PubMed Health⌕ Search

Biomedical subjects

W W Just

Publications and source records attributed to W W Just.

At least 37 records · Page 2Linked to original sources
PubMed1992

Peroxisomal protein import. In vivo evidence for a novel translocation competent compartment.

In homogeneous of isolated hepatocytes separated by Nycodenz density gradient centrifugation, two peroxisomal populations are identified that differ in buoyant density. Organelles present in a high density fraction (1.22-1.23 g/cm3) represent mature peroxisomes. Vesicles of intermediate density (1.16-1.17 g/cm3) represent mature peroxisomes. Vesicles of intermediate density (1.16-1.17 g/cm3) are present in much lower concentration and seem to play a particular role in the import and distribution of newly synthesized peroxisomal proteins. In a typical pulse-chase experiment with a 7.5 min pulse, peroxisomal acyl-CoA oxidase is first imported into the peroxisomal fraction of intermediate density. After a chase of up to 30 min, the enzyme is found in mature peroxisomes.

Acyl-CoA Oxidase↗
PubMed1992

Turnover of peroxisomal vesicles by autophagic proteolysis in cultured fibroblasts from Zellweger patients.

Previous studies have shown that in fibroblasts from patients with the Zellweger syndrome (ZS) aberrant membrane structures are present which contain peroxisomal membrane proteins (Santos, M. J. et al., Science 239, 1536-1538 (1988)). In order to characterize these structures we have performed double labeling immunoelectron microscopy experiments using antisera directed against the 69 kDa peroxisomal integral membrane protein (PMP) and lysosomal hydrolases. The results indicate that at least 80% of the structures earlier referred to as 'peroxisomal ghosts' contain lysosomal hydrolases. In addition, we have studied the effect of culture of ZS fibroblasts in the presence of 3-methyladenine, an inhibitor of autophagy, on the intracellular distribution of the 69 kDa PMP. Immunofluorescence experiments showed that in the presence of 3-methyladenine there is an increase in fluorescent spots and a change in the distribution of the spots from mainly perinuclear to randomly distributed throughout the cytoplasm. Double labeling immunoelectron microscopy revealed that after culture in the presence of 3-methyladenine the 69 kDa PMP also accumulates mainly in compartments containing lysosomal hydrolases. In one ZS cell line we found that after culture in the presence of 3-methyladenine there was also an accumulation of structures which were as small as normal microperoxisomes. We conclude that in ZS fibroblasts the 69 kDa PMP is mainly present in lysosomal compartments, presumably degradative autophagic vacuoles. Furthermore, in ZS fibroblasts peroxisomes of apparently normal morphology may be synthesized, but they are degraded by autophagic proteolysis.

Adenine↗
PubMed1991

Peroxisomes of normal morphology but deficient in 3-oxoacyl-CoA thiolase in rhizomelic chondrodysplasia punctata fibroblasts.

Rhizomelic Chondrodysplasia Punctata (RCDP) is an autosomal recessive disorder in which plasmalogen biosynthesis and phytanate catabolism are impaired. Peroxisomal structure and the intracellular localization of catalase, the 69 kDa peroxisomal integral membrane protein (PMP), and 3-oxoacyl-CoA thiolase were studied in cultured skin fibroblasts from control subjects and patients with RCDP. A punctate fluorescence pattern characteristic for peroxisomes was seen in control cells incubated with either anti-(catalase), anti-(69 kDa PMP) or anti-(3-oxoacyl-CoA thiolase). Incubation of mutant cells with anti-(catalase) or anti-(69 kDa PMP) resulted in the same pattern. However, when RCDP fibroblasts were incubated with a monoclonal anti-(3-oxoacyl-CoA thiolase) antibody no punctate fluorescence could be observed. Cryosections from control and RCDP cells were examined by electron microscopy using double immunogold labelling. RCDP fibroblasts contained structures indistinguishable from control peroxisomes, the membranes reacting with anti-(69 kDa PMP) and the matrix with anti-(catalase). However, the matrix of RCDP peroxisomes, unlike control peroxisomes, did not react with anti-(3-oxoacyl-CoA thiolase). We conclude that RCDP fibroblasts contain regularly shaped peroxisomes, comparable to control peroxisomes in number as well as in content of catalase and 69 kDa PMP. However, in RCDP peroxisomes the amount of 3-oxoacyl-CoA thiolase protein proved to be below the limit of detection.

Acetyl-CoA C-Acetyltransferase↗
PubMed1990

Rhizomelic chondrodysplasia punctata. Deficiency of 3-oxoacyl-coenzyme A thiolase in peroxisomes and impaired processing of the enzyme.

The rhizomelic form of chondrodysplasia punctata (RCDP) is a peroxisomal disorder characterized biochemically by an impairment of plasmalogen biosynthesis and phytanate catabolism. We have now found that the maturation of peroxisomal 3-oxoacyl-CoA thiolase is impaired in fibroblasts from RCDP patients. To establish the subcellular localization of the 3-oxoacyl-CoA thiolase precursor protein, cultured skin fibroblasts were fractionated on a continuous Nycodenz gradient. Only a small amount of 3-oxoacyl-CoA thiolase activity was present in the catalase-containing (peroxisomal) fractions of RCDP fibroblasts in comparison with control fibroblasts. Moreover, the amount of thiolase protein in immunoblots of the catalase-containing fractions was below the limit of detection. Finally, the beta-oxidation of [14C]palmitoyl-CoA was found to be reduced in these fractions. We conclude that the mutation in RCDP leads to a partial deficiency of 3-oxoacyl-CoA thiolase activity in the peroxisomes and, concomitantly, an impairment in the ability to convert the precursor of this protein to the mature form. The reduction of 3-oxoacyl-CoA thiolase activity results in a decrease in the rate of peroxisomal beta-oxidation of palmitoyl-CoA. However, the capacity of the peroxisomes to oxidize very-long-chain fatty acids must be sufficient to prevent excessive accumulation of these compounds in vivo.

Acetyl-CoA C-Acyltransferase↗
PubMed1989

Biochemical effects and zonal heterogeneity of peroxisome proliferation induced by perfluorocarboxylic acids in rat liver.

Rats were treated for 5 to 14 days with perfluoroacetate, perfluorobutyrate and perfluorooctanoate. Alterations in hepatic morphology with special reference to the peroxisomal compartment were investigated by light and electron microscopy following cytochemical staining of catalase activity with the alkaline 3,3'-diaminobenzidine medium. All three compounds induced hepatomegaly and peroxisome proliferation. Perfluorobutyrate and perfluorooctanoate were found to be more active than perfluoroacetate. Perfluorooctanoate-induced peroxisome proliferation was more prevalent in centrilobular than in periportal hepatocytes. Peroxisomes in centrilobular liver cells frequently were of round shape, exhibited diameters of up to 1.5 microns and were predominantly located within smooth endoplasmic reticulum-glycogen areas. In periportal cells, however, clusters of polymorphous peroxisomes ranging from 250 to 1,100 nm in diameter were observed at the periphery of smooth endoplasmic reticulum-glycogen regions. Peroxisome proliferation was accompanied by a change of peroxisomal and mitochondrial enzyme activities, in particular an increase in peroxisomal palmitoyl-CoA oxidation. Significant alterations in the concentration of peroxisomal matrix and membrane polypeptides were also noted. Within the first 2 days, perfluorooctanoate treatment exerted a strong hypolipidemic activity and both compounds perfluorooctanoate and perfluorobutyrate raised the level of hepatic free acid-soluble CoA nearly 10-fold as compared with control livers. The results suggest perfluorinated carboxylic acids to be model substances suitable to correlate biochemical and morphological parameters with the zonal heterogeneity of the peroxisomal compartment in rat liver. Due to the manifold hepatic effects, contact of humans with perfluorinated carboxylic acids or their metabolic precursors may represent a severe health risk.

Animals↗
PubMed1989

Presence of peroxisomal membrane proteins in liver and fibroblasts from patients with the Zellweger syndrome and related disorders: evidence for the existence of peroxisomal ghosts.

The presence and intracellular localization of peroxisomal integral membrane proteins (PMP) were investigated in liver and cultured skin fibroblasts from control subjects and patients with the Zellweger syndrome and related disorders in which peroxisomes are virtually absent. Immunoblotting experiments showed that 22, 36 and 69 kDa PMPs were present and were confined to the membranous fraction both in the control liver and in the livers from the Zellweger patients. The 22 and 36 kDa PMPs were present in significantly lower amounts in the patients' livers than in the control liver. A reduced amount of the 69 kDa PMP was found in liver from one Zellweger but not in liver from another. The subcellular localization in fibroblasts of catalase and the 69 kDa PMP was studied by indirect immunofluorescence. A characteristic punctate fluorescence was seen in control cells incubated with either anti-(catalase) or with anti-(69 kDa PMP). Incubation of mutant cells with anti-(catalase) resulted in a diffuse fluorescence, whereas with anti-(69 kDa PMP) fluorescent particles were visualized which, in some cell lines, were larger and fewer in number than in control cells. Cryosections of control and mutant cells were examined by electron microscopy using immunogold labeling. Control cells contained small structures consisting of a single membrane enclosing a homogeneous matrix; the membranes reacted with anti-(69 kDa PMP) and the matrix with anti-(catalase). The mutant cell lines contained spherical or ellipsoidal structures whose membranes reacted with anti-(69 kDa PMP); no labeling was observed with anti-(catalase). We conclude that peroxisomal ghosts, the membranes of which contain the 69 kDa PMP, are present in peroxisome-deficient cell lines from all complementation groups studied so far.

Cell Line↗
PubMed1987

Integral membrane polypeptides of rat liver peroxisomes: topology and response to different metabolic states.

Rats were treated with clofibrate, a hypolipidemic drug, and with thyroxine. Both drugs which are known to cause peroxisome proliferation, and a concomitant increase in peroxisomal fatty acid beta-oxidation activity in liver increased one of the major integral peroxisomal membrane polypeptides (PMPs), with apparent molecular mass of 69-kDa, six- and twofold, respectively. On the other hand hypothyroidism caused a decrease in peroxisomal fatty acid beta-oxidation activity and considerably lowered the concentration of PMP 69 in the peroxisomal membrane. Two other PMPs with apparent molecular masses of 36 and 22 kDa were not influenced by these treatments. The PMPs with apparent molecular masses of 42, 28, and 26 kDa were shown to be derived from the 69-kDa polypeptide by the activity of a yet uncharacterized endogenous protease during isolation of peroxisomes. Limited proteolysis of intact peroxisomes using proteinase K and subtilisin further substantiated that some portion of the 69-kDa polypeptide extends into the cytoplasm. The 36- and the 22-kDa polypeptides were accessible to proteolytic attack to a much lower extent and, therefore, are supposed to be rather deeply embedded within the peroxisomal membrane. It is demonstrated that peroxisomal acyl-CoA synthetase, an integral PMP extending partially into the cytoplasm, and PMP 69 are not identical polypeptides. Comparison of the peroxisomal membrane with that of mitochondria and microsomes revealed that the 69- and 22-kDa polypeptides as well as the bifunctional protein of the peroxisomal fatty acid beta-oxidation pathway were specifically located only in peroxisomes. Considerable amounts of a polypeptide cross-reacting with the antiserum against the 36-kDa polypeptide were found in mitochondria.

Animals↗
PubMed1987

Permeability of the peroxisomal membrane to cofactors of beta-oxidation. Evidence for the presence of a pore-forming protein.

Peroxisomes were purified from livers of clofibrate-treated rats. Permeability measurements on the isolated organelles revealed that peroxisomes are permeable to small solutes, including sucrose and the cofactors for fatty acid oxidation NAD+, CoA, ATP, and carnitine. The intraperoxisomal distribution volume was equal for all solutes. Peroxisomal solute uptake was rapid, not saturable and not visibly influenced by temperature. NAD+ and carnitine uptake in the solute accessible volume was not diminished by a variety of analogs and inhibitors. Subfractionation of peroxisomes and reconstitution of the subfractions into liposomes preloaded with solutes made the liposomes reconstituted with the integral membrane protein fraction, but not those reconstituted with the other subperoxisomal protein fractions, permeable to the same solutes that entered intact peroxisomes. Solute leakage from the preloaded liposomes was rapid and not visibly influenced by temperature. Leakage activity was destroyed by heat treatment of the integral membrane protein fraction and was not present in lipid extracts of the membrane. Separation of the integral membrane proteins on sucrose density gradients and reconstitution of the gradient fractions into liposomes indicated that the leakage activity was caused by a polypeptide of rather low molecular weight. The gradient distribution of leakage activity corresponded most closely to the presence of a 22- and a 28-kDa polypeptide. Our experiments indicate that the nonspecific permeability of the peroxisomal membrane to small solutes is based on the presence in the membrane of a nonselective pore-forming protein.

Adenosine Triphosphate↗
PubMed1986

In vitro synthesis of peroxisomal membrane polypeptides.

Peroxisomal membranes containing predominantly integral peroxisome membrane polypeptides were obtained from a highly purified peroxisomal fraction. Following sodium dodecylsulfate polyacrylamide gel electrophoresis three polypeptides with apparent molecular weights of 69, 36, and 22 kDa were isolated and used to raise antibodies in rabbits. Cell-free synthesis of these polypeptides was carried out in an in vitro translational system derived from rabbit reticulocytes. By subjecting peroxisomal membranes to reductive methylation [14C]-radiolabeled mature membrane polypeptides were obtained. The comparison of the three mature integral peroxisome membrane polypeptides with their corresponding in vitro synthesis products revealed no size differences indicating the lack of recognizable presequences for these peroxisomal membrane polypeptides.

Animals↗
PubMed1985

Improved isolation and purification of rat liver peroxisomes by combined rate zonal and equilibrium density centrifugation.

Two different peroxisome preparations were isolated from male rat liver by using total homogenate (TH) as the starting material for one and the light mitochondrial (L) fraction for the other. The technique worked out is based on rate zonal (RZ) centrifugation in a sucrose gradient and subsequent isopycnic centrifugation in a Nycodenz gradient. The peroxisome fraction isolated from the L fraction consisted of 97-98% peroxisomal protein with catalase activity 49-fold enriched over TH. The peroxisome preparation isolated directly from TH represented about 55% of the total liver peroxisome population and had catalase activity 43-fold enriched compared with TH. The contribution of peroxisome protein to the liver protein was calculated to be in the range 1.82-2.02%. Peroxisomes isolated from TH were considerably more heterogeneous in size than peroxisomes isolated from the L fraction. Comparison of the polypeptide patterns of both preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed some quantitative differences. Several major polypeptides were found to be exclusively located in the peroxisome membrane. These polypeptides migrated in the gel with apparent molecular masses of 69, 42.5, 36, 26, 21, and 15 kDa.

Animals↗
PubMed1983

Rat liver peroxisomes, II. Stimulation of peroxisomal fatty-acid beta-oxidation by thyroid hormones.

From liver homogenates of control and thyroxine-treated rats the light mitochondrial fractions were prepared by differential centrifugation. Mitochondria and peroxisomes of these fractions were further separated by isopycnic sucrose density gradient centrifugation. Analysis of the activities of the peroxisomal marker enzymes catalase and urate oxidase throughout the gradient exhibited completely corresponding distributions with that of peroxisomal palmitoyl-CoA oxidation. Thyroxine treatment did not change the proportion of mitochondria and peroxisomes present in the differential fractions of the homogenate. Consequently, the activities of peroxisomal beta-oxidation in the light mitochondrial fractions of control and thyroxine-treated animals were directly comparable with each other. A 2.5-fold increase in peroxisomal palmitoyl-CoA oxidation raising the activity from 0.089 to 0.220 U/g as estimated in the light mitochondrial fractions was found following treatment of male rats with daily doses of 25 micrograms L-thyroxine (s.c.) over a period of 10 days. Total hepatic peroxisomal fatty-acyl-CoA oxidation was calculated based on estimations in the light mitochondrial fractions. The difficulties encountered herein are discussed. The present result replenishes the broad spectrum of cellular effects exerted by thyroid hormones and indicates that both the mitochondrial and the peroxisomal system contribute to the well known hormone effects such as increase in fatty-acid oxidation, O2 consumption and heat production.

Acyl Coenzyme A↗
PubMed1982

Rat liver peroxisomes. I. New peroxisome population induced by thyroid hormones in the liver of male rats.

The paper presents results that a hyperthyroid state for 5 to 10 days induces a new peroxisome population in the liver of male rats. This assumption is based on the following observations: 1. Treatment of male rats for five consecutive days with 25 micrograms thyroxine per day resulted in an increase of catalase and D-amino acid oxidase activities by 32 and 61%, respectively. On the other hand, urate oxidase activity declined by 20%. Prolonged treatment for ten days raised catalase activity by 68% and lowered urate oxidase activity by 35%. 2. Morphological examinations on the light and electron microscopic level revealed that the increase in peroxisomal enzyme activities was accompanied by an augmentation of peroxisomal profiles. "Microperoxisomes" and peroxisomes lacking the electron dense core were frequently observed. 3. The peroxisomes of the livers of thyroxine-treated animals exhibited an altered centrifugal behaviour in sucrose density gradients. The mean equilibrium density of normal liver peroxisomes was 1.23, whereas peroxisomes of thyroxine-treated livers equilibrated at the density of 1.20. It is discussed that peroxisomal metabolism may contribute considerably to the late biochemical effects of thyroid hormones, that means enhancement of lipid oxidation and heat production. Up to now these late effects were attributed to a stimulated mitochondrial metabolism only.

Animals↗
PubMed1980

Lack of ornithine decarboxylase activity in isolated rat liver parenchymal cells.

Polyamines are associated with fundamental metabolic and functional steps in cell metabolism. The activity of ornithine decarboxylase, the key enzyme in polyamine metabolism, was followed during the preparation of rat liver parenchymal cells and in the isolated cells during incubation. In experiments in which ornithine decarboxylase was not induced in vivo, enzyme activity dropped to barely measurable values during the preparation. An even more drastic loss of enzyme activity was noted in livers in which ornithine decarboxylase activity was stimulated in vivo 20-40fold by previous injection of bovine growth hormone, or thioacetamide or elevated because of circadian rhythmical changes of the enzyme activity. Within the first 20 min of liver perfusion to disintegrate the tissue, ornithine decarboxylase activity decreased by up to 80%. The presence of bovine growth hormone during cell preparation cannot prevent the loss of enzyme activity. Incubation of the isolated cells for periods of up to 240 min did not restore the enzyme activity. Furthermore, incubation of the cells with bovine growth hormone did not induce ornithine decarboxylase, even though the medium was supplemented with amino acids in physiological concentrations. During normal liver perfusion and in contrast to the situation with isolated cells, there is no loss of enzyme activity but a small rise. Following pretreatment of the animals with bovine growth hormone or thioacetamide the highly stimulated activity of ornithine decarboxylase declined slowly during liver perfusion, but never dropped to values lower than normal for perfusion periods of up to 240 min. Moreover, in the intact perfused organ ornithine decarboxylase remains responsive to bovine growth hormone. The experiments demonstrate that enzymatic tissue dispersion by collagenase in particular or the preparation of isolated cells in general drastically alters the metabolic and functional state of rat liver parenchymal cells.

Animals↗