Ecdysome 20-hydroxylase from the midgut of the tobacco hornworm (Manduca sexta L.).

An ecdysone 20-hydroxylase enzyme system that converts alpha-ecdysone to 20-hydroxyecdysone was prepared from the midgut of the tobacco hornworm prepupa. This partially purified enzyme is NADPH dependent and is localized in the mitochondrial fraction of the midgut tissue.     

Studies on carbon turnover in the freshwater snailAncylus fluviatilis (Basommatophora)

Summary Carbon turnover rates of young specimens of the freshwater snailAncylus fluviatilis are nearly equal for all organs, whereas in adult specimens intestinal organs (midgut gland and others) show considerably higher turnover rates than the rest animal (animal without organs of the pallial complex).Supported by the Deutsche Forschungsgemeinschaft.     

Effect of α- and β-ecdysone on DNA synthesis inAeshna cyanea (Insecta,Odonata) midgut

Summary InAeshna cyanea larvae, - and -ecdysone stimulate DNA synthesis in the midgut regenerative cells. In last larval instar, the number of cells obtained after the imaginal epithelium genesis is greater after - than after -ecdysone supply. Such a result should be compared with the imaginal epithelium differentiation which occurs earlier after -ecdysone injection.     

Acidic metabolite of prednisolone

Summary The metabolic fate of the 17-ketol side chain of (21-³H) prednisolone was studied with an enzyme preparation from male golden hamster liver. The acidic metabolite of prednisolone was identified by mass spectrometry as 11, 17,20-trihydroxy-3-oxo-1,4-pregnadien-21-oic acid. The enzyme showed substrate specificity, depending on the nature of substituent on the steroid nucleus.This investigation was supported in part by USPHS Grant CA 2515.     

Androgen dependency of hepatic hydroxysteroid dehydrogenases in the rat: Prepubertal responsiveness and unresponsiveness towards exogenous testosterone

Summary The prepubertal responsiveness of 3 typical androgen-dependent enzyme activities, namely 20-ketoreductase, 3- and ⁴-3-hydroxysteroid dehydrogenase, towards a large dose of testosterone was investigated. The androgen induced the activity of the 3-enzyme prepubertally in both sexes.This investigation was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 87, Endokrinologie).     

Absence of low molecular weight DNA polymerase activity from the nuclei ofAmoeba discoides

Summary Amoeba discoides nuclear protein partially purified by passage through Sephadex G-200 showed 3 high-mol.-wt DNA polymerase activities which eluted in and just following the void volume. No low-mol.-wt (45,000 daltons) DNA polymerase activity was detected. Nuclear protein layered on 5–20% sucrose gradients also showed an absence of lowmol.-wt DNA polymerase . The void volume enzyme showed deoxyribonuclease activity, but no low-mol.-wt nuclease activity was detected.     

Canonical protein inhibitors of serine proteases

Serine proteases and their natural protein inhibitors are among the most intensively studied protein complexes. About 20 structurally diverse inhibitor families have been identified, comprising -helical, sheet, and / proteins, and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished based on their mechanism of action: canonical (standard mechanism) and non-canonical inhibitors, and serpins. The canonical inhibitors bind to the enzyme through an exposed convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is always very similar and resembles that of an ideal substrate. The non-canonical inhibitors interact through their N-terminal segment. There are also extensive secondary interactions outside the active site, contributing significantly to the strength, speed, and specificity of recognition. Serpins, similarly to the canonical inhibitors, interact with their target proteases in a substrate-like manner; however, cleavage of a single peptide bond in the binding loop leads to dramatic structural changes.Received 28 March 2003; received after revision 12 May 2003; accepted 16 May 2003     

Enzymatic basis for protection of fish embryos by the fertilization envelope

The mechanism by which the fertilization envelope (FE) is able to protect the embryo of fish until hatching is almost unknown, except for its function as a physical barrier. FE extract from activated or fertilized eggs of the fishSalmo gairdneri was demonstrated to contain enzyme activities using an agar plate enzyme assay. The enzymes apparently active were carboxymethylcellulase (cellulase; EC 3.2.1.4), laminaranase (endo-1,3(4)--glucanase; EC 3.2.1.6), carboxymethylchitinase (chitinase; EC 3.2.1.14), xylanase (endo-1,4--xylanase; EC 3.2.1.8), mannanase (mannan 1,2-(1,3)--mannosidase; EC 3.2.1.77), dextranase (EC 3.2.1.11), a protease and lysozyme (EC 3.2.1.17). The FE extract exerted an antifungal or fungicidal action on the fungusSaprolegnia parasitica, whereas an extract from the vitelline envelopes (VE) has no apparent enzyme activity nor antifungal or fungicidal action. Enzymes acquired by the FE through the cortical reaction may have an important defensive role, protecting the embryo against invaders or pathogens.     

Purification and characterization of aβ-glucosidase (linamarase) from the haemolymph ofZygaena trifolii Esper, 1783 (Insecta,Lepidoptera)

Summary A -glucosidase (linamarase) was purified 52-fold with a recovery of 27% from the haemolymph of the larvae ofZygaena trifolii, ESPER, 1783 (Lepidoptera, Zygaenidae). The final enzyme preparation was found to be nearly homogeneous on both disc polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined to be about 130 kDa; it consisted of two subunits of about 66 kDa. The enzyme showed an optimum between pH 4.5 and 5 with linamarin and a broad optimum between pH 3.5 and 6.5 for p-nitrophenyl--D-glucoside; the temperature optimum was 40°C. The -glucosidase showed a high specificity for its endogenous substrates linamarin and lotaustralin. Among the other natural and artificial substrates tested, only prunasin and p-nitrophenyl--D-glucoside were hydrolyzed by the enzyme, whereas linustatin, salicin, cellobiose and trehalose were not. The enzyme is strongly inhibited by -glucosylpiperidine.     

Alpha-crystallin: an ATP-independent complete molecular chaperone toward sorbitol dehydrogenase

-Crystallin, the major component of the vertebrate lens, is known to interact with proteins undergoing denaturation and to protect them from aggregation phenomena. Bovine lens sorbitol dehydrogenase (SDH) was previously shown to be completely protected by -crystallin from thermally induced aggregation and inactivation. Here we report that -crystallin, in the presence of the SDH pyridine cofactor NAD(H), can exert a remarkable chaperone action by favoring the recovery of the enzyme activity from chemically denaturated SDH up to 77%. Indeed, even in the absence of the cofactor, -crystallin present at a ratio with SDH of 20:1 (w:w) allows a recovery of 35% of the enzyme activity. The effect of ATP in enhancing -crystallin-promoted SDH renaturation appears to be both nonspecific and to not involve hydrolysis phenomena, thus confirming that the chaperone action of -crystallin is not dependent on ATP as energy donor.Received 28 October 2004; received after revision 22 December 2004; accepted 10 January 2005     

Occurrence of 3-epi-22-deoxy-20-hydroxyecdysone and its phosphoric ester in diapause eggs of the silkworm,Bombyx mori

Ecdysteroids in diapause eggs of the silkworm,Bombyx mori, were analyzed using high-performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). A relatively large amount of an unidentified free ecdysteroid and its phosphoric ester (conjugated form) were detected. These two compounds were isolated by a combination of column chromatography on silicic acid, thin-layer chromatography (TLC), and HPLC using a reverse-phase (RP) column. The purified compounds were identified as 3-epi-22-deoxy-20-hydroxyecdysone (22d20E) and 3-epi-22-deoxy-20-hydroxyecdysone 2-phosphate (22d20E2P) by means of mass spectrometry and nuclear magnetic resonance spectroscopy. to our knowledge, this is the first report of 22d20E and 22d20E2P.     

Quantification of digoxin by enzyme immunoassay: synthesis of a maleimide derivative of digoxigenin succinate for enzyme coupling

Summary We synthesized the m-maleimidobenzoyl derivative of digoxigenin-3-0-succinate (through a p-phenylenediamine bridge) as a hapten derivative directed towards coupling to sulfhydryl groups of -galactosidase. Prepared enzyme conjugate had about 97% of the enzyme labeled with the hapten derivative while retaining full enzyme activity. The enzyme immunoassay for digoxin we prepared showed a maximum sensitivity of 30 pg per assay (c.v.=3%) with minimal cross-reaction with digotoxin (3.8%). Our method for hapten conjugation to -galactosidase is highly efficient and is simple and easily replicated.     

Effect of tea consumption on the levels ofα-ketoglutarate and pyruvate dehydrogenase in rat brain

Summary Administration of tea to rats fed on a normal diet results in a marked drop in brain levels of total thiamine as well as of -ketoglutarate and pyruvate dehydrogenase activities. The patterns of decrease in both enzyme activities are similar to that of total thiamine content; they drop to about 65% of the control at 14–20 weeks after continuous consumption of tea.Supported by a grant from the International Foundation for Science (IFS), Sweden.     

Potentiating effect of phenylephrine on isoproterenol activation of thyroxine type II deiodinase in the pineal gland of adult rats

In the present study we show, for the first time, that phenylephrine (PHE), an -adrenergic receptor agonist, potentiates the effect of isoproterenol (ISO), a -adrenergic agonist, in activating pineal type II5-deiodinase (5-D) activity. The potentiating effect of PHE was observed only at doses of ISO which induce submaximal activation of the enzyme. However, at doses which lead to maximal activation of the enzyme, PHE was ineffective. The results suggest that not only -, but also -adrenergic receptors, are involved in the sympathetic noradrenergic regulation of pineal 5-D activity in the adult rat.     

Steroidal sapogenins. XXX stereochemistry of the side chain

Zusammenfassung Die optischen Drehungen einiger 20, 25d; 20, 25l; 20, 25d; und 20, 25-l-Sapogenine wurden bestimmt. Die ersten drei Serien gaben übereinstimmend linksdrehende Werte, aber die letztere Gruppe erwies sich als rechtsdrehend.Die Struktur der vier Serien der Sapogenine folgte aus der Analyse dieser Befunde. Der mögliche Mechanismus bei der Entstehung dieser Verbindungen aus Pseudosapogeninen wurde besprochen. Die Autoren gelangen zum Schluss, dass sterische Faktoren an C₂₀ und C₂₅ die Richtung der Ringschliessung beeinflussen.

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Steroidal sapogenins. XXX stereochemistry of the side chain

     

The biotransformation of tritiated 3-dehydroecdysone by crayfish,Procambarus clarkii

The injection of 3-dehydroecdysone (3dhE, 5 /g), the major ecdysteroid secreted by the Y-organ of crayfishProcambarus clarkii, resulted in apolysis within about 5 days. The hormonal response at the molecular level was investigated by injection of the radio-labeled compound; within 3 h of injection of [³H]3dhE, most radio-isotope was found in the extracted epidermal tissues and identified as ecdysone, 20-hydroxyecdysone (20E), and their 3-hydroxy epimers. The biotransformation was undoubtedly performed in the peripheral area of the Y-organ. Cleavage of the polar conjugates, using an enzyme fromHelix pomatia, gave all of the above ecdysteroids including 3dhE. It was also found that the biosynthetic site of 3dhE was different from that of ecdysone at the subcellular level of the Y-organ.     

Diphasic action of the aliphatic amide,HOE 17879, on hepatic microsomal drug metabolizing enzymes in the mouse

Summary -Hydroxy--ethylbutyryl-diethylamide (HOE 17879) influences the hepatic microsomal drug hydroxylating enzyme system of mice in a two-phasic way: First it reduces the overall reaction of drug hydroxylation but not the single enzymes whilst in the second period it increases enzyme activities.Acknowledgments. The authors are indebted to their colleague, Dr.K. Schmitt, for making the substance HOE 17879 available. They wish to thank to Mr.Mayer, Mr.Paulusch Mrs.Fey, Mrs.Stein, and MissFehlings for their skilful assistance in the project.     

Hormonal induction of steroid sulphatase in the mouse

Summary By comparing steroid sulphatase levels per se, and also ratios to -galactosidase, in 6 sets of mice — normal females, entire and castrated males both with and without exogenous testosterone administration — we obtained support for the contention that induction of this enzyme is in part controlled by male hormones.     

Purification of 20α-hydroxysteroid dehydrogenase from human erythrocytes

Summary 20-hydroxysteroid dehydrogenase was found in the supernatant after hemolysis of human erythrocytes, and the enzyme was isolated from the membrane-free hemolysate on a DEAE-cellulose column. The NADPH-generating system was essential for the reaction.Acknowledgments. We wish to thank Dr A. Tomoda, Department of Biochemistry, Kanazawa University School of Medicine, and Dr T. Yubisui, Department of Biochemistry, Medical College of Oita, for their advice and useful discussions.     

An enzyme immunoassay for mouse epidermal growth factor utilizing a liquid phase double-antibody system

Summary An enzyme immunoassay for mouse epidermal growth factor (EGF) involving a liquid phase double-antibody system was developed. The EGF--galactosidase conjugate prepared was stable for at least 8 months. By this method, EGF was detectable at a concentration as low as 20 pg per tube. The concentrations of EGF in various tissues of mice are also presented.This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.