Lipid composition of liver peroxisomes isolated from untreated and clofibrate-treated mice and rats

Peroxisomes were isolated from liver tissue of control and clofibrate-treated adult male NMRI mice and Sprague-Dawley rats. Phospholipids, cholesterol, triglycerides and free fatty acids were measured in the peroxisomes. The fatty acid profiles of the phosphatidylethanolamine, the phosphatidylcholine, the triglyceride and the free fatty acid fractions were also analyzed. Phosphatidylethanolamine was the dominating phospholipid in peroxisomes from untreated animals. The fatty acid profiles of phosphatidylethanolamine, free fatty acids and triglycerides were similar for untreated mice and rats but differences between the species were observed in the pattern derived from phosphatidylcholine. Phosphatidylcholine was the most abundant phospholipid after clofibrate treatment. Clofibrate treatment caused an increase in the concentrations of phospholipids and unsaturated long-chain fatty acids and a decrease in the concentrations of triglycerides, free fatty acids, cholesterol and shorter saturated fatty acids.     

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats.

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.     

Liver peroxisomes in newborns from clofibrate-treated rats. I. A morphometric study of the recovery period.

Morphological and morphometric parameters (volume density (Vv), numerical density (NA) and mean diameter (D)) of newborn liver peroxisomes were measured throughout the first week of life in rats born to mothers treated with clofibrate (ethyl 2 p-chlorophenoxy isobutyrate) during the last five days of pregnancy. In control studies the same analyses were carried out in newborns from untreated rats. At birth (day 0), treated animals exhibited a proliferated, pleiomorphic peroxisomal population (higher Vv, NA and D, and a spread distribution of profile diameter with respect to the controls). In the subsequent two days, many peroxisomes disappeared (decrease of Vv and NA to values even lower than controls), with a persisting high pleiomorphism (no change of D and diameter distribution) in residual ones. Starting from day 3, and up to day 6, larger peroxisomes were no longer detectable in test animals, and a significant, not pleiomorphic proliferation took place (D and diameter distributions strictly comparable to the controls and progressively increasing Vv and NA). The correlation analysis validated these morphological results, from which it can be surmised that the postnatal peroxisome recovery period consists of a destructive phase followed by a proliferative one. The possible mechanism(s) of disposal of the excess of drug-induced peroxisomes are discussed.     

Distribution of albumin in normal and regenerating livers of mice

Summary The conditions affecting the immunohistochemical identification of albumin in livers of male NMRI-mice were investigated by light microscopy. In normal livers albumin is randomly distributed, revealing a pancytoplasmic nearly homogen reaction in groups of hepatocytes or single parenchymal cells. However, combined autoradiographic studies after pulse labelling with ³H-valin and perfusion experiments with human albumin indicate that this distribution is caused by albumin from blood plasma and does not reflect true protein synthesis. After perfusion of the livers followed by immunohistochemical amplification techniques which allowed to dilute the primary antibody up to 1:30,000, albumin could be detected nearly in all liver parenchymal cells as granular deposits decreasing in its density from periportal fields towards the terminal hepatic venules.In regenerating livers due to partial hepatectomy no remarkable differences in granular albumin deposits between G₁- and S-phase of the cell cycle could be detected as was demonstrated by combined immunohistochemistry and ³H-dThd-autoradiography. However, during mitosis the content of albumin was often considerably reduced.Supported by a grant from the Robert-Bosch-Foundation, Stuttgart, Federal Republic of Germany     

Liver peroxisomes in newborns from clofibrate-treated rats. II. A biochemical study of the recovery period.

The fatty-acyl-CoA beta-oxidation (FAO) and catalase activities, as well as membrane fluidity of liver peroxisomes of newborns from normal and clofibrate-treated rats were studied during the recovery period, ie, throughout the first week of postnatal life. In the test animals the enzyme activities, which are significantly higher than controls at birth return to normal levels showing a somewhat different time course with FAO rapidly decreasing to control values within three days but with catalase still higher than controls at day 6. The half-life and degradation rate (Kd) of FAO are identical to those calculated by us for the whole organelles and to those reported by others for total catalase in normal or clofibrate-treated adult animals in the presence of catalase inhibitors. Soluble catalase shows turnover values which are similar though not identical to those of FAO, while total catalase has a very long half-life and a low Kd. Peroxisomal membrane fluidity, as determined by fluorescence anisotropy of 1-anilinonaphthalene-8-sulfonate (ANS) bound to purified peroxisomal fractions is higher in tests than in controls, recovering normal values within 6 days. Our results demonstrate that liver peroxisomes of rats prenatally exposed to clofibrate return to control conditions within about 1 week. The turnover parameters of enzymes and the membrane fluidity values are discussed in terms of disposal mechanism(s) for the excess of induced peroxisomes.     

Purification and immuno-electron microscopical characterization of crystalline inclusions from plant peroxisomes

Summary This paper describes the first purification method for crystalline inclusions (cores) from plant peroxisomes and an ultrastructural characterization of these isolated cores. 5-day-old sunflower (Helianthus annuus L.) cotyledon fractions which were highly enriched in cores showed negligible activity of the matrix enzyme glycolate oxidase but high catalase activity. As proven by electron microscopy, crystalline particles were surrounded neither by matrix material nor by membranes. Their geometrical outlines and ultrastructure were identical to those of cores in tissue sections, as was their reactivity with three different polyclonal catalase antibodies in the immunogold technique. Three-dimensional reconstruction, based on the geometrical outlines and ultrastructure of sectioned isolated cores from sunflower, suggested that they were quadrangular blocks. Ultrastructural analysis revealed an even periodic arrangement of repeating units which are probably cubes with 20 nm long edges. Isolated peroxisomal cores from potato (Solanum tuberosum L.) tubers had outlines which suggested that they were even rhomboidal prisms. They showed a granular ultrastructure without any repeating units and contained catalase, demonstrated by immunogold labelling and enzyme activity measurement. The results presented here suggested the hypothesis that the structural elements in plant peroxisomal cores are made of enzymatically active catalase, although the substructure may vary from species to species.Abbreviations ACOx acyl-CoA oxidase - BSA bovine serum albumin - EDTA ethylenediamine-tetraacetate - GDH glutamate dehydrogenase - GOx glycolate oxidase - KPB potassium phosphate buffer     

Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrate-treated mice. Purification procedure and physiochemical characterization of the pure enzymes

Cytosolic epoxide hydrolase was purified from the liver of untreated and clofibrate-treated male C57Bl/6 mice. The purification procedure involves chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxyapatite, takes two days to perform and results in a 120-fold purification and approximately 35% yield of the enzyme from untreated mice. The purified enzyme is a dimer with a molecular mass of 120 kDa, a Stokes' radius of 4.2 nm, a frictional ratio of 1.0 and an isoelectric point of 5.5. The subunits behave identically upon isoelectric focusing in 8 M urea and only one band with a molecular mass of 60 kDa is seen after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The form purified from clofibrate-treated mice had very similar properties and was apparently identical to the control form as judged by amino acid analysis and peptide mapping as well. These analyses also demonstrated that the cytosolic enzyme is clearly different from microsomal epoxide hydrolase isolated from rat liver. Furthermore, Ouchterlony immunodiffusion using antibodies raised in rabbits towards the control form of cytosolic epoxide hydrolase revealed identity between the two forms of cytosolic epoxide hydrolase, but no reaction with the microsomal epoxide hydrolase was observed. These findings indicate large structural differences between the cytosolic and microsomal forms of epoxide hydrolase in the liver.     

Purification of peroxisomes and mitochondria from spinach leaf by percoll gradient centrifugation

A procedure was developed to purify simultaneously peroxisomes and mitochondria from spinach (Spinacia oleracea L.) leaf under isoosmotic and low viscosity conditions. This method involved differential centrifugation and density gradient centrifugation on four layers of Percoll. Chlorophyll-free preparations of highly intact and active organelles were obtained and cross-contamination was negligible. Both organelles were stable for several hours, even if they remained in Percoll. Purified mitochondria were able to carry out the oxidation of different substrates with excellent respiratory control and ADP:O ratios. The method described in the present work was also suitable to purify mitochondria and peroxisomes from potato (Solanum tuberosum L.) tubers.     

Five-in-One: Simultaneous isolation of multiple major liver cell types from livers of normal and NASH mice

NASH is a chronic liver disease that affects 3%–6% of individuals and requires urgent therapeutic developments. Isolating the key cell types in the liver is a necessary step towards understanding their function and roles in disease pathogenesis. However, traditional isolation methods through gradient centrifugation can only collect one or a few cell types simultaneously and pose technical difficulties when applied to NASH livers. Taking advantage of identified cell surface markers from liver single-cell RNAseq, here we established the combination of gradient centrifugation and antibody-based cell sorting techniques to isolate five key liver cell types (hepatocytes, endothelial cells, stellate cells, macrophages and other immune cells) from a single mouse liver. This method yielded high purity of each cell type from healthy and NASH livers. Our five-in-one protocol simultaneously isolates key liver cell types with high purity under normal and NASH conditions, enabling for systematic and accurate exploratory experiments such as RNA sequencing.     

Purification of plant peroxisomes in iso-osmotic metrizamide

Methods were developed for isolating highly-purified peroxisomes under iso-osmotic conditions from 3 plant parts, namely cotyledons of cotton ( Gossypium hirsutum L.) seedlings, endosperm of castor bean ( Ricinus communis L.) seedlings and leaves of mature spinach ( Spinaca oleracea L.) plants. Purification was achieved by sedimentation of the organelles into metrizamide gradients centrifuged in a vertical rotor (VTi 50). Gradients consisted of an upper transition layer (1:1 mixture of homogenizing medium and 0.25 M metrizamide), a linear 0.25–0.76 M metrizamide gradient and a 0.76 M metrizamide pad. Peroxisomes from all 3 plant parts were recovered in a major band at a density ranging from 1.24 to 1.27 g cm⁻³, which is a density range similar to that for peroxisomes isolated in sucrose gradients. The percent of the total gradient cytochrome c oxidase (mitochondria marker) activity recovered in peroxisome fractions ranged from 1.5% in endosperm to 2.8% in leaves, while a plastid marker (chlorophyll or galactosyl transferase activity) ranged from undetectable in leaf peroxisome fractions to 3.6% in endosperm peroxisome fractions. Intactness of the peroxisomes was judged to be 69%, 89% and 78% for the cotyledon, endosperm and leaf peroxisomes, respectively. Isolated peroxisomes were stable for at least 5 h in metrizamide medium. Microscopic (bright-field and transmission electron microscopy) assessments verified that the peroxisomes were morphologically intact and fractions were essentially free of contaminating organelles. Metrizamide is an excellent iso-osmotic medium for purifying peroxisomes from these plant organs and tissue.     

Isolation and characterization of peroxisomes from the liver of normal untreated rats

The classic method of Leighton et al. [(1968) J. Cell Biol. 37, 482-513] for the isolation of peroxisomes from rat liver involves the use of Triton WR-1339 which alters the biochemical properties of this organelle and requires the specialized type Beaufay-rotor which is not easily available. We have employed Metrizamide as the gradient medium and a commercial type vertical rotor to obtain highly purified and structurally well-preserved peroxisomes from normal untreated animals. The livers were homogenized in buffered 0.25 M sucrose and a slightly modified 'light mitochondrial fraction' was prepared by differential centrifugation. This was loaded on top of a linear Metrizamide gradient (1.12-1.26 g/cm3) and subjected to an integrated force of 1.252 X 10(6) X (g X min) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at the density of 1.245 g/cm3. In the isolated fraction 95% of the protein was contributed by peroxisomes, which exhibited a strong activity for cyanide-insensitive lipid beta-oxidation. The purity of fractions was also confirmed by morphometry, which revealed that 98% of isolated particles consisted of peroxisomes. The latency for catalase was about 90% indicating a high degree of peroxisomal integrity. This corresponded to the low level of extraction of catalase in 3,3'-diaminobenzidine-stained filter preparations. The entire procedure took about five hours. Highly purified and structurally well preserved peroxisomes should be useful in further elucidation of the function of this organelle and especially in studies of peroxisomal enzymes with multiple intracellular localizations.     

Cytosolic epoxide hydrolase from liver of control and clofibrate-treated mice. Structural comparison by HPLC peptide mapping

Cytosolic epoxide hydrolases purified from livers of control and clofibrate-induced male C57B1/6 mice were compared. The proteins were reduced, alkylated and cleaved with trypsin and chymotrypsin. The digests were analyzed by HPLC and no qualitative differences were observed in the peptide mapping profiles of the two types of epoxide hydrolase preparation. The amino acid compositions and N-terminal residues of selected tryptic peptides also gave identical results for the control and clofibrate-induced mice. Both intact proteins have e-amino-blocked N-termini. The two enzyme forms are concluded to have highly similar, if not identical, primary structures.Abbreviations HPLC high-performance liquid chromatography - DABITC dimethylaminoazobenzene isothiocyanate     

Polyamine metabolism in regenerating livers from normal and hypocalcemic rats

There is a marked increase in the concentration of putrescine during the first ten hours following partial hepatectomy in rats. The concentration of spermidine also increases but to a smaller degree. Putrescine levels return to normal between 10 and 24 hours after the operation, whereas the increased spermidine level is maintained. The production of putrescine and spermidine appears to be initiated by the induction of ornithine decarboxylase which shows a single peak of activity at four hours after hepatectomy. The activity of S-adenosylmethionine decarboxylase shows little change following hepatectomy. The changes in polyamine levels and the activities of the enzymes of polyamine metabolism are not affected by thyroparathyroidectomy 72 hours prior to hepatectomy. Thus although these hypocalcemic conditions considerably reduce and delay DNA synthesis and mitosis, the prereplicative changes in polyamine metabolism still occur. These data suggest that the hepatocytes in hypocalcemic animals have become activated and moved to an advanced stage of prereplicative development before being blocked.     

Purification and properties of acyl/alkyl dihydroxyacetone-phosphate reductase from guinea pig liver peroxisomes

The peroxisomal acyl/alkyl dihydroxyacetone-phosphate reductase (EC 1.1.1.101) was solubilized and purified 5500-fold from guinea pig liver. The enzyme could be solubilized by detergents only at high ionic strengths in presence of the cosubstrate NADPH. Peroxisomes, isolated from liver by a Nycodenz step density gradient centrifugation, were first treated with 0.2% Triton X-100 to remove the soluble and a large fraction of the membrane-bound proteins. The enzyme was solubilized from the resulting residue by 0.05% Triton X-100, 1 M KCl, 0.3 mM NADPH, and 2 mM dithiothreitol in Tris-HCl buffer (10 mM) at pH 7.5. The enzyme was further purified after precipitating it by dialyzing out the KCl and then resolubilized with 0.8% octyl glucoside in 1 M KCl (plus NADPH and dithiothreitol). The second solubilized enzyme was purified to homogeneity (370-fold from peroxisomes) by gel filtration in a Sepharose CL-6B column followed by affinity chromatography on an NADPH-agarose gel matrix. NADPH-agarose was prepared by reacting periodate-oxidized NADP+ to adipic acid dihydrazide-agarose and then reducing the immobilized NADP+ with NaBH4. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed a single homogeneous band with an apparent molecular weight of 60,000. The molecular weight of the native enzyme was estimated to be 75,000 by size exclusion chromatography. Amino acid analysis of the purified protein showed that hydrophobic amino acid comprised 27% of the molecule. The Km value of the purified enzyme for hexadecyldihydroxyacetone phosphate (DHAP) was 21 microM, and the Vmax value in the presence of 0.07 mM NADPH was 67 mumol/min/mg. The turnover number (Kcat), after correcting for the isotope effect of the cosubstrate NADP3H, was calculated to be 6,000 mol/min/mol of enzyme, assuming the enzyme has a molecular weight of 60,000. The purified enzyme also used palmitoyldihydroxyactone phosphate as a substrate (Km = 15.4 microM, and Vmax = 75 mumol/min/mg). Palmitoyl-DHAP competitively inhibited the reduction of hexadecyl-DHAP, indicating that the same enzyme catalyzes the reduction of both acyl-DHAP and alkyl-DHAP. NADH can substitute for NADPH, but the Km of the enzyme for NADH (1.7 mM) is much higher than that for NADPH (20 microM). The purified enzyme is competitively (against NADPH) inhibited by NADP+ and palmitoyl-CoA. The enzyme is stable on storage at 4 degrees C in the presence of NADPH and dithiothreitol.     

Selenium content of livers from sex-linked dwarf and normal broiler breeders

Plasma concentrations of cGH, T₃, and T₄ were not different between dwarf and normal broiler breeders. Normal hens had a liver selenium content of 710±35 ng/g, and dwarf hens 656 ±nine ng/g (n=8). Following injections into a wing vein of different doses (1.5, 3, 6, 12, and 24 μg/kg) of the hypothalamic hormone TRH, GH was increased after 15 min. This effect seemed to last longer in dwarf chickens. Plasma concentrations of T₃ increased significantly 1 h after TRH in normal hens, but TRH was ineffective in raising T₃ levels in dwarf animals. The selenium content of livers obtained following decapitation after 2 h was also increased in normal hens up to 902±42 ng/g using the highest dose of TRH (24 μg/kg). This seemed not to be the case for dwarf animals. A much smaller. number of hepatic cGH receptors was also found in dwarf hens, whereas the affinity of the hepatic GH receptor was not influenced by the genotype. It is concluded that the sex-linked dwarf hens are unable to increase their hepatic T₄ into T₃ conversion following a TRH challenge probably because of a deficiency in hepatic GH receptors. The lower content of selenium in dwarfs and their inability to increase its uptake after TRH seem therefore to support the hypothesis that selenium has a direct role in the activity of the 5′-deiodinase complex.