-Catalase (CAT), glutathione S-transferase (GST) and superoxide dismutase (SOD) activities were determined in postmitochondrial fractions of tissue homogenates (spermathecae, muscle and ventriculi), in hemolymph plasma, and in semen of honey bees. The highest CAT activity was found in semen (4.8 mU/µg fresh weight), and the enzyme was confined to the spermatozoa. CAT and GST activities of ventriculi exceeded those of other tissues and hemolymph, CAT being highest in mated queen ventriculi (2.7 mU/µg) and GST highest in worker ventriculi (10 mU/mg). Spermathecae of mated queens had higher CAT and GST activities (0.84 mU/µg, and 2.4 mU/mg, respectively) than virgin spermathecae (0.15 mU/µg, and 1.6 mU/mg). SOD activities (15-59 mU/µg) varied less than activities of CAT or GST between tissues. Seminal plasma contained two thirds of the total SOD activity of semen and one third was in the spermatozoa. The substantial activities of all three enzymes in spermathecae of mated queens suggest their involvement in the long-term protection of the spermatozoa from oxidative stress.Apis mellifera / catalase / glutathione S-transferase / superoxide dismutase

No abstract

In incubations with 80,OOOg supernatant of Manduca sexta midgut homogenates, [3H]ecdysone was converted to 3-[3H]epiecdysone and tritiumlabeled highly polar metabolites. CI8 SEP-PAK cartridges were found suitable for the separation and purification of the free ecdysteroids and of the highly polar metabolites. Eighty to ninety percent of the metabolites were hydrolyzed by enzyme mixtures (mainly 0-glucuronidase, sulphatase, and acid phosphatase) from molluscs, even when 0-glucuronidase activity was completely inhibited by D-saccharic acid 1,4lactone, or various human acid phosphatases (free of sulphatase activity). In each experiment, the hydrolysate contained a much higher proportion of 3-epiecydsone than the free (unconjugated) ecdysteroid fraction. [3H]ecdysone was not metabolized in anaerobic incubations of midgut supernatant that had been filtered through Sephadex C-25. Addition of 5 m M ATP and 5 m M Mg2+ restored the conjugate formation in incubations of Sephadex-filtered supernatant. Four ecdysone conjugates and two 3-epiecdysone conjugates were resolved by reversedphase ion-pair high-performance liquid chromatography. It i s concluded that the midgut cytosol contains several ATP:ecdysteriod phosphotransferases. This is the first demonstration of the formation of ecdysteroid phosphoconjugates in a cell-free system.

Ecdysone and 20-hydroxyecdysone are converted to their 3-epimers by enzymes in the midgut cytosol o f Manduca sexta larvae. A partially purified cytosol preparation has been used to analyze the nature of and the interaction between these enzymes. The cytosol was shown to contain ecdysone oxidase, one or more 3-oxoecdysteroid 3wreductase(s), and one o r more 3-oxoecdysteroid 3P-reductase(s). The reductases reacted at different velocities with NADH and NADPH. W i t h NADH, 3a-reduction was the major reaction; with NADPH, 3P-reduction was the major reaction. The apparent kinetic parameters for the enzymes support the assumed two-step mechanism for the 3-epimerization with a 3-oxoecdysteroid as intermediate.