Ether Phospholipid Biosynthesis Research Articles

Peroxisomes in adrenal steroidogenesis.

Peroxisomes, cytoplasmic organelles limited by a single membrane and with a matrix of moderate electron density, are present in a great number of cells, namely in adrenal cortex and other steroid-secreting organs. Presently peroxisomes are considered to be involved in important metabolic processes. They intervene in: (1) the production and degradation of H2O2; (2) biosynthesis of ether-phospholipids, cholesterol, dolichol, and bile acids; (3) oxidation of very long chain fatty acids, purines, polyamines, and prostaglandins; (4) catabolism of pipecolic, phythanic and glyoxylic acids; and (5) gluconeogenesis. Recent studies demonstrated that the experimental alterations in the normal steroidogenesis, produce significant morphological and biochemical changes in peroxisomes. Besides this, the presence of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (the key enzyme in the de novo cholesterol synthesis from acetate) and of sterol carrier protein-2 (SCP2), which is involved in the cholesterol metabolism and steroid metabolic pathways, are located in peroxisomes of steroid-secreting cells. In addition, patients with peroxisome diseases present deficiency in steroidogenesis, as well as reduced levels of SCP2. These data pointed out the important role of peroxisomes in steroid biosynthesis.

Alkyl dihydroxyacetone phosphate synthase in glycosomes of Trypanosoma brucei

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.

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.

Ether lipid synthesis: Purification and identification of alkyl dihydroxyacetone phosphate synthase from guinea-pig liver

Alkyl-dihydroxyacetone phosphate synthase, the second enzyme involved in ether phospholipid biosynthesis from dihydroxyacetone phosphate and responsible for glycero-ether bond formation, has been purified from guinea-pig liver. Alkyl-dihydroxyacetone phosphate synthase was solubilized from a membrane fraction prepared from an enriched peroxisome fraction with Triton X-100 and potasssium chloride. The solubilized enzyme was further purified by chromatography on QAE-Sephadex, Matrex Red, Phosphocellulose and Concanavalin A. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis alkyl-dihydroxyacetone phosphate synthase appears as a 65 kDa band. Chromatofocusing revealed an isoelectric point of pH 5.9 for the enzyme. The pH optimum of alkyl-dihydroxyacetone phosphate synthase was found to be between pH 7 and 8 in a 50 mM potassium phosphate buffer. The specific activity of the enzyme was estimated to be at least 350 nmol · min −1 · mg −1, corresponding to a purification of at least 13 000-fold.

Effect of peroxisome proliferation on ether phospholipid biosynthesizing enzymes in rat liver

1. 1. The effect of the peroxisome proliferators clofibrate and plasticizer on the activities of the first two enzymes involved in ether phospholipid biosynthesis, i.e. dihydroxyacetone-phosphate acyltransferase (DHAP-AT) and alkyldihydroxyacetone-phosphate synthase, were studied in rat liver homogenates and purified peroxisomes. 2. 2. DHAP-AT in homogenates increased by 2 to 3-fold both in total and specific activity. However, the specific activity in purified peroxisomes showed no significant increase demonstrating for the first time that there is no specific induction of this enzyme that exceeds the induction of total peroxisomal protein. 3. 3. Alkyldihydroxyacetone-phosphate synthase showed no significant increase in total and specific activity in homogenates and a slight decrease of its specific activity in purified peroxisomes was observed. 4. 4. The total amount of plasmalogens did not increase upon proliferation and a slight decrease in the percentage plasmalogens in total phospholipids was observed. 5. 5. Proliferation did not influence the phospholipid composition of the peroxisomal membrane.

Topography of ether phospholipid biosynthesis

The enzyme, dihydroxyacetone-phosphate acyltransferase (DHAP-AT) is localized at the inner surface of the peroxisomal membrane but shows no latency. The product, i.e., acyl-DHAP, is the first lipidic intermediate in ether lipid biosynthesis that is converted by alkyldihydroxyacetone-phosphate synthase into alkyl-DHAP. This step and the reductions of acyl-DHAP and alkyl-DHAP to the glycerol 3-phosphate analogs can still take place in peroxisomes. The further conversions of these intermediates into ether-linked choline or ethanolamine glycerophospholipids or plasmalogens take place in the endoplasmic reticulum. In this paper we describe studies to localize acyl-DHAP in the transversal plane of the peroxisomal membrane. To enable these studies, the usually employed assay conditions for DHAP-AT were modified by omission of BSA and by lowering the temperature and the palmitoyl-CoA concentration. Using these modified conditions, we were able to label peroxisomes with endogenously generated acyl-DHAP, with retention of catalase within the peroxisomal membrane. Endogenously generated acyl-DHAP was rapidly extractable from the outer surface of peroxisomes with BSA at 0°C, suggesting that even at this temperature the product was transported very fast across the membrane. Trypsin treatment of peroxisomes did not affect this behaviour. Only after short incubation periods, an increase in the proportion of non-extractable acyl-DHAP was observed. In large unilamellar vesicles made from peroxisomal phospholipids no transmembrane movement of acyl-DHAP was found. Despite the apparently rapid transbilayer movement of acyl-DHAP in the presence of BSA, it could still serve as a substrate for the enzyme alkyl-DHAP synthase, which is also localized at the inner surface of the peroxisomal membrane. In addition it was shown that endogenously generated substrate is used at a higher efficiency compared to exogenously added acyl-DHAP, suggesting a close interaction of the two enzymes in the peroxisomal membrane.

Zellweger syndrome: biochemical procedures in diagnosis, prevention and treatment.

In patients with cerebro-hepato-renal (Zellweger) syndrome, the absence of peroxisomes results in an impairment of metabolic processes in which peroxisomes are normally involved. These include the catabolism of very long chain (greater than C22) fatty acids, the biosynthesis of ether-phospholipids and of bile acids, the catabolism of phytanic acid and the catabolism of pipecolic acid. Many diagnostic tests for Zellweger syndrome have become available in recent years. In classic Zellweger syndrome abnormal C27-bile acids, very long chain fatty acids, dicarboxylic acids and pipecolic acid accumulate in the plasma of the patients. Moreover, depending upon the diet, plasma phytanic acid concentrations may be elevated. In platelets the activity of acyl-CoA: dihydroxyacetone phosphate acyltransferase is deficient; in erythrocytes from young (less than 4 months) patients the plasmalogen content of the phospholipids is decreased. In cultured fibroblasts from skin and from chorionic villus and cultured amniotic fluid cells from Zellweger patients the plasmalogen level is lowered; there is a decreased activity of acyl-CoA: dihydroxyacetone phosphate acyltransferase, alkyl dihydroxyacetonephosphate synthase and phytanic acid oxidase; the de novo biosynthesis of plasmalogens and the peroxisomal beta-oxidation of fatty acids are impaired and the intracellular localization of catalase is abnormal. Dietary treatment of patients with Zellweger syndrome has not so far resulted in an objective clinical improvement. As Zellweger syndrome is usually fatal in early life, prenatal diagnosis of the disease is important.

Acyl-CoA: dihydroxyacetone phosphate acyltransferase in human skin fibroblasts: study of its properties using a new assay method

In relation to the finding that human skin fibroblasts are capable of de novo ether phospholipid biosynthesis, we have studied the properties of acyl-CoA: dihydroxyacetone phosphate acyltransf erase in fibroblast homogenates using a new assay method. The results indicate that the acylation of dihydroxyacetone phosphate shows an optimum at pH 5.5 with a broad shoulder of activity up to pH 6.4 and a decline in activity up to pH 8.2. At pH 5.5 the acyltransferase accepts dihydroxyacetone phosphate, but not glycerol 3-phosphate as a substrate. Furthermore, the transferase activity was found to be membrane-bound and inactivated by Triton X-100 at concentrations above 0.025% (w/v). Similar properties have been described for the enzyme as present in rat-liver and guinea-pig liver peroxisomes. These data, together with the finding that acyl-CoA:dihydroxyacetone phosphate acyltransferase is deficient in cultured skin fibroblasts from patients without peroxisomes (Zellweger syndrome), suggest that in cultured skin fibroblasts the enzyme is primarily located in peroxisomes.

Deficiency of acyl-CoA: dihydroxyacetone phosphate acyltransferase in thrombocytes of Zellweger patients: a simple postnatal diagnostic test

Activity of acyl-CoA: dihydroxyacetone phosphate acyltransferase, a membrane-bound peroxisomal enzyme in mammalian liver cells catalyzing the first step in the biosynthesis of etherphospholipids, is detectable in thrombocytes isolated from blood of human controls. However, in thrombocytes from patients affected by the cerebro-hepato-renal (Zellweger) syndrome, the activity of this enzyme is severely reduced, permitting rapid postnatal biochemical detection of this severe inborn disease, by measuring the enzyme activity in patients' thrombocytes.

Alkyl dihydroxyacetone phosphate synthase in human fibroblasts and its deficiency in Zellweger syndrome.

The cerebro-hepato-renal (Zellweger) syndrome is an autosomal recessive disorder biochemically characterized by the absence of morphologically distinguishable peroxisomes. Key enzymes involved in the biosynthesis of ether phospholipids, i.e., dihydroxyacetone phosphate acyltransferase and alkyl dihydroxyacetone phosphate synthase, are located in mammalian (micro)peroxisomes. We have previously shown a strikingly reduced activity of dihydroxyacetone phosphate acyltransferase in liver, brain, and cultured skin fibroblasts from Zellweger patients (Schutgens et al. 1984. Biochim. Biophys. Res. Commun. 120: 179-184). We have now extended these investigations by studying alkyl dihydroxyacetone phosphate synthase in cultured human skin fibroblasts. Enzymatic activity was determined by measuring the formation of radioactive alkyl dihydroxyacetone phosphate from palmitoyl dihydroxyacetone phosphate and [1-14C]hexadecanol as substrates. The enzyme was optimally active at pH 8.5 and was stimulated (about 2-3-fold) by the presence of 0.05% (v/v) Triton X-100. The apparent KM values for the enzyme in control fibroblasts amounted to 35 microM for palmitoyl dihydroxyacetone phosphate and 90 microM for hexadecanol. The reaction became inhibited at higher concentrations of both Triton X-100 and palmitoyl dihydroxyacetone phosphate. Control skin fibroblasts showed alkyl dihydroxyacetone phosphate synthase activity of 69 +/- 28 pmol X min-1 X mg-1 (n = 7), while fibroblasts from patients had an activity of only 6.3 +/- 1.7 pmol X min-1 X mg-1 (n = 7). Alkyl dihydroxyacetone phosphate synthase was also found to be deficient in tissue homogenates of Zellweger patients. The specific activity of this enzyme in liver, kidney, and brain homogenates from Zellweger patients was less than 15% of that in the corresponding tissues from controls.

Peroxisomal matrix enzymes in Zellweger syndrome: activity and subcellular localization in liver.

The cerebro-hepato-renal (Zellweger) syndrome (McKusick 21410) can be considered as the prototype of a newly recognized group of inherited diseases, the peroxisomopathies, in which one or more peroxisomal functions are impaired. In contrast to earlier suggestions, it is now clear that peroxisomes fulfil an essential role in a number of metabolic processes, including ether phospholipid biosynthesis, oxidation of very long chain fatty acids, bile acid synthesis and oxidation of dicarboxylic acids (reviewed in Borst, 1983; Kelley, 1983). The absence of peroxisomes in Zellweger syndrome is accompanied by a generalized impairment of these peroxisomal functions.