Influence of chloroquine treatment on enzymes and phospholipids from rat liver cell fractions

Rats, treated for 12 days with chloroquine show a threefold increase of arylsulfatase activity in the mitochondrial-lysosomal mixed fraction, whereas the succinate: cytochrome c reductase activity is decreased to about 50% in this fraction. Purified lysosomes possess a 35 fold higher arylsulfatase activity, compared with homogenate, whereas neither NADPH: - nor succinate: cytochrome c reductase activity can be detected. In these lysosomes, one third of the phospholipids consists of bis (monoacylglycero) phosphate. The neutral phospholipids — mainly phosphatidylethanolamine — are drastically reduced in these cell organelles during the treatment. Our results indicate that chloroquine is nearly exclusively present in the lysosomal fraction. Furthermore we conclude from our data that bis (monoacylglycero) phosphate — isolated from lysosomal phospholipids — forms complexes with chloroquine.     

In vitro insertion of the 22-kD peroxisomal membrane protein into isolated rat liver peroxisomes

The membrane insertion of the 22-kD integral peroxisomal membrane protein (PMP 22) was studied in a system in which peroxisomes isolated from rat liver were incubated with the [35S]methionine-labeled in vitro translation product of PMP 22 mRNA. Membrane insertion of PMP 22 was demonstrated by protease treatment of peroxisomes in the absence and presence of detergent. Approximately 35% of total in vitro translated PMP 22 became protease resistant after a 1-h incubation at 26 degrees C. Import was dependent on time and temperature, did not require ATP or GTP and was not inhibited by N-ethylmaleimide treatment of neither the soluble components of the translation mixture nor of the isolated peroxisomes. In contrast to these results it was recently shown that the import of the peroxisomal marker, firefly luciferase, into peroxisomes of permeabilized cells was dependent on ATP hydrolysis and was blocked by N-ethylmaleimide pretreatment of the cytosol-depleted cells (Rapp et al., 1993; Wendland and Subramani, 1993). Therefore, the present data suggest that insertion of PMP 22 into the peroxisomal membrane and translocation of firefly luciferase into peroxisomes follow distinct mechanisms. At low temperature binding of PMP 22 to the peroxisomal membrane was not influenced whereas insertion was strongly inhibited. Pretreatment of peroxisomes with subtilisin reduced binding to a low level and completely abolished insertion. Therefore it is suggested that binding is prerequisite to insertion and that insertion may be mediated by a proteinaceous receptor.     

A study of some enzymes of glycerolipid biosynthesis in rat liver after subtotal hepatectomy

1. The accumulation of triglyceride in the liver remnant after subtotal hepatectomy (removal of 82% of the liver) exceeded that described for partial hepatectomy (removal of 70% of the liver). 2. Palmitoyl-CoA synthetase, glycerol phosphate acyltransferase and diglyceride acyltransferase activities were measured in the microsomal fraction, and phosphatidate phosphohydrolase activity was measured in the particle-free supernatant fraction, prepared from the liver remnant at various times after subtotal hepatectomy. 3. The only enzyme showing a significant change in specific activity was phosphatidate phosphohydrolase. The specific activity was approximately fivefold that of the control value at 6h after operation and threefold that of the control at 10, 16 and 24h after operation. A smaller increase in the specific activity of the enzyme in sham-operated animals occurred only at 6h after operation. 4. However, at this time the total phosphohydrolase activity of the remaining liver in the sham-operated rats was approximately threefold that in hepatectomized rats. 5. Injection of actinomycin D prevented the increase in activity of phosphatidate phosphohydrolase but did not prevent the accumulation of triglyceride.     

Effects of antilipolytic agents on rat liver peroxisomes and peroxisomal oxidative activities

The mechanisms involved in the inhibitory effects of antilipolytic agents on rat liver peroxisomal fatty acid oxidative activity have been explored. Treatment of fasting rats with antilipolytic drugs (either 3,5-dimethylpyrazole (12 mg/kg body weight) or Acipimox (25 mg/kg body weight)) resulted in a decrease in free fatty acid and glucose plasma levels within 5–10 and in a significant increase in the plasma glucagon to insulin ratio within 15. Changes in the fatty acid oxidative activity appeared with a 2.5–3 h delay and were then very rapid (a 30–40% decrease in the activity occured in additional 2 h). Many peroxisomal enzyme activities (including non-β-oxidative activities such as uricase and D-amino acid oxidase) exhibited similar changes with the same delay. Simultaneously with the enzyme changes, at the electron microscope level many autophagic vacuoles were detected in the liver cells, often containing peroxisomal structures. Glutamine, an inhibitor of proteolysis in vivo, prevented the decrease in enzyme activities. It was concluded that the decrease in peroxisomal enzyme activities may be the consequence of enhanced peroxisome degradation due to the stimulation of autophagic processes in liver cells.     

Investigation of peroxisomal lipid beta-oxidation enzymes in guinea pig liver peroxisomes by immunoblotting and immunocytochemistry.

We investigated the immunoreactivity of the peroxisomal lipid beta-oxidation enzymes acyl-CoA oxidase, trifunctional protein, and thiolase in guinea pig liver and compared it with that of homologous proteins in rat, using immunoblotting of highly purified peroxisomal fractions and monospecific antibodies to rat proteins. In addition, the immunocytochemical localization of beta-oxidation enzymes in guinea pig liver was compared with that of catalase. All antibodies showed crossreactivity between the two species, indicating that these peroxisomal proteins have been well conserved, although all exhibited some differences with respect to molecular size and, in the case of acyl-CoA oxidase, in frequency of the immunoreactive bands. In the latter case, a distinct second band in the 70 KD range was observed in guinea pig, in addition to the regular band due to subunit A present in rat liver. This novel band could be due either to trihydroxycoprostanoyl-CoA oxidase or to the non-inducible branched chain fatty acid oxidase described recently. All three beta-oxidation enzymes were immunolocalized by light and electron microscopy to the matrix of peroxisomes, in contrast to catalase, which is also found in the cytoplasm and the nucleus of hepatocytes in guinea pig liver.     

Postnatal development of peroxisomal and mitochondrial enzymes in rat liver.

Subcellular organellles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks. The peroxisomal and mitochondrial fatty acid beta-oxidation and the peroxisomal and supernatant activities of catalase and glycerol-3-phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively. Fatty acid beta-oxidation in both peroxisomes and mitochondria and peroxisomal glycerol-3-phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal beta-oxidation accounted for at least 10% of the total beta-oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial beta-oxidation after two weeks of age. The greatest change in beta-oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much beta-oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol-3-phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week. Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5- to 3-fold, and all of the increased activity was in the supernatant fraction.     

Phosphoinositide synthesis and degradation in isolated rat liver peroxisomes

Analyzing peroxisomal phosphoinositide (PId(#)) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P(2) and PtdIns(3,5)P(2). PtdIns3P was hardly detected in vitro, however, was observed in vivo after [(32)P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of phosphatase inhibitors enhanced the amount of PtdIns4P. The results obtained provide evidence that isolated rat liver peroxisomes synthesize PIds and suggest the association of PId 4-kinase and PId 5-kinase and PId 4-phosphatase activities with the peroxisomal membrane.     

Effect of triton WR-1339 on peroxisomal enzymes of rat liver

The effect of Triton WR-1339 on peroxisomal enzymes of rat liver was studied. The dose vs. response relationships of peroxisomal enzyme activities to Triton WR-1339 were first examined 3.5 days after injection. Catalase activity was reduced to 50% of that of the control at a dose of 200 mg per 100 g body weight; it was found that the decrease depended on the dose of this compound. Urate oxidase activity was not significantly affected. D-Amino acid oxidase activity showed intermediate behavior. The activities of these enzymes were found to be reduced more markedly at 2 days than at 3.5 days after injection, and subsequently the levels of the activities recovered. At 2 days after injection of a dose of 200 mg per 100 g body weight, the activities of catalase, D-amino acid oxidase and urate oxidase had decreased to 40, 60 and 60%,respectively, of the control values.It was found that the decreases in the activities of these enzymes caused by Triton WR-1339 had occurred in the large granule fraction, but not in the cytoplasm.Measurement of the specific activity, Ouchterlony gel diffusion and quantitative immunoprecipitation suggested that there was a similarity between the Triton WR-1339-treated and untreated rats in the nature of purified catalases.These results suggest that Triton WR-1339 depresses the activities of liver peroxisomal enzymes, especially the catalase activity.     

Turnover of enzymes of peroxisomal β-oxidation in rat liver

Male Wistar rats were given a diet containing 2% (w/w) di-(2-ethylhexyl)-phthalate (DEHP), a peroxisomal proliferator, for 4 weeks. The activities of enzymes of peroxisomal β-oxidation and of catalase were markedly increased by the DEHP administration. The time required to reach halfway to the maximal induction for enzymes of peroxisomal β-oxidation was 5–7 days, whereas that for catalase was 3 days. A separate DEHP group was placed on the control diet after 14 days of feeding with the DEHP diet. On the withdrawal of DEHP, activities of enzymes of the β-oxidation system and of catalase decreased to the control levels with a half-life of 2–3 days. Responses of some mitochondrial enzymes involved in fatty acid oxidation are also described.     

Differential induction of peroxisomal populations in subcellular fractions of rat liver

In rat liver, peroxisome proliferators induce profound changes in the number and protein composition of peroxisomes, which upon subcellular fractionation is reflected in heterogeneity in sedimentation properties of peroxisome populations. In this study we have investigated the time course of induction of the peroxisomal proteins catalase, acyl-CoA oxidase (ACO) and the 70 kDa peroxisomal membrane protein (PMP70) in different subcellular fractions. Rats were fed a di(2-ethylhexyl)phthalate (DEHP) containing diet for 8 days and livers were removed at different time-points, fractionated by differential centrifugation into nuclear, heavy and light mitochondrial, microsomal and soluble fractions, and organelle marker enzymes were measured. Catalase was enriched mainly in the light mitochondrial and soluble fractions, while ACO was enriched in the nuclear fraction (about 30%) and in the soluble fraction. PMP70 was found in all fractions except the soluble fraction. DEHP treatment induced ACO, catalase and PMP70 activity and immunoreactive protein, but the time course and extent of induction was markedly different in the various subcellular fractions. All three proteins were induced more rapidly in the nuclear fraction than in the light mitochondrial or microsomal fractions, with catalase and PMP70 being maximally induced in the nuclear fraction already at 2 days of treatment. Refeeding a normal diet quickly normalized most parameters. These results suggest that induction of a heavy peroxisomal compartment is an early event and that induction of 'small peroxisomes', containing PMP70 and ACO, is a late event. These data are compatible with a model where peroxisomes initially proliferate by growth of a heavy, possibly reticular-like, structure rather than formation of peroxisomes by division of pre-existing organelles into small peroxisomes that subsequently grow. The various peroxisome populations that can be separated by subcellular fractionation may represent peroxisomes at different stages of biogenesis.     

Biogenesis of peroxisomes: immunocytochemical investigation of peroxisomal membrane proteins in proliferating rat liver peroxisomes and in catalase-negative membrane loops

Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554-564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment.     

Effect of thyroid hormone on peroxisomal flavin enzymes in rat liver and kidney

The effect of thyroid hormone on peroxisomal enzyme activity was studied in thyroidectomized- and T4-administered-thyroidectomized rats. In liver, the activities of isozyme A of L-alpha-hydroxyacid oxidase, D-amino acid oxidase, urate oxidase and catalase were decreased by thyroidectomy, and the diminished enzyme activities were restored by T4 administration to rats. These modifications induced by thyroidectomy or by T4 administration, however, were prominent only in immature animals (20-day-old rats). Although the changes in-alpha-hydroxyacid oxidase and D-amino acid oxidase activities, induced by thyroidectomy or by T4 administration, were also observed in 40-day-old rats, those in urate oxidase and catalase activities were not significant in 40-day-old rats. Acyl CoA oxidase activity was not affected by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. In the kidney, isozyme B of L-alpha-hydroxyacid oxidase activity was reduced by thyroidectomy and the diminished enzyme activity was restored by T4 administration in both 20- and 40-day-old rats. D-Amino acid oxidase and catalase activities in kidney, however, were not significantly modified by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. The results suggest that thyroid hormone can modify the peroxisomal enzyme activity, which is prominent in immature animals.     

Determination of the cross-points of rat liver peroxisomes, peroxisomal core and the core components by cross-partition.

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same cross-point, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, D-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2 : 1. The theoretical value of cross-point of the core calculated from from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

Bicarbonate ion-ATPase in rat liver cell fractions.

The distribution of HCO3MINUS-ATPase activity was studied in cell fractions prepared from homogenates of rat liver. The level of mitochondrial contamination in the microsomal fraction depended on the fractionation procedure and on the method of homogenization. With proper care, microsomes with undetectable mitochondrial contamination could be prepared. These microsomes had no detectable HCO3MINUS-ATPase activity. Approximately 85% of the total HCO3minus-ATPase activity of the post 6000 times g-min supernatant was recovered in the mitochondrial fraction. The properties of this mitochondrial HCO3minus-ATPase were not distinguishable from those of the various microsomal HCO3minus-ATPases previously described by other investigators.     

Enhancement of long-chain acyl-CoA hydrolase activity in peroxisomes and mitochondria of rat liver by peroxisomal proliferators

The present study has confirmed previous findings of long-chain acyl-CoA hydrolase activities in the mitochondrial and microsomal fractions of the normal rat liver. In addition, experimental evidence is presented in support of a peroxisomal localization of long-chain acyl-CoA hydrolase activity. (a) Analytical differential centrifugation of homogenates from normal rat liver revealed that this activity (using palmitoyl-CoA as the substrate) was also present in a population of particles with an average sedimentation coefficient of 6740 S, characteristic of peroxisomal marker enzymes. (b) The subcellular distribution of the hydrolase activity was greatly affected by administration of the peroxisomal proliferators clofibrate and tiadenol. The specific activity was enhanced in the mitochondrial fraction and in a population of particles with an average sedimentation coefficient of 4400 S, characteristic of peroxisomal marker enzymes. Three populations of particles containing lysosomal marker enzymes were found by analytical differential centrifugation, both in normal and clofibrate-treated rats. Our data do not support the proposal that palmitoyl-CoA hydrolase and acid phosphatase belong to the same subcellular particles. In livers from rats treated with peroxisomal proliferators, the specific activity of palmitoyl-CoA hydrolase was also enhanced in the particle-free supernatant. Evidence is presented that this activity at least in part, is related to the peroxisomal proliferation.