PLA2 activity in rat liver nuclei

1. Subcellular fractions of rat liver were assayed for PLA2 activity. 2. The PLA2 assay measures the release of [3H]oleic acid from phospholipids, using labeled E. coli as substrate. 3. Nuclear fractions contained PLA2 activity, which was Ca2+ dependent and could not be explained from mitochondria, microsomal or plasma membrane contamination. 4. The Vmax value of nuclear PLA2 is 0.30 +/- 0.04 pmol oleic acid/min/mg protein; its Km value is 0.86 +/- 0.12 microM, similar to that of mitochondrial PLA2. 5. We conclude that rat liver nuclei contain PLA2 activity.     

Intracellular localisation of urea-cycle enzymes in liver (author's transl)

Enzymes of the Krebs-Henseleit urea-cycle were localized by means of differential centrifugation and fractional tissue extraction in rat liver and in human liver. Argininosuccinatlyase (ASAL) and Argininosuccinatsynthetase (ASAS) represent enzymes of the soluble cytoplasmic fraction. Ornithine-ketoacid-transaminase(OKT), carbamyl-phosphate-synthetase (CPS) and ornithine-carbamyl-transferase (OCT) are localized in the mitochondrial and nuclei fractions of the liver cell. Most of the arginase activity is bound to subcellular structures (probably to nuclei). A small portion of arginase-activity was found in the soluble cytoplasmatic fraction. The enzymes of the Krebs-Henseleit urea-cycle are equally distributed in rat liver and in human liver. Differences in the subcellular localisation of (mitochondrial) enzymes in human liver could be attributed to mitochondrial breakage during tissue preparation and do not represent in-vivo conditions.     

Effect of phenobarbital treatment on characteristics determining susceptibility to oxidants of homogenates, mitochondria and microsomes from rat liver

This study was designed to investigate the possible oxidative changes associated with alterations in cytochrome P450 levels in rat liver. Accordingly, extent of peroxidative processes, cytochrome and antioxidant content, capacity to face an oxidative stress were determined in liver microsomes, mitochondria, and homogenates from normal and phenobarbital (PB)-treated rats. Liver content of microsomal and mitochondrial proteins was also determined by the values of the activities of marker enzymes (glucose-6-phosphatase and cytochrome oxidase, respectively) in liver homogenate and in two cellular fractions. The increase in the liver content of microsomal and mitochondrial proteins indicated that PB caused proliferation of both smooth endoplasmic reticulum and mitochondrial population. Treatment with PB also gave rise to a general increase in peroxidative reactions (evaluated measuring malondialdehyde and hydroperoxides (HPs)), in the different cell compartments, even though HPs were not found significantly increased in mitochondrial fraction. The increase in peroxidative processes was associated with significant decreases in antioxidant concentration (expressed in terms of equivalent concentration of an antioxidant, such as the desferrioxamine), in all preparations from PB-treated rats. The response to oxidative stress in vitro (evaluated determining the parameters characterizing light emission from preparations stressed with sodium perborate) showed a substantial PB-induced increase in the susceptibility to oxidative challenge only in liver homogenate. The lack of changes in the mitochondrial preparations is likely due to decrease in concentration of both free radical producing species and antioxidants. The lack of changes in microsomal fraction is apparently in contrast with its lower oxidant capacity and higher content of cytochromes which are able to determine sensitivity to pro-oxidants. However, it could be due to the ability of cytochrome P450 to interact with the active oxygen species formed at its active center.     

Delta-Aminolevulinic acid synthetase in the heart

The regultion of cardiac delta-aminolevulinic acid synthetase activity was studied in rat heart homogenates. Optimal conditions were determined for the measurement of delta-aminolevulinic acid and an appropriate assay was established for the heart. The activity of cardiac delta-aminolevulinic acid synthetase was determined in rats either fed ad libitum or starved for 24 or 48 h. Marked decreases in delta-aminolevulinic acid synthetase activity were observed in homogenates or mitochondrial fractions prepared from hearts of fasted animals and an explanation for previous findings that the enzyme is undetectable in heart tissue is provided. Dexamethasone treatment was effective in reversing the decreases brought about by fasting but had no effect on the delta-aminolevulinic acid synthetase activity in heart homogenates from fed rats. ACTH treatment had no effect in fed or starved rats. Decreases in delta-aminolevulinic acid synthetase activity induced by fasting were not reversed in homogenates or mitochondrial preparations by succinyl-CoA-generating systems or when alpha-ketoglutarate was substituted for succinate in homogenate preparations. Cardiac delta-aminolevulinic acid dehydratase levels are not altered by fasting. Agents such as allylisopropylacetamide or 3,5-dicarbethoxy-1,4-dihydrocollidine, which produce marked increases in hepatic delta-aminolevulinic acid synthetase activity, have no effects on the activity of this enzyme in the heart.     

Elevated expression and altered pattern of activity of DNA methyltransferase in liver tumors of rats fed methyl-deficient diets

DNA methyltransferase (MTase) activity in nuclear extracts from neoplastic and preneoplastic livers of rats fed a methyl-deficient diet (MDD) is elevated compared with that seen in the livers of control rats. Nuclear proteins were prepared in the presence of protease inhibitors including trans-epoxy succinyl-L-leucylamido-(4-guanido)butane and were fractionated by isoelectric focusing. In normal, control liver, two distinct MTase fractions were observed. In MDD-induced malignant liver, a third fraction, in addition to the previous two, was also seen. Both the DNA substrate and the cytosine site specificities of the third MTase fraction differ from those of the other two fractions. The distinct MTase activity in liver tumor has significantly more de novo MTase activity than do the MTase fractions of normal, control liver. Thus, normal and neoplastic rat livers differ in DNA MTase fractionation patterns and site specificities. The altered DNA MTase activity observed in rat liver tumors caused by MDDs may be one of the critical factors contributing to cancer formation through abnormal DNA methylation.     

Glucocorticoid receptor binding to rat liver nuclei occurs without nuclear transport

The binding of cell-free activated glucocorticoid receptor-steroid complexes from HTC cells to various preparations of HTC and rat liver nuclei has been examined under conditions that did or did not support the nuclear translocation of macromolecules via nuclear pores. To the best of our knowledge, this is the first such study with functionally active isolated nuclei. Conventionally prepared HTC nuclei were found to be porous, as determined from their inability to exclude the fluorescent macromolecule phycoerythrin (PE) at 4 degrees C. Thus the nuclear binding of activated complexes to these nuclei can not involve nuclear translocation. Further studies, using established conditions with sealed nuclei prepared from rat liver, revealed that nuclear translocation of PE containing a covalently linked, authentic nuclear translocation sequence could be obtained at 22 degrees C, but not at 4 degrees C. However, under the same conditions, activated glucocorticoid complexes displayed equal levels of nuclear binding at both temperatures. We therefore conclude that the current translocation conditions with intact rat liver nuclei are not sufficient to reproduce the nuclear transport of glucocorticoid complexes observed in intact cells. The nuclear binding that was seen with intact rat liver nuclei was not affected by aurintricarboxylic acid, which selectively inhibits protein-nucleic acid interactions. The antibody AP-64, shown to be specific for amino acids 506-514 of the nuclear translocation sequence of the rat glucocorticoid receptor, inhibited the nuclear binding of activated complexes, apparently by blocking receptor access to the nuclear membrane. Collectively, these data argue that activated complex binding to nuclei capable of nuclear translocation involves only an association with nuclear membrane components such as nuclear pores. Thus this system, and these reagents, may be useful in future studies of activated complex binding to nuclear pores.     

Membrane and nuclear protein kinase C activation in the early stages of diethylnitrosamine-induced rat hepatocarcinogenesis

The activity and expression of Ca(2+)-dependent cPKC alpha and beta isoenzymes in the particulate, soluble (cytosolic) and nuclear fractions of rat liver and the expression of Ca(2+)-independent nPKC delta and aPKC zeta were examined during the early stages (30 and 60 min, 24 and 96 h and 7 and 60 days post-hepatectomy) of the Solt-Farber 'resistant hepatocyte' model of diethylnitrosamine (DENA)-induced hepatocarcinogenesis in Fischer 344 rats and related to the presence of gamma-glutamyl transpeptidase (GGT)-positive hyperplastic cell foci and persistent nodules in rat liver. Total PKC activity was unmodified by the carcinogenic treatment. In contrast, the PKC activity in the particulate, as well as nuclear fractions increased with time, reaching a maximum 60 days post-hepatectomy, with a decrease in the cytosolic activity. In carcinogen-treated animals maximal expression of cPKC alpha and beta isoenzymes was present 7 days post-hepatectomy, while no changes in nPKC delta and aPKC zeta immunoreactivity were detected. In the nucleus, no cPKC alpha isoform expression was observed, the cPKC beta expression being maximal at 60 days. Seven and 60 days post-hepatectomy GGT-positive hyperplastic cell foci and persistent nodules were present in rat liver respectively. Taken together, the results of this study suggest a role for nuclear cPKC beta and for cPKC alpha in promoting the selective growth of carcinogen-initiated hepatocytes in rat liver. No evidence for a role of Ca(2+)-independent nPKC delta and aPKC zeta isoenzymes in the early stages of DENA-induced liver carcinogenesis could be demonstrated.     

Role of female sex steroids in regulating cholesteryl ester transfer protein in transgenic mice

The role of sex steroids in the regulation of cholesteryl ester transfer protein (CETP) was examined in the following groups of female transgenic mice carrying the human CETP gene: (1) normal, (2) ovariectomized, (3) ovariectomized and treated with estrogen; (4) ovariectomized and treated with progesterone; (5) ovariectomized and treated with both hormones, and (6) ovariectomized and treated with tamoxifen. CETP activity was measured in the plasma, and in the particulate and the soluble fractions of liver, muscle, and adipose tissue. Human CETP specific activity was determined by taking the difference of cholesterol ester transfer in the presence and absence of an antibody (TP2) against human CETP Ovariectomy reduced hormone levels, but did not completely abolish them from the circulation. Plasma CETP activity was significantly reduced in the tamoxifen group. There were significant reductions in CETP in liver homogenate and the soluble fraction, as well as in the particulate fraction of adipose with ovariectomy. Hormone replacement did not restore CETP activity in either the plasma or the tissues. Tamoxifin treatment resulted in a decrease in CETP activity in both fractions of liver, but had no effect on adipose. In the soluble fraction of adipose tissue and both fractions of muscle, only trace CETP activity was detected. We conclude that (1) minimal amounts of sex steroid hormones may be sufficient to affect CETP expression; (2) the effects of sex steroid hormones vary among tissues; and (3) in addition to the sex steroids, factor(s) from the ovary are needed for the full expression of CETP in this animal model.     

Distribution of nitrazepam and its metabolites in the subcellular fractions of several white rat organs

After intraperitoneal administration into rats at a dose of 100 mg per kg of body weight nitrazepame (mogadone, eunoktine) was enzymatically reduced with the subsequent acetylation. Derivatives of nitrazepame were found in cellular fragments and nuclei, in mitochondrial, microsomal and soluble fractions of liver, lungs, heart and brain tissues. Reduction of the substrate was shown to occur in soluble and microsomal fractions of liver tissue and acetylation--in mitochondria of lungs and liver tissue. Nitrazepame metabolites were quite uniformly distributed over cell organelles of heart and brain tissues; this suggests that they originate in the organs from other tissues, where the processes of reduction and acetylation take place. Nitrazepame and its derivatives penetrated into brain very effectively; this phenomenon is considered as an essential one for their pharmocological activity.     

Cadmium and cardiac muscle cell - biomarkers of oxidative stress - experimental work

OBJECTIVE: To study in several tissues (heart, kidney and liver) of Halobatrachus didactylus the cellular response induced by an acute exposure to a sublethal cadmium concentration. DESIGN: Fifteen species of H. didactylus (marine teleost) were divided in to three groups: CTRL: control group, the fish were injected with a saline solution; 24 H: 1 mg/kg of cadmium chloride was injected and the fish were sacrificed after 24 hours; 7 D: the fish were subjected to the same cadmium concentration and sacrificed 7 days after injection. INTERVENTIONS: Superoxide dismutase--SOD (McCord & Fridovich, 1969) and catalase--CAT (Clairborne, 1985) activities were determined in the cytosolic and mitochondrial fractions of these three tissues. The lipid degradation products were also determined by the tiobarbithuric acid (TBA) test. MEASUREMENTS AND RESULTS: Cadmium induced an increase in SOD activity in both fractions (cytosolic and mitochondrial) of these H. didactylus tissues. The highest levels of activity observed were located at mitochondrial fraction and in the heart. There was a significant increase in CAT activity in both liver and heart tissue fractions after cadmium exposure. The highest values were observed in the liver. The kidney presented a different response: there was a rise in CAT activity only in the mitochondrial fraction after seven days of exposure. There were no significant changes in lipid degradation products in any of these tissues after cadmium exposure. CONCLUSIONS: The two antioxidant enzymes studied in the heart, kidney and liver of H. didactylus demonstrated a high sensitivity to oxidative stress induced by cadmium and presented a high potential as cellular biological makers. The results indicate membrane lesion caused by lipid peroxidation did not occur, which suggests an efficient response of the cellular protection mechanisms against cadmium cytotoxicity.     

Protease activities during preparation and handling of nuclear particles containing hnRNA

Conditions were devised to avoid protease activity during the preparation and the subsequent handling of nuclear particles containing hnRNA. During all the steps of preparation of rat liver particles, the presence of phenylmethyl sulfonyl fluoride (PMSF) was required for the reproducibility of the results. It probably inhibited the cellular serine proteases before the separation of the particles from the other cellular structures. Protease activity was detected in the rat liver particles. The enzyme(s) preferentially hydrolyzed a few particle polypeptides. It was not inhibited by PMSF, nor by two trypsin and chymotrypsin-like protease inhibitors, nor by iodoacetamide, but was inhibited by sodium bisulfite and para-hydroxymercury benzoate (PHMB). PHMB was preferred above bisulfite because it could be used at lower concentration. It proved useful when particles were to be incubated at 37 degrees C. A protease hydrolysing the same polypeptides as the liver enzyme was also detected in rat brain particles. However, its activity was much lower in this tissue and the presence of protease inhibitors was not absolutely required under the standard conditions of preparation and handling of brain particles.     

An oxidative damage-specific endonuclease from rat liver mitochondria

Reactive oxygen species have been shown to generate mutagenic lesions in DNA. One of the most abundant lesions in both nuclear and mitochondrial DNA is 7,8-dihydro-8-oxoguanine (8-oxoG). We report here the partial purification and characterization of a mitochondrial oxidative damage endonuclease (mtODE) from rat liver that recognizes and incises at 8-oxoG and abasic sites in duplex DNA. Rat liver mitochondria were purified by differential and Percoll gradient centrifugation, and mtODE was extracted from Triton X-100-solubilized mitochondria. Incision activity was measured using a radiolabeled double-stranded DNA oligonucleotide containing a unique 8-oxoG, and reaction products were separated by polyacrylamide gel electrophoresis. Gel filtration chromatography predicts mtODE's molecular mass to be between 25 and 30 kDa. mtODE has a monovalent cation optimum between 50 and 100 mM KCl and a pH optimum between 7.5 and 8. mtODE does not require any co-factors and is active in the presence of 5 mM EDTA. It is specific for 8-oxoG and preferentially incises at 8-oxoG:C base pairs. mtODE is a putative 8-oxoG glycosylase/lyase enzyme, because it can be covalently linked to the 8-oxoG oligonucleotide by sodium borohydride reduction. Comparison of mtODE's activity with other known 8-oxoG glycosylases/lyases and mitochondrial enzymes reveals that this may be a novel protein.     

Effects of alkali cations on the nuclear magnetic resonance intensity of 23Na in rat liver homogenate

Effects of alkali cations on the nuclear magnetic resonance intensity of 23Na were studied in rat liver homogenate. The loss in the resonance intensity of 23Na in the homogenate was able to be divided into two components, one of which is abolished by the addition of Cs+ ("Cs-sensitive component"), the other being insensitive to Cs+ ("Cs-insensitive component"). Both components were sensitive to guanidinium ion. In a pH range of 7.4-4.9, the Cs-sensitive component varied remarkably, but the Cs-insensitive component remained virtually unchanged. The sequence of effectiveness of alkali cations (300 mmol/kg sample) in restoring the fractional intensity of 23Na was: Cs approximately Na greater than Li approximately Rb greater than K. It was suggested that the sequences of effectiveness of alkali cations in abolishing the two components are quite different from each other. The present results were examined within the framework of a simple model. Within this framework, the results suggest that there occur, in particulate fractions, sites whose affinity for Cs+ is sufficiently lower than that for Na+.     

Pulmonary arterial lesions in Takayasu arteritis: relationship of inflammatory activity to scintigraphic findings and sequential changes

Peroxisomal proliferators (HPP), such as ciprofibrate and clofibric acid, are species-specific drugs. Since HPP-coenzyme A derivatives might be involved in their action, we studied the subcellular distribution of liver ciprofibroyl-CoA hydrolase in rat and in two HPP-unresponsive species, humans and guinea pig. Total activity was similar in the three species and was not induced by clofibric acid treatment. In guinea pig, as in humans, the enzyme is localized in the mitochondrial and soluble fractions and no changes are observed after drug treatment. In the rat, the enzyme has a microsomal localization, but upon clofibric acid treatment it changes to a mitochondrial and soluble distribution, as in unresponsive species. These results raise the possibility that drug-induced hydrolases in rats might be normally expressed in humans and guinea pigs.     

Interactions of calcium and magnesium with the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae

The activity of the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae was measured in the presence of increasing concentrations of magnesium and calcium. Calcium pyrophosphate (dissociation constant Kd = 1.9 microM) inhibited pyrophosphatase by competition with magnesium pyrophosphate (Kd = 50 microM). The small movements of calcium detected in mitochondria from yeast may be physiologically significant for the control of inorganic pyrophosphatase activity and the concentration of pyrophosphate in the matrix of yeast mitochondria.     

Change in lipid composition and lipid peroxidation rate in brain tissue under conditions of thymic hormone deficiency

Experiments on mature male rats have shown that 3 and 6 months after removal of the brain thymus, the level of total lipids in all cell centrifugate fractions (homogenate, supramitochondrial and mitochondrial fractions), which was followed by the higher rate of lipid peroxidation and the lower activity of cellular compartments, the spectrum imbalance of lipid fractions with predominant increases in the levels of phospholipids and free cholesterol. It is suggested that these changes make a contribution to the dysfunction of brain structures and participate in regulatory processes in thymectomy.     

Identification and partial characterization of a glycoprotein species with high affinity for methylmercury in peripheral nervous tissues of man and experimental animals

A small amount of a glycoprotein species (21-kDa glycoprotein) with high affinity for methylmercury (MeHg) was detected in the post-nuclear or post-mitochondrial supernatant fraction of the homogenate of rat sciatic nerve on electrophoresis and autoradiography after binding of Me203Hg to the fraction. The 21-kDa glycoprotein was also found in the subcellular fractions of mouse, hamster, guinea pig, rabbit and human peripheral nervous tissues. Experiments with the cellular fractions of the tissues revealed that the 21-kDa glycoprotein is localized mainly in the myelin fraction, whereas it was not found in the cellular fractions of brain, spinal cord and nonneural tissues, such as kidney and liver. The specific binding activity of the 21-kDa glycoprotein with MeHg was 12-15 fold that of the major myelin protein, Po. It was shown that the interaction of the 21-kDa glycoprotein with MeHg was mediated through sulfhydryl groups in experiments with iodoacetamide and dithiothreitol. The amino acid compositions of the rat and human 21-kDa glycoproteins were similar but very different from that of a typical metallothionein. The N-terminal amino acid sequences of the two components of the rat 21-kDa glycoprotein were identical to those of P0 and PMP-22, respectively. The in vitro binding of MeHg was also observed in the myelin fraction obtained from the sciatic nerves of MeHg-dosed rats.     

Inhibition of insulin degradation by nonsuppressible insulin-like activity

Nonsuppressible insulin-like activity, provided by three sources, was evaluated for its effect on the proteolytic degradation of insulin utilizing insulin protease obtained from rat liver homogenate as well as liver cell membranes. All three preparations of nonsuppressible insulin-like activity were found to be competitive inhibitors of insulin degradation. In addition human plasma was fractionated yielding an acetone precipitate which was found to have nonsuppressible insulin-like activity and to be a competitive inhibitor of insulin protease.     

Partial purification and some properties of human liver alkaline phosphatase

1. Alkaline phosphatase (EC 3.1.3.1) from human liver was solubilized from the homogenate using 0.2% Triton X-100 containing 0.2 M lithium 3,5-diiodosalicylate, and the pellet obtained was resolubilized with 20% n-butanol. The procedure resulting in 3842-fold purification included acetone fractionation, ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration, hydroxyapatite gel chromatography and further concanavalin A/Sepharose 4B affinity chromatography. 2. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 8.6, and exhibited one-seventh of the alkaline p-nitrophenylphosphatase activity in the hepatic enzyme preparation contains of the alkaline pyrophosphatase activity. 3. The highly purified enzyme was a sialic-acid containing glycoprotein. 4. Sialidase-treated hepatic enzyme clearly presented the phenomenon of delayed mobility, and the delayed enzyme fraction stained more strongly than that of non-treated hepatic alkaline phosphatase. 5. In order to investigate the role of the carbohydrate region(s) of the hepatic alkaline phosphatase molecule on substrate binding, the effect of sialidase treatment on the rate of substrate inhibition of alkaline phosphatase was studied. In the case of hepatic enzyme without sialidase, substrate inhibition of alkaline phosphatase activity was clearly shown, while in the case of the hepatic enzyme with sialidase, there was hardly any substrate inhibition in the range of 1-8 mM p-nitrophenylphosphate.     

Purification, identification and subcellular distribution of three predominant protein-tyrosine phosphatase enzymes in skeletal muscle tissue

Protein-tyrosine phosphatases (PTPases) play a key role in the regulation of insulin action. In order to identify PTPases in skeletal muscle, the major site of insulin-mediated glucose disposal in vivo, we purified PTPases from rat muscle tissue fractions by a series of column chromatographic techniques. PTPase activities were assayed by measuring the dephosphorylation of a rat insulin receptor kinase domain, derivatized lysozyme and p-nitrophenylphosphate, and the enzymes were further characterized by immunoblotting. Of the total PTPase activity in muscle homogenates, 51-64% was localized to the solubilized particulate fraction, with the specific PTPase activity 3.3-fold and 5.6-fold higher in the particulate fraction towards RCM-lysozyme or the insulin receptor, respectively. The major peak (> 75%) of PTPase activity in the particulate fraction was purified further to 700-fold; 75% of this activity passed through a Blue-3GA column and revealed immunoreactivity for both LAR and SH-PTP2. PTPase activity retained on the Blue-3GA column contained PTPase1B. The major peak (> 70%) from muscle cytosol was further purified to 1500-fold. After the Blue-3GA step, immunoblotting revealed both SH-PTP2 and PTPase1B in the cytosol fraction, but LAR was absent from this fraction. LRP (RPTP-alpha) was not detected by blotting the PTPase activities from the purified particulate or cytosol fractions. Immunodepletion studies demonstrated that LAR, SH-PTP2 and PTPase1B were quantitatively major PTPase activities in the initial muscle homogenate, together accounting for over 70% of the total activity towards RCM-lysozyme. These studies provide insight into the relative abundance and subcellular distribution of specific PTPases in muscle tissue that are involved in the regulation of reversible tyrosine phosphorylation in this tissue.