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J M Tager

Publications and source records attributed to J M Tager.

At least 91 records · Page 5Linked to original sources
PubMed1988

Identification of L-pipecolate oxidase in human liver and its deficiency in the Zellweger syndrome.

The ability of human liver to oxidize L-pipecolic acid was investigated. Liver from control subjects was found to contain L-pipecolic acid oxidase, an H2O2-producing enzyme not previously demonstrated in mammals. In livers from patients with the cerebro-hepato-renal syndrome of Zellweger, a genetic disease characterized by the absence of morphologically distinguishable peroxisomes, L-pipecolic acid oxidase was found to be deficient. These results indicate that L-pipecolic acid oxidase is a peroxisomal enzyme in man and provide an explanation for the fact that elevated levels of L-pipecolic acid are found in body fluids of patients with the Zellweger syndrome.

Acyltransferases↗
PubMed1988

Direct demonstration that the deficient oxidation of very long chain fatty acids in X-linked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long chain fatty acids.

A method was developed to prepare peroxisome-enriched fractions depleted of microsomes and mitochondria from cultured skin fibroblasts. The method consists of differential centrifugation of a postnuclear supernatant followed by density gradient centrifugation on a discontinuous Metrizamide gradient. The activity of hexacosanoyl-CoA synthetase was subsequently measured in postnuclear supernatants and peroxisome-enriched fractions prepared from cultured skin fibroblasts from control subjects and patients with X-linked adrenoleukodystrophy. Whereas the hexacosanoyl-CoA synthetase activity in postnuclear supernatants of X-linked adrenoleukodystrophy fibroblasts was only slightly decreased (77.8 +/- 4.4% of control (n = 15], enzyme activity was found to be much more markedly reduced in peroxisomal fractions isolated from the mutant fibroblasts (19.6 +/- 6.7% of control (n = 5]. This is a direct demonstration that the defect in X-linked adrenoleukodystrophy is at the level of a deficient ability of peroxisomes to activate very long chain fatty acids, as first suggested by Hashmi et al. [Hashmi, M., Stanley, W. and Singh, I. (1986) FEBS Lett. 86, 247-250].

Adrenoleukodystrophy↗
PubMed1988

Kinetics of the assembly of peroxisomes after fusion of complementary cell lines from patients with the cerebro-hepato-renal (Zellweger) syndrome and related disorders.

We have recently identified four complementation groups in fibroblasts from patients deficient in peroxisomes. Here we describe a kinetic analysis of the complementation process. The kinetics of peroxisome assembly was assessed in heterokaryons of complementary cell lines by measuring the rate of incorporation of catalase, initially present in the cytosol, into particles. In two combinations of cell lines assembly was rapid and insensitive to cycloheximide. Thus the components required for peroxisome assembly must have been present in the parental cell lines, at least one of which presumably contained peroxisomal ghosts. In three other combinations of cell lines assembly of peroxisomes was slow and sensitive to cycloheximide.

Brain Diseases↗
PubMed1988

Heterogeneity in human acid beta-glucosidase revealed by cellulose-acetate electrophoresis.

Cellulose-acetate gel electrophoresis, a technique commonly used for the separation of human acid hydrolases, was applied to study heterogeneity in acid beta-glucosidase (EC 3.2.1.45). With this technique, three forms of beta-glucosidase were distinguishable in extracts of several tissues. The most anodic beta-glucosidase activity (band 3) represents the broad-specificity beta-glucosidase that is not deficient in Gaucher disease and is not inhibited by conduritol B-epoxide (CBE). The beta-glucosidase activity was deficient in Gaucher disease. A third beta-glucosidase activity with an intermediate mobility (band 2) was also inhibited by CBE and deficient in Gaucher disease. Band 1 and band 2 beta-glucosidase thus represent different forms of glucocerebrosidase. By adding phosphatidylserine and sphingolipid activator protein (SAP-2), monomeric glucocerebrosidase could be completely converted into a form that comigrated with band 2 beta-glucosidase of tissue extracts. The addition of phosphatidylserine only also resulted in a changed mobility of the monomeric enzyme, but the migration in this case differed from that of band 2 beta-glucosidase of tissue extracts. The electrophoretic profile of beta-glucosidase activity of tissue extracts changed upon ethanol/chloroform extraction: the two glucocerebrosidase forms were converted into a band with a mobility identical to that of band 1 beta-glucosidase. Our findings indicate that the interaction of glucocerebrosidase with phospholipid and SAP-2 has major effects on the mobility of the enzyme in the cellulose-acetate gel electrophoresis system. The findings with the cellulose-acetate gel electrophoretic system are discussed in relation to the heterogeneity in glucocerebrosidase observed with sucrose density gradient analysis, immunochemical methods and isoelectric focussing studies.

Electrophoresis↗
PubMed1988

Glucocerebrosidase, a lysosomal enzyme that does not undergo oligosaccharide phosphorylation.

Labelling of cultured human skin fibroblasts from either control subjects or patients with mucolipidosis II (I-cell disease) with [32P]phosphate resulted in tight association of phosphate with immunoprecipitated glucocerebrosidase, a membrane-associated lysosomal enzyme. Endoglycosidase F digestion of the immunoprecipitated glucocerebrosidase did not release labelled phosphate, suggesting that the phosphate was not associated with the oligosaccharide moiety of this glycoprotein. Purification of the enzyme from cells labelled with [32P]phosphate and [35S]methionine by an immunoaffinity chromatography procedure, which included a washing step with detergent, resulted in complete separation of the phosphate label from the peak of glucocerebrosidase activity and methionine labelling. We conclude that oligosaccharide phosphorylation, which is essential for transport of soluble lysosomal enzymes to the lysosomes in fibroblasts, does not occur in glucocerebrosidase.

Cells, Cultured↗
PubMed1988

Characterization of glucocerebrosidase in peripheral blood cells and cultured blastoid cells.

We have characterized glucocerebrosidase in various cell types of peripheral blood of control subjects and in cultured human blastoid cells. The intracellular level of glucocerebrosidase in cultured blastoid cells (10-30 nmol substrate hydrolyzed/h.mg protein) resembles closely values observed for leukocyte cell types and various tissues and is significantly lower than that observed in cultured fibroblasts (150-500 nmol substrate hydrolyzed/h.mg protein). Glucocerebrosidase is extracted from leukocyte cell types and cultured blastoid cells almost exclusively in a monomeric, nonactivated form with enzymatic properties identical to those of the tissue enzyme. In contrast, extracts of platelets are rich in an aggregated, activated form of the enzyme. Glucocerebrosidase in blood cells and cultured blastoid cells is heterogeneous with respect to Mr and pI due to a heterogeneous oligosaccharide composition of the enzyme. The different forms seen represent intermediates in the biosynthesis and maturation of the enzyme. Blastoid cells should thus be an attractive model system for studying the natural history of glucocerebrosidase in a cell type related to those cells involved in the pathology of Gaucher disease.

Cell Line↗
PubMed1988

Peroxisomal functions in classical Refsum's disease: comparison with the infantile form of Refsum's disease.

The infantile and classical forms of Refsum's disease are generally considered to belong to the newly recognized group of peroxisomal disorders. In this study we carried out a detailed investigation into different peroxisomal functions in classical Refsum's disease by analyses of plasma (very long chain fatty acids, di- and trihydroxycoprostanoic acid and pipecolic acid) and cultured skin fibroblasts from the patients (de novo plasmalogen biosynthesis, very long chain fatty acid oxidation and amount of particle-bound catalase). The results obtained indicate that, except for a deficient phytanic acid oxidation, peroxisomal functions were found to be normal in classical Refsum's disease in contrast with the findings in infantile Refsum's disease, in which there is a general impairment of peroxisomal functions. Based on these results it is concluded that peroxisomal biogenesis is normal in classical (but not in infantile) Refsum's disease and that the classical and infantile form of Refsum's disease hence represent distinct entities. Since available evidence suggests that phytanic acid is oxidized in mitochondria rather than in peroxisomes, at least in rat liver, it remains to be established whether classical Refsum's disease is a peroxisomal disorder or not.

Acyltransferases↗
PubMed1988

Peroxisomal disorders in neurology.

Although peroxisomes were initially believed to play only a minor role in mammalian metabolism, it is now clear that they catalyse essential reactions in a number of different metabolic pathways and thus play an indispensable role in intermediary metabolism. The metabolic pathways in which peroxisomes are involved include the biosynthesis of ether phospholipids and bile acids, the oxidation of very long chain fatty acids, prostaglandins and unsaturated long chain fatty acids and the catabolism of phytanate and (in man) pipecolate and glyoxylate. The importance of peroxisomes in cellular metabolism is stressed by the existence of a group of inherited diseases, the peroxisomal disorders, caused by an impairment in one or more peroxisomal functions. In the last decade our knowledge about peroxisomes and peroxisomal disorders has progressed enormously and has been the subject of several reviews. New developments include the identification of several additional peroxisomal disorders, the discovery of the primary defect in several of these peroxisomal disorders, the recognition of novel peroxisomal functions and the application of complementation analysis to obtain information on the genetic relationship between the different peroxisomal disorders. The peroxisomal disorders recognized at present comprise 12 different diseases, with neurological involvement in 10 of them. These diseases include: (1) those in which peroxisomes are virtually absent leading to a generalized impairment of peroxisomal functions (the cerebro-hepato-renal syndrome of Zellweger, neonatal adrenoleukodystrophy, infantile Refsum disease and hyperpipecolic acidaemia); (2) those in which peroxisomes are present and several peroxisomal functions are impaired (the rhizomelic form of chondrodysplasia punctata, combined peroxisomal beta-oxidation enzyme protein deficiency); and (3) those in which peroxisomes are present and only a single peroxisomal function is impaired (X-linked adrenoleukodystrophy, peroxisomal thiolase deficiency (pseudo-Zellweger syndrome), acyl-CoA oxidase deficiency (pseudo-neonatal adrenoleukodystrophy) and probably, the classic form of Refsum disease.

Animals↗
PubMed1988

Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis.

We have used complementation analysis after somatic cell fusion to investigate the genetic relationships among various genetic diseases in humans in which there is a simultaneous impairment of several peroxisomal functions. The activity of acyl-coenzyme A:dihydroxyacetonephosphate acyltransferase, which is deficient in these diseases, was used as an index of complementation. In some of these diseases peroxisomes are deficient and catalase is present in the cytosol, so that the appearance of particle-bound catalase could be used as an index of complementation. The cell lines studied can be divided into at least five complementation groups. Group 1 is represented by a cell line from a patient with the rhizomelic form of chondrodysplasia punctata. Group 2 consists of cell lines from four patients with the Zellweger syndrome, a patient with the infantile form of Refsum disease and a patient with hyperpipecolic acidemia. Group 3 comprises one cel line from a patient with the Zellweger syndrome, group 4 one cell line from a patient with the neonatal form of adrenoleukodystrophy, and group 5 one cell line from a patient with the Zellweger syndrome. We conclude that at least five genes are required for the assembly of a functional peroxisome.

Acyltransferases↗
PubMed1988

Immunocytochemical demonstration of the lysosomal enzyme alpha-glucosidase in the brush border of human intestinal epithelial cells.

In investigations on the intracellular transport route(s) of lysosomal enzymes in polarized epithelial cells, we used immunocytochemical methods to localize lysosomal alpha-glucosidase in human small-intestinal epithelial cells. Two monoclonal antibodies which can discriminate between different biosynthetic forms of this enzyme were used. One monoclonal antibody, 43D1, which recognizes all forms of the enzyme, showed labeling of the Golgi apparatus, the lysosomes and, unexpectedly, of the brush border of the cells. Multivesicular bodies were free of label. In contrast, monoclonal antibody 43G8, which recognizes all forms except the 110,000 Da precursor of alpha-glucosidase, showed labeling of the lysosomes only. This leads us to conclude that the 110,000 Da precursor form of alpha-glucosidase is present in the Golgi apparatus and the brush border of human small-intestinal epithelial cells. Moreover, biochemical experiments show that this precursor copurifies with sucrase, a typical brush-border marker, when a partially purified microvilli fraction is prepared.

Antibodies, Monoclonal↗
PubMed1988

Aberration in de novo ether lipid biosynthesis in peroxisomal disorders.

The Zellweger syndrome is a rare inborn error of metabolism characterized by the absence of morphologically distinguishable peroxisomes. As a consequence tissues and cells from Zellweger patients contain severely reduced levels of ether phospholipids. These are replaced by diacylphospholipids while keeping the polar headgroup composition of the cellular phospholipids constant. Both peroxisomal enzymes involved in glycero-ether lipid bond formation appear to be deficient. The experiments clearly establish that peroxisomes are indispensible for ether lipid biosynthesis. The peroxisomal deficiency in de novo ether lipid biosynthesis in fibroblasts and amniotic fluid cells can be applied in diagnostic assays. The mutation can be by-passed by feeding the cells with alkylglycerol. Similar characteristics as found for plasmalogen biosynthesis in Zellweger syndrome were assessed in other diseases with a general impairment of peroxisomal functions such as infantile Refsum disease and neonatal adrenoleukodystrophy as well as in rhizomelic chondrodysplasia punctata, a disease characterized by the absence of some, but not all, peroxisomal functions. Complementation analysis after somatic cell fusion has revealed that at least three genes must be involved in the biogenesis of fully functional peroxisomes.

Glyceryl Ethers↗