Phospholipid hydroperoxide glutathione peroxidase: specific activity in tissues of rats of different age and comparison with other glutathione peroxidases

The tissue distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPX) was studied in rats of different ages. In the same samples the activities of Se-dependent glutathione peroxidase (GPX), and non-Se-dependent glutathione peroxidase (non Se-GPX) were also determined using specific substrates for each enzyme. Enzymatically generated phospholipid hydroperoxides were used as substrate for PHGPX, hydrogen peroxide for GPX, and cumene hydroperoxide for non-Se-GPX (after correction for the activity of GPX on this substrate). PHGPX specific activity in different organs is as follows: liver = kidney greater than heart = lung = brain greater than muscle. Furthermore, this activity is reasonably constant in different age groups, with a lower specific activity observed only in kidney and liver of young animals. GPX activity is expressed as follows: liver greater than kidney greater than heart greater than lung greater than brain = muscle, and substantial age-dependent differences have been observed (adult greater than old greater than young). Non-Se-GPX activity was present in significant amount only in liver greater than lung greater than heart and only in adult animals. These results suggest a tissue- and age-specific expression of different peroxidases.     

A comparative study of superoxide dismutase in various animal species

1. Superoxide dismutase activity has been determined in liver homogenates of five species. There were no significant differences in the activities of the enzyme in the rat, rainbow lizard, wall gecko and chicken. The activity was significantly lower in the fish. 2. The order of activity of the enzyme in the organs/tissues of the rat was liver greater than kidney greater than heart greater than skeletal muscle greater than brain greater than lung greater than spleen greater than spinal cord greater than retina greater than pancrease greater than lens greater than small intestine. 3. Inhibition studies with cyanide showed that the enzyme in the liver of the various animal species was inhibited by cyanide. 4. The developmental pattern for the enzyme showed no significant changes in the liver of the rat from birth and up to 7 weeks after. However, the activity increased at about 8 weeks and remained constant to adult life.     

Catalase enzymatic activity and electrophoretic patterns in adult amphibians--a comparative study

Catalase electrophoretic patterns and enzymatic activities were measured in four organs of two anuran species, Rana ridibunda perezii and Discoglossus pictus. The D. pictus enzyme appeared as two distinguishable bands, whereas R. ridibunda catalase was monomorphic. Electrophoretic mobility of the major D. pictus catalase band was greater than that of R. ridibunda. Enzymes from both species showed slower mobility than that from bovine liver. Catalase activities did not show significant differences according to sex in any of the organs tested in R. ridibunda. Enzyme activities were similar in liver, kidney and brain when both species were compared. Only the heart showed much higher activity in D. pictus than in R. ridibunda. The catalase activity levels followed the order: liver greater than kidney greater than heart in both species. The heart showed higher activity than the brain in D. pictus but not in R. ridibunda.     

Tissue distribution and molecular heterogeneity of bovine thiol:protein-disulphide oxidoreductase (disulphide interchange enzyme)

1. The distribution of thiol:protein-disulphide oxidoreductase (disulphide interchange enzyme) in 17 bovine tissue extracts was determined by rocket immunoelectrophoresis and by measuring the reductive cleavage of insulin. 2. The relative concentration (per mg total protein) was found to be in the order: Pancreas greater than liver greater than lymph node greater than testes, fat tissue greater than parotid gland, brain, spleen, lung greater than small intestine, spinal cord, large intestine, kidney greater than paunch, aorta greater than skeletal muscle greater than heart. 3. The distribution of specific activity showed a similar pattern, irrespectively of whether glutathione or L-cysteine was used as cosubstrate. 4. The concentration varied 200-fold and the specific activity 400-fold between pancreas and heart muscle, respectively. 5. Crossed immunoelectrophoresis demonstrated that a fast-migrating form of the enzyme was the only one present in almost all tissues, but 15% of the enzyme in liver was a slow-migrating form and 50% in heart muscle a medium-migrating form. 6. The lung contains a species having partial immunological identity to the enzyme. 7. Purified enzyme from bovine liver has a somewhat lower mobility than the fast-migrating form in extract. 8. The results seem to support the general view that the enzyme is involved in synthesis of disulphide-bonded extracellular proteins, although the presence of the enzyme in tissues like fat, brain, spinal cord, skeletal muscle and heart indicates other cellular functions as well.     

Tissue concentrations of MPTP and MPP+ in relation to catecholamine depletion after the oral or subcutaneous administration of MPTP to mice

One hour after MPTP was given to mice at a dose of 30 mg/kg s.c., its concentration in tissues varied in the order kidney greater than liver greater than lung greater than brain greater than heart. When the same dose of MPTP was given orally, concentrations in most tissues were much lower at 1 hr than after s.c. administration, although the MPTP concentration in liver was only slightly lower. The concentrations of MPP+ (a metabolite of MPTP) at 1 hr were as high or higher than those of MPTP in all tissues except kidney, and MPP+ disappeared from the various tissues with half-lives from 3-20 hrs. The highest concentrations of MPP+, both absolute and relative to MPTP, were in heart. After oral administration of MPTP, no MPP+ was found in brain, and MPP+ concentrations in other tissues were lower than those after s.c. dosing. The depletion of heart norepinephrine was similar after MPTP administration by either route of administration even though MPTP and MPP+ concentrations in heart were lower after oral administration, suggesting that other metabolites of MPTP might also contribute to heart norepinephrine depletion.     

Cytosolic enzymes from rat tissues that activate the cooked meat mutagen metabolite N-Hydroxyamino-1-methyl-6- phenylimidazo[4,5-b]pyridine (N-OH-PhIP)

Heterocyclic amines are formed during the cooking of foods rich in protein and can be metabolically converted into cytotoxic and mutagenic compounds. These "cooked-food mutagens" constitute a potential health hazard because DNA damage arising from dietary exposure to heterocyclic amines can modify cell genomes and thereby affect future organ function. To determine enzymes responsible for heterocyclic amine processing in mammalian tissues, we performed studies to measure genotoxic activation of the N-hydroxy form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) --a common dietary mutagen. O-Acetyltransferase, sulfotransferase, kinase, and amino-acyl synthetase activities were assayed using substrate-specific reactions and cytosolic enzymes from newborn and adult rat heart, liver, spleen, kidney, brain, lung, and skeletal muscle. The resultant enzyme-specific DNA adduct formation was quantified via (32)P-postlabeling techniques. In biochemical assays with rat tissue cytosolic proteins, O-acetyltransferases were the enzymes most responsible for N-hydroxy-PhIP (N-OH-PhIP) activation. Compared to O-acetyltransferase activation, there was significantly less kinase activity and even lesser amounts of sulfotransferase activity. Proyl-tRNA synthetase activation of N-OH-PhIP was not detected. Comparing newborn rat tissues, the highest level of O-acetyltransferase mutagen activation was observed for neonatal heart tissue with activities ranked in the order of heart > kidney > lung > liver > skeletal muscle > brain > spleen. Enzymes from cultured neonatal myocytes displayed high O-acetyltransferase activities, similar to that observed for whole newborn heart. This tissue specificity suggests that neonatal cardiac myocytes might be at greater risk for damage from dietary heterocyclic amine mutagens than some other cell types. However, cytosolic enzymes from adult rat tissues exhibited a different O-acetyltransferase activation profile, such that liver > muscle > spleen > kidney > lung > brain > heart. These results demonstrated that enzymes involved in catalyzing PhIP-DNA adduct formation varied substantially in activity between tissues and in some tissues, changed significantly during development and aging. The results further suggest that O-acetyltransferases are the primary activators of N-OH-PhIP in rat tissues.     

Distribution in tissues and effects by acute hypoxia on angiotensin I converting enzyme activity in guinea pig and chicken

Angiotensin I converting enzyme activity was measured in homogenates of guinea pig and chicken organs (lung, kidney, heart, ileum, diaphragm and liver), using a spectrophotometric assay for hydrolysis of hippuryl-L-histidyl-L-leucine. High specific activities were found in lung, kidney and diaphragm, but the highest corresponded to guinea pig lung and chicken kidney. Acute hypoxia decreased angiotensin I converting enzyme activity in guinea pig lung and chicken diaphragm, but the changes in kidney were considered non-significant in both the guinea pig and chicken.     

Effect of cigarette smoke on lipid peroxidation and antioxidant enzymes in albino rat

Effect of cigarette smoke on lipid peroxidation (LPX) and antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) in various organs like brain, heart, lung, liver and kidney of the albino rats exposed to cigarette smoke for 30 min/day for a period of 30 days were assayed. It was observed that the lipid peroxide levels in liver, lung and kidney were enhanced in case of animals exposed to cigarette smoke, whereas brain and heart did not show any change as compared to control animals. The activity of the antioxidant enzymes was also elevated in liver, lung and kidney of the test animals whereas, brain and heart did not show any change in the activities of all of these antioxidant enzymes except glutathione-s-transferase which was increased in brain also. The level of reduced glutathione (GSH) was lowered in liver, lung and kidney of the tested animals when compared with the control animals but there was no significant change in brain and heart. The results of our study suggest that cigarette smoke induces lipid peroxidation in liver, lung and kidney, and the antioxidant enzymes levels were enhanced in order to protect these tissues against the deleterious effect of the oxygen derived free radicals. The depletion of reduced glutathione in these organs could be due to it's utilization by the tissues to mop off the free radicals.     

Detection and distribution of scorpion (Heterometrus bengalensis) venom in rabbit tissues by enzyme-linked immunosorbent assay (ELISA).

ELISA was carried out to detect distribution of scorpion venom in experimental animal tissues. The venom content of different tissues was in the order, liver greater than kidney greater than spleen greater than lung greater than heart greater than diaphragm greater than brain. Tissue distribution of venom antigen in the envenomental subject by ELISA will provide a better approach for serotherapy.     

Occurrence and subcellular location of NADH- and NADPH-linked aquacobalamin reductases in human liver

1. Both activities of NADH- and NADPH-linked aquacobalamin reductases were found in some human tissues, liver, kidney pancreas, heart, spleen, lung, cerebrum, cerebellum, adrenal glands, stomach, duodenum, jejunum, ileum, colon and bone marrow. 2. Human liver contained both enzymes with higher specific activities than any other tissues. 3. The liver NADH-linked enzyme was distributed in both mitochondrial (approx. 60%) and microsomal (40%) fractions; similar to the distribution of the NADPH-linked enzyme, but of which 40% activity was found in the mitochondria and the remaining activity was recovered in the microsomes. 4. The results suggest that the synthetic systems of the cobalamin coenzymes occur in both mitochondria and microsomes of human liver.     

Physiological significance of catalase and glutathione peroxidases,and in vivo peroxidation,in selected tissues of the toadDiscoglossus pictus (Amphibia) during acclimation to normobaric hyperoxia

1. Various parameters related to oxidative stress were measured in adult Discoglossus pictus acclimated for 15 days to either normoxia or hyperoxia (PO2 = 710 mmHg). 2. Total weight of the toads and total and relative wet weight of liver, kidneys, lungs and heart were not changed by hyperoxic acclimation. 3. In vivo tissue peroxidation increased in lung, decreased in skeletal muscle, and was not changed in liver, kidney, heart and skin after hyperoxic exposure. 4. Hyperoxic acclimation increased catalase activities in the lung, liver, kidney and heart but not in skeletal muscle and skin. 5. Liver showed higher GSH-peroxidase activity with cumene-OOH than with H2O2 as substrate, whereas lung, skeletal muscle and skin presented similar GSH-peroxidase activities with both substrates. 6. GSH-peroxidase activities did not change between hyperoxic and normoxic animals in liver, lung, skeletal muscle and skin. 7. These results show that catalase, not GSH-peroxidase, is the principal H2O2 detoxifying enzyme involved in the adaptation of D. pictus to hyperoxia.     

Changes in drug-metabolizing activities in the livers of suckling rats as a result of treatment of the lactating mothers with phenobarbitone and chlorpromazine

1. The activities in rat tissues of 3-oxo acid CoA-transferase (the first enzyme involved in acetoacetate utilization) were found to be highest in kidney and heart. In submaxillary and adrenal glands the activities were about one-quarter of those in kidney and heart. In brain it was about one-tenth and was less in lung, spleen, skeletal muscle and epididymal fat. No activity was detectable in liver. 2. The activities of acetoacetyl-CoA thiolase were found roughly to parallel those of the transferase except for liver and adrenal glands. The high activity in the latter two tissues may be explained by additional roles of thiolase, namely, the production of acetyl-CoA from fatty acids. 3. The activities of the two enzymes in tissues of mouse, gerbil, golden hamster, guinea pig and sheep were similar to those of rat tissues. The notable exception was the low activity of the transferase and thiolase in sheep heart and brain. 4. The activities of the transferase in rat tissues did not change appreciably in starvation, alloxan-diabetes or on fat-feeding, where the rates of ketone-body utilization are increased. Thiolase activity increased in kidney and heart on fat-feeding. 5. The activity of 3-hydroxybutyrate dehydrogenase did not change in rat brain during starvation. 6. The factors controlling the rate of ketone-body utilization are discussed. It is concluded that the activities of the relevant enzymes in the adult rat do not control the variations in the rate of ketone-body utilization that occur in starvation or alloxan-diabetes. The controlling factor in these situations is the concentration of the ketone bodies in plasma and tissues.     

Sex- and species-related differences in the biotransformation of isosorbide dinitrate by various tissues of the rabbit and rat

The biotransformation of isosorbide dinitrate (ISDN) by various tissues of the rabbit and rat was examined. Incubation of 2 X 10(-7) M ISDN at 37 degrees C with tissue homogenates of liver, lung, kidney, intestine, skeletal muscle, aorta, and erythrocytes from the rabbit and rat resulted in a significant disappearance of ISDN after a 30-min incubation (also, 5-min incubation for liver). The disappearance of ISDN in each tissue homogenate was accompanied by an equimolar production of the mononitrate metabolites, isosorbide-2-mononitrate (2-ISMN) and isosorbide-5-mononitrate (5-ISMN), with the exception of liver homogenates where the loss of ISDN could not be accounted for by mononitrate formation. The relative rate of ISDN disappearance in various tissue homogenates was for the male rabbit, liver greater than lung approximately intestine greater than kidney greater than erythrocytes approximately skeletal muscle approximately aorta; for the female rabbit, liver greater than kidney approximately lung approximately intestine greater than erythrocytes approximately skeletal muscle approximately aorta; and for the male rat, liver greater than intestine greater than erythrocytes greater than skeletal muscle greater than lung approximately kidney. A sex difference in the percent disappearance of ISDN was observed in homogenates of lung and intestine from male and female rabbits. In addition, a sex difference in the ratio of metabolite (2-ISMN/5-ISMN) formed by denitration of ISDN was seen in homogenates of lung, skeletal muscle, and erythrocyte lysate.(ABSTRACT TRUNCATED AT 250 WORDS)     

猪激素敏感脂酶和甘油三酯水解酶基因组织表达特性研究

以八眉猪为研究对象,采用RT-PCR和Western blot方法对猪激素敏感酯酶(HSL)和甘油三酯水解酶(TGH)基因组织表达特点进行了研究。RT-PCR半定量检测显示,HSL基因的mRNA在检测的7种组织中都有表达,其中在脂肪组织表达量较高,中等程度表达于心脏、肝脏、肺、脾和肾脏。TGH基因在7种组织也均有表达,其中肝脏和脂肪组织表达量较高,心脏和肾脏次之,脾脏和肺脏表达量较低。Western blot检测显示,HSL基因在大网膜脂肪和皮下脂肪表达量最高,而在肾脏中没有检测到表达,其他组织中中度表达;TGH基因在大网膜脂肪、皮下脂肪、肝脏、肺脏和脾脏组织中表达,其中在脂肪组织和肝脏组织中表达量最高,而在心脏和肾脏中没有检测到表达。以上结果表明:HSL和TGH基因存在转录后调控,这可能与其在不同组织中的功能差异有关。     

Organ distribution of rat histidine-pyruvate aminotransferase isoenzymes.

The organ distribution of rat histidine-pyruvate aminotransferase isoenzymes 1 and 2 was examined by using an isoelectric-focusing technique. Isoenzyme 1 (pI8.0) is present only in the liver and its activity is increased by the injection of glucagon, whereas isoenzyme 2 (pI5.2) is distributed in all tissues (liver, kidney, brain and heart) tested, and is not affected by glucagon injection. Isoenzyme 2 of the liver, kidney, brain and heart was purified by the same procedure and characterized. Isoenzyme 2 preparations from these four tissues were nearly identical in physical and enzymic properties. These properties differed from those previously found for the highly purified isoenzyme 1 preparation of rat liver. Isoenzyme 2 was active with pyruvate but not with 2-oxoglutarate as amino acceptor. Amino donors were effective in the following order of activity: tyrosine greater than histidine greater than phenylalanine greater than kynurenine greater than tryptophan. Very little activity was found with 5-hydroxytryptophan. The apparent Km for histidine was about 0.45 mM. The Km for pyruvate was about 4.5 mM with histidine as amino donor. The amino-transferase activities of isoenzyme 2 towards phenylalanine and tyrosine were inhibited by histidine. The ratio of aminotransferase activities towards these three amino acids was constant through gel filtration, electrophoresis, isoelectric focusing and sucrose-density-gradient centrifugation of the purified isoenzyme 2 preparations. These results suggest that these three activities are properties of the same enzyme protein. Sephadex G-150 gel filtration and sucrose-density-gradient centrifugation yielded mol.wts. of approx. 95000 and 92000 respectively. The pH optimum was between 9.0 and 9.3.     

Release of ethane and pentane from rat tissue slices: effect of vitamin E, halogenated hydrocarbons, and iron overload

The effects of in vitro addition of halogenated hydrocarbons on the susceptibility of various rat tissues to lipid peroxidation, and of iron overload and dietary vitamin E in the intact rat on subsequent lipid peroxidation in rat tissue slices were examined. The ease and speed of tissue slice preparation allowed testing of multiple tissues from the same animals. Total ethane and pentane (TEP) released from the slices was as reliable as and more sensitive than thiobarbituric acid-reactive substances as an index of lipid peroxidation. TEP was released by tissues from vitamin E-deficient rats in the following order of magnitude:intestine = brain = kidney greater than liver = lung greater than heart greater than testes = diaphragm greater than skeletal muscle. The potency of halogenated hydrocarbons for causing increased TEP release from vitamin E-deficient rat liver slices was CBrCl3 greater than CCl4 = 1,1,2,2-tetrabromoethane = 1,1,2,2-tetrachloroethane greater than perchloroethylene. CBrCl3 also stimulated TEP release from kidney, intestine, and heart slices, thus identifying these as potential target organs for CBrCl3 toxicity. Dietary vitamin E decreased TEP release from liver and, to a lesser extent, from kidney. Iron overload in the rat increased TEP release by slices from all tissues tested except the brain.     

Incomplete palmitate oxidation in cell-free systems of rat and human muscles

The palmitate oxidation capacity was determined in whole homogenates, postnuclear fractions and mitochondrial fractions of various rat and human muscles and in rat liver, kidney, brain and lung. The oxidation rate (production of 14CO2 and 14C-labeled acid-soluble intermediates) was [1-14C]palmitate greater than [U-14C]palmitate greater than [16-14C]palmitate in all cell-free systems. Oxidation rates were highest in rat heart and liver, intermediate in kidney, diaphragm and m. quadriceps, and low in brain and lung. The capacity of human heart was much lower than that of rat heart and about twice that of human skeletal muscles. Omission of L-carnitine and addition of malonyl-CoA, KCN or antimycin A decreased the oxidation rates in whole homogenates and mitochondrial fractions. Antimycin or KCN increased and malonyl-CoA decreased the ratio of the oxidation rates with [1-14C]- and [16-14C]palmitate. The carnitine concentration had no significant effect on the ratio. 14C-labeled dodecanoic and tetradecanoic acids were identified in homogenates and mitochondrial fractions of m. quadriceps and liver of rat as acid-insoluble intermediates of [16-14C]palmitate oxidation in the presence and absence of antimycin A. Their amounts recovered can account for the differences in oxidation rates found with [1-14C]- and [16-14C]palmitate. The incomplete palmitate oxidation in cell-free systems appears to be mainly caused by an inadequate mitochondrial degradation of peroxisomal oxidation products.     

Subcellular and organ distribution of cholesterol epoxide hydrolase in the rat

The subcellular and organ distributions of microsomal epoxide hydrolases measured with cis-stilbene oxide and cholesterol 5,6 alpha-epoxide as substrates have been investigated. These two enzyme activities were found to have essentially the same subcellular distribution, with the highest total and specific activities localized in rough and smooth endoplasmic reticulum. Among the tissues studied (i.e., liver, kidney, lung, testis, spleen, brain and intestinal epithelium), the highest specific activities were recovered in liver microsomes, where the activities were at least 5-fold greater than in any of the other microsomal preparations.     

Inducing effects of clofibric acid on 1-acylglycerophosphorylcholine acyltransferase in kidney and intestinal mucosa of rats

Administration of p-chlorophenoxyisobutyric acid (clofibric acid) markedly increased the activity of microsomal 1-acylglycerophosphorylcholine (1-acyl-GPC) acyltransferase in kidney, intestinal mucosa and liver, but not in brain, heart, lung, spleen, testis or skeletal muscle. In both kidney and liver, a marked dose-dependent increase in the activities of both microsomal 1-acyl-GPC acyltransferase and peroxisomal beta-oxidation was observed. In the rats treated with clofibric acid at a relatively low dose, the increase in the activity of 1-acyl-GPC acyltransferase in kidney was more marked than that in liver. The extent of the relative increase in the activity of 1-acyl-GPC acyltransferase to the activity of peroxisomal beta-oxidation in kidney was more marked than that in liver. The increased activity of 1-acyl-GPC acyltransferase in both kidney and liver lasted throughout the 8-week treatment period of rat with clofibric acid.