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Enzymatic synthesis of penicillin V (penV) by acylation of 6-aminopenicillanic acid (6-APA) was carried out using methyl phenoxyacetate (MPOA) as activated acyl donor and soluble penicillin acylase...

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.

Anabolic androgenic steroids are used in the sport context to enhance muscle mass and strength and to increase muscle fatigue resistance. Since muscle fatigue has been related to oxidative stress caused by an exercise-linked reactive oxygen species (ROS) production, we investigated the potential effects of a treatment with the anabolic androgenic steroid stanozolol against oxidative damage induced on rat skeletal muscle mitochondria by an acute bout of exhaustive exercise. Mitochondrial ROS generation with complex I- and complex II-linked substrates was increased in exercised control rats, whereas it remained unchanged in the steroid-treated animals. Stanozolol treatment markedly reduced the extent of exercise-induced oxidative damage to mitochondrial proteins, as indicated by the lower levels of the specific markers of protein oxidation, glycoxidation, and lipoxidation, and the preservation of the activity of the superoxide-sensitive enzyme aconitase. This effect was not due to an enhancement of antioxidant enzyme activities. Acute exercise provoked changes in mitochondrial membrane fatty acid composition characterized by an increased content in docosahexaenoic acid. In contrast, the postexercise mitochondrial fatty acid composition was not altered in stanozolol-treated rats. Our results suggest that stanozolol protects against acute exercise-induced oxidative stress by reducing mitochondrial ROS production, in association with a preservation of mitochondrial membrane properties.

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.

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.

Isolated Langendorff-perfused hearts from sedentary and prolonged (24 weeks) treadmill-trained rats were subjected to 30 min of normoxic perfusion either alone or followed by 20 min of global ischaemia, or by 20 min of global ischaemia and 15 min of normoxic reperfusion. Pre-ischaemic values of antioxidant enzyme activities and ecto-5'-nucleotidase activity were not different in sedentary and trained hearts but a 5-fold increase of 72-kDa heat shock protein (HSP72) levels was detected in trained myocardium. After ischaemia and reperfusion (I/R), metabolic recovery was better in trained than in sedentary hearts as indicated by higher ATP and creatine phosphate levels. However, antioxidant enzymatic activities, glutathione reductase, and total and mitochondrial superoxide dismutase decreased in trained rats after I/R, whereas they remained unchanged in the sedentary ones. Ecto-5'-nucleotidase activity was modified by I/R in sedentary as well as in trained hearts while HSP72 content did not change. Ecto-5'-nucleotidase activity and HSP72 content increased in parallel by the 30-min perfusion period. In conclusion, the cardioprotection induced by long-term training could be mediated by the exercise-induced increase in HSP72 levels and is not related to enhanced antioxidant systems or ecto-5'-NT activity.

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.

The purpose of this work was to study whether exercise training induces changes in the lipid profile of rat aorta and nervous system and in the in vitro intrinsic responsiveness of these tissues to endothel in-1 (ET-1) treatment. The exercise program performed successfully produced the characteristic metabolic alterations of the trained state. Exercise training induced a large and significant increase in the levels of both aortic ethanolamine plasmalogens (PlasEtn) and glucosylceramides. In contrast, a decrease of aortic ceramide and cholesterol levels was evoked by exercise training. ET-1 increased PlasEtn content only in sedentary animals. An exercise-induced increase in cerebellum levels of ceramides and ceramide monohexosides was found. The cerebellum ceramide content was increased by ET-1 more noticeably in sedentary rats than in trained animals. In contrast, cerebral cortex was observed to be largely insensitive to both exercise training and ET-1 treatment. It was concluded that exercise training (i) induces changes in both vascular and cerebellar lipid profiles, the former being much more pronounced than the latter, and (ii) diminishes the aortic and cerebellar sensitivity to ET-1 action.

Transverse tubule (T-tubule) ecto-ATPase from chicken skeletal muscle is an integral membrane glycoprotein that seems to exist as a homodimer and exhibits unusual properties. Treatment of T-tubule membranes with concanavalin A (Con A) did not significantly affect the thermal variation of the fluorescence anisotropy of vesicles labelled with 1,6-diphenyl-1,3,5-hexatriene or trimethylammonium-1,6-diphenyl-1,3,5-hexatriene. Cross-linking of membrane components with glutaraldehyde elicited effects on ecto-ATPase activity very similar to those of Con A treatment: a severalfold increase in activity, a decrease in Triton X-100 sensitivity and a requirement to be present before ATP to exert its action. In addition, glutaraldehyde and Con A normalized the temperature dependence and the kinetic behaviour of the enzyme. Membrane-perturbing agents (detergents, alcohols and cholesterol oxidase), with the sole exception of digitonin, caused a marked decrease in ecto-ATPase activity; the prior presence of Con A prevented this inhibition, whereas when the lectin was added after the membrane perturbing agent, recovery of the activity was not always possible. The addition of nucleotides before Con A led to a suppression of ecto-ATPase stimulation; it occurred when the nucleotide was hydrolysed (ATP or UTP) and when it was not (adenosine 5′-[β,γ-imido]triphosphate) and even in the presence of 3mM Pi. A model is proposed for the complex regulatory mechanisms of chicken T-tubule ecto-ATPase that involves the occurrence of two different catalytic states in an equilibrium modulated by lectins and cross-linking agents, by the structure of the membrane and by the presence of ligands for a regulatory site.

The purpose of this investigation was to examine the effects of chronic and acute exercise on the main components involved in excitation-contraction coupling and relaxation in rat heart. Sixty male Wistar rats were divided into a sedentary (S) and three 12-wk treadmill-trained groups (T-1, moderate intensity; T-2, high intensity; T-3, interval running). After 12-wk, 15 rats from the S group and 15 rats from the T-2 group were subjected to a single treadmill-exercise session until exhaustion before being killed at 0, 24, or 48 h (acute exercise). The remaining animals were killed 48 h after the last standard exercise session (chronic exercise). The efficacy of the training programs was confirmed by an increase in treadmill endurance time and in skeletal muscle citrate synthase activity. None of the exercise programs modified heart weight or cardiac oxidative capacity. [3H]PN200–110 and [3H]ryanodine binding to cardiac homogenates indicated that the density of L-type and sarcoplasmic reticulum (SR) Ca2+channels was the same in S and trained rats. The SR Ca2+-ATPase activity was also unmodified. Finally, the activities of the ectoenzymes Mg2+-ATPase and 5′-nucleotidase, which are involved in degradation of extracellular nucleotides, were not affected by either of the running programs. After the acute exercise session, no changes were detected in either of the tested parameters in heart homogenates of S and T-2 animals. We conclude that neither treadmill-exercise training for 12 wk nor exhaustive exercise alters the density of Ca2+channels involved in excitation-contraction coupling or the SR Ca2+-ATPase and the ectonucleotidase activities in rat heart.

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.

Background and aims: Exercise training can improve health of patients with metabolic syndrome(MetS).However,whichMetS factors aremost responsive to exercise training remains unclear. We studied the time-course of changes in MetS factors in response to training and detraining. Methods and results: Forty eightMetS patients (52 8.8 yrs old; 33 4 BMI) underwent 4months (3 days/week) of supervised aerobic interval training (AIT) program. After 1 month of training, there were progressive increases in high density lipoprotein cholesterol (HDL-c) and reductions in waist circumference and blood pressure (12 3, 3.9 0.4, and 12 1%, respectively after 4 months; all P < 0.05). However, fasting plasma concentration of triglycerides and glucose were not reduced by training. Insulin sensitivity (HOMA), cardiorespiratory fitness (VO2peak) and exercisemaximal fat oxidation (FOMAx) also progressively improved with training ( 17 5; 21 2 and 31 8%, respectively, after 4months; all P < 0.05). Vastus lateralis samples fromseven subjects revealed that mitochondrial O2 flux was markedly increased with training (71 11%) due to increased mitochondrial content. After 1 month of detraining, the training-induced improvements in waist circumference and blood pressure were maintained. HDL-c and VO2peak returned to the values found after 1e2 months of training while HOMA and FOMAx returned to pre-training values. Conclusions: The health related variables most responsive to aerobic interval training in MetS patients arewaist circumference, blood pressure and the muscle and systemic adaptations to consume oxygen and fat. However, the latter reverse with detraining while blood pressure andwaist circumference are persistent to one month of detraining.

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.

Calcium overload is a fundamental pathogenic event associated with chronic muscle degeneration in muscular dystrophies. The possibility that L-type voltage-dependent calcium channels were involved in the etiology of chicken muscular dystrophy was investigated by studying the dihydropyridine receptors in transverse tubule membranes isolated from skeletal muscle of normal (line 412) and dystrophic (line 413) chickens. The yield of T-tubular protein from dystrophic muscle was considerably increased compared with that from normal muscle (2.51 +/- 0.18 vs 1.04 +/- 0.31 mg protein x 100 g muscle-1). The binding of the calcium channel antagonist (+) [3H]PN200-110 to the dihydropyridine receptor in transverse tubule preparations was relatively slow, markedly affected by temperature and required divalent cations. (+) [3H]PN200-110 equilibrium binding assays revealed a single class of high-affinity sites and showed that maximum binding capacity (Bmax) (3.17 +/- 0.47 for normal and 3.51 +/- 0.52 pmol x mg protein-1 for dystrophic transverse tubules) and dissociation constant (Kd) (0.32 +/- 0.07 and 0.26 +/- 0.09 nM, respectively) were not significantly different in normal and dystrophic membranes. Kinetic studies indicated that normal and dystrophic transverse tubules did not differ significantly in association (2.54 x 10(6) and 2.27 x 10(6) M(-1)s(-1), respectively) and dissociation (8.5 x 10(-4) and 9.3 x 10(-4)s(-1), respectively) rate constants. Since dissociation kinetics for both preparations were monoexponential under all the experimental conditions employed, no low-affinity binding sites for (+) [3H]PN200-110 could be detected in chicken transverse tubules membranes. However, immunoblot assay, using a monoclonal antibody, revealed that dystrophic transverse tubules as compared with normal membranes were enriched twofold with the alpha 1-subunit of the dihydropyridine receptor. Therefore, although dihydropyridine-binding sites were not altered in transverse tubule membranes from dystrophic chicken skeletal muscle, both the increased yield in T-tubule vesicles and the enhanced immunodetection of the alpha 1-subunit of the dihydropyridine receptor, suggest that total content in dihydropyridine receptor is higher in dystrophic than in normal muscle.

To examine the influence of exercise training on the expression of dihydropyridine (DHP)-sensitive Ca2+ channels in skeletal and cardiac muscle, we have determined DHP receptor levels by [3H]PN200-110-binding and immunoblot analysis in homogenates and microsomal fractions of slow- and fast-twitch muscles and heart from rats subjected to a 12-week programme of moderate endurance training. We found that exercise increases the amount of DHP receptor in homogenates of the slow-twitch soleus (42%) and the fast-twitch extensor digitorum longus (60%). Comparable increases in DHP receptor density with training were also observed in the microsomal fractions isolated from both skeletal muscles; these increases were not due to differences in the membrane composition of the microsomal fractions, since the relative proportion of specific enzyme markers was not affected by exercise training. Levels of DHP receptor were not modified in cardiac muscle as a result of the exercise programme. These data suggest an up-regulation of the DHP receptor in the skeletal muscle as a consequence of exercise training, which may play a role in the adaptation of skeletal muscle to increased contractile activity.

The effects of anabolic-androgenic steroid administration and exercise training on various aspects of hepatic function were investigated in sedentary and trained (treadmill for 12 wk) male and female rats treated orally with fluoxymesterone or methylandrostanolone (2 mg.kg-1 body weight, 5 d.wk-1 for 8 wk). The mean values of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total and direct bilirubin, and total- and high-density lipoprotein-cholesterol remained within normal range in all groups of male animals. The same is true for female rats, except for an increase in alkaline phosphatase activity in the steroid-treated groups. Hepatic microsomal aniline p-hydroxylase activity was reduced in male and increased in female rats by either steroid, whereas no significant effect was detected on 7-ethoxycoumarin deethylase activity. The levels of cytochrome P-450 and cytochrome b5 were markedly decreased by the anabolic-androgenic steroid treatment in male rat microsomes, but neither the steroid administration nor exercise training induced significant changes in the cytochrome levels of female rat livers. Taking into account the significant increase in microsomal protein yield elicited by fluoxymesterone or methylandrostanolone treatment both in males and females, it is noteworthy that the total monooxygenase activities and cytochrome P-450 content, expressed on a per gram liver basis, were significantly increased in female whereas they were apparently unchanged in male rats. In conclusion, the present data show that the prolonged ingestion of high doses of anabolic-androgenic steroids, either with or without concurrent exercise training, can modify in a sex-dependent manner the capacity of rat liver to metabolize drugs without affecting classical serum indicators of hepatic function.

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.

The purpose of this study was to analyze the short-term effects of a marathon race (Madrid Marathon) on both markers of cardiac damage and echocardiographic parameters in a group of 22 runners (17 male and 5 female; 34 +/- 5 yr; VO2max: 55.7 +/- 9.1 mL x kg(-1) x min(-1) with a wide range of fitness levels.Venous blood samples were collected from each subject 48 h before the race, at race finish, and 6, 24, and 48 h postexercise for the determination of myoglobin, total creatine kinase catalytic activity (total CK), mass concentration of creatine kinase isoenzyme MB (CK-MB mass), and cardiac isoforms of troponin T and I (TnT-c and TnI-c, respectively). In addition, echocardiographic parameters (M-mode two-dimensional and Doppler analysis) indicative of both left ventricular (LV) systolic and diastolic function were obtained three times from each runner: 2-5 d before the race, at race finish, and 24-36 h after exercise.Except in one subject, levels of TnT-c and TnI-c were within normal limits (0.1 ng x mL(-1)) in all the samples collected before or after the race. Overall LV systolic function was not altered by marathon running. Finally, LV diastolic function was transiently altered after the race since the ratio between peak early and late transmitral filling velocities (E/A) was significantly reduced at race finish (P0.01) and returned to resting levels after 24-36 h.Our findings suggest that marathon running does not adversely affect the hearts of healthy individuals independently from their training status.

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.