Characterization of γ-glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze-tolerant wood frog Rana sylvatica

The freeze tolerant wood frog Rana sylvatica was studied to determine the impact of the freezing and thawing of this frog on the activity of γ-glutamyltranspeptidase in the liver. On exposure to ?2·5°C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4°C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at ?2·5°C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at ?2·5°C on cooling; a 10·5-fold increase after 12 h at ?2·5°C on the initiation of freezing; and a 22-fold increase after 24 h at ?2·5°C in the fully frozen state. Blood glucose levels remained elevated four-fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1·8-fold in the thawed state, while plasma ketone levels were increased 1·8-fold in the frozen state and 1·5-fold in the thawed state. Plasma total T₃ levels were decreased by 22 per cent in the frozen state and normalized on thawing. In homogenates and plasma membranes isolated from the livers of Rana sylvatica, the activity of γ-glutamyltranspeptidase was found to be elevated at all stages of the freeze–thaw process. After 1, 12 and 24 h at ?2·5°C, activities were increased 2·5-, 2·3-, 2·4-fold respectively in the homogenates and 2·5-, 2·2-, 2·4-fold respectively in the plasma membranes. After thawing, activities were still increased 1·9-fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys of Rana sylvatica, the activity of γ-glutamyltranspeptidase was increased 1·4-fold after 1 h at ?2·5°C after which it returned to normal. The role of thyroid hormone in producing the increase in γ-glutamyltranspeptidase in the liver of Rana sylvatica in response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 2. Response to season,temperature and thyroid hormone in Rana pipiens

The impact of season and temperature on frog liver γ-glutamyltranspeptidase was assessed by measuring the activity of this enzyme in plasma membranes isolated from the livers of Rana pipiens obtained as summer and winter frogs; subjected to short-term (3 weeks) temperature acclimation; and subjected to multiple-temperature shifts. Plasma levels of T₃ were determined. γ-Glutamyltranspeptidase was found to be 2·2-fold higher in the summer frog relative to the winter frog; decreased by 44 percent in the summer frog by cold acclimation and increased by 1·7-fold in the winter frog by warm acclimation; and increased by 1·9-fold in the summer frog and 2·8-fold in the winter frog subjected to multiple-temperature shifts. Plasma T₃ levels were found to be 42-fold higher in the summer frog relative to the winter frog; decreased by 42 percent by cold acclimation and increased by 2·9-fold by warm acclimation; and decreased by 39 percent and 38 percent in the summer and winter frogs subjected to multiple temperature shifts. T₃ replacement during the last phase of the multiple-temperature shift protocol, restored the plasma T₃ levels to 75 percent of the control levels and prevented the increase evoked by the multiple-temperature shifts in γ-glutamyl-transpeptidase activity. Indeed, enzyme activity in the T₃ replaced state was 19 percent lower than in the control state. The involvement of thyroid hormone as a negative regulator of enzyme activity is discussed.     

A characterization of gamma-glutamyltranspeptidase in normal, perinatal, premalignant and malignant rat liver

gamma-Glutamyltranspeptidase displays the following order of activity in tissues of the Fischer 344 rat: kidney much greater than small intestine much greater than cerebral cortex = testis greater than lung much greater than liver = heart. The activity of the hepatic enzyme in rats is: 4-fold higher in females than males; 4-fold higher in male Wistar, Sprague-Dawley and Zucker rats than male Fischer 344 rats; increased 10-fold in very old vs young male Fischer 344. The hepatic enzyme displays significant species variation: the mouse and rat liver enzymes are similar and low in activity, while duck, dog, pig and beef enzymes are 7, 13, 86 and 92-fold higher, respectively, in activity than the male Fischer rat liver enzyme. A liver plasma membrane isolation procedure has been devised which selects for the sinusoidal face of the liver parenchymal cell as assessed by marker enzyme analysis: for these plasma membranes the purification of gamma-glutamyltranspeptidase is 21.5 and the recovery is 42% indicating that this is the cellular and subcellular locus of the enzyme in rat liver. The characteristics of the liver plasma membrane from female rats are: pH optimum of 8.0; classical Michaelis-Menten kinetics; Km of 1.43 mM and Vmax of 33.3 nmol X mg-1 X min-1. In Fischer 344 rats, gamma-glutamyltranspeptidase activities are elevated over adult levels in perinatal liver: in fetal liver homogenates and plasma membranes the activities are increased 179 and 109-fold, respectively. The activity peaks just after birth and declines rapidly over the first 15 postnatal days. The activity of the liver enzyme in the male Fischer 344 rat exhibits a progressive increase throughout diethylnitrosamine-induced hepatocarcinogenesis: it is increased 7.8-fold in homogenates and 5.4-fold in plasma membranes at the early premalignant stage; 74-fold in homogenates and 31-fold in plasma membranes at the later hyperplastic nodular premalignant stage; and 174-fold in homogenates and 61-fold in plasma membranes at the hepatoma stage. The gradual drop in purification during hepatocarcinogenesis is associated with the appearance of the enzyme in the blood.     

The influence of clinostat rotation on the fertilized amphibian egg

Unrestrained, fertilized eggs ofRana pipiens andXenopus laevis were rotated in a plane parallel to the normal gravity vector. InR. pipiens rotation at 1/4 rpm for 5 days at 18°C produced a significantly increased number of commonly occurring abnormalities. Rotation at 1/15, 1/8, 1, 2, 5 and 10 rpm did not significantly affect normal development.X. laevis eggs reacted similarly.R. pipiens eggs were most sensitive to rotation at 1/4 rpm when exposure was initiated before first cleavage. Mixing of intracellular constituents apparently occurred only at 1/4 rpm inR. pipiens (of the clinostat speeds studied), and may have been the cause of the increased abnormality observed at this rate.     

The impact of type I diabetes on rat liver γ-glutamyltranspeptidase

The impact of type 1 diabetes mellitus on liver -glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, -glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13,2 fold in Strague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, -glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5-nuleohdase was found to be similar: 9–14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing -glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T₃ (0.6 g/Kg) once a day and T₄ (6.0 g/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20–40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in -glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold ever control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in -glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma.     

Δ53β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate-binding co-operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Δ⁵3β hydroxysteroid dehydrogenase activity transforms biologically inactive Δ⁵3β hydroxy steroids into the active Δ⁴3-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for Δ⁵3β HSD we have analysed the initial velocity rates (V_o) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20α hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 μm) of the dioestrous and proestrous rat ovary. The results are mean ± S.E.M. The relationship between DHEA concentration (0 to 50 μM) and Δ⁵3β HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/V_o versus 1/S plot, the x intercept being positive. Using a 1/V_o versus 1/S² plot the V_max was determined to be 1·0 ± 0·08 μmol min^?1 mg^?1 CL (n = 6). The Hill constant was 2·7 ± 0·02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 ± 1 μmoles (n = 6). In the corpora lutea cells of the proestrous ovary, the V_max for DHEA transformation was unchanged (0·95 ± 0·04 μmol min^?1 mg^?1, n = 3) whilst the S_0·5 was significantly increased to 27 ± 0·1 (p < 0·01, n = 3). The Hill constant remained positive being 2·9 ± 0·2 (n = 3). NAD⁺ binding to 3β HSD in regressing corpora lutea of the proestrous ovary has been demonstrated previously to be hyperbolic and fit the classical Michaelis-Menten model.¹ Extending the analysis of NAD⁺ binding to the regressing corpus luteum of the dioestrous rat ovary revealed similar kinetic characteristics to that seen with the proestrous enzyme, the apparent V_max and K_m being 0·84 ± 0·04 μmol min^?1 mg^?1 CL (n = 3) and 27 ± 7 μmol 1^?1 (n = 3) respectively. The Hill constant was 1·1 ± 0·03 (n = 3), indicating no co-operativity of co-factor binding.     

Purification and kinetic characterization of γ-aminobutyraldehyde dehydrogenase from rat liver

Oxidative deamination of putrescine, the precursor of polyamines, gives rise to γ-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD⁺-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3–8.4. Temperatures higher than 28°C promote slow activation and the process is favoured by the presence of at least one substrate. K_m for aliphatic aldehydes decreases from 110 μM for ABAL and acetaldehyde to 2–3 μM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD⁺ is affected by the aldehyde present at the active site: K_m for NAD⁺ is 70 μM with ABAL, 200 μM with isobutyraldehyde and capronaldehyde, and>800 μM with acetaldehyde. The kinetic behaviour at 37°C is quite complex; according to enzymatic models, NAD⁺ activates the enzyme (K_act 500 μM) while NADH competes for the regulatory site (K_in 70 μM). In the presence of high NAD⁺ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (K_act 10 mM). The data show that the enzyme is probably an E₃ aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.     

Consequence of aflatoxin B1 ingestion on the membrane-bound liver endoplasmic reticulum Ca2+-ATPase of protein-undernourished Fischer F344 rats

Ingestion of the mycotoxin Aflatoxin B₁ (AFB₁) by protein-undernourished Fischer F344 rats for twelve weeks resulted in significant (p<0.05) increases in the proliferation of liver cells, reduced body weight and increased microsomal Ca²⁺-ATPase activity. Cyt. P₄₅₀ content, -Glutamyl Transferase (GGT) and Glutathione Transferase (GST) activities were unaffected. The ingestion of AFB₁ by normal rats had no effect on all the parameters investigated. It appears that the microsomal Ca²⁺-pumping ATPase of Protein-Undernourished (PU) Fischer F344 rats is more sensitive to the mycotoxin AFB₁ than its counterpart in well-nourished rats. The use of PU rat as animal model for studies on AFB₁ toxicity, particularly the effect of AFB₁ on the regulation of intracellular calcium ion concentration ([Ca²⁺]_i), is suggested.     

Comparative enzymological study of catalytic properties of liver monoamine oxidases in frogs

Comparative enzymological study of catalytical properties of monoamine oxidase (MAO) of liver of the marsh frog Rana ridibunda and common frog Rana temporaria has revealed certain features of similarity and differences between these enzymes. The MAOs from both studied biological sources show catalytic properties resembling those of the classical MAO of terrestrial vertebrates: they deaminate tyramine, tryptamine, serotonin, and benzylamine and do not deaminate histamine, have sensitivity to clorgyline, the specific inhibitor of the MAO A form, and deprenyl, the specific inhibitor of the MAO B form, and are not inhibited by 10⁻² M semicarbazide. Based on data of substrate-inhibitor analysis, a suggestion is put forward about the existence of two molecular forms of the enzyme in liver of the studied frog species. Quantitative interspecies differences have been revealed between liver MAO of Rana ridibunda and Rana temporaria in values of kinetic parameters of reactions of deamination of several substrates and in sensitivity to the inhibitors, deprenyl and clorgyline. In the species Rana temporaria the MAO activity in reaction of deamination of serotonin and benzylamine were virtually identical, whereas in the species Rana ridibunda these parameters for serotonin were almost one order higher than for benzylamine. In the species Rana ridibunda, selectivity of action of deprenyl was expressed many times weaker, while selectivity of the clorgyline—one order of magnitude stronger than in the species Rana temporaria. The catalytic activities towards all studied substrates of liver MAO of both studied amphibian species were several times lower as compared with the enzyme of rat liver.     

Aging-Associated Changes in the Pharmacological Properties of the Benzodiazepine (ω) Receptor Isotypes in the Rat Hippocampus

Abstract: The aging-associated changes in hippocampal benzodiazepine (ω) receptor isotypes have been investigated in rats of the Wistar and Fischer 344 strains. Displacement experiments of [³H]flunitrazepam binding by zolpidem demonstrated that in hippocampal membranes from adult (3-month-old) Wistar strain rats, high (type I; ω₁)-, intermediate (type II_M; ω₂)-, and low (type II_L; ω₅)- affinity sites for this imidazopyridine account for 27.1 ± 7.5, 44.2 ± 7.5, and 28.8 ± 5.1%, respectively. In hippocampal membranes from aged (24-month-old) rats of the same strain, the relative abundance of these sites was 42.8 ± 9.3, 26.3 ± 4, and 36.0 ± 5.9%, respectively. In contrast, no significant difference was observed in the whole benzodiazepine (ω) binding site density between adult and aged rats. The increase in type I (ω₁) binding site density in the hippocampus of aged rats was also demonstrated in saturation experiments with [³H]zolpidem. This aging-induced increase in [³H]zolpidem binding was also observed in hippocampal membranes from Fischer 344 rats. Moreover, in both rat strains, GABA induced a greater enhancement of [³H]zolpidem (5 nM) binding to type I (ω₁) sites (GABA shift) in aged than in adult hippocampal membranes. Quantitative autoradiographic analysis of [³H]zolpidem binding to coronal brain sections from adult and aged Fischer 344 rats demonstrated that the aging-associated increases in the density of type I (ω₁) binding sites were restricted to the hippocampus. Moreover, increases in binding density were larger in the dentate gyrus and in the CA2 field than in the CA1 and CA3 fields.     

The Arrhenius plot behaviour of rat liver 5′-nucleotidase in different lipid environments

The plasma membrane enzyme 5′-nucleotidase (EC 3.1.3.5) was prepared from rat liver as a complex with sphingomyelin or in detergent-solubilized forms. Each preparation exhibited a K_m and Arrhenius break temperature indistinguishable from that of 5′-nucleotidase in plasma membranes. Measurement of fatty acid profiles, cholesterol and phospholipid content however showed a very wide variation between these preparations. We conclude that the biphasic nature of the Arrhenius plot of 5′-nucleotidase may be a property of the enzyme rather than its lipid environment.     

Testicular and androgen dependence of skin gland morphology in the anurans,Xenopus laevis and Rana pipiens

In anuran amphibians, there is increasing evidence that exocrine glands dispersed throughout the general integument are secondary sex characters (SSC). Following the recent discovery of sexually dimorphic “breeding glands” in the dorsum of male Rana pipiens, we studied the effects of castration and testosterone treatment on the dorsal skin glands of male Xenopus laevis and R. pipiens to determine whether the dorsal breeding glands, or any other dorsal skin glands, are androgen dependent. The dorsal skin glands of X. laevis were unaffected by androgen status. By contrast, in R. pipiens, breeding, mucous, and seromucous glands responded to testosterone stimulation. Mucous glands were significantly (P < 0.05) larger in testosterone-treated frogs than in castrates. There was a large, but statistically insignificant, increase in the size of the dorsal breeding glands. Testosterone treatment also increased the epithelial cell height of breeding and seromucous glands (P < 0.05). In the skins of castrated and testosterone-treated frogs, there was a reciprocal relationship between the abundance of seromucous and breeding glands: in castrates, seromucous glands were abundant and breeding glands virtually absent, whereas in testosterone-treated frogs, breeding glands were abundant and seromucous glands less common. The total number of the two gland types was similar in both treatment groups. Glands that appeared to be intermediate in form between seromucous and breeding glands were observed in some frogs. These data suggest that seromucous glands may be the regressed form of breeding glands in the dorsal skin of R. pipiens and that the dorsal skin of R. pipiens is a SSC. © 1993 Wiley-Liss, Inc.     

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo

Summary High yields of human hepatocytes (up to 23×10⁶ viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10⁻⁸ M insulin, and 10⁻⁸ M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50±0.17 nmol glucose·mg⁻¹·min⁻¹) similar to that reported for human liver. Insulin at 10⁻⁸ M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10⁻⁹ M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, α₁-antitrypsin, α₁-antichymotrypsin, α₁-acid glycoprotein, haptoglobin, α₂-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10⁻⁹ M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg⁻¹ cell protein·min⁻¹, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol·mg⁻¹). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.     

The effects of (±)-, (+)-, and (−)-atenolol,sotalol, and amosulalol on the rat left atria and portal vein

The effects of (±)-, (+)-, and (?)-atenolol, sotalol, and amosulalol alone on the rat left atria and portal vein and on the respective β₁- and β₂-adrenoceptor-mediated responses to isoprenaline have been determined. (±)-Atenolol at 10^?6 M had no effect whereas high concentrations of (+)- and (?)-sotalol, 10^?5–10^?4 M, and (±)-, (+)-, and (?)-amosulalol depressed the response of the rat left atria to cardiac stimulation which indicates membrane stabilizing activity. None of the drugs tested had any effect alone on the rat portal vein. The order of potency as antagonists was (±)-amosulalol > (±)-atenolol > (±)-sotalol at β₁-adrenoceptors and (±)-amosulalol > (±)-sotalol > (±)-atenolol at β₂-adrenoceptors. (±)-Atenolol and (±)-amosulalol are β₁-selective whereas (±)-sotalol is β₂-selective. For each of the racemic β-blockers, the β₁- and β₂-adrenoceptor blocking activity was predominantly due to the (?)-enantiomer. © 1993 Wiley-Liss, Inc.     

Regulation of cation transport pathways and glycolytic enzyme activity by alterations in red cell volume

In the presence of NH₄Cl and hypotonic solutions, Rana balcanica red cells respond by increasing their volume. The stimulation of cellular volume by hypotonicity is more rapid than that of NH₄Cl, while the maximum value is less than that observed in the presence of NH₄Cl. Depending on the cause of swelling, (net uptake of NH₄Cl or decrease in external osmolality) cells show specific responses. The NH₄Cl treatment causes a significant increase in intracellular Na⁺, from 5·14±0·78 to 29·84±0·47 mmoles l⁻¹ cell, while hypotonicity leads to a significant decrease of this cation, to 3·85±0·25 mmoles l⁻¹ cell in relation to the control, after 30 min of incubation of Rana balcanica erythrocytes. In addition, amiloride significantly reverses the NH₄Cl effect with respect to intracellular Na⁺. Both treatments cause a significant K⁺ loss in comparison with controls. Two glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) of Rana balcanica haemolysate were found to respond to the NH₄Cl effect by significantly decreasing their activity. Copyright © 1999 John Wiley & Sons, Ltd.     

Functional expression of basolateral Cl−/HCO3− exchange from rat jejunum in Xenopus laevis oocytes

Poly(A)⁺ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of Cl⁻/HCO₃⁻ antiport was investigated by means of ³⁶Cl⁻ uptake. Two days after injection of 50 ng of poly(A)⁺ RNA, Cl⁻ uptake was significantly increased with respect to water-injected oocytes. The expressed transport was inhibited by 0·2 mM DIDS, whereas endogenous Cl⁻ uptake was unaffected by this disulphonic stilbene. After sucrose density gradient fractionation, the highest expression of DIDS-sensitive Cl⁻ uptake was detected with mRNA size fraction of about 2–4 kb in length. The expressed Cl⁻ uptake can occur against a Cl⁻ concentration gradient and is unaffected by the known Cl⁻ channel blocker anthracene-9-carboxylic acid. Cl⁻ transport mechanism has properties similar to jejunal basolateral Cl⁻/HCO₃⁻ exchange with regard to Na⁺ dependence. © 1998 John Wiley & Sons, Ltd.     

Extraction and analysis of superoxide free radicals (·O−2) from whole mammalian liver

Extraction of whole lobes of normal rat liver with dimethyl sulphoxide (DMSO) under N₂ gives extracts which contain 5—10 μmol/l·O^?₂ (50-100 nmol·O^?₂ per 10 ml extract per 4 g liver; 1.25-2.50 nmol·O^?₂ per millilitre per gram liver). Evidence for ·O^?₂ in the extracts is given by: (1) electron spin resonance signals (ESR), (2) differential pulse polarography (DPP), (3) chemiluminescence (CL), and (4) nitroblue tetrazolium reduction (NBT). All tests yield results identical with those obtained with authentic ·O^?₂. Extraction of ·O^?₂ is enhanced by tetrabutyl ammonium ion, and is maximal at 1-3 min. These results raise the possibility that substantial amounts of ·O^?₂ are normally sequestered in protective membranous sites in vivo.     

Enalapril maleate affects 2-oxoglutarate metabolism in mitochondria from the rat kidney cortex

Enalapril maleate (EM) is the salt of N-{(S)-1-(ethoxycarbonyl)-3-phenylpropyl}-L -alanyl-L -proline, used therapeutically as an anti-hypertensive agent. The effects of EM on some aspects of the energy metabolism and membrane properties of mitochondria from rat liver and kidney cortex were studied, but only the latter were significantly affected. With 0·8 mM of EM and 2-oxoglutarate as oxidizable substrate for isolated mitochondria from rat kidney cortex, the findings were: (a) inhibition of the respiratory rate in state III (37 per cent) and decrease (45 per cent) in respiratory control ratio (RCR), with only one addition of ADP; (b) reinforcement of the inhibition when a second addition of ADP was made; (c) no significant effect either on the rate of respiration in state IV or on the ADP/O ratio; (d) no effect on the ATPase activity of mitochondria from liver or kidney cortex; (e) inhibition of the transmembrane potential (Δψ) after a second addition of ADP; (f) inhibition of the 2-oxoglutarate dehydrogenase complex. It is suggested that in kidney mitochondria, EM interferes in the gluconeogenesis dependence of at least five substrates: 2-oxoglutarate, glutamine, glutamate, lactate, and pyruvate. Also EM may inhibit Na⁺/H⁺ exchange causing natriuresis.     

Corrections by melatonin of liver mitochondrial disorders under diabetes and acute intoxication in rats

The aim of the present work was to investigate the mechanisms of oxidative damage of the liver mitochondria under diabetes and intoxication in rats as well as to evaluate the possibility of corrections of mitochondrial disorders by pharmacological doses of melatonin. The experimental (30 days) streptozotocin‐induced diabetes mellitus caused a significant damage of the respiratory activity in rat liver mitochondria. In the case of succinate as a respiratory substrate, the ADP‐stimulated respiration rate V₃ considerably decreased (by 25%, p < 0·05) as well as the acceptor control ratio (ACR) V₃/V₂ markedly diminished (by 25%, p < 0·01). We observed a decrease of the ADP‐stimulated respiration rate V₃ by 35% (p < 0·05), with glutamate as substrate. In this case, ACR also decreased (by 20%, p < 0·05). Surprisingly, the phosphorylation coefficient ADP/O did not change under diabetic liver damage. Acute rat carbon tetrachloride‐induced intoxication resulted in considerable decrease of the phosphorylation coefficient because of uncoupling of the oxidation and phosphorylation processes in the liver mitochondria. The melatonin administration during diabetes (10 mg·kg^‐1 body weight, 30 days, daily) showed a considerable protective effect on the liver mitochondrial function, reversing the decreased respiration rate V₃ and the diminished ACR to the control values both for succinate‐dependent respiration and for glutamate‐dependent respiration. The melatonin administration to intoxicated animals (10 mg·kg⁻¹ body weight, three times) partially increased the rate of succinate‐dependent respiration coupled with phosphorylation. The impairment of mitochondrial respiratory plays a key role in the development of liver injury under diabetes and intoxication. Melatonin might be considered as an effector that regulates the mitochondrial function under diabetes. Copyright © 2011 John Wiley & Sons, Ltd.     

Characterization of an ethanol‐tolerant 1,4‐β‐xylosidase produced by Pichia membranifaciens

Aims: The purification and biochemical properties of the 1,4‐β‐xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol‐tolerant 1,4‐β‐xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G‐100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS‐PAGE. The activity of 1,4‐β‐xylosidase was optimum at pH 6·0 and at 35°C. The activity had a K_m of 0·48 ± 0·06 mmol l^?1 and a V_max of 7·4 ± 0·1 μmol min^?1 mg^?1 protein for p‐nitrophenyl‐β‐d ‐xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4‐β‐xylosidase from P. membranifaciens to be used in the bioethanol production process.