ABSTRACT
Drosophila diaphorase-1 (DIA-1) is an enzyme similar to mammalian DT-diaphorase and is inhibited in vitro by dicoumarol. However, a ten-fold increase in DIA-1 activity was observed when third instar Drosophila virilis larvae were fed on a diet containing 0.1 M dicoumarol for 48 h. This induction was shown to be dose dependent and immunoprecipitation experiments with DIA-1 anti-serum demonstrated an increase in the DIA-1 protein level in dicoumarol-treated larvae. The induction of DIA1 by dicoumarol was found to be blocked by actinomycin D, which suggests a transcriptional mechanism of regulation. The opposite effect of dicoumarol on DIA-1 in vitro vs. in vivo suggests that a metabolic conversion takes place after the ingestion of this compound by D. virilis larvae.
Subject(s)
Dicumarol/pharmacology , Drosophila/enzymology , NAD(P)H Dehydrogenase (Quinone)/biosynthesis , Animals , Dactinomycin/pharmacology , Dicumarol/antagonists & inhibitors , Drosophila/drug effects , Electrophoresis, Starch Gel , Enzyme Induction , Larva/drug effects , Larva/enzymology , NAD(P)H Dehydrogenase (Quinone)/drug effects , Weight LossABSTRACT
Diaphorase-1 and diaphorase-2 were isolated from two Drosophila species, D. virilis and D. melanogaster, and purified by gel filtration, affinity chromatography, immunoaffinity chromatography, and ion-exchange chromatography. The molecular weights of both enzymes were the same in each species. The molecular weight of diaphorase-1 was the same under both denaturating and nondenaturating conditions, close to 60,000, indicating a monomeric structure. Sodium dodecyl sulfate (SDS) electrophoresis of the purified diaphorase-2 revealed the presence of a single protein band of 55,000 Da, while the molecular weight of the native enzyme was found to be 67,000. The two diaphorases were further characterized by their pH optima, isoelectric points, and kinetic parameters, and antibodies were raised in rabbits against the purified enzymes from D. virilis. The antibodies showed no cross-reactions but recognized the corresponding diaphorases in D. melanogaster and D. novamexicana as well as D. virilis. The data obtained confirmed the hypothesis of an independent genetic control of diaphorase-1 and diaphorase-2 in Drosophila.
Subject(s)
Dihydrolipoamide Dehydrogenase/metabolism , Drosophila/enzymology , Isoenzymes/metabolism , Animals , Blotting, Western , Dihydrolipoamide Dehydrogenase/isolation & purification , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Isoenzymes/isolation & purification , Kinetics , Precipitin Tests , Species SpecificityABSTRACT
Histones and histone acetylation have been investigated during three stages of Drosophila hydei embryogenesis--early gastrula, late gastrula and organogenesis. No essential changes in the electrophoretic pattern of the histones have been revealed during the stages examined. However, we established an enhanced level of [14C]acetate incorporation at the time of extensive gene activation during gastrulation as well as some quantitative differences in the pattern of acetylation during gastrula and organogenesis. We consider most of them to be related to chromatin assembly during the stage of gastrulation and suggest that the correlation between histone acetylation and gene activity during Drosophila embryogenesis concerns histone H3 acetylation. The involvement of both acetylation and deacetylation in the steady-state acetylation level has been examined as well. We have found that the higher acetyltransferase activity is responsible for the enhanced level of acetate incorporation during gastrulation.
Subject(s)
Drosophila/metabolism , Histones/metabolism , Acetylation , Animals , Drosophila/embryology , Drosophila/genetics , Kinetics , Transcription, GeneticABSTRACT
Histone H1 phosphorylation was examined during embryonic development of Drosophila hydei. A changing pattern of H1 phosphorylation upon separation on an acid-urea polyacrylamide gel was observed in the course of Drosophila embryogenesis. It is considered to be related to the decrease of the mitotic activity of the cells as development proceeds.