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Biomedical subjects

D Hardeman

Publications and source records attributed to D Hardeman.

10 recordsLinked to original sources
PubMed1993

Ether lipid synthesis and its deficiency in peroxisomal disorders.

This paper deals with the discovery of plasmalogen deficiency in the cerebro-hepato-renal (Zellweger) syndrome and discusses how this has led to the development of postnatal and prenatal diagnostic procedures for this and a number of related peroxisomal disorders in man that show a general impairment in the biosynthesis of ether glycerophospholipids. The results have clearly shown an indispensable role for peroxisomes in the total process of ether lipid synthesis as evidenced by a description of the cellular topography of this process. Platelet-activating factor is a bioactive phospholipid in which the glycero-ether linkage is essential for its biological activities. The deficient formation of this lipid mediator can be correlated to the residual amounts of ether phospholipids found in patients with impaired ether lipid production. Evidence is provided to demonstrate that the extent to which cells upon stimulation produce platelet-activating factor and its 1-acyl counterpart is not caused by enzyme selectivities for ether-linked versus ester-linked phospholipid species. Rather, the relative production of these compounds appears to be mainly governed by the relative abundance of ether-linked and ester-linked precursor molecules and the activity of cellular enzymes, such as lysophospholipases, that catabolize the acyl analog of platelet-activating factor through deacylation.

Humans↗
PubMed1991

Localization of enzymes involved in glycero-ether bond formation in rat liver.

Studies on the localization of alkyldihydroxyacetone-phosphate synthase in rat liver are described. Less than 5% of the total activity was found in the cytosolic fraction, suggesting that the enzyme is membrane-bound. The ratio of the enzymatic (specific) activity over that of dihydroxyacetone-phosphate acyltransferase, a peroxisomal enzyme, is 10-fold higher in the microsomal fraction, when compared to the peroxisomal fraction. Studying the distribution of the enzyme in a linear density gradient, two activity peaks were found in the peroxisomal and the microsomal fraction indicating a bimodal localization of alkyldihydroxyacetone-phosphate synthase. Rabert et al. (Rabert, U., Völkl, A. and Debuch, H. (1986) Biol. Chem. Hoppe-Seyler 367, 215-222) have presented evidence that the activity in the microsomal fraction was mainly caused by a different enzyme that preferentially converted acyldihydroxyacetone to alkyldihydroxyacetone. We also found a radioactive product, different from alkyldihydroxyacetone-phosphate, upon incubation of microsomal protein in the presence of [14C]hexadecanol. However, it was shown that this product was formed independently of the presence of acyldihydroxyacetone. The product yielded [14C]hexadecanol upon alkaline hydrolysis, clearly showing that it did not contain an ether-bond. Upon incubation of microsomal protein with [14C]palmitic acid and hexadecanol the product was also observed and its chromatographic behaviour resembled that of a synthetically prepared palmitoyl ester of hexadecanol. From these data it was concluded that the product formed is most likely a wax and that the enzyme responsible for this conversion is clearly different from the alkyldihydroxyacetone-phosphate synthase. The implication is that acyldihydroxyacetone-phosphate cannot be replaced by acyldihydroxyacetone in the process of glycero-ether bond formation.

Alkyl and Aryl Transferases↗
PubMed1990

Studies on peroxisomal membranes.

The phospholipid/protein ratios of rat liver peroxisomes, mitochondria and microsomes were determined and found to be 257 +/- 26, 232 +/- 20 and 575 +/- 20 nmol.mg-1, respectively. After correction for the loss of soluble protein, a peroxisomal ratio of 153 nmol.mg-1 was calculated. Organelle fractions were treated with sodium carbonate, whereafter membrane fragments containing integral membrane proteins were pelleted. For the membrane fractions of peroxisomes, mitochondria and microsomes phospholipid/protein ratios of 1054 +/- 103, 1180 +/- 90 and 1050 +/- 50 nmol.mg-1 were found, whereas 26 +/- 2, 20 +/- 2 and 49 +/- 2% of the organelle protein was recovered in these membrane fractions, respectively. The phospholipid composition of the different organelle fractions were determined, but no large differences were obtained, except for cardiolipin that was found only in the mitochondrial fraction. After sodium carbonate treatment virtually all enzymatic activity of the enzymes tested was lost. Therefore Triton X-114 phase separation was used to obtain the peroxisomal membrane components. In this fraction 42.9 +/- 3.5% of the protein and 90.2 +/- 3.7% of the phospholipid was found. Enzymatic activity of two integral membrane proteins was recovered for over 90% in the membrane fraction, whereas activity of two matrix proteins was mainly found in the soluble fraction. Urate oxidase, the peroxisomal core protein, behaved differently and was recovered mainly with the membrane components. Recoveries of enzymatic activities after the Triton X-114 phase separation varied from 45 to 116%, and together with the good separation that was obtained between soluble proteins and integral membrane proteins this method provides a useful alternative for the isolation of membrane components.

Animals↗
PubMed1990

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

1. The effect of the peroxisome proliferators clofibrate and plasticizer on the activities of the first two enzymes involved in either phospholipid biosynthesis, i.e. dihydroxyacetone-phosphate acyltransferase (DHAP-AT) and alkyldihydroxyacetone-phosphate synthase, were studied in rat liver homogenates and purified peroxisomes. 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. 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. The total amount of plasmalogens did not increase upon proliferation and a slight decrease in the percentage plasmalogens in total phospholipids was observed. 5. Proliferation did not influence the phospholipid composition of the peroxisomal membrane.

Acyltransferases↗
PubMed1989

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 degree 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.

Acyltransferases↗
PubMed1988

Rat liver dihydroxyacetone-phosphate acyltransferase: enzyme characteristics and localization studies.

Peroxisomes were isolated from rat liver by pelleting a light mitochondrial (L) fraction over a 30% (w/v) Metrizamide layer. Peroxisomes were recovered as a loose pellet from the bottom of the tube and the purity of the peroxisomal fraction was calculated to be about 90%. The characteristics of dihydroxyacetone-phosphate acyltransferase (DHAP-AT) in the light mitochondrial fraction and the purified peroxisomal fraction were compared. The behaviour of the enzyme in the two fractions was very similar, except for the effect of sodium fluoride, which stimulated the activity in the L fraction 5-10-fold and in the peroxisomal fraction only 1.6-fold. This difference could be explained by the action of fluoride-sensitive acid phosphatases present in the L fraction that dephosphorylate palmitoyl-coenzyme A, a substrate for DHAP-AT. The localizations of DHAP-AT and alkyldihydroxyacetone-phosphate synthase in the rat liver peroxisomal membrane were studied. It is shown that in intact peroxisomes, DHAP-AT and alkyl-DHAP synthase are resistant to proteolytic inactivation by trypsin, as is fatty acid beta-oxidation activity, which served as a marker for the intactness of the peroxisomal membrane. Catalase was found not to be a suitable marker to assess peroxisome intactness in view of its relative insensitivity to trypsin. In 1-lauroyllysophosphatidylcholine-permeabilized peroxisomes, DHAP-AT, alkyl-DHAP synthase and beta-oxidation activities were rapidly inactivated by trypsin. It is concluded that in rat liver peroxisomes, at least the active sites of the integral membrane proteins DHAP-AT and alkyl-DHAP synthase are localized exclusively at the inner surface of the peroxisomal membrane.

Acyltransferases↗
PubMed1987

Isolation of protein components from rat lung lamellar bodies.

Lamellar bodies isolated from 10% (w/v) rat lung homogenates by discontinuous sucrose gradient centrifugation were shown to contain variable amounts of adhering proteins. These contaminating proteins could be removed by either Sepharose 4B gel filtration or precipitation of the crude preparation at pH 11.5. Both purification methods yielded membrane preparations with a phospholipid-to-protein ratio of 10.0 mumol/mg. Nearly complete separation of lamellar body phospholipid and protein could be achieved upon application of the purified membranes to DEAE-cellulose in the presence of 0.2% (v/v) Triton X-100. Phospholipid analyses showed that 83% of total lipid phosphorus was recovered in phosphatidylcholine. In phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and phosphatidylinositol recoveries amounted to 4, 8, 2 and 2%, respectively. Molecular mass determinations of the isolated protein component of lamellar bodies by means of SDS polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue revealed the presence of three protein bands with molecular masses of 64, 33 and 31 kDa. Upon staining with silver a 16 kDa protein was also visible. Sephadex G-100 gel filtration showed only one protein peak corresponding to a molecular mass of 64 kDa when protein was assayed with Coomassie brilliant blue.

Animals↗
PubMed1987

Phospholipid-protein interactions in rat lung lamellar bodies.

We investigated the specificity of the cytosol-mediated phosphatidylcholine transfer between isolated rat lung microsomes and rat lung lamellar bodies. For that purpose we labeled the microsomes with 1-acyl-2-[1-14C]palmitoyl- and 1-acyl-2-[9,10-3H]oleoylphosphatidylcholine through protein-catalyzed phosphatidylcholine exchange. Incubation in buffer resulted in 3-5% transfer of label from microsomes to lamellar bodies. Lung cytosol stimulated this transfer about 2-fold and the presence of 12 micrograms/ml phosphatidylcholine-transfer protein from bovine liver resulted in a 30 to 35% recovery of radioactivity in the lamellar bodies. When microsomal donor membranes with a 3H/14C ratio of 2.6 were used, the 3H/14C ratios of the lamellar bodies were 3.9, 3.7 and 3.7, after incubation in buffer, with cytosol and with bovine liver exchange protein, respectively. Doubling the amount of lamellar body acceptor membranes resulted in 3H/14C ratios in the lamellar bodies of 4.6 and 4.1, after incubation in buffer and with cytosol, respectively. Furthermore, we isolated the protein component from rat lung lamellar bodies and performed reconstitution experiments with phospholipids. Reconstituted and non-reconstituted phospholipid and protein were separated by either Sepharose 4B gel filtration or discontinuous sucrose gradient centrifugation. The presence of lamellar body protein in the reconstitution mixture resulted in the formation of larger structures with higher density than those formed in control experiments without protein. When 1-acyl-2-[1-14C]palmitoyl- and 1-acyl-2-[9,10-3H]oleoylphosphatidylcholine were included in the reconstitution mixture, the structures containing lamellar body protein had 2- to 4-fold lower 3H/14C ratios than initially present in the incubation. These results suggest that lamellar body proteins associate preferentially with disaturated phosphatidylcholine species.

Animals↗