkylations in insect abdomen homogenates. Phenolphthalein and eosin (50 muM) were 2-3 times more effective than SKF 525-A and piperonyl butoxide (50 muM) as inhibitors of biphenyl hydroxylation in vitro. 2. The phthaleins, Aurin and Aluminon, inhibited both epoxidation and hydroxylation to similar extents, but fluoresceins, Rhodamine B, Malachite Green, and basic diphenylmethane derivatives preferentially inhibited hydroxylation. 3. Tetrabromophenolphthalein ethyl ester and bis-(N-dimethyl-4-aminophenyl-methane inhibited biphenyl hydroxylation in vivo. Bis-(N-dimethyl-4-aminophenyl) methane synergized the toxic effects of 1-naphthyl N-methylcarbamate in live houseflies.

Asunto(s)

RESUMEN

Double labelling experiments using [phenyl-14C], [2,6-p-nitro-phenyl-14C]- or, nonradioactive EPN with [35S]-, [glycine-3H]- or nonradioactive glutathione in the presence of the 100,000g supernatant fraction from the Rutgers strain of houseflies resulted in the formation of a radiolabelled conjugate. Hydrolysis of the [phenyl-14C] conjugate formed the phenyl-[14C]-phosphonothioic acid. The proposed structure of the conjugate is S-(O-ethyl phenylphosphonothionyl) glutathione. The conjugate is formed with racemic, (+), or (-) EPN. Structure activity studies indicate the following: a decrease in the electron withdrawing properties of the substituents on the O-p-nitrophenyl moiety results in a decrease in the formation of the conjugates; substituting the phenylphosphonothioic moiety with the ethyl-phosphonothioate, inhibits the formation of the corresponding conjugate' substituting the O-ethyl group with other alkyl groups did not effect the formation of the corresponding conjugate.

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

A nitroreductase, reducing parathion to aminoparathion, was found in the soluble fraction that was obtained from abdomens of female houseflies. The reaction required reduced nicotinamide adenine dinucleotide phosphate (NADPH), but was not affected by the presence or absence of oxygen. Further degradation of aminoparathion into water-soluble compounds occurred in NADPH-fortified incubation mixtures over prolonged incubation periods. The effect of sesamex or SKF 525-A on these reactions is described.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

Phospholipase B has been found in the mosquito Culex pipiens fatigans, and some of its properties have been studied. The enzyme had a high optimum temperature (45 degrees C) and broad alkaline pH optimum (8-9). It was inactive toward diacylphospholipids, and hydrolyzed lysolecithin at a higher rate than lysophosphatidyl ethanolamine. The enzyme was heat labile, but lysolecithin protected it against heat inactivation. Phosphatidyl ethanolamine, phosphatidyl choline, deoxycholate, Fe(+++), and Hg(++) inhibited the enzyme markedly. The enzyme was present mainly in larvae; little enzyme activity was detected in pupae or adults. The total and specific activities were highest in IV instar (6 day) and I instar (1st day) larvae, respectively. It was localized in the microsomal fraction and was distributed mainly in the abdomen and thorax of the insect. The enzyme was present at much higher levels of activity in larvae of the mosquitoes Anopheles stephensi and Aedes aegypti.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

1. Optimum conditions were established for determining the activities of the NADP(+)-linked enzymes, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase, in mosquito tissues. 2. The activity of each dehydrogenase was determined in samples of mosquitoes of different ages throughout the life-span. The specific-activity curves attained maximal values in the pupal or early adult period. From these maxima an 81% decrease in glucose 6-phosphate-dehydrogenase and 67% decrease in 6-phosphogluconate-dehydrogenase activities occurred after the tenth day of adult life; a 77% decrease in isocitrate-dehydrogenase activity occurred before the fifth day. 3. The activity differences were found in different body regions as well as in whole organisms. 4. Starvation of the larva or adult did not result in decreases in enzyme activity. 5. These findings support the hypothesis that the activities of enzymes that form NADPH are related to the biosynthetic activity, for the enzyme activities increased during the period of cellular growth and decreased during the aging period.

Asunto(s)