RESUMO
Identification of fifth instar larval Manduca sexta fat body and epidermis as sites of synthesis of a hemolymph protein (hemolymph trophic factor or HTF) was achieved using in vitro 3H-leucine incorporation into protein and subsequent immunoprecipitation of tissue homogenates. Fat body is the primary site of HTF synthesis with a maximal rate on Day 1; epidermis is a secondary site with peak synthesis on Day 0. In vitro radiolabelling followed by TCA precipitation of general protein of fat body and epidermal homogenates suggest that fat body actively elaborates protein on Days 0-5 with peak rates on Days 1 and 4, while epidermis is active on Days 0-5 with a peak rate on Day 3. Based on Anti-HTF ELISA estimates, HTF [500 to 1000 micrograms/ml] was found in the hemolymph of representatives of the insect orders Blattodea, Hemiptera, Orthoptera, and Lepidoptera and in the class Crustacea, but not in the class Merostomata. These studies suggest a possible fundamental role for HTF among modern arthropods in cuticular deposition involving both epidermis and fat body. The physiological role of HTF is undetermined.
Assuntos
Insetos/metabolismo , Manduca/metabolismo , Filogenia , Biossíntese de Proteínas , Animais , Epiderme/metabolismo , Corpo Adiposo/metabolismo , Hemípteros/metabolismo , Hemolinfa/metabolismo , Técnicas de Imunoadsorção , Marcação por Isótopo , Lepidópteros/metabolismo , Ortópteros/metabolismo , Proteínas/análise , Especificidade da Espécie , Distribuição Tecidual , TrítioRESUMO
Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone at C-20 of the side-chain to form ecdysterone, has been characterized in the fat body of early last instar larvae of the tobacco hornworm, Manduca sexta, using a radioenzymological assay. Ecdysterone was demonstrated to be the product of the enzyme system by high-pressure liquid chromatography, gas-liquid chromatography and mass spectrometry. Differential centrifugation, sucrose-gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with the mitochondria. The enzymatic properties of ecdysone 20-monooxygenase are that it is most active in a 0.05 M phosphate buffer, is inhibited by Mg2+ and exhibits pH and temperature optima at 7.5 and 30 degrees C, respectively. The enzyme complex has an apparent Km for ecdysone of 1.60 x 10(-7) M and is competitively inhibited by its product, ecdysterone, with an apparent Ki of 2.72 x 10(-5) M. The cytochrome P-450 nature of this insect steroid hydroxylase was initially suggested by its obligate requirement for NADPH and its inhibition by carbon monoxide, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Difference spectroscopy revealed the presence of cytochrome P-450 in the fat-body mitochondrial fraction. A photochemical action spectrum of ecdysone 20-monooxygenase activity confirmed the involvement of cytochrome P-450 in this monooxygenase system.