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Many intercellular communication processes, known as quorum sensing (QS), are regulated by the autoinducers N-acyl-l-homoserine lactones (AHLs) in Gram-negative bacteria. The inactivation of these QS processes using different quorum quenching (QQ) strategies, such as enzymatic degradation of the autoinducers or the receptor blocking with non-active analogs, could be the basis for the development of new antimicrobials. This study details the heterologous expression, purification, and characterization of a novel N-acylhomoserine lactone acylase from Actinoplanes utahensis NRRL 12052 (AuAHLA), which can hydrolyze different natural penicillins and N-acyl-homoserine lactones (with or without 3-oxo substitution), as well as synthesize them. Kinetic parameters for the hydrolysis of a broad range of substrates have shown that AuAHLA prefers penicillin V, followed by C12-HSL. In addition, AuAHLA inhibits the production of violacein by Chromobacterium violaceum CV026, confirming its potential use as a QQ agent. Noteworthy, AuAHLA is also able to efficiently synthesize penicillin V, besides natural AHLs and phenoxyacetyl-homoserine lactone (POHL), a non-natural analog of AHLs that could be used to block QS receptors and inhibit signal of autoinducers, being the first reported AHL acylase capable of synthesizing AHLs.

Application of polyester-degrading enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. Many hydrolases from several fungi and bacteria have been discovered and successfully evaluated for their activity towards different aliphatic polyesters (PHA, PBS, PBSA, PCL, PLA), aromatic polyesters (PET, PBT, PMT) as well as their co-polyesters (PBST, PBAT, PBSTIL). This revision gives an up-to-date overview on the main biochemical features and biotechnological applications of those reported enzymes which are able to degrade polyester-based plastics, including different microbial polyester depolymerases, esterases, cutinase-like enzymes and lipases. Summarized information includes available protein sequences with the corresponding accession numbers deposited in NCBI server, 3D resolved structures, and data about optimal conditions for enzymatic activity and stability of many of these microbial enzymes that would be helpful for researchers in this topic. Although screening and identification of new native polyester hydrolases from microbial sources is undeniable according to literature, we briefly highlight the importance of the design of improved enzymes towards recalcitrant aromatic polyesters through different approaches that include site-directed mutagenesis and surface protein engineering.

Testosterone and its synthetic derivatives anabolic-androgenic steroids have been shown to increase skeletal muscle work capacity and fatigue resistance, but the molecular basis for these effects remains uncertain. Since muscle performance has been related to redox status of exercising muscles, this investigation was aimed at testing whether a treatment with suprapharmacological doses of the anabolic-androgenic steroid stanozolol, (2 mg/kg body weight, 5 days/week, for 8 weeks), either alone or in conjunction with treadmill training (12 weeks), enhanced antioxidant defences in rat muscles. Stanozolol treatment did not modify thiobarbituric acid reactive substances and glutathione content in soleus and extensor digitorum longus (EDL) homogenates. In soleus from sedentary rats, superoxide dismutase and glutathione reductase activities were increased by 25% (P < 0.05) and by 40% (P < 0.01) after stanozolol administration, whereas catalase and glutathione peroxidase activities were not modified. This response was similar to that induced by training alone. In EDL from sedentary rats, stanozolol increased only superoxide dismutase activity (20%, P < 0.05). In no case, the effects of steroid administration and training were additive. HSP72 levels were up-regulated in soleus (1.5-fold, P < 0.01) and EDL (threefold, P < 0.001) following training but remained unchanged after stanozolol treatment. Endurance capacity, assessed in a treadmill endurance test, was similar for treated and control rats. We conclude that stanozolol treatment increases antioxidant capacity in selected skeletal muscles from sedentary rats. However, the steroid was not effective in improving endurance capacity or enhancing the training effects on muscle antioxidant defence systems.

[Background] Serine proteinase inhibitors (serpins) finely regulate serine proteinase activity via a suicide substrate-like inhibitory mechanism. In parasitic nematodes, some serpins interact with host physiological processes; however, little is known about these essential molecules in Anisakis. This article reports the gene sequencing, cloning, expression and preliminary biochemical and bioinformatically-based structural characterization of a new Anisakis serpin (ANISERP)., [Methods] The full AniSerp gene was cloned by specific RACE-PCR after screening an Anisakis simplex (L3) cDNA library. For biochemical assays, the AniSerp gene was subcloned into both prokaryotic and eukaryotic vectors, and the recombinant proteins were purified. The inhibitory properties of the proteins were tested in classical biochemical assays using human serine peptidases and AMC substrates. Immunolocalization of ANISERP, theoretical structural analysis and bioinformatically-based structural modelling of the ANISERP protein were also conducted., [Results] The AniSerp gene was found to have 1194 nucleotides, coding for a protein of 397 amino acid residues plus a putative N-terminal signal peptide. It showed significant similarity to other nematode, arthropod and mammalian serpins. The recombinant ANISERP expressed in the prokaryotic and eukaryotic systems inhibited the human serine proteases thrombin, trypsin and cathepsin G in a concentration-dependent manner. No inhibitory activity against Factor Xa, Factor XIa, Factor XIIa, elastase, plasmin or chymotrypsin was observed. ANISERP also acted on the cysteine protease cathepsin L. ANISERP was mainly localized in the nematode pseudocoelomic fluid, somatic muscle cell bodies and intestinal cells. The findings of molecular dynamics studies suggest that ANISERP inhibits thrombin via a suicide substrate-like inhibitory mechanism, similar to the mechanism of action of mammalian coagulation inhibitors. In contrast to findings concerning human antithrombin III, heparin had no effect on ANISERP anticoagulant inhibitory activity., [Conclusions] Our findings suggest that ANISERP is an internal Anisakis regulatory serpin and that the inhibitory activity against thrombin depends on a suicide substrate-like inhibitory mechanism, similar to that described for human antithrombin (AT)-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in the latter of five of the six positively charged residues usually seen at the AT-III-heparin binding site., This work was funded by the Spanish Ministry of Science and Innovation (SAF2002-04057-CO2-01) and the Instituto de Salud Carlos III (ISCIII) as part of project MPY 1404/09, and ISCIII-AESI project MPY 1279/15. Carolina Hurtado and Pamela Campioli were supported by grants from the ISCIII (Fondo de Investigaciones Sanitarias), through VI NP of I + D + I (2008–2011), ISCIII General Sub-Direction of Networks and Centers for Collaborative Research (RETIC-RICET, RD06/0021/0019, RD12/0018/011). Victoria Martínez Sernández holds a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (Programa de Formación del Profesorado Universitario).

Isolated mitochondria respiring on physiological substrates, both in state 4 and 3, are reported to be or not to be a source of reactive oxygen species (ROS). The cause of these discrepancies has been investigated. As protein concentration was raised in in vitro assays at 37 degrees C, the rate of H2O2 release by rat heart mitochondria supplemented with pyruvate/malate or with succinate (plus rotenone) was shown to increase (0.03-0.15 mg protein/ml), to decrease (0.2-0.5 mg protein/ml) and to be negligible (over 0.5 mg protein/ml). The inhibition of mitochondrial respiration (with rotenone or antimycin A) or the increase in the oxygen concentration dissolved in the assay medium allowed an enhancement of ROS production rate throughout the studied range of protein concentrations. In mitochondria respiring in state 3 on pyruvate/malate or on succinate (plus rotenone), ROS release vanished for protein concentrations over 0.5 or 0.2 mg/ml, respectively. However, ROS production rates measured with low protein concentrations (below 0.1 mg/ml) or in oxygen-enriched media were similar or even slightly higher in the active respiratory state 3 than in the resting state 4 for both substrates. Consequently, these findings indicate that isolated mitochondria, respiring in vitro under conditions of forward electron transport, release ROS with Complex I- and II-linked substrates in the resting condition (state 4) and when energy demand is maximal (state 3), provided that there is sufficient oxygen dissolved in the medium.

The present study was conducted to investigate the effects of long-term exercise training on the main components involved in excitation-contraction coupling and relaxation in rat myocardium. Twenty male Wistar rats were divided into sedentary (S) and treadmill-trained (T) groups. Group T was trained for 24 weeks, 5 days/week (25 m/min, 45-60 min, 0% slope). 48 h after the last exercise session, animals were killed and ventricular and soleus muscle homogenates were obtained. The citrate synthase activity in soleus muscle was significantly increased (163%) in T compared with S rats ( P0.01), confirming the exercise training efficacy. Although heart weight and cardiac oxidative capacity were not modified by exercise training, the binding of [(3)H] ryanodine and the dihydropyridine [(3)H]PN200-110 to cardiac homogenates, and sarcoplasmic reticulum Ca(2+)-ATPase activity were increased significantly in the ventricular homogenates from T compared with S animals ( P0.01). Western blot analysis of ventricular homogenates failed to show significant alterations in dihydropyridine receptor and Ca(2+)-ATPase levels in T animals, but revealed an increase of ryanodine receptor density in this group ( P0.01). The activity of the ectoenzymes 5'-nucleotidase and Mg(2+)-ATPase was not affected by training ( P0.05). In conclusion, long-term treadmill training induces adaptive changes in some of the components of myocardial rat excitation-contraction coupling and relaxation systems that could contribute to the improvement of cardiac function.

Purpose The aim of this study was to examine the separate and combined effects of an 8-wk treatment with high doses of 17alpha-alkylated anabolic-androgenic steroids (AAS) and exercise training on selected lysosomal and mitochondrial enzyme activities in rat liver. Methods Sedentary and treadmill-trained (25 m x min(-1), 45 min x d(-1), 5 d x wk(-1)) male rats were treated with fluoxymesterone, methylandrostanolone, or stanozolol (2 mg x kg body weight(-1), 5 d x wk(-1)) for 8 wk. Results Acid phosphatase, arylsulfatase, beta-glucuronidase, and beta-galactosidase activities were increased in liver homogenates of sedentary and trained AAS-treated rats. The mitochondrial respiratory chain activities rotenone-sensitive NADH-cytochrome c reductase (NCCR), succinate cytochrome c reductase (SCCR), and cytochrome oxidase (COX) showed a significant decrease in steroid-administered rats, whereas citrate synthase (CS), a matrix enzyme, exhibited no changes in activity, pointing to a selective effect of AAS on mitochondrial membrane complexes. In vitro studies in mitochondrial fractions isolated from the liver of control rats showed that COX and CS activities were insensitive to the AAS, whereas NCCR and SCCR activities were partly inhibited. On the other hand, the mean values of serum parameters related to hepatic function were within normal ranges in all the experimental groups of animals. Conclusions The present data show that 8-wk ingestion of three different anabolic-androgenic steroids, either with or without concurrent exercise training, affects lysosomal hydrolases and mitochondrial respiratory chain electron transport in rat liver without modifying classical serum indicators of hepatic function.

The measurement of individual respiratory chain complexes is an important component of the investigation of diseases due to mitochondrial dysfunction. We have evaluated assays which measure complexes I to IV in human skeletal muscle mitochondria and in addition optimized these assays to provide sensitive and reliable diagnostic techniques, particularly in situations where a partial interruption at a single complex needs to identified. Using several established methods of membrane disruption we have found that optimal activities of complexes I and II are obtained by freeze-thawing the mitochondria in hypotonic potassium phosphate buffer, whereas complex III and IV activities are markedly increased by the addition of the detergent n -dodecyl-β- D -maltoside. Complex I activity is measured in the presence of 2.5 mg · ml −1 bovine serum albumin, which increases rotenone sensitivity, and we have shown that NADH-cytochrome b 5 reductase makes an important contribution to the rotenone-insensitive NADH-ubiquinone oxidoreductase activity. Complex II activity is measured after preincubation of the mitochondrial fraction with succinate to fully activate the complex. Complex I and III activities are dependent upon the length of the isoprenoid chain of the ubiquinone and ubiquinol, respectively. These assays have been used to establish a control range.

The orientation of the enzyme Mg(2+)-ATPase (EC 3.6.1.3) in the transverse tubule (TT) membranes of skeletal muscle was investigated using highly purified chicken and rabbit TT vesicles. The percentage of sealed vesicles present in these preparations averaged 88 and 78%, respectively, as calculated from the detergent-induced increase in ouabain-sensitive (Na+, K+)-ATPase activity, ATP-dependent ouabain binding, and lactate dehydrogenase activity (sarcoplasmic enzyme trapped in the TT vesicles). Sidedness of the sealed vesicles, estimated from latency of 5'-nucleotidase, acetylcholinesterase, and adenylate cyclase, was predominantly right-side out (69-76%, chicken TT and 62-70%, rabbit TT). In both chicken and rabbit native vesicles, high Mg(2+)-ATPase activity was detected by addition of ATP to the extravesicular medium; this activity was increased 14-12% by alamethicin pointing to the external localization of the active site. Furthermore, the enzymatic activity resulted partially inhibited by treatment of the chicken TT vesicles with proteinase K or p-hydroxymercuribenzoate. Concanavalin A stimulated 4-fold the chicken TT Mg(2+)-ATPase activity, an effect not potentiated by detergent permeabilization of the intact vesicles, indicating that lectin-binding sites were also solvent accessible. This stimulatory effect was not observed in native or permeabilized rabbit TT vesicles. From these results we conclude that the TT Mg(2+)-ATPase is an ectoenzyme with its nucleotide-hydrolyzing site and glycosylated regions facing the extracellular space. Inhibitors of ion-motive ATPases did not modify the enzyme activity, suggesting a different physiological role for the TT Mg(2+)-ATPase which may be involved in the regulation of muscle fiber functions affected by extracellular ATP levels.

The abuse of anabolic-androgenic steroids (AAS) to enhance physical performance is widespread in sport communities despite their reported side effects. Since the biochemical bases for the hepatotoxic effects of these compounds are largely unknown, this investigation was aimed at testing whether prolonged (8 weeks) treatment with high doses (2 mg kg(-1) body weight; 5 d wk(-1)) of stanozolol (ST), either alone or in conjunction with treadmill-exercise training, induced changes in oxidative stress biomarker levels and antioxidant defence systems in rat liver. After ST oral administration, the mean values of serum parameters related to hepatic function were within normal ranges. No changes in protein carbonyl content and in the reduced to oxidized glutathione (GSH/GSSG) ratio were detected in liver homogenates of ST-treated rats, whereas thiobarbituric acid-reactive substances (TBARS) levels resulted increased (P

Treatment of male rats with the anabolic steroids fluoxymesterone or methylandrostanolone increased the activity of the outer carnitine palmitoyltransferase in liver and fast-twitch muscle mitochondria. This effect was not potentiated by physical exercise and was not observed in heart and slow-twitch muscle mitochondria. Anabolic steroids did not affect the sensitivity of the liver enzyme to inhibition by malonyl-CoA. The data presented herein suggest that androgens may have an important physiological role in the regulation of fatty acid oxidation in liver and fast-twitch muscle mitochondria. In addition, our results are at odds with the notion that (most of) the metabolic effects of anabolic steroids on muscle are only evident when physical training is parallely performed.

The purpose of this investigation was to analyze the short-term effects of marathon running on serum levels of cardiac markers in a group of master runners (> 60 yrs). Ten marathoners (9 male and 1 female; 63 ± 4 yrs) were enrolled in the study. All of them completed the Madrid Marathon (1998). Venous blood was drawn from each subject three times during the study (48 h before the race, immediately after the race, and 24 h post-competition) for the determination of the several biochemical markers, such as total creatine kinase catalytic activity (total CK), mass concentration of creatine kinase isoenzyme MB (CK-MB mass), and cardiac troponin I (Tnl-c). The most important finding was that in each sample (pre- or post-race) serum Tnl-c was below the commonly accepted level of 0.1 ng × ml -1 indicative of myocardial injury. Although further research is needed using more complete methodology, our results suggest that marathon running does not have an acute deleterious effect on the hearts of the elderly.

The presence of a high and nonlinear Ca2+-independent (or basal) ATPase activity in rat heart preparations makes difficult the reliable measurement of sarcoplasmic reticulum (SR) Ca2+-ATPase activity by usual methods. A spectrophotometric assay for the accurate determination of SR Ca2+-ATPase activity in unfractionated homogenates from rat heart is described. The procedure is based on that reported by Simonides and van Hardeveld (1990, Anal. Biochem. 191, 321-331) for skeletal muscle homogenates. To avoid overestimation of the Ca2+-ATPase activity of cardiac homogenates that occurs when sequential measurements of total and basal ATPase activities are performed, two parallel and independent assays are required: one with low (micromolar) and other high (millimolar) calcium concentration. Addition of thapsigargin (0.2 microM) blocked totally the activity considered as Ca2+-ATPase activity. Using this method, the rat heart homogenate Ca2+-ATPase activity was 10.5 +/- 2.0 micromol. min-1 x g-1 tissue wet weight (n = 8). Likewise, a spectrophotometric assay for measuring E-type Mg2+-ATPase activity in cardiac total homogenates has been developed, comparing the following characteristics of the enzymatic activity in homogenate and a membrane-enriched fraction: first-order rate constant for ATP-dependent inactivation, Km for ATP, and effects of concanavalin A, Triton X-100, and specific inhibitors.

The chicken T-tubule Mg2+-ATPase is an integral membrane glycoprotein that presents properties different from those of other ATPases located in skeletal muscle cells and exhibits ATP-hydrolysing activity on the extracellular side of the transverse tubule (TT) membranes. In this study we demonstrate that TT vesicles purified from chicken skeletal muscle possess ecto-ADPase and ecto-5ʹ-nucleotidase activities that, along with ecto-ATPase, are able to sequentially degrade extracellular ATP to ADP, AMP and adenosine. Characterization studies of these TT ectonucleotidases revealed remarkable differences between ecto-ATPase and ecto-ADPase activities with respect to thermal stability, temperature dependence of the hydrolytic activity, effect of ionic strength, kinetic behaviour, divalent cation preference and responses to azide, N-ethylmaleimide, NaSCN, Triton X-100 and concanavalin A. Ecto-ATPase, but not ecto-ADPase, was inhibited by a polyclonal antibody against the chicken TT ecto-ATPase. On the basis of these results we propose that ATP and ADP hydrolysis are accomplished by two distinct enzymes and therefore the TT ecto-ATPase is not an apyrase. 5ʹ-Nucleotidase activity was inhibited by adenosine 5ʹ-[α,β-methylene]diphosphate and concanavalin A, followed simple Michaelis-Menten kinetics and was released from the membranes by treatment with phosphatidylinositol-specific phospholipase C, indicating that AMP hydrolysis in T-tubules is catalysed by a typical ecto-5ʹ-nucleotidase. Results obtained from electrophoresis experiments under native conditions suggest that ecto-ATPase, ecto-ADPase and 5ʹ-nucleotidase might be associated, forming functional complexes in the T-tubule membranes. The TT ectonucleotidases constitute an enzymic cascade for the degradation of extracellular ATP that might be involved in the regulation of purinergic signalling in the muscle fibre.

Soleus and extensor digitorum longus (EDL) mitochondria and sarcotubular system were examined in sedentary and trained (treadmill for 12 wk) male rats that were treated with fluoxymesterone or methandrostanolone (2 mg/kg, 5 days/wk, for 8 wk). Neither physical exercise nor anabolic/androgenic steroid administration resulted in a significant change in muscle wet weight. Treatment with the anabolizing androgens increased succinate dehydrogenase activity in fast-twitch muscle mitochondria; this effect was not enhanced by training and was not observed in soleus mitochondria. On the other hand, the content of the slow-twitch muscle in sarcotubular fraction was increased in sedentary rats by fluoxymesterone or methandrostanolone treatment, whereas no significant changes were found in EDL. The training program affected adenosinetriphosphatase (ATPase) activities in the sarcotubular fraction; Mg2(+)-ATPase was increased in both soleus and EDL, but Ca2(+)-ATPase was decreased only in soleus. However, in sedentary animals only the Mg2(+)-dependent activity of EDL was increased by anabolizing androgen treatment, and this change was not potentiated by additional training. The present data indicate that anabolic/androgenic steroids can affect mitochondrial and sarcotubular enzymes in skeletal muscle. The effects are muscle-type specific.

Purified transverse tubule membranes from normal and dystrophic chicken skeletal muscle were isolated by a calcium-loading procedure. Normal and dystrophic T-tubules were similar in cholesterol content and (Na + ,K + )-ATPase and 5′-nucleotidase activities but a significant decrease of Mg 2+ -ATPase activity was observed in dystrophic membranes. A comparative analysis of the enzyme properties revealed that the kinetic parameters were altered in dystrophic T-tubules and the ATP-hydrolyzing activity was differently affected by the ionic strength. However, the influence of temperature and the regulatory effect of concanavalin A were the same as in normal T-tubules. Membrane fluidity was similar in both preparations as estimated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium diphenylhexatriene. These results point to an impairment in the function of Mg 2+ -ATPase due to structural alterations of the enzyme.

1. 1. Different non-hydrolytic procedures for the solubilization of NADH-cytochrome b5 reductase and cytochrome b5 from Ceratitis capitata larvae microsomes have been compared. 2. 2. Solubilization of both proteins by detergents was a more adequate method than detergent extraction after delipidation with acetone of native microsomes or phosphate buffer extraction. Treatment with acetone produced an irreversible denaturation of these proteins. 3. 3. CHAPS was the most effective of all detergents assayed in the solubilization of cytochrome b5 whereas Triton X-100 and Triton N-101 were more suitable for the solubilization of NADH-cytochrome b5 reductase. The relation between solubilizing power and HLB number of non-ionic detergents is discussed. 4. 4. CHAPS appears to be efficient in breaking protein-protein interactions and thus at 6 mM induced disaggregation of purified NADH-cytochrome b5 reductase from the insect to a dimeric form.

1. 1. Activity of NADH-cytochrome b 5 reductase from larval microsomes of Ceratitis capitata has been studied with respect to protein concentration, temperature, pH and ionic strength. 2. 2. The enzymatic reduction of potassium ferricyanide was specific for NADH whereas the reduction of endogenous cytochrome b 5 did not exhibit specific requirements for either NADH or NADPH. 3. 3. NADH cytochrome b 5 reductase from the insect was competitively inhibited with respect to NADH by NAD + , ADP and Blue Dextran. 4. 4. Enzymatic activity was inhibited by sodium p -hydroxymercuribenzoate and 5,5′-dithiobis(2-nitrobenzoic acid) and the inhibition was reversed by mercaptoethanol. NADH, but not NADPH, was able to protect the enzyme against inactivation, suggesting that -SH groups are involved in the NADH-binding. 5. 5. Treatment of microsomal preparations of Ceratitis capitata with phospholipase C decreased the enzymatic activity toward endogenous cytochrome b 5 as substrate. It suggests that an appropriate phospholipid environment is essential for an optimal interaction between cytochrome b 5 and its NADH-dependent reductase.

Cytochrome b5 has been purified to electrophoretic homogeneity from larvae microsomes of the insect Ceratitis capitata. The purification procedure involves its solubilization with Triton X-100 and column chromatography on DEAE-cellulose and Ultrogel AcA-44. On the basis of sodium dodecyl sulfate polyacrylamide gel electrophoresis the apparent molecula weight of the purified protein is 21 000. The molecular weight estimated by gel filtration on Sepharose 6B in the absence of detergents is 125 000, indicating that it exists as an aggregate in aqueous media. The prosthetic group has been identified as protoheme IX. The isolated cytochrome possesses an acidic character and contains 31% hydrophobic amino acids and four tryptophan residues as determined by amino acid analysis after mineral or organic acid hydrolysis. The absorption spectrum of the oxidized hemoprotein shows an absorption maximum at 413 nm whereas the dithionite-reduced form has absorption maxima at 555, 527 and 423 nm. From the circular dichroism spectra it is concluded that (1) heme is located in an asymmetric environment and (2) the secondary structure of the insect cytochrome is composed of 25% α-helix, 17% β-structure and 58% random. Its fluorescence emission spectrum, having a maximum at 340 nm, indicates a predominant contribution from tryptophan residues. The cytochrome interacts efficiently with NADH-cytochrome-b5 reductase purified from larvae microsomes. These results suggest that the insect cytochrome b5 is similar to that of mammals, although some differences have been found between proteins from the two sources.

1. 1. NADH-cytochrome b 5 reductase was purified from microsomes of Ceratitis capitata larvae by a procedure involving its solubilization with Triton X-100, DEAE-cellulose anion exchange and hydroxylapatite adsorption chromatography, and affinity chromatography on 5′-ADP-agarose. The overall purification from isolated microsomes was 172-fold and the yield was 13%. 2. 2. The purified enzyme was free from cytochrome b 5 , cytochrome P450 and NADPH-cytochrome c(P450) reductase. The preparation showed a major protein band with an apparent molecular weight of 31,000 D by SDS-polyacrylamide slab gel electrophoresis, whereas the estimated molecular weight ranged between 240,000 D and 260,000 D by chromatography on Sepharose-6B gels in the presence of 0.2% Triton X-100. 3. 3. The enzyme was capable to reduce potassium ferricyanide and 2,6-dichlorophenolindophenol with NADH as electron donor. NADPH was ineffective as electron donor. Reduction of cytochrome c required the presence of NADH and cytochrome b 5 which has been purified from larval microsomes. 4. 4. Influence of ionic strength, pH, temperature and thiol reagents on the enzyme activity is reported. 5. 5. The purified flavoprotein exhibited membrane-binding capacity confirming its amphypathic nature. Reductase bound to microsomal membrane preparations appears to be functionally similar to the endogenous proteins.