Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes

Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin- KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes.     

Ascorbate-Fe2+ lipid-peroxidation of rat liver microsomes: Effect of vitamin E and cytosolic proteins

In the present study, we examined the effect of the intraperitoneal administration of vitamin E (100 mg/kg weight/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of rat liver microsomes . We also analyzed the effect of hepatic cytosolic proteins on this process. The results indicate that the ascorbate induced light emission was 76% lower in microsomes (1 mg protein) obtained from vitamin E treated animals when compared with controls. In the presence of cytosolic protein (1 mg) the chemiluminescence of control microsomes diminished 55.8 and 59.5% when cytosol from controls and treated animals was used, respectively. The chemiluminescence of vitamin E microsomes diminished 25.03 and 22.08% when both types of cytosol were added to the medium. Dialyzed or treated at 70°C cytosol was also able to inhibit the lipid peroxidation of either control or vitamin E rat liver microsomes. By means of gas chromatography we analyzed the fatty acid composition of native and peroxidated microsomes from both animal groups. The peroxidation affected principally arachidonic acid and its diminution was more evident in the control microsomes than in the microsomes from the vitamin E treated group. By HPLC we analyzed the vitamin E content in all subcellular fractions employed. In microsomes from the vitamin E-group, the content of vitamin was 11 times higher than in the control ones (0.678 ± 0.1038 vs. 0.062 ± 0.0045 g -tocopherol/mg protein, respectively), while levels in the cytosol from the vitamin E-group were only 2 times higher than in the control cytosol (0.057 ± 0.0051 vs. 0.025 ± 0.0015 g -tocopherol/mg protein, respectively).     

Zonal changes in the rat liver following an acute dose of phenobarbitone: An ultrastructural,morphometric and biochemical correlation

Alterations in the liver of rats 6 h after a dose of phenobarbitone have been studied by subcellular fractionation, conventional electron microscopy and morphometric analysis. The area immediately surrounding the central vein was the only area to undergo any alterations. There was a morphometrically measurable but not observable cellular hypertrophy of 71% whilst the hepatocyte complement of rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) was increased by 72% and 93% respectively. The increases in RER and SER were not apparent by observation and it is assumed that they have been diluted by the cell hypertrophy to 1% and 22% which must be below the threshold for detection by subjective observation. Following subcellular fractionation and measurement of microsomal protein, there was no significant difference in the level of microsomes isolated from control or treated rats. Therefore, the morphometrically measured increase in RER and SER would appear to be restricted to a relatively small population of hepatocytes adjacent to the central vein. Such an increase would represent only a small percentage of total microsomes in a homogenate and would almost certainly be masked by variation in animals and techniques. Disruption of RER was also observed in hepatocytes that would proliferate their SER should phenobarbitone treatment have been continued. Therefore this RER disruption would seem in no way to interfere with the process of membrane and enzyme synthesis.     

Amino acid composition of rat and human liver microsomes in normal and pathological conditions

The amino acid composition of proteins from liver microsomes has been studied in rats and in human subjects with normal liver, with obstructive jaundice or liver cirrhosis. The pattern of the amino acid composition of microsomes appeared to be species-specific. Phenylalanine, threonine, serine, proline, histidine and [aspartic acid plus asparagine] were increased, while alanine, tyrosine, glycine and arginine were decreased in the human compared to the rat microsomes. In patients with obstructive jaundice of short duration (less than two months) only a slight decrease in leucine and phenylalanine could be noticed, while in the case of liver cirrhosis amino acid composition was markedly changed.     

Stereoselective metabolism of benalaxyl in liver microsomes from rat and rabbit

Benalaxyl (BX), methyl‐N‐phenylacetyl‐N‐2,6‐xylyl alaninate, is a potent acylanilide fungicide and consist of a pair of enantiomers. The stereoselective metabolism of BX was investigated in rat and rabbit microsomes in vitro. The degradation kinetics and the enantiomer fraction (EF) were determined using normal high‐performance liquid chromatography with diode array detection and a cellulose‐tris‐(3,5‐dimethylphenylcarbamate)‐based chiral stationary phase (CDMPC‐CSP). The t_1/2 of (?)‐R‐BX and (+)‐S‐BX in rat liver microsomes were 22.35 and 10.66 min of rac‐BX and 5.42 and 4.03 of BX enantiomers. However, the t_1/2 of (?)‐R‐BX and (+)‐S‐BX in rabbit liver microsomes were 11.75 and 15.26 min of rac‐BX and 5.66 and 9.63 of BX enantiomers. The consequence was consistent with the stereoselective toxicokinetics of BX in vitro. There was no chiral inversion from the (?)‐R‐BX to (+)‐S‐BX or inversion from (+)‐S‐BX to (?)‐R‐BX in both rabbit and rat microsomes. These results suggested metabolism of BX enantiomers was stereoselective in rat and rabbit liver microsomes. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane

Summary The mechanism of steroid uptake by the cell remains controversial. [³H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (K _d = 33 ± 4 nm, B _max = 32 ± 2 pmol/mg). [³H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.This work was supported by grants PB85-0461 from the Comisión Asesora de Investigatión Científica y Técnica and PGV-8612 from the Departamento de Educatión, Universidades e Investigation del Gobierno Vasco. We thank Roussel-Uclaf (France) for the nonradioactive RU-steroids kindly provided.     

Zonal changes in the rat liver after chronic administration of phenobarbitone in ultrastructural, morphometric and biochemical correlation.

Alterations in the liver of rats subjected to 24 days of continuous administration of phenobarbitone have been supplied bu subcellular fractionation, conventional electron microscopy and morphometric analysis. The increase in wet weight of the liver was found to result from a combination of cellular hypertrophy, hyperplasia and an enlarged hepatic blood space. In the centrilobular zone all the hepatocytes underwent a substantial proliferation of total ER, became enlarged and had an increased blood supply. However, in the periportal zone phenobarbitone caused changes in only 45% of the hepatocytes, the remainder being apparently resistent or tardy. An overall dramatic increase in hepatic RER was both measured and observed but the response involved hepatocytes in which the RER had proliferated as well as those which were depleted of RER or had stacks and cisternae that were severely shortened and dispersed. These alterations are discussed in relation to changes in RER after administration of agents causing hepatonecrosis. Possible reasons for the inability of other workers to detect a phenobarbitone-induced increase in RER are also put forward. After subcellular fractionation and corection for centrifugation losses into the 9500 g pellet, using the microsomal marker cytochrome P-450, phenobarbitone-induced increase in total ER was substantially less than that found by morphometric analysis. This indicates that during the preparation of microsomes a substantial proportion of intracellular membranes, having different metabolic and synthetic properties to those finally isolated, are discarded and emphasizes the need to exercise care when using microsomal preparations.     

Membrane integrity and phospholipid movement influence the base exchange reaction in rat liver microsomesý

Properties of Ca2+-stimulated incorporation of amincalcohols, serine and ethanolamine, into phospholipids, and factors regulating the reaction were studied in endoplasmic reticulum membranes isolated from rat liver. In contrast to apparent Km values for either aminoalcohol, maximal velocities of the reaction were significantly affected by Ca2+ concentration. No competition between these two soluble substrates used at equimolar concentrations close to their Km values was observed, suggesting the existence of two distinct phospholipid base exchange activities. The enzyme utilizing the electrically neutral serine was not sensitive to changes of membrane potential evoked by valinomycin in the presence of KCI. On the other hand, when positively charged ethanolamine served as a substrate, the enzyme activity was inhibited by 140 mM KCI and this effect was reversed by valinomycin. The rates of inhibition of phospholipid base exchange reactions by various thiol group modifying reagents were al so found to differ. Cd2+ and lipophylic p-chloromercuribenzoic acid at micromolar concentrations were most effective. It can be suggested that -SH groups located within the hydrophobic core of the enzymes molecules are essential for the recognition of membrane substrates. However, the influence of the -SH group modifying reagents on the protein-facilitated phospholipid motion across endoplasmic reticulum membranes can not be excluded, since an integral protein-mediated transverse movement of phospholipids within the membrane bilayer and Ca2+-mediated changes in configuration of the phospholipid polar head groups seem to be a regulatory step of the reaction. Indeed, when the membrane integrity was disordered by detergents or an organic solvent, the reaction was inhibited, although not due to the transport of its water-soluble substrates is affected, but due to modulation of physical state of the membrane bilayer and, in consequence, the accessibility of phospholipid molecules.     

Vitellogenin precursors in the liver of the oviparous lizard,Podarcis sicula

In reptiles, as in the other oviparous vertebrates, vitellogenin (VTG) synthesis is stimulated in the liver by ovarian estrogens. In this article, the presence of VTG precursors was detected in liver subcellular fractions of the oviparous lizard, Podarcis sicula, in the reproductive period. The rough endoplasmic reticulum (RER) and the smooth microsomal fraction (SMF), which includes smooth endoplasmic reticulum and Golgi complex, were separated by means of two different sucrose gradients. The successful separation was controlled at the electron microscope. The contents of the different compartments were extracted by means of n-octyl-beta-D-glucopiranoside detergent and subjected to SDS-PAGE. Western Blotting with homologous anti/VTG antibody revealed two immunoreactive proteins of about 84 and 70 kDa in the RER, and four proteins of about 180, 150, 60, 50 kDa in the SMF; all these proteins appeared phosphorylated and glycosylated. The differences in the molecular weight of these VTG precursors are discussed.     

Studies on spice principles as antioxidants in the inhibition of lipid peroxidation of rat liver microsomes

Polyunsaturated fatty acids (PUFA) are vulnerable to peroxidative attack. Protecting PUFA from peroxidation is essential to utilize their beneficial effects in health and in preventing disease. The antioxidants vitamin E, t-butylhydroxy toluene (BHT) and t-butylhydroxy anisole (BHA) inhibited ascorbate/Fe²⁺-induced lipid peroxidation in rat liver microsomes. In addition, a number of spice principles, for example, curcumin (5–50 µM) from turmeric, eugenol (25–150 µM) from cloves and capsaicin (25–150 µM) from red chillies inhibited lipid peroxidation in a dose-dependent manner. Zingerone from ginger inhibited lipid peroxidation at high concentrations (> 150 µM) whereas linalool (coriander), piperine (black pepper) and cuminaldehyde (cumin) had only marginal inhibitory effects even at high concentrations (600 µM). The inhibition of lipid peroxidation by curcumin and eugenol was reversed by adding high concentrations of Fe²⁺.     

Rat hepatoma 5123TC albumin mRNA directs the synthesis of pre-proalbumin identical to rat liver pre-proalbumin.

Effect of hemin, mild periodate oxidation and concanavalin A (Con A) on $\text{in vitro}$ biosynthesis of membrane proteins and hemoglobin, in the rabbit reticulocyte, was examined. Whereas addition of hemin to the incubation medium stimulates synthesis of both hemoglobin and membrane proteins, addition of Con A, at concentrations which agglutinate cells, selectively stimulates membrane protein biosynthesis. Mild periodate treatment of cells inhibits synthesis of hemoglobin and membrane proteins; this inhibition is not related to oxidation of a membrane component since hemoglobin synthesis in a cell free lysate of treated cells is similarily inhibited.     

Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites

Two proteins (ribophorins I and II), which are integral components of rough microsomal membranes and appear to be related to the bound ribosomes, were shown to be exposed on the surface of rat liver rough microsomes (RM) and to be in close proximity to the bound ribosomes. Both proteins were labeled when intact RM were incubated with a lactoperoxidase iodinating system, but only ribophorin I was digested during mild trypsinization of intact RM. Ribophorin II (63,000 daltons) was only proteolyzed when the luminal face of the microsomal vesicles was made accessible to trypsin by the addition of sublytical detergent concentrations. Only 30--40% of the bound ribosomes were released during trypsinization on intact RM, but ribosome release was almost complete in the presence of low detergent concentrations. Very low glutaraldehyde concentrations (0.005--0.02%) led to the preferential cross-linking of large ribosomal subunits of bound ribosomes to the microsomal membranes. This cross-linking prevented the release of subunits caused by puromycin in media of high ionic strength, but not the incorporation of [3H]puromycin into nascent polypeptide chains. SDS-acrylamide gel electrophoresis of cross-linked samples a preferential reduction in the intensity of the bands representing the ribophorins and the formation of aggregates which did not penetrate into the gels. At low methyl-4-mercaptobutyrimidate (MMB) concentrations (0.26 mg/ml) only 30% of the ribosomes were cross-linked to the microsomal membranes, as shown by the puromycin-KCl test, but membranes could still be solubilized with 1% DOC. This allowed the isolation of the ribophorins together with the sedimentable ribosomes, as was shown by electrophoresis of the sediments after disruption of the cross-links by reduction. Experiments with RM which contained only inactive ribosomes showed that the presence of nascent chains was not necessary for the reversible cross-linking of ribosomes to the membranes. These observations suggest that ribophorins are in close proximity to the bound ribosomes, as may be expected from components of the ribosome-binding sites.     

Modulation of biosynthesis of phosphatidylcholine via CDP-choline in rat liver: Influence of ethanol on the microsomal cholinephosphotransferase activity

We have studied in vitro the effects of ethanol on the different enzymes involved in the biosynthesis of phosphatidylcholine (PC) via CDP-choline. Ethanol alters neither choline kinase (CK) nor CTP:phosphocholine cytidylyltransferase (CT) activities but, at levels higher than 50 mM, it does significantly inhibit microsomal cholinephosphotransferase (CPT) activity concomitantly with an increase in the ethanol concentration. A study of the kinetics of the reaction catalysed by CPT shows that ethanol decreases V_max without altering K_m, indicating a non-competitive inhibitory effect. An analysis of the thermodependence of CPT activity in the absence of ethanol reveals a break in the Arrhenius plot and thus a straight relationship between enzyme activity and the physico-chemical state of the microsomal membrane. Incubation of microsomes in the presence of ethanol increased the transition temperature from 25.8–28.2°C. Microsomes were also incubated with n-alkanols with chain-lengths of fewer than five carbon atoms at concentrations which, according to their partition coefficients, produce equimolar levels in the membrane. Under these conditions all the alkanols caused the same inhibitory effect. All these results demonstrate that ethanol modulate the PC biosynthesis at the level of CPT activity and does not affect the CT enzyme. The inhibition found on CPT is clearly dependent on the alteration produced by ethanol on the hepatic microsomal membrane.     

Enantioselective analyses of chloroquine and hydroxychloroquine in rat liver microsomes through chiral liquid chromatography–tandem mass spectrometry

An efficient, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) chiral analysis method was established for determination of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes. Effects of polysaccharide chiral stationary phases and basic additives on chiral separations of two analytes were discussed in detail. Amylose tris(3, 5-dimethylphenylcarbamate)-coated chiral stationary phase showed the best separation performance for them with acetonitrile-diethylamine-ethanol-diethylamine mixture (90:0.1:10:0.1, v/v/v/v) among four chiral stationary phases. Then, multiple reaction monitoring mode was selected as the data acquisition for determination of two pairs of enantiomers. The proposed LC–MS/MS chiral analysis method was validated in terms of linearity, accuracy, precision, and specificity. Good linearity with correlation coefficient over 0.998 was obtained in the concentration range of 0.05–5 μM. Limits of quantification for chloroquine and hydroxychloroquine enantiomers were 5.0 and 1.0 nM, respectively. The recoveries ranged from 81.14% to 111.09%. The intra-day and inter-day relative standard deviation were less than 6.5%. Moreover, concentrations of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes were determined through the proposed LC–MS/MS analysis method. After incubated with rat liver microsomes for 10 min, the enantiomeric factor of hydroxychloroquine decreased from 0.50 to 0.45 (p < 0.001). In brief, our developed determination method for chloroquine and hydroxychloroquine enantiomers through LC–MS/MS spectrometry showed the characteristics of high-efficiency, fast speed, and very low detection limit, and would be greatly beneficial for screening and quantitation of them in biological matrices.     

Characterization of regucalcin effect on proteolytic activity in rat liver cytosol: relation to cysteinyl-proteases

The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1–0.5 M) in the absence of Ca²⁺. This increase was not significantly altered by the presence of diisopropylfluorophsophate (DPF;2.5 mM)—although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 M) additively enhanced the dithiothreitol (DTT; 1.0 mM)—increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 M) enhanced the effect of Ca²⁺ (10 M) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca²⁺ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 M) was significantly decreased by the presence of calpastatin (24 g/ml), an inhibitor of Ca²⁺-activated neutral protease (calpain). Now, regucalcin (0.25 M) increased about 7-fold the activity ofm-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.     

Metabolism of halazepam by rat liver microsomes: stereoselective formation and N-dealkylation of 3-hydroxyhalazepam

Metabolism of halazepam [7-chloro-1,3-dihydro-5-phenyl-1-(2,2,2-trifluoroethyl)-2H-1,4-benzod iazepin- 2-one, HZ] was studied by incubation with liver microsomes prepared from untreated, phenobarbital (PB)-treated, and 3-methylcholanthrene (3MC)-treated male Sprague-Dawley rats. Metabolites of HZ were separated by normal-phase HPLC. Relative rates of HZ metabolism by liver microsomes prepared from untreated and treated rats were PB-treated much greater than untreated greater than 3MC-treated at low concentration of microsomal enzymes (0.25 mg protein per ml of incubation mixture) and PB-treated much greater than 3MC-treated approximately untreated at high concentration of microsomal enzymes (2 mg protein per ml of incubation mixture). The relative amounts of major metabolites were found to be 3-hydroxy-HZ (3-OH-HZ) greater than N-desalkylhalazepam (NDZ, also known as N-desmethyldiazepam and nordiazepam) much greater than oxazepam (OX) for all three rat liver microsomal preparations and the distribution of metabolites was independent of microsomal enzyme concentrations. Enantiomers of 3-OH-HZ were resolved by HPLC on a Chiralcel OC column (cellulose trisphenylcarbamate coated on silica gel, particle size 10 microns). 3-OH-HZ enantiomeres have racemization half-lives of approximately 150 min in pH 4, 7.5, and 10 aqueous solutions. 3-OH-HZ formed in the metabolism of HZ by liver microsomes prepared from untreated and treated rats were found to have 3R/3S enantiomer ratios of 37/63 (untreated), 55/45 (PB-treated), and 36/64 (3MC-treated), respectively. N-dealkylation of 3-OH-HZ by liver microsomes from PB-treated rats was substrate enantioselective; the 3R-enantiomer was N-dealkylated faster than 3S-enantiomer.(ABSTRACT TRUNCATED AT 250 WORDS)     

The smooth endoplasmic reticulum in neurohypophysial axons of the rat: Possible involvement in transport,storage and release of neurosecretory material

Summary The intra-axonal organization of the smooth endoplasmic reticulum was studied in the neurohypophysis of rats during and after water deprivation. Parallel to conventional electron microscopy, the material was treated with a double impregnation staining technique specifically designed to contrast the intracellular membranous system. In conventionally stained ultrathin sections from severely dehydrated rats most axons appeared to be free of membranous organelles, whereas corresponding axons treated with the double-impregnation technique generally exhibited a highly developed system of smooth endoplasmic reticulum. In axonal endings, both techniques revealed a profusion of microvesicles in intimate relationship with tubular elements of the smooth endoplasmic reticulum. In short-term (12 h) rehydrated rats, a similarly developed system of smooth endoplasmic reticulum was still observed at all axonal levels with both procedures. After 24 to 48 h of rehydration the tubules of the smooth endoplasmic reticulum exhibited, in double impregnated material, numerous dilatations which resembled the adjacent neurosecretory granules. In conventionally stained ultrathin sections, an accumulation of electron dense material occurred within tubules of the smooth endoplasmic reticulum in the more proximal axonal segments, while in the more terminal segments, which contained numerous elongated granules, membrane continuity was frequently observed between newly formed granules and the smooth endoplasmic reticulum. After 7 days of rehydration the general pattern of the axonal smooth endoplasmic reticulum was comparable to that in untreated rats. These results are discussed in the light of a suggested involvement of the axonal smooth endoplasmic reticulum in the non-granular transport of neurosecretory material in connection with (1) storage in distally formed granules, and (2) release via microvesicles. Acknowledgements: The authors wish to express their gratitude to Mrs. M. Balmefrézol for her skillful technical assistance