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Ronald J.a. Wanders - One of the best experts on this subject based on the ideXlab platform.

  • Prenatal Diagnosis of Zellweger Syndrome: Case Report
    Gynecology Obstetrics and Reproductive Medicine, 2016
    Co-Authors: Bilgin Kutukcu, Meral Topçu, Sinan Beksaç, Ronald J.a. Wanders
    Abstract:

    Zellweger syndrome (ZS) (Cerebro-Hepato-Renal syndrome) is a rare autosomal recessive disorder characterized by an absence or marked decrease in peroxisomes, resulting in profound muscular hypotonia and death in the neonatal period. The clinical presentation of ZS is dominated by craniofacial dysmorphic features, neurological abnormalities, hepatomegaly, and chondrodysplasia punctata. Prenatal diagnosis is possible by analysis of Dihydroxyacetone-Phosphate Acyltransferase (DHAPAT) activity, which catalyzes the first step in the biosynthesis of ether-phospholipids, in chorionic villi or amniotic fluid cells. We report the prenatal diagnosis of three pregnancies of a mother who had lost two children previously due to ZS.

  • Etherphospholipid biosynthesis and dihydroxyactetone-Phosphate Acyltransferase: resolution of the genomic organization of the human gnpat gene and its use in the identification of novel mutations.
    Biochemical and biophysical research communications, 2001
    Co-Authors: Rob Ofman, Shahin Lajmir, Ronald J.a. Wanders
    Abstract:

    Etherphospholipids are characterised by the occurrence of an alkyl- or alkenyl-group at the sn-1 position of the glycerol backbone. Peroxisomes play an essential role in the formation of etherphospholipids since the first two enzymes of the biosynthetic pathway are strictly peroxisomal. The function of plasmalogens is still an enigma but the recent identification of patients suffering from an isolated defect in either Dihydroxyacetone Phosphate Acyltransferase (GNPAT) or alkylDihydroxyacetone Phosphate synthase provides conclusive evidence that plasmalogens play an essential role for human survival and functioning. In this paper we report the complete genomic organisation of the GNPAT gene coding for the peroxisomal Dihydroxyacetone Phosphate Acyltransferase. The gene is located on chromosome 1q42.12-43. It spans approximately 28 kb and consists of 16 exons and 15 introns. This information was used to analyse the GNPAT gene in 12 patients with GNPAT deficiency. All patients analysed were found to have mutations in their GNPAT gene. Of the 9 different mutations found, 2 were missense mutations, 2 small deletions, 1 insertion and 3 mutations were within splice donor/acceptor-sites. Another mutation created an alternative splice donor-site causing the partial deletion of an exon. The data obtained provide conclusive evidence for the major role of GNPAT in etherphospholipid biosynthesis.

  • Defective PEX gene products correlate with the protein import, biochemical abnormalities, and phenotypic heterogeneity in peroxisome biogenesis disorders
    Journal of medical genetics, 1999
    Co-Authors: Nobuyuki Shimozawa, Ronald J.a. Wanders, Yasuyuki Suzuki, Tadao Orii, Yukio Fujiki, Atsushi Imamura, Zhongyi Zhang, Toshiro Tsukamoto, Takashi Osumi, Guy T N Besley
    Abstract:

    Peroxisome biogenesis disorders (PBD) comprise three phenotypes including Zellweger syndrome (ZS) (the most severe), neonatal adrenoleucodystrophy, and infantile Refsum disease (IRD) (the most mild), and can be classified into at least 12 genetic complementation groups, which are not predictive of the phenotypes. Several pathogenic genes for PBD groups have been identified, but the relationship between the defective gene products and phenotypic heterogeneity has remained unclear. We identified a mutation in the PEX2 gene in an IRD patient with compound heterozygosity for a missense mutation and the known nonsense mutation detected in ZS patients. In transfection experiments using the peroxisome deficient CHO mutant, Z65 with a nonsense mutation in the PEX2 gene, we noted the E55K mutation had mosaic activities of peroxisomal protein import machinery and residual activities of peroxisomal functions, including Dihydroxyacetone Phosphate Acyltransferase and β oxidation of very long chain fatty acids. The nonsense mutation severely affects these peroxisomal functions as well as the protein import. These data suggest that allelic heterogeneity of the PEX gene affects the peroxisomal protein import and functions and regulates the clinical severity in PBD.

  • identification and characterization of the mouse cdna encoding acyl coa Dihydroxyacetone Phosphate Acyltransferase
    Biochimica et Biophysica Acta, 1999
    Co-Authors: Rob Ofman, Eveline M Hogenhout, Ronald J.a. Wanders
    Abstract:

    Abstract We used the amino acid sequence of human acyl-CoA:Dihydroxyacetone Phosphate Acyltransferase (DHAPAT) as bait to screen the database of expressed sequence tags (dbEST) and identified several partial mouse cDNA clones showing high identity. Primers were selected based on the dbEST sequences and used for amplification of this transcript from cDNA prepared from mouse skin fibroblasts. The complete nucleotide sequence was then determined and revealed an open reading frame (ORF) of 2034 bp encoding a protein consisting of 678 amino acids with a calculated molecular mass of 76 870. The deduced amino acid sequence showed high identity (80%) with that of human DHAPAT and also revealed a typical peroxisomal targeting signal type 1 (PTS1) at its extreme carboxy-terminus (alanine-lysine-leucine, AKL). Definitive evidence that this cDNA indeed codes for DHAPAT was obtained by heterologous expression in the yeast Saccharomyces cerevisiae. Northern blot analysis revealed high expression of DHAPAT especially in mouse heart, liver and testis.

  • measurement of Dihydroxyacetone Phosphate Acyltransferase dhapat in chorionic villous samples blood cells and cultured cells
    Journal of Inherited Metabolic Disease, 1995
    Co-Authors: Ronald J.a. Wanders, G J Romeijn, Petra A W Mooijer, C Dekker, Rob Ofman, Ruud B.h. Schutgens, H. Bosch
    Abstract:

    Dihydroxyacetone-Phosphate Acyltransferase (DHAPAT) is a peroxisomal enzyme catalysing the first step in ether-phospholipid biosynthesis. DHAPAT is deficient in cells from patients suffering from a variety of peroxisomal disorders. Accurate measurement of the activity of this enzyme is of great importance, especially since it is a central parameter in the prenatal diagnosis of the disorders of peroxisome biogenesis, rhizomelic chondrodysplasia punctata and DHAPAT-deficiency. We describe a straightforward and accurate assay allowing the activity of DHAPAT to be measured reliably in chorionic villus samples, blood cells, cultured skin fibroblasts, cultured chorionic villus fibroblasts and cultured amniocytes.

Amiya K. Hajra - One of the best experts on this subject based on the ideXlab platform.

  • PDFlib PLOP: PDF Linearization, Optimization, Protection Page inserted by evaluation version www.pdflib.com –
    2016
    Co-Authors: Lipid Biosynthesis In Peroxisomes, Amiya K. Hajra, K. Das
    Abstract:

    In 1977, it was discovered that peroxisomes contain Dihydroxyacetone Phosphate Acyltransferase (DHAPAT), a lipid biosynthetic enzyme. This was surprising be-cause until then, it was thought that peroxisomes contained mainly catabolic en-zymes, especially the oxidases which catalyze the oxidation of various metabolite

  • American Journal of Medical Genetics 24:69-82 (1986) Zellweger Syndrome: Diagnostic Assays, Syndrome Delineation, and Potential Therapy
    2014
    Co-Authors: Golder N. Wilson, Ronald G. Holmes, Joseph Custer, Jeffrey L. Lipkowitz, Joan Stover, Nabanita Datta, Amiya K. Hajra
    Abstract:

    Patients with the cerebrohepatorenal syndrome of Zellweger lack peroxisomes and certain peroxisomal enzymes such as Dihydroxyacetone Phosphate Acyltransferase in their tissues. Deficiency of this enzyme, which is necessary for glycerol ether lipid synthesis, provides a biochemical method for recognizing patients with subtle manifestations of Zellweger syndrome and suggests the utility of exogenous ether lipid precursors as a therapeutic strategy for these children. We describe the results of glycerol ether lipid supplementation to two children, one with classic Zellweger syndrome and 9 % of control fibroblast Dihydroxyacetone Phosphate Acyltransferase activity, and one with mild facial manifestations, wide sutures, hypotonia, developmental delay, hepatomegaly, peripheral retinal pigmentation, and 50 % of control fibroblast Dihydroxyacetone Phosphate Acyltransferase activity. An increase in erythrocyte plasmalogen levels following therapy was clearly demonstrated in the milder patient, and neither patient showed evidence of toxicity. Evaluation of therapy by comparison to the usual clinical course of Zellweger syndrome was not helpful because of the variability and incomplete documentatio

  • Dihydroxyacetone Phosphate Acyltransferase
    Biochimica et Biophysica Acta, 1997
    Co-Authors: Amiya K. Hajra
    Abstract:

    Abstract In this article the properties, assay, distribution, subcellular localization, deficiency in congenital peroxisomal disorders, purification and physiological functions of Dihydroxyacetone Phosphate Acyltransferase (EC 2.3.1.42) are reviewed.

  • Regulation of phosphatidic acid biosynthetic enzymes in Saccharomyces cerevisiae
    1994
    Co-Authors: Stacey A. Minskoff, Amiya K. Hajra, Patricia V. Racenis, Miriam L. Greenberg
    Abstract:

    Abstract Phosphatidic acid is the biosynthetic precursor of all glycerolipids. To understand how phosphatidic acid biosynthesis is controlled in Saccharomyces cereuisiae, we studied the regulation of three enzyme activities involved in the synthesis of this glycerolipid precursor, Le., glyceroPhosphate Acyltransferase (GPAT), Dihydroxyacetone Phosphate Acyltransferase (DHAPAT), and acyl DHAP reductase. GPAT activity was increased S-fold, while DHAPAT activity was increased up to 9-fold in wild type cells grown in a nonfermentable carbon source compared to that of glucose-grown cells. The ratio of GPAT/DHAPAT activity was 12 in glucose-grown cells but only 4 in cells grown in glycerol/ethanol. In the previously characterize

  • purification of Dihydroxyacetone Phosphate Acyltransferase from guinea pig liver peroxisomes
    Archives of Biochemistry and Biophysics, 1993
    Co-Authors: Keith O Webber, Amiya K. Hajra
    Abstract:

    Abstract Dihydroxyacetone Phosphate Acyltransferase (EC 2.3.1.42), a peroxisomal enzyme which initiates the biosynthesis of glycerolipids (especially the ether-linked glycerolipids) in higher eukaryotes, has been purified by over 3250-fold from guinea pig liver. Initial stages of purification entailed isolation of liver peroxisomes by a combination of differential and density-gradient centrifugation. Dihydroxyacetone Phosphate Acyltransferase was solubilized from peroxisomal membranes with 3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate at moderate ionic strength (0.15 M NaCl). The solubilized enzyme was further purified by a regimen of size-exclusion chromatography, cation-exchange chromatography, and hydroxylapatite chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of different fractions during the purification of the enzyme, a 69-kDa protein band copurified with the enzyme activity, indicating that the monomeric enzyme may have a M r of 69,000. This was verified by further purifying the enzyme by chromatofocusing, when a single 69-kDa band was observed on SDS-PAGE. The M r of Dihydroxyacetone Phosphate Acyltransferase determined by gel filtration is 90 kDa. The V max of the purified enzyme was 4 pmol acylDihydroxyacetone Phosphate (acylDHAP) formed per minute per milligram protein and the K m (DHAP) is 70 μM when assayed at saturating concentrations of palmitoylCoA. Free coenzyme A inhibits the Acyltransferase reaction with an inhibition constant ( K i ) of approximately 0.76 mM. To date, this is the most highly purified DHAP Acyltransferase(>3200-fold) of mammalian origin.

Ruud B.h. Schutgens - One of the best experts on this subject based on the ideXlab platform.

  • measurement of Dihydroxyacetone Phosphate Acyltransferase dhapat in chorionic villous samples blood cells and cultured cells
    Journal of Inherited Metabolic Disease, 1995
    Co-Authors: Ronald J.a. Wanders, G J Romeijn, Petra A W Mooijer, C Dekker, Rob Ofman, Ruud B.h. Schutgens, H. Bosch
    Abstract:

    Dihydroxyacetone-Phosphate Acyltransferase (DHAPAT) is a peroxisomal enzyme catalysing the first step in ether-phospholipid biosynthesis. DHAPAT is deficient in cells from patients suffering from a variety of peroxisomal disorders. Accurate measurement of the activity of this enzyme is of great importance, especially since it is a central parameter in the prenatal diagnosis of the disorders of peroxisome biogenesis, rhizomelic chondrodysplasia punctata and DHAPAT-deficiency. We describe a straightforward and accurate assay allowing the activity of DHAPAT to be measured reliably in chorionic villus samples, blood cells, cultured skin fibroblasts, cultured chorionic villus fibroblasts and cultured amniocytes.

  • Rhizomelic chondrodysplasia punctata with isolated DHAP-AT deficiency.
    Archives of disease in childhood, 1993
    Co-Authors: D. G. D. Barr, Ronald J.a. Wanders, J. M. Kirk, M. Al Howasi, Ruud B.h. Schutgens
    Abstract:

    An infant with the characteristic phenotype of classical rhizomelic chondrodysplasia punctata was found to have an isolated deficiency of the peroxisomal enzyme acyl CoA Dihydroxyacetone Phosphate Acyltransferase (DHAP-AT). All other peroxisomal functions measured were found to be normal. Previously described in one other case report, this confirms the existence of another distinct form of peroxisomal disorder characterised biochemically by a deficiency in de novo plasmalogen biosynthesis only.

  • Genetic and biochemical heterogeneity in patients with the rhizomelic form of chondrodysplasia punctata — a complementation study
    Human Genetics, 1992
    Co-Authors: Judith C. Heikoop, Ronald J.a. Wanders, Ruud B.h. Schutgens, Anneke Strijland, Rally Purvis, Joseph M. Tager
    Abstract:

    The genetic relationship between 10 patients with clinical manifestations of rhizomelic chondrodysplasia punctata (RCDP) was studied by complementation analysis after somatic cell fusion. Biochemically, 9 out of the 10 patients were characterized by a partial deficiency of acyl-CoA: Dihydroxyacetone Phosphate Acyltransferase (DHAP-AT) and an impairment of plasmalogen biosynthesis, phytanate catabolism and the maturation of peroxisomal 3-oxoacyl-CoA thiolase; 3-oxoacyl-CoA thiolase was strongly reduced in the peroxisomes of these patients. Fusion of fibroblasts from these 9 patients with Zellweger fibroblasts resulted in complementation as indicated by the restoration of DHAP-AT activity, plasmalogen biosynthesis, and punctate fluorescence after staining with a monoclonal antibody to peroxisomal thiolase. No complementation was observed after fusion of different combinations of the 9 RCDP cell lines, suggesting that they belong to a single complementation group. The tenth patient was characterized biochemically by a deficiency of DHAP-AT and an impairment of plasmalogen biosynthesis. However, maturation and localization of peroxisomal thiolase were normal. Fusion of fibroblasts from this patient with fibroblasts from the other 9 patients resulted in complementation as indicated by the restoration of plasmalogen biosynthesis. We conclude that mutations in at least two different genes can lead to the clinical phenotype of RCDP.

  • Zellweger syndrome in a preterm, small for gestational age infant
    Journal of Inherited Metabolic Disease, 1992
    Co-Authors: J. F. Samsom, Ruud B.h. Schutgens, C. Jakobs, J. Klei-van Moorsel, L. M. E. Smit, Ronald J.a. Wanders
    Abstract:

    A preterm (gestational age 34 weeks), small for gestational age infant (birth weight < P _2,3) is described. Because of unexplained slightly disturbed liver function tests at age 2 months, extensive metabolic examinations were performed. Elevated blood levels of very long-chain fatty acids, pipecolic acid and abnormal levels of bile acid intermediates were detected, suggesting a peroxisomal disorder. The plasmalogen content of erythrocytes was decreased. Morphologically distinct peroxisomes were absent in the liver. In fibroblasts an accumulation of very long-chain fatty acids, decreased activity of acyl-CoA: Dihydroxyacetone Phosphate Acyltransferase and impaired de novo biosynthesis of plasmalogens was found. In summary, a mild variant of the classical cerebro-hepato-renal syndrome of Zellweger was found without the characteristic clinical facial signs.

  • Genetic and biochemical heterogeneity in patients with the rhizomelic form of chondrodysplasia punctata--a complementation study.
    Human genetics, 1992
    Co-Authors: Judith C. Heikoop, Ronald J.a. Wanders, Ruud B.h. Schutgens, Anneke Strijland, Rally Purvis, Joseph M. Tager
    Abstract:

    The genetic relationship between 10 patients with clinical manifestations of rhizomelic chondrodysplasia punctata (RCDP) was studied by complementation analysis after somatic cell fusion. Biochemically, 9 out of the 10 patients were characterized by a partial deficiency of acyl-CoA: Dihydroxyacetone Phosphate Acyltransferase (DHAP-AT) and an impairment of plasmalogen biosynthesis, phytanate catabolism and the maturation of peroxisomal 3-oxoacyl-CoA thiolase; 3-oxoacyl-CoA thiolase was strongly reduced in the peroxisomes of these patients. Fusion of fibroblasts from these 9 patients with Zellweger fibroblasts resulted in complementation as indicated by the restoration of DHAP-AT activity, plasmalogen biosynthesis, and punctate fluorescence after staining with a monoclonal antibody to peroxisomal thiolase. No complementation was observed after fusion of different combinations of the 9 RCDP cell lines, suggesting that they belong to a single complementation group. The tenth patient was characterized biochemically by a deficiency of DHAP-AT and an impairment of plasmalogen biosynthesis. However, maturation and localization of peroxisomal thiolase were normal. Fusion of fibroblasts from this patient with fibroblasts from the other 9 patients resulted in complementation as indicated by the restoration of plasmalogen biosynthesis. We conclude that mutations in at least two different genes can lead to the clinical phenotype of RCDP.

H. Bosch - One of the best experts on this subject based on the ideXlab platform.

  • Alkyl‐Dihydroxyacetone Phosphate synthase and Dihydroxyacetone Phosphate Acyltransferase form a protein complex in peroxisomes
    FEBS Journal, 1999
    Co-Authors: Jan Biermann, Wilhelm W Just, H. Bosch
    Abstract:

    Dihydroxyacetone Phosphate (GrnP) Acyltransferase and alkyl-GrnP synthase are the key enzymes involved in the biosynthesis of ether phospholipids. Both enzymes are located on the inside of the peroxisomal membrane. Here we report evidence for a direct interaction between these enzymes obtained by the use of chemical cross-linking. After cross-linking and immunoblot analysis alkyl-GrnP synthase could be detected in a 210-kDa complex which was located entirely on the lumenal side of the peroxisomal membrane. Two-dimensional SDS/PAGE demonstrated that GrnP-Acyltransferase is also cross-linked in a 210-kDa complex. Co-immunoprecipitation confirmed that the two enzymes interact, in a heterotrimeric complex. Furthermore, alkyl-GrnP synthase can form a homotrimeric complex in the absence of GrnP-Acyltransferase as was demonstrated by immunoblot analysis after cross-linking experiments with either GrnP-Acyltransferase deficient human fibroblast homogenates or recombinant (His)6-tagged alkyl-GrnP synthase. We conclude that alkyl-GrnP synthase interacts selectively with GrnP-Acyltransferase in a heterotrimeric complex and in the absence of GrnP-Acyltransferase can also form a homotrimeric complex.

  • alkyl Dihydroxyacetone Phosphate synthase and Dihydroxyacetone Phosphate Acyltransferase form a protein complex in peroxisomes
    FEBS Journal, 1999
    Co-Authors: Jan Biermann, Wilhelm W Just, H. Bosch
    Abstract:

    Dihydroxyacetone Phosphate (GrnP) Acyltransferase and alkyl-GrnP synthase are the key enzymes involved in the biosynthesis of ether phospholipids. Both enzymes are located on the inside of the peroxisomal membrane. Here we report evidence for a direct interaction between these enzymes obtained by the use of chemical cross-linking. After cross-linking and immunoblot analysis alkyl-GrnP synthase could be detected in a 210-kDa complex which was located entirely on the lumenal side of the peroxisomal membrane. Two-dimensional SDS/PAGE demonstrated that GrnP-Acyltransferase is also cross-linked in a 210-kDa complex. Co-immunoprecipitation confirmed that the two enzymes interact, in a heterotrimeric complex. Furthermore, alkyl-GrnP synthase can form a homotrimeric complex in the absence of GrnP-Acyltransferase as was demonstrated by immunoblot analysis after cross-linking experiments with either GrnP-Acyltransferase deficient human fibroblast homogenates or recombinant (His)6-tagged alkyl-GrnP synthase. We conclude that alkyl-GrnP synthase interacts selectively with GrnP-Acyltransferase in a heterotrimeric complex and in the absence of GrnP-Acyltransferase can also form a homotrimeric complex.

  • Alkyl Dihydroxyacetone Phosphate synthase in glycosomes of Trypanosoma brucei.
    Biochimica et Biophysica Acta, 1995
    Co-Authors: Anna W. M. Zomer, Frederik Opperdoes, H. Bosch
    Abstract:

    Alkyl-Dihydroxyacetone Phosphate synthase (E.C. 2.5.1.26), the key enzyme in ether phospholipid biosynthesis, was demonstrated to be present in Trypanosoma brucei. The distribution of alkyl-Dihydroxyacetone Phosphate synthase was found to be identical to that of Dihydroxyacetone Phosphate Acyltransferase (E.C. 2.3.1.42), which has previously been shown to be exclusively associated with the glycosome fraction (Opperdoes, F.R. (1984) FEBS Lett. 169, 35-39). Studies with gradient purified glycosomes indicated that the formation of alkyl-Dihydroxyacetone Phosphate was completely dependent on the presence of acyl-Dihydroxyacetone Phosphate. The glycosomal alkyl-Dihydroxyacetone Phosphate synthase activity was characterized with respect to its pH optimum, Triton X-100 sensitivity and the dependency on the concentration of the substrates palmitoyl-Dihydroxyacetone Phosphate and hexadecanol. Using thin-layer chromatographic and alkaline hydrolysis procedures the reaction product was identified as alkyl-Dihydroxyacetone Phosphate. Alkyl-Dihydroxyacetone Phosphate synthase was resistant to proteolytic inactivation by trypsin in intact glycosomes but not in Triton X-100 disrupted glycosomes. It is concluded that T. brucei glycosomes contain the enzymes responsible for glycero-ether bond formation analogous to mammalian peroxisomes.

  • measurement of Dihydroxyacetone Phosphate Acyltransferase dhapat in chorionic villous samples blood cells and cultured cells
    Journal of Inherited Metabolic Disease, 1995
    Co-Authors: Ronald J.a. Wanders, G J Romeijn, Petra A W Mooijer, C Dekker, Rob Ofman, Ruud B.h. Schutgens, H. Bosch
    Abstract:

    Dihydroxyacetone-Phosphate Acyltransferase (DHAPAT) is a peroxisomal enzyme catalysing the first step in ether-phospholipid biosynthesis. DHAPAT is deficient in cells from patients suffering from a variety of peroxisomal disorders. Accurate measurement of the activity of this enzyme is of great importance, especially since it is a central parameter in the prenatal diagnosis of the disorders of peroxisome biogenesis, rhizomelic chondrodysplasia punctata and DHAPAT-deficiency. We describe a straightforward and accurate assay allowing the activity of DHAPAT to be measured reliably in chorionic villus samples, blood cells, cultured skin fibroblasts, cultured chorionic villus fibroblasts and cultured amniocytes.

Joseph M. Tager - One of the best experts on this subject based on the ideXlab platform.

  • Genetic and biochemical heterogeneity in patients with the rhizomelic form of chondrodysplasia punctata — a complementation study
    Human Genetics, 1992
    Co-Authors: Judith C. Heikoop, Ronald J.a. Wanders, Ruud B.h. Schutgens, Anneke Strijland, Rally Purvis, Joseph M. Tager
    Abstract:

    The genetic relationship between 10 patients with clinical manifestations of rhizomelic chondrodysplasia punctata (RCDP) was studied by complementation analysis after somatic cell fusion. Biochemically, 9 out of the 10 patients were characterized by a partial deficiency of acyl-CoA: Dihydroxyacetone Phosphate Acyltransferase (DHAP-AT) and an impairment of plasmalogen biosynthesis, phytanate catabolism and the maturation of peroxisomal 3-oxoacyl-CoA thiolase; 3-oxoacyl-CoA thiolase was strongly reduced in the peroxisomes of these patients. Fusion of fibroblasts from these 9 patients with Zellweger fibroblasts resulted in complementation as indicated by the restoration of DHAP-AT activity, plasmalogen biosynthesis, and punctate fluorescence after staining with a monoclonal antibody to peroxisomal thiolase. No complementation was observed after fusion of different combinations of the 9 RCDP cell lines, suggesting that they belong to a single complementation group. The tenth patient was characterized biochemically by a deficiency of DHAP-AT and an impairment of plasmalogen biosynthesis. However, maturation and localization of peroxisomal thiolase were normal. Fusion of fibroblasts from this patient with fibroblasts from the other 9 patients resulted in complementation as indicated by the restoration of plasmalogen biosynthesis. We conclude that mutations in at least two different genes can lead to the clinical phenotype of RCDP.

  • Genetic and biochemical heterogeneity in patients with the rhizomelic form of chondrodysplasia punctata--a complementation study.
    Human genetics, 1992
    Co-Authors: Judith C. Heikoop, Ronald J.a. Wanders, Ruud B.h. Schutgens, Anneke Strijland, Rally Purvis, Joseph M. Tager
    Abstract:

    The genetic relationship between 10 patients with clinical manifestations of rhizomelic chondrodysplasia punctata (RCDP) was studied by complementation analysis after somatic cell fusion. Biochemically, 9 out of the 10 patients were characterized by a partial deficiency of acyl-CoA: Dihydroxyacetone Phosphate Acyltransferase (DHAP-AT) and an impairment of plasmalogen biosynthesis, phytanate catabolism and the maturation of peroxisomal 3-oxoacyl-CoA thiolase; 3-oxoacyl-CoA thiolase was strongly reduced in the peroxisomes of these patients. Fusion of fibroblasts from these 9 patients with Zellweger fibroblasts resulted in complementation as indicated by the restoration of DHAP-AT activity, plasmalogen biosynthesis, and punctate fluorescence after staining with a monoclonal antibody to peroxisomal thiolase. No complementation was observed after fusion of different combinations of the 9 RCDP cell lines, suggesting that they belong to a single complementation group. The tenth patient was characterized biochemically by a deficiency of DHAP-AT and an impairment of plasmalogen biosynthesis. However, maturation and localization of peroxisomal thiolase were normal. Fusion of fibroblasts from this patient with fibroblasts from the other 9 patients resulted in complementation as indicated by the restoration of plasmalogen biosynthesis. We conclude that mutations in at least two different genes can lead to the clinical phenotype of RCDP.

  • factors influencing the latency of the peroxisomal enzyme Dihydroxyacetone Phosphate Acyltransferase dhap at in permeabilized human skin fibroblasts
    Biochimica et Biophysica Acta, 1991
    Co-Authors: Ernst J Wolvetang, Joseph M. Tager, Ronald J.a. Wanders
    Abstract:

    Abstract In selectively permeabilized fibroblasts suspended in a medium mimicking the composition of the cytosol the peroxisomal enzyme Dihydroxyacetone-Phosphate Acyltransferase (DHAP-AT) wasfound to exhibit about 80% latency (Wolvetang, E.J., Tager, J.M. and Wanders, R.J.A. (1990) Biochem. Biophys. Res. Commun. 1035, 6–11). We investigated which components of the cytosol mimicking medium are important for latency of DHAP-AT and unmasking of latent DHAP-AT activity by ATP. We show that the latency of DHAP-AT is critically dependent upon the presence of reduced glutathione in the medium and that the in vivo prevailing GSH/GSSG ratio is sufficient to maintain DHAP-AT latency. Although thiol-groups in the peroxisomal membrane seem to be essential for the integrity of peroxisomes in selectively permeabilized fibroblasts no latency of DHAP-AT is observed in buffered sucrose media or in cell homogenates, irrespective of the presence of GSH in the medium used. We suggest that during homogenization irreversible damage is inflicted upon the peroxisomal membrane and/or that more factors than at present investigated are involved in maintaining peroxisomal integrity. Furthermore, we demonstrate that cations play a role in the stimulatory effect of ATP on latent DHAP-AT activity while a proton gradient is not directly involved in the stimulatory effect of ATP on latent DHAP-AT activity.

  • Factors influencing the latency of the peroxisomal enzyme Dihydroxyacetone-Phosphate Acyltransferase (DHAP-AT) in permeabilized human skin fibroblasts.
    Biochimica et biophysica acta, 1991
    Co-Authors: Ernst J Wolvetang, Joseph M. Tager, Ronald J.a. Wanders
    Abstract:

    Abstract In selectively permeabilized fibroblasts suspended in a medium mimicking the composition of the cytosol the peroxisomal enzyme Dihydroxyacetone-Phosphate Acyltransferase (DHAP-AT) wasfound to exhibit about 80% latency (Wolvetang, E.J., Tager, J.M. and Wanders, R.J.A. (1990) Biochem. Biophys. Res. Commun. 1035, 6–11). We investigated which components of the cytosol mimicking medium are important for latency of DHAP-AT and unmasking of latent DHAP-AT activity by ATP. We show that the latency of DHAP-AT is critically dependent upon the presence of reduced glutathione in the medium and that the in vivo prevailing GSH/GSSG ratio is sufficient to maintain DHAP-AT latency. Although thiol-groups in the peroxisomal membrane seem to be essential for the integrity of peroxisomes in selectively permeabilized fibroblasts no latency of DHAP-AT is observed in buffered sucrose media or in cell homogenates, irrespective of the presence of GSH in the medium used. We suggest that during homogenization irreversible damage is inflicted upon the peroxisomal membrane and/or that more factors than at present investigated are involved in maintaining peroxisomal integrity. Furthermore, we demonstrate that cations play a role in the stimulatory effect of ATP on latent DHAP-AT activity while a proton gradient is not directly involved in the stimulatory effect of ATP on latent DHAP-AT activity.

  • Topography of very-long-chain-fatty-acid-activating activity in peroxisomes from rat liver.
    Biochemical Journal, 1991
    Co-Authors: Wessel Lageweg, Joseph M. Tager, Ronald J.a. Wanders
    Abstract:

    We have investigated the localization of palmitoyl-CoA (hexadecanoyl-CoA) synthetase (EC 6.2.1.3) and cerotoyl-CoA (hexacosanoyl-CoA) synthetase in peroxisomes isolated from rat liver. Palmitoyl-CoA and cerotoyl-CoA synthetases, like acyl-CoA: Dihydroxyacetone Phosphate Acyltransferase (EC 2.3.1.42), are present in the peroxisomal membrane. Trypsin treatment of intact peroxisomes led to the disappearance of both palmitoyl-CoA and cerotoyl-CoA synthetase activities but had little, if any, effect on L-alpha-hydroxy-acid oxidase (EC 1.1.3.15), D-amino acid oxidase (EC 1.4.3.3) or acyl-CoA:Dihydroxyacetone Phosphate Acyltransferase. The latter three enzymes were inactivated if the trypsin treatment was preceeded by disruption of the peroxisomes by sonication. These results show that the active site, or at least domains essential for the activity of cerotoyl-CoA synthetase, like that of palmitoyl-CoA synthetase, is located on the cytosolic face of the peroxisomal membrane.