Effect of depot medroxyprogesterone acetate and testosterone ananthate on the testis of albino rats: ultrastructural and biochemical studies.

Male albino rats were treated with depot medroxyprogesterone acetate (1 mg/animal/day) + testosterone ananthate (100 micrograms/100 g body weight/day) for 30 and 60 days. After 30 days of treatment, all the testicular enzymes like beta-glucuronidase, hyaluronidase, sorbitol dehydrogenase, lactate dehydrogenase, acid and alkaline phosphatase, registered non-significant decrease in their values. Fifty percent of the treated animals achieved sterility after 30 days of treatment. After 60 days of treatment the testis showed degenerative changes in Golgi phase and late spermatids. Changes in the Golgi phase spermatids were related with degeneration of the nuclear membrane. Changes in the late phase spermatids included mitochondrial hypertrophy of the midpieces, membrane lysis, absence of cristae and degeneration of annulus leading to detachment of tail. Cytoplasm of luminal area displayed hypertrophied mitochondria devoid of cristae, prominent appearance of Golgi bodies, intense lysosomal activity and ample vacuolation. Tail fragments of degenerated spermatids prevailed in luminal cytoplasm. Except for beta-glucuronidase which registered a significant decrease, levels of all the other testicular enzymes, viz. hyaluronidase, lactate dehydrogenase, sorbitol dehydrogenase, acid phosphatase and alkaline phosphatase were within their control limits. The ultrastructural and biochemical changes are correlated.     

Pyruvate dehydrogenase phosphate (PDHb) phosphatase in brain: activity, properties, and subcellular localization

The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg . ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho-PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K-phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K-phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.     

Use of toluene-permeabilized mitochondria to study the regulation of adipose tissue pyruvate dehydrogenase in situ. Further evidence that insulin acts through stimulation of pyruvate dehydrogenase phosphate phosphatase.

Rat epididymal-adipose-tissue mitochondria were made selectively permeable to small molecules without the loss of matrix enzymes by treating the mitochondria with toluene under controlled conditions. With this preparation the entire pyruvate dehydrogenase system was shown to be retained within the mitochondrial matrix and to retain its normal catalytic activity. By using dilute suspensions of these permeabilized mitochondria maintained in the cuvette of a spectrophotometer, it was possible to monitor changes of pyruvate dehydrogenase activity continuously while the activities of the interconverting kinase and phosphatase could be independently manipulated. Permeabilized mitochondria were prepared from control and insulin-treated adipose tissue, and the properties of both the pyruvate dehydrogenase kinase and the phosphatase were compared in situ. No difference in kinase activity was detected, but increases in phosphatase activity were observed in permeabilized mitochondria from insulin-treated tissue. Further studies showed that the main effect of insulin treatment was a decrease in the apparent Ka of the phosphatase for Mg2+, in agreement with earlier studies with mitochondria made permeable to Mg2+ by using the ionophore A23187 [Thomas, Diggle & Denton (1986) Biochem. J. 238, 83-91]. No effects of spermine were detected, although spermine diminishes the Ka of purified phosphatase preparations for Mg2+. Since effects of insulin on pyruvate dehydrogenase phosphatase activity are not evident in mitochondrial extracts, it is concluded that insulin may act by altering some high-Mr component which interacts with the pyruvate dehydrogenase system within intact or permeabilized mitochondria, but not when the mitochondrial membranes are disrupted.     

Effects of phospholipases C on membrane-bound enzymes of yeast

Alkaline phosphatase was released from protoplasts of the yeast Saccharomyces cerevisiae without cell lysis not only by phosphatidylinositol (PI)-specific phospholipase C but also by phosphatidylcholine (PC)-hydrolyzing phospholipase C. Activities of mitochondrial enzymes such as succinate dehydrogenase, antimycin-sensitive NADH-cytochrome c reductase, and oligomycin-sensitive ATPase were decreased by the action of PC-hydrolyzing phospholipase C. Hydrolysis of microsomal PC or PI did not cause any decrease in the activities of NADPH-cytochrome c reductase and antimycin-insensitive NADPH-cytochrome c reductase. In the requirement of phospholipids, the properties of yeast mitochondrial enzymes were very close to those of mammalian mitochondrial enzymes, whereas those of yeast microsomal enzymes were completely different from those of mammalian microsomal enzymes.     

Hepatotoxic effects of acetaminophen. Protective properties of tryptophan derivatives

Rat intoxication with acetaminophen (APAP) (500–1500 mg/kg body weight, intragastrically) caused a considerable dose-dependent decrease in reduced glutathione (GSH) level in both liver cell cytoplasm and mitochondria (at the dose 1500 mg/kg body weight by 60% and 33%, respectively). The decrease in cytoplasmic GSH level was more pronounced than in mitochondria. Despite of significant mitochondrial GSH depletion we did not observe any inactivation of the mitochondrial enzymes: succinate dehydrogenase, α-ketoglutarate dehydrogenase, glutathione peroxidase, and also any decrease in the respiratory activity of liver mitochondria isolated from APAP-intoxicated rats. We have investigated hepatoprotector properties of tryptophan derivatives, melatonin and N-acetyl-nitrosotryptophan (a nitric oxide donor). The pineal gland hormone, melatonin, a known antioxidant (10 mg/kg body weight), did not prevent intramitochondrial GSH, but decreased the APAP hepatotoxicity evaluated as the decrease in the activity of marker enzymes of hepatic damage, ALT and AST and total bilirubin content in blood plasma of intoxicated rats, whereas NNT did not exhibit any hepatoprotective effects.     

Adriamycin induced myocardial failure in rats: Protective role of Centella asiatica

Generation of reactive oxygen species and mitochondrial dysfunction has been implicated in adriamycin induced cardiotoxicity. Mitochondrial dysfunction is characterized by the accumulation of oxidized lipids, proteins and DNA, leading to disorganization of mitochondrial structure and systolic failure. The present study was aimed to evaluate the efficacy of Centella asiatica on the mitochondrial enzymes; mitochondrial antioxidant status in adriamycin induced myocardial injury. Adriamycin (2.5 mg/kg body wt., i.p.) induced mitochondrial damage in rats was assessed in terms of decreased activities (p< 0.05) of cardiac marker enzymes (lactate dehydrogenase, creatine phosphokinase, amino transferases), TCA cycle enzymes (isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, respiratory marker enzymes (NADH-dehydrogenase, cytochrome-C-oxidase), mitochondrial antioxidant enzymes (GPx, GSH, SOD,CAT) and increased (p< 0.05) level of lipid peroxidation. Mitochondrial damage was confirmed by transmission electron microscopic examination. Pre-co-treatment with aqueous extract of Centella asiatica (200 mg/kg body wt, oral) effectively counteracted the alterations in mitochondrial enzymes and mitochondrial defense system. In addition, transmission electron microscopy study confirms the restoration of cellular normalcy and accredits the cytoprotective role of Centella asiatica against adriamycin induced myocardial injury. Our results demonstrated elevated oxidative stress and mitochondrial dysfunction in adriamycin treated rats. Moreover, on the basis of our findings it may be concluded that the aqueous extract of C. asiatica not only possesses antioxidant properties but it may also reduce the extent of mitochondrial damage     

Activity of several erythrocyte enzymes in chick embryos and chicks after gamma-irradiation

After irradiation of chick embryos and chicks (1,000 rad), the activity of some erythrocyte enzymes undergoes significant changes. During the 1st day after irradiation of chick embryos, the activity of lactate dehydrogenase leucine aminopeptidase and glutamate pyruvate transaminase decreases. At the 3rd day, the decrease in the activity of glucose-6-phosphate dehydrogenase and acid phosphatase is also observed. In irradiated chicks, the activity of lactate dehydrogenase, leucine aminopeptidase and aldolase decreases within the 1st and the 3rd days, the decrease being most significant for the former two enzymes. At later period (10 and 15 days after irradiation), most significant decrease was found in the activity of glucose-6-phosphate dehydrogenase. The activity of the same enzymes in the blood plasma of irradiated embryos and chicks increases, the increase being most evident for glucose-6-phosphate dehydrogenase.     

Buffer-induced rat liver mitochondrial aggregation during differential centrifugation

Use of buffers in homogenization media can result in loss of considerable particulate enzyme activity even with low-speed centrifugation. Addition of tris chloride buffer to 0.25 M sucrose homogenization media resulted in precipitation of 80 to 95% of the activity of two mitochondrial marker enzymes (3-hydroxy-3-methylglutaryl CoA lyase and citrate synthase) with the nuclear fraction during differential centrifugation. Lactate dehydrogenase, a cytoplasmic marker, was not precipitated under the same conditions, indicating that the precipitated enzymes were not associated with intact cells. Photomicrographs showed that tris chloride buffers resulted in mitochondrial aggregation. Isolated mitochondria resuspended in tris chloride or potassium phosphate buffer also aggregated, which resulted in a marked decrease in assayable mitochondrial enzyme activity.     

S-allylcysteine ameliorates isoproterenol-induced cardiac toxicity in rats by stabilizing cardiac mitochondrial and lysosomal enzymes

This study was aimed to evaluate the preventive role of S-allylcysteine (SAC) on mitochondrial and lysosomal enzymes in isoproterenol (ISO)-induced rats. Male albino Wistar rats were pretreated with SAC (50, 100 and 150 mg/kg) daily for a period of 45 days. After the treatment period, ISO (150 mg/kg) was subcutaneously injected to rats at an interval of 24 h for two days. The activities of heart mitochondrial enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and alpha-ketoglutarate dehydrogenase) and respiratory chain enzymes (NADH dehydrogenase and cytochrome C oxidase) were decreased significantly (p<0.05) in ISO-induced rats. The activities of lysosomal enzymes (beta-glucuronidase, beta-N-acetyl glucosaminidase, beta-galactosidase, cathepsin-D and acid phosphatase) were increased significantly (p<0.05) in serum and heart of ISO-induced rats. Pretreatment with SAC (100 mg/kg and 150 mg/kg) for a period of 45 days increased significantly (p<0.05) the activities of mitochondrial and respiratory chain enzymes and decreased the activities of lysosomal enzymes significantly (p<0.05) in ISO-induced rats. Oral administration of SAC (50, 100 and 150 mg/kg) for a period of 45 days to normal rats did not show any significant (p<0.05) effect in all the parameters studied. The altered electrocardiogram (ECG) of ISO-treated rats was also restored to near normal by treatment with SAC (100 and 150 mg/kg). These results confirm the efficacy of SAC in alleviating ISO-induced cardiac damage.     

Role of DL α-lipoic acid in gentamicin induced nephrotoxicity

The effect of DL -lipoic acid on the nephrotoxic potential of gentamicin was examined. Intraperitoneal injection of gentamicin (100 mg/kg/day) to rats resulted in decreased activity of the glycolytic enzymes-hexokinase, phosphoglucoisomerase, aldolase and lactate dehydrogenase. The two gluconeogenic enzymes—glucose-6-phosphatase and fructose-1, 6-diphosphatase, the transmembrane enzymes namely the Na⁺, K⁺-ATPase, Ca²⁺-ATPase, Mg²⁺-ATPase and the brushborder enzyme alkaline phosphatase, also showed decreased activities. This decrease in the activities of ATPases and alkaline phosphatase suggests basolateral and brush border membrane damage. Decreased activity of the TCA cycle enzymes isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH), suggests a loss in mitochondrial integrity. These biochemical disturbances were effectively counteracted by lipoic acid administration. Lipoic acid administration by gastric intubation at two different concentrations (10 mg and 25 mg/kg/day) brought about an increase in the activity of the glycolytic enzymes, ATPases and the TCA cycle enzymes. The gluconeogenic enzymes however showed a further decrease in their activities at both the concentrations of lipoic acid administered. These observations shed light on the nephroprotective action of lipoic acid against experimental aminoglycoside toxicity and the protection afforded at 25 mg/kg/day of lipoic acid was noted to be higher than that at 10 mg level.     

Effect of Coscinium fenestratum on hepatotoxicity in rats

Anti-hepatotoxic activity of methanol extract of Coscinium fenestratum stem (MEC) was investigated against carbon tetrachloride-induced hepatopathy in rats. Hepatotoxic rats were treated with MEC for a period of 90 days (60mg/kg body weight, daily, orally by intubation). Anti-hepatotoxic effect was studied by assaying the activities of serum marker enzymes like aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, lactate dehydrogenase etc. and glucose (6) phosphate dehydrogenase in liver. We also estimated the concentrations of total proteins, total lipids, triglycerides, phospholipids and cholesterol in serum, liver and kidney. The activities of all the marker enzymes registered a significant elevation in carbon tetrachloride-treated rats, which were significantly recovered towards an almost normal level in animals co-administered with MEC. Other biochemical changes induced by carbon tetrachloride too showed reliable signs of retrieving towards the normalcy. Histopathological analysis confirmed the biochemical investigations. This study unravels the anti-hepatotoxic activity of MEC.     

Preventive effect of naringin on cardiac mitochondrial enzymes during isoproterenol-induced myocardial infarction in rats: a transmission electron microscopic study

Dietary flavonoids intake has been reported inversely related to the incidence of cardiovascular diseases (CVD). The present study is undertaken to evaluate the preventive role of naringin on mitochondrial enzymes in isoproterenol (ISO)-induced myocardial infarction in male albino Wistar rats. Rats subcutaneously injected with ISO (85 mg/kg) at an interval of 24 h for 2 days, resulting in significant (p < 0.05) increase in the levels of mitochondrial lipid peroxides. ISO-induction also showed significant (p < 0.05) decrease in the activities of mitochondrial tricarboxylic acid cycle enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, and alpha-ketoglutarate dehydrogenase) and respiratory chain enzymes (NADH dehydrogenase and cytochrome c oxidase). Oral pretreatment with naringin (10, 20, and 40 mg/kg) to ISO-induced rats daily for a period of 56 days significantly (p < 0.05) minimized the alterations in all the biochemical parameters and restored the normal mitochondrial function. Transmission electron microscopic (TEM) observations also correlated with these biochemical findings. Thus, our findings demonstrate that naringin prevents the mitochondrial dysfunction during ISO-induced myocardial infarction in rats.     

β-adrenergic receptors and enzymes in rat myocardial membranes: implications of fractionation procedures andβ-adrenoceptor antagonists

1. We performed an enzymatic characterization of two different fractionation procedures of ventricles from rat hearts. The enzymatic assays covered succinic dehydrogenase as a marker for inner mitochondrial membranes, monoamine oxidase as a marker for outer mitochondrial membranes, NADPH-cytochrome c reductase and RNA as endoplasmatic reticular markers, acid phosphatase as a lysosomal marker, and lactic dehydrogenase as a marker for the "soluble" compartment; DNA was estimated for nuclear contamination. 2. The plasma membrane markers 5'-nucleotidase, Ca2+-ATPase, Mg2+-ATPase, Na+-K+-ATPase, and adenylate cyclase were determined. 3. The roughly prepared membrane fractions showed increased yields of the membrane markers; the number of beta receptors, determined with (-)-[3H] dihydroalprenolol and DL-propranolol, amounted to 68 +/- 6 fmol/mg protein (KD = 3390 +/- 450 pmol, Hill coefficient = 1.5). 4. The membrane fraction prepared with a linear sucrose gradient showed an increased inner mitochondrial membrane marker; presumably the outer mitochondrial membrane was stripped off. The beta-receptor number was 39 +/- 3 fmol/mg protein (KD = 6250 +/- 300 pmol; Hill coefficient = 1.2).     

SEPARATION OF MITOCHONDRIAL MEMBRANES OF NEUROSPORA CRASSA : II. Submitochondrial Localization of the Isoleucine-Valine Biosynthetic Pathway

Separation of Neurospora mitochondrial outer membranes from the inner membrane/matrix fraction was effected by digitonin treatment and discontinuous density gradient centrifugation. The solubilization of four isoleucine-valine biosynthetic enzymes was studied as a function of digitonin concentration and time of incubation in the detergent. The kinetics of the appearance of valine biosynthetic function in fractions outside of the inner membrane/matrix fraction, coupled with enzyme solubilization patterns similar to that for the matrix marker, mitochondrial malate dehydrogenase, indicate that the four isoleucine-valine pathway enzymes are localized in the mitochondrial matrix.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cells. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ' Crabtree effect', was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate dehydrogenase was subject to glucose inactivation.     

Mitochondrial malate dehydrogenase and malic enzyme: Mendelian inherited electrophoretic variants in the mouse

Malate dehydrogenase and malic enzyme each possess supernatant and mitochondrial molecular forms which are structurally and genetically independent. We describe electrophoretic variants of the mitochondrial enzymes of malate dehydrogenase and malic enzyme in mice. Progeny testing from genetic crosses indicated that the genes which code for mitochondrial malate dehydrogenase and malic enzyme were not inherited maternally but as independent unlinked nuclear autosomal genes. The locus for mitochondrial malic enzyme was located on linkage group I. Linkage analysis with a third mitochondrial enzyme marker, glutamic oxaloacetic transaminase, showed that the nuclear genes which code for the three mitochondrial enzymes were not closely linked to each other. This evidence suggests that clusters of nuclear genes coding for mitochondrial function are unlikely in mice.Supported by U.S. Public Health Service grants 5F2 HD-35,531 and GM-09966.     

Use of enzymes for the diagnosis of alcohol-related organ damage

Elevated levels of serum enzymes are frequently associated not only with alcohol-related organ damage but also with excessive alcohol consumption and alcoholism without significant tissue injury. However, both in the early detection of alcoholism as well as also in the diagnosis of alcohol-related diseases the sensitivities and specificities of these enzyme markers vary considerably. They may be influenced by nonalcohol-related diseases, enzyme-inducing drugs, nutritional factors, metabolic disorders, age, smoking, etc. Consequently, we have neither a single laboratory test--enzyme marker--nor a test combination that is reliable enough for the exact diagnosis between alcohol- and nonalcohol-related organ damage. In most cases it is possible to determine the tissue from which the elevated enzyme is derived, but only occasionally enzyme changes reflect the quantity of the tissue injury. Gamma-glutamyltransferase (GGT) is the most widely used laboratory marker of alcoholism and heavy drinking, detecting 34-85% of problem drinkers and alcoholics. However, the unspecificity of increased serum GGT limits its use for general screening purposes. Its value in the follow-up of various treatment programs, however, is well established. An elevated level of serum aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) in an alcoholic or a heavy consumer indicates alcohol-induced organ damage. The use of test combinations significantly improves the information received with single serum enzyme determinations. An ASAT/ALAT ratio greater than 1.5 can be considered as highly suggestive for the alcoholic etiology of the liver injury. Still better discrimination between alcoholic and nonalcoholic origin of the liver disease may be achieved by the determination of the ratio of GGT to alkaline phosphatase. If this ratio exceeds 1.4 the specificity of the finding in favor for alcoholic liver injury is 78%. The determination of the mitochondrial isoenzyme of ASAT also improves the diagnostic value of ASAT determination. The ratio of mitochondrial isoenzyme to total over 4 is highly suggestive for alcohol-related liver injury. In general, however, the determination of serum activities of other enzymes such as ornithine carbamyl transferase, lactate dehydrogenase, isocitrate dehydrogenase, sorbitol dehydrogenase, alcohol dehydrogenase, guanase, aldolase, alkaline phosphatase or glutathione S-transferase do not significantly improve the diagnostic information obtained with more conventional laboratory markers of liver injury.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitative morphologic changes in nephron structures and urinary enzyme activity pattern in sodium-maleate-induced renal injury

The morphologic changes in sodium-maleate-induced acute renal injury in the rat were quantified by a stereologic analysis. The major changes were confined to an increase in endocytic vacuoles and a decrease in mitochondrial inner membrane surface area. These results were found to be linked to significantly increased urinary activities of the cytosolic of the cytosolic enzymes fructose-1,6-bisphosphatase (FBP) and lactate dehydrogenase, the lysosomal enzyme N-acetyl-beta-glucosaminidase (NAG) and the NAD-dependent mitochondrial isocitrate dehydrogenase (ICDH). The highest increase was found for NAG, followed by FBP and ICDH.     

Fractionation by differential and zonal centrifugation of spheroplasts prepared from a glucose-repressed fission yeast Schizosaccharomyces pombe 972h-.

A method is described for the preparation of spheroplasts in high yield from Schizosaccharomyces pombe, by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome c oxidase activity and spectroscopically-detectable cytochromes a + a3) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with Saccharomyces carlsbergensis, cytochrome c oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mM-MgCl2 or 0-4 mM-EDTA. Mitochondria were apparently smaller and of lower buoyant density in gradients containing EDTA. The bulk of the total units of malate dehydrogenase and NADH; cytochrome c oxidoreductase sedimented with mitochondria, whereas NADPH: cytochrome c oxidoreductase was located in fractions containing no mitochondria. The distributions of mitochondrial enzymes were heterogeneous in populations of mitochondria separated on the basis of size or density. The possible origins of mitochondrial heterogeneity in extracts of S. pombe are discussed with special reference to changes in the enzyme activities of cells during the cell cycle.