Immunochemical identification of hepatic protein adducts derived from estragole

Hepatic protein adducts derived from the allylbenzene food flavor estragole, which is hepatocarcinogenic when given to rodents at high doses, have been identified using immunochemical approaches. Male Fischer 344 rats were given estragole orally and hepatic protein adducts were detected by immunoblotting, using antisera raised by immunizing rabbits with 4-methoxycinnamic acid-modified rabbit serum albumin. A major 155-kDa adduct was expressed in livers of animals that had been treated with estragole at 100, 300, or 500 mg/kg. Levels of expression of the adduct increased disproportionately with respect to dose, and other adducts (170, 100, 44, and 35 kDa) were detected also in the high-dose group. Rats given estragole for 5 days, at 300 mg/kg/day, expressed predominantly 155- and 44-kDa adducts. The 155-, 100-, 44-, and 35-kDa adducts were detected in greatest abundance in liver microsomal fractions, while the 170-kDa adduct was most abundant in the nuclear fraction. Interestingly, whereas the 170-, 155-, 100-, and 35-kDa adducts were detected in cytosolic fractions, relatively low levels of the 44-kDa adduct were detected in nuclear fractions but not in cytosolic fractions. The various adducts were solubilized when microsomal fractions were extracted with sodium carbonate and were digested by trypsin. This implies that the target proteins are peripheral membrane proteins bound to the outer surface of microsomal membranes. Experiments undertaken with isolated rat hepatocytes and with V79 cells transfected with human monoamine phenol sulfotransferase cDNA revealed that adduct formation required 1'-hydroxylation of estragole, followed by sulfation. The pattern of adducts expressed when the transfected V79 cells were incubated with 1'-hydroxyestragole was very similar to that expressed in livers of estragole-treated rats. These cells should constitute a valuable in vitro model system for investigation of toxicological consequences arising from estragole-induced protein adduct formation.     

Visualization of a covalent intermediate between microsomal epoxide hydrolase, but not cholesterol epoxide hydrolase, and their substrates

Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of alpha/beta-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with [1-(14)C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude microsomes showed a single radioactive signal of the expected molecular mass that could be suppressed by inclusion of the competitive inhibitor 1,1,1-trichloropropene oxide in the incubation mixture. In a similar manner, 4-fluorochalcone-oxide-sensitive binding of epoxystearic acid to rat soluble epoxide hydrolase could be demonstrated in rat liver cytosol. Under similar conditions, no covalent binding of [26-(14)C]cholesterol-5alpha,6alpha-epoxide to microsomal proteins or solubilized fractions tenfold enriched in cholesterol epoxide hydrolase activity could be observed. Our data provide definitive proof for the formation of an enzyme-substrate-ester intermediate formed in the course of epoxide hydrolysis by microsomal epoxide hydrolase, show no formation of a covalent intermediate between cholesterol epoxide hydrolase and its substrate under the same conditions as those under which an intermediate was shown for both microsomal and soluble epoxide hydrolases and therefore indicate that the cholesterol epoxide hydrolase apparently does not act by a similar mechanism and is probably not structurally related to microsomal and soluble epoxide hydrolases.     

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.     

Phospholipid requirement of epididymal testosterone 5 alpha-reductase and phospholipid composition of epididymal microsomes

We have investigated phospholipid requirement for testosterone 5 alpha-reductase solubilized from microsomal and nuclear fractions of rat epididymis. The 5 alpha-reductase from microsomal fraction was stimulated by phosphatidylcholine (PC) with long acyl-chain lengths, but inhibited by short chain PC. The nuclear enzyme activity was weakly activated by PC with various acyl-chain lengths tested. Synthetic phosphatidylserine (PS), such as dioleoylPS, most strongly stimulated the microsomal enzyme activity, but did not exhibit any activation of the nuclear enzyme activity. Endogenous phospholipids, such as PC, PS, and phosphatidylethanolamine (PE) separated from bovine epididymal microsomes were tested for their stimulatory effects on microsomal and nuclear enzymes. Among these endogenous phospholipids, PS most greatly stimulated the microsomal 5 alpha-reductase activity, whereas both PC and PE weakly activated the enzyme activity. On the other hand, endogenous PC and PS had no ability to support the nuclear enzyme activity. The fatty acid compositions of PC and PS from bovine epididymal microsomes were determined, in order to elucidate the relationship between 5 alpha-reductase activation by these phospholipids and the structure of their acyl chains. The relative content of fatty acids in PC, in a decreasing order, was palmitate > linoleate > oleate; that in PS was stearate > oleate > palmitate. Based on these observations, the roles of microsomal PS and PC in epididymal 5 alpha-reductase reaction will be discussed.     

Influence of verapamil and digoxin on the in vitro binding of doxorubicin to the rat heart

A study has been carried out to characterize the binding of doxorubicin to heart homogenates and subcellular fractions. The technique chosen to perform the study was equilibrium dialysis and the levels of anthracycline in the samples obtained from the dialysis were assessed using HPLC. Doxorubicin has high affinity to heart homogenates and subcellular fractions such as nuclear, mitochondrial and microsomal. The binding was saturable and dose-dependent. Doxorubicin binding is decreased in the presence of digoxin and especially verapamil.     

Species heterogeneity of Gly-11 gramicidin A incorporated into sodium dodecyl sulfate micelles

Evidence is presented for species heterogeneity of the gly-11 analog of gramicidin A incorporated into sodium dodecyl sulfate (SDS) micelles. The evidence for species heterogeneity has been obtained using one-dimensional (1D) 1H NMR spectroscopy. The 1D spectra of the indole NH moiety of tryptophans 9, 13, and 15 show the presence of more than one species. It has been found that the heterogeneity is dependent upon the gly-11/SDS molar ratio. At high SDS concentration (i.e., gly-11/SDS of 3 mM/700 mM) the heterogeneity almost completely disappears. The temperature dependence of these 1H NMR signals suggests that the two species do not interconvert. The results of nuclear Overhauser effect spectroscopy NMR experiments indicate that one species is embedded within the micelle, while the other is nearer the aqueous interface. The importance of side chain interactions with the membrane environment in producing stable, solubilized species of small peptides in SDS micelles is illustrated.     

Distribution of glutathione and technetium-99m-meso-HMPAO in normal and diethyl maleate-treated mouse brain mitochondria

The aim of this study was to explain the contribution of mitochondria to the accumulation of 99mTc-meso-hexamethyl propyleneamine oxime (HMPAO) in the brain, after examinations were performed. METHODS: We studied subcellular distribution of 99mTc-meso-HMPAO and glutathione (GSH) in normal and diethyl maleate (DEM)-administered mice. RESULTS: In normal brain, major radioactivity was found in the mitochondrial (49.0%) and cytosolic fractions (33.0%), while the GSH content was high in the cytosol (63.2%) and mitochondria (30.6%). The radioactivity in mitochondrial, cytosolic, microsomal and nuclear fractions was decreased in a dose-dependent manner by DEM, a GSH depleting agent, to 32.2% (mitochondrial) and 24.7% (cytosolic) of the control by a dose of 550 mg/kg. The GSH content in mitochondrial and cytosolic fractions also decreased in a dose-dependent manner on DEM treatment to 29.3% (mitochondrial) and 30.0% (cytosolic) of the control by 550 mg/kg of DEM. A good correlation was found between the uptake of 99mTc-meso-HMPAO and GSH content in mitochondrial, cytosolic and nuclear fractions, with a correlation coefficient (r) of 0.814, 0.834 and 0.784, respectively. CONCLUSION: Mitochondria are a major subcellular fraction for the uptake of 99mTc-meso-HMPAO by the brain, and GSH in mitochondria contributes to the accumulation of 99mTc-meso-HMPAO.     

Subcellular distribution of oxygen free radical scavenging enzymes in goat ovary

Oxygen free radical scavenging enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase were heterogeneously distributed in goat ovary. Activities of SOD and catalase were predominantly located in cytosolic fractions compared to mitochondrial and microsomal fractions. Most of the peroxidase activity was observed in microsomal fractions with little activity in cytosolic and mitochondrial fractions. Higher activities of all these enzymes were in luteal phase as compared to follicular phase. Most of the activities of these enzymes in luteal phase were restricted to luteal cells while in follicular phase these were mainly present in the follicles of the ovary.     

Cell surface expression of paraneoplastic encephalomyelitis/sensory neuronopathy-associated Hu antigens in small-cell lung cancers and neuroblastomas

BACKGROUND: Serum from patients with small-cell lung cancer-associated paraneoplastic encephalomyelitis/sensory neuronopathy contains autoantibodies recognizing 35- to 40-kDa nuclear antigens present in neurons, small-cell lung cancers, and some neuroblastomas (anti-Hu). AIM: Because the mechanisms by which Hu autoantibodies may contribute to the paraneoplastic syndrome are largely unknown, we sought to examine if Hu antigens are expressed at the plasma membrane of cultured cells from Hu-expressing tumors. METHODS AND RESULTS: Hu-related molecules of 35 to 41 kDa were detected in the membrane of small-cell lung cancers and neuroblastomas using: (1) immunofluorescence, (2) absorption assays, (3) Western blotting on membrane fractions, and (4) surface biotinylation. The antibodies recognizing these membrane components were specifically absorbed by recombinant HuD protein. There was a perfect correlation between nuclear and membrane Hu expression. To determine the purity of the subcellular fractions, their reactivity with antibodies recognizing the A2 nuclear ribonucleoprotein and the cytoplasmic mitogen-activated protein kinase was examined. None of them was detected in the membrane fractions reactive with sera containing Hu antibodies. CONCLUSIONS: Hu-related antigens can be detected both in the nucleus and the membrane of small-cell lung cancer and neuroblastomas. IMPLICATIONS: These results provide an experimental basis for surface binding-mediated pathogenic mechanisms in paraneoplastic encephalomyelitis/sensory neuronopathy and in Hu-expressing tumors.     

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.     

The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane

The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation.     

Zinc and cadmium analysis in human prostate neoplasms

The objective of this study was to test the hypothesis that prostatic cancer is associated with the changes of zinc (Zn) and cadmium (Cd) concentration. Normal prostate, benign prostatic hyperplasia (BPH), and prostatic carcinoma (PCA) were analyzed for Zn and Cd by atomic absorption spectrometry. Cd level was measured using a graphite furnace and Zn level was measured by flame mode. Metal content was assessed in whole tissues and in nuclear, plasma membrane, and cytosolic fractions. An increase of Zn content in BPH, but a decrease in PCA as compared to normal tissue, was observed. Cd concentration appeared to be higher in BPH and PCA than in normal tissue. No correlation between Zn and Cd level was found in BPH specimens obtained from the same patients. Probability values of p < or = 0.05 were considered to indicate significant differences. Obtained results seem to support the hypothesis of Cd carcinogenicity and preventing function of Zn in prostatic cancer. Plasma membrane fraction corresponding to lysosomal, mitochondrial, and microsomal subcellular compartments are probably critical in Zn and Cd participation in human prostate neoplasms.     

Guinea worm disease--a chance for successful eradication in the Volta Region, Ghana

Parsley cells recognize the fungal phytopathogen Phytophthora sojae through a plasma membrane receptor. A 13 amino acid oligopeptide fragment (Pep-13) of a 42 kDa fungal cell wall glycoprotein was shown to bind to the receptor and stimulate a complex defense response in cultured parsley cells. The Pep-13 binding site solubilized from parsley microsomal membranes by non-ionic detergents exhibited the same ligand affinity and ligand specificity as the membrane-bound receptor. Chemical crosslinking and photoaffinity labeling assays with [125I]Pep-13 revealed that a monomeric 100 kDa integral plasma membrane protein is sufficient for ligand binding and may thus constitute the ligand binding domain of the receptor. Ligand affinity chromatography of solubilized microsomal membrane protein on immobilized Pep-13 yielded a 5000-fold enrichment of specific receptor activity.     

C-21- and 6-hydroxylation of progesterone by rabbit liver subcellular fractions

In vitro C-21-hydroxylation of [3H]progesterone (P) has been demonstrated for the first time with rabbit liver microsomes and mitochondria. Deoxycorticosterone (DOC) was rigorously characterized as a metabolite of both mitochondrial and microsomal metabolism, whereas 6alpha-hydroxy DOC and 6alpha-hydroxy P were only identified as microsomal metabolites. 6beta-Hydroxy metabolites were also detected but were of less quantitative significance. Formation of 6alpha-hydroxy P and 6alpha-hydroxy DOC increased steadily between 5 and 120 min of incubation with the microsomal fraction, whereas DOC increased up to 30 min of incubation and then declined. Maximal yield of DOC was 25.9 and 22.5 pmol/mg protein with the mitochondrial and microsomal fractions, respectively.     

Characterization of fatty acid synthesis by cow mammary subcellular fractions

Cellular site of fatty acid synthesis was investigated with mammary tissue from lactating cows. Cytosol, mitochondrial, and microsomal fractions were obtained by differential centrifugation and characterized by measurement of marker enzymes. Two incubation media were utilized to quantitate acetate incorporation into fatty acids by the subcellular fractions. The cytosol fraction synthesized fatty acids from 4 to 16-carbons in chain length with the pattern similar to those synthesized in vivo. No significant acetate incorporation into fatty acids was obtained with mitochondria or microsomal incubations. The malonyl-CoA pathway was the predominant pathway of fatty acid synthesis in cow mammary cytosol as evidenced by inhibitory studies with avidin.     

Subcellular distributions of lipids in cultured BHK cells: evidence for the enrichment of lysobisphosphatidic acid and neutral lipids in lysosomes

Homogenates of cultured hamster fibroblasts (BHK 21 cells) were fractionated by differential centrifugation into six main fractions: nuclear, mitochondrial, light mitochondrial, microsomal, soluble, and floating. The contents of several lipids and some marker enzymes were measured. According to the enzyme distributions, lysosomes were enriched both in the floating fraction and in the light mitochondrial fraction. Lysobisphosphatidic acid was enriched in the floating fraction more than tenfold relative to phospholipid. Cholesteryl esters and triglycerides were the main constituents of the fraction (70% of total lipids). Lysobisphosphatidic acid, triglycerides, and cholesteryl esters were enriched also in the light mitochondrial fraction. Their distribution patterns were different from those of the other lipids. Electron microscopy showed that the floating fraction contained numerous lipofuscin-like particles with darkly stained peripheries and with core regions staining like droplets of neutral lipids. Similar particles, frequently containing prominent multilamellar formations, were also common in intact cells. They contained cytochemically identified acid phosphatase. We conclude that lysobisphosphatidic acid was enriched in the lysosomes of the BHK cells and that the lysosomes also contained variable amounts of neutral lipids in the form of intralysosomal droplets.     

Effect of papain-induced emphysema on the distrubtion of pleural surface pressure

In vivo experiments using 203Pb and radioactively labelled precursors such as [14C] arginine and [3H] tryptophan were performed to identify lead binding components in rat liver. The distribution of lead in 9 tissues and the intracellular distribution in liver and kidney was also investigated. Male rats were injected intravenously with 18 mug of 203Pb/rat and the 203Pb radioactivity was measured in whole tissues as well as in nuclei, mitochondria, lysosomes, microsomes and soluble fractions obtained by centrifugation of liver and kidney homogenates. The subcellular fractions from liver were purified and fractionated into macromolecular components by ultracentrifugation, gel filtration, ion exchange chromatography and solvent extraction. Nuclei were fractionated into membranes, chromatin proteins (histone and residual non-histone proteins) and DNA. Most of the lead was detected in the nuclear membrane fraction bound exclusively to membrane proteins and absent in phospholipids. The intranuclear lead was associated with histone fractions and other basic or very weakly acid proteins as indicated by the incorporation of [14C] arginine and [3H] tryptophan. Lead was present in the chromatographically purified DNA fraction but whether lead was really bound to the nucleic acid was not determined. Mitochondria were fractionated into heavy, soluble and light subfractions representing the inner membranes, the intramitochondrial matrix and the outer membranes respectively. These subfractions contained appreciable quantities of lead. No appreciable lead was present in lipids of the mitochondrial membranes. Significant quantities of lead were associated with the endoplasmic reticulum. Fractionation of microsomes into rough and smooth membranes showed that lead was almost exclusively bound to membranes of rough-surfaced microsomes associated with the heavy rough membrane subfraction. No significant lead was present in the free polysome subfraction or in lipids from the endoplasmic reticulum. More than one lead binding site was identified in the soluble fraction, the high molecular weight components representing the most important lead binding site.     

Development and characterization of antibodies directed against the mouse D4 dopamine receptor

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H(+)-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H(+)-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H(+)-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H(+)-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H(+)-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H(+)-ATPase antiserum and with an anti-14-3-3 antiserum indicated an FC-induced association of FCBP with the PM H(+)-ATPase. Analysis of the activation state of PM H(+)-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.     

14C]7-ethoxycoumarin metabolism by precision-cut rat hepatic slices

It is recognized that iodine-123-labelled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (123I-BMIPP) slowly washes out of the myocardium. The mechanism for the washout was investigated in normal rat hearts by analyses of the subcellular distribution and lipid classes based on the BMIPP metabolism. Rat hearts were excised at 1-120 min after intravenous injection of 123I-BMIPP. After counting the radioactivity, the hearts were digested with Nagarse and homogenized, and then fractionated into the cytosolic, mitochondrial, microsomal and crude nuclear fractions by centrifugations. The radioactivity of each fraction was counted, and the lipid classes were analysed by radio-thin-layer chromatographic and high-performance liquid chromatographic methods. The heart uptake of 123I-BMIPP was maximal at 5 min (6.81%+/-0.36% ID/g), and 41% of the radioactivity disappeared within 120 min. The myocardial radioactivity was immediately distributed into the cytosolic, mitochondrial, microsomal and crude nuclear fractions. The distribution (%) of each fraction was almost identical from 5 min through 120 min. The cytosolic fraction was always the major site of radioactivity deposition (60%), and the time-activity curve of the cytosolic fraction paralleled that of the whole heart throughout the 120-min study period. In the cytosolic fraction, most of the radioactivity was incorporated into the triglyceride class, and the rest was present in the free fatty acid, phospholipid (phosphatidylcholine) and diglyceride classes. In the mitochondrial fraction, the radioactivity was mostly incorporated into the phospholipid class (phosphatidylethanolamine), followed by free fatty acids. The final metabolite of 123I-BMIPP, 123I-p-iodophenylacetic acid (123I-PIPA), initially appeared in the mitochondrial fraction as early as 1 min, and subsequently in the cytosolic fraction at 5 min. Another intermediary metabolite, 123I-p-iodophenyldodecanoic acid (123I-PIPC12), was found only in the mitochondrial fraction after 5 min. In conclusion, the slow washout kinetics of 123I-BMIPP from the myocardium mainly reflects the turnover rate of the triglyceride pool in the cytosol. The BMIPP metabolism, i.e. initial alpha-oxidation followed by subsequent cycles of beta-oxidation, was confirmed in vivo. The participation of the mitochondria in the metabolism was also proven.