Mutants of Saccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase. Simultaneous loss of dihydroxyacetone phosphate acyltransferase indicates a common gene

Fourteen independent mutants of Saccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase activity were isolated using a colony autoradiographic screening technique. All 14 mutants were similarly defective in dihydroxyacetone phosphate acyltransferase activity. The mutations were recessive and fell into a single complementation group. Tetrad analysis gave results consistent with mutations in a single nuclear gene affecting both activities. sn-Glycerol-3-phosphate acyltransferase activity from different mutant strains exhibited different substrate dependencies and differing responses to temperature, detergent, and pH. In each case, the response of the dihydroxyacetone phosphate acyltransferase activity was similar to that of the sn-glycerol-3-phosphate acyltransferase. These results are consistent with the mutations occurring in the structural gene. The data also establish that the predominant dihydroxyacetone phosphate acyltransferase activity in yeast is a second activity of the sn-glycerol-3-phosphate acyltransferase.     

Postnatal Development and Isolation of Peroxisomes from Brain

We analyzed the postnatal peroxisome development in rat brain by measuring the enzyme activities of catalase and acyl-CoA oxidase and beta-oxidation of [1-14C]lignoceric acid. These enzyme activities were higher between 10 and 16 days of postnatal life and then decreased. We developed and compared two different methods for isolation of enriched peroxisomes from 10-day-old rat brain by using a combination of differential and density gradient centrifugation techniques. Peroxisomes in Percoll (self-generating gradient) banded at a density of 1.036 +/- 0.012 g/ml and in Nycodenz continuous gradient at 1.125 +/- 0.014 g/ml. Acyl-CoA oxidase, D-amino acid oxidase, L-pipecolic acid oxidase, and dihydroxyacetone phosphate acyltransferase activities and activities for the oxidation of very long chain fatty acid (lignoceric acid) were almost exclusively associated with catalase activity (a marker enzyme for peroxisomes) in the gradient. The postnatal increase in peroxisomal activity with the onset of myelination and the presence of enzyme for the biosynthesis of plasmalogens and oxidation of very long chain fatty acid (both predominant constituents of myelin) suggest that brain peroxisomes may play an important role in the assembly and turnover of myelin.     

Peroxisomal dihydroxyacetone phosphate acyltransferase. Effect of acetaldehyde on the intact and solubilized activity

The peroxisomal enzyme dihydroxyacetone phosphate (DHAP) acyltransferase shows a differential response to acetaldehyde. Employing whole peroxisomes, the enzyme displays a 130-400% stimulation of activity when assayed in the presence of 10-250 mM acetaldehyde. Following taurocholate solubilization of the enzyme the response to 0.25 M acetaldehyde is one of almost total inhibition. This inhibition of the taurocholate-solubilized enzyme is not observed at acetaldehyde concentrations below 200 mM. The stimulation of DHAP acyltransferase by acetaldehyde is solely a response of the peroxisomal enzyme as evidenced by its insensitivity to N-ethylmaleimide and 5 mM glycerol 3-phosphate. Furthermore, microsomal dihydroxyacetone phosphate acyltransferase activity is inhibited at all acetaldehyde concentrations. The activation of membrane-bound DHAP acyltransferase by acetaldehyde appears to be specific for this enzyme in comparison to several other peroxisomal and microsomal enzymes. The specificity of activation and differential response of the peroxisomal enzyme to acetaldehyde indicates that the microenvironment of the peroxisomal membrane is important for normal enzymatic function of this enzyme.     

Partial phenotypic suppression of a peroxisome-deficient animal cell mutant treated with aminoglycoside G418.

Certain enzymes normally associated with peroxisomes, such as the dihydroxyacetone phosphate (DHAP) acyltransferase involved in plasmalogen biosynthesis, are present at low levels in peroxisome-deficient mutants of Chinese hamster ovary (CHO) cells. We now show that the aminoglycoside G418 increases the residual DHAP acyltransferase in mutant ZR-82 by 60-fold. This is accompanied by a dose- and time-dependent restoration of the plasmalogen content. G418 treatment of ZR-82 also increases residual peroxisomal beta-oxidation activity by 3.8-fold. G418 does not affect wild-type CHO cells (CHO-K1) or a different peroxisome-deficient mutant, ZR-78.1. The effects of G418 on ZR-82 are transient, since plasmalogens and DHAP-acyltransferase decline to basal levels 5 days after G418 withdrawal. Other aminoglycosides and lysosomotropic agents do not alter plasmalogen levels in ZR-82. The subcellular distribution of catalase (an enzyme of the peroxisomal matrix which is present in normal amounts in peroxisome-deficient mutants but is mislocalized in the cytosol) is unaffected by G418 treatment of ZR-82, demonstrating that G418 does not restore peroxisomes. Localization of catalase by immunofluorescence microscopy confirms a total absence of intact peroxisomes in ZR-82, either before or after exposure to G418. This study is the first to demonstrate that some peroxisome-deficient mutants can be induced to accumulate functional DHAP acyltransferase and other peroxisomal enzymes, usually missing in the absence of peroxisomes. G418 may have some therapeutic value in selected patients with inborn errors of peroxisome assembly, such as Zellweger syndrome.     

Mutants in a macrophage-like cell line are defective in plasmalogen biosynthesis, but contain functional peroxisomes.

We have used a fluorescence-activated cytotoxicity protocol, 9-(1'-pyrene)nonanol (P9OH)/UV selection (Morand, O. H., Allen, L.-A. H., Zoeller, R. A., and Raetz, C. R. H. (1990) Biochim. Biophys. Acta 1034, 132-141), to isolate a series of plasmalogen-deficient mutants in a murine, macrophage-like cell line, RAW 264.7. Three of these mutants, RAW.7, RAW.12, and RAW.108, displayed varying degrees of plasmalogen deficiency (48, 17, and 14% of wild-type levels, respectively), and all three mutants were deficient in peroxisomal dihydroxyacetone phosphate (DHAP) acyltransferase activity (5% of wild-type). Unlike previously described Chinese hamster ovary (CHO) cell mutants, the RAW mutants contained intact, functional, peroxisomes and normal levels of alkyl-DHAP synthase activity, a peroxisomal, membrane-bound enzyme. In RAW.7 and RAW.108 cells, the loss of peroxisomal DHAP acyltransferase is the primary lesion. RAW.12 displayed not only a deficiency in the DHAP acyltransferase activity, but also displayed a second lesion in the biosynthetic pathway, a deficiency in delta 1'-desaturase activity (plasmanylethanolamine desaturase, EC 1.14.99.19), the final step in plasmenylethanolamine biosynthesis. The deficiencies expressed in the mutants represent unique lesions in plasmalogen biosynthesis. Since the RAW cell line is a macrophage-like responsive cell line, these mutants can be used to examine the role of plasmalogens in cellular functions such as arachidonic acid metabolism, prostaglandin synthesis, protein secretion, and signal transduction.     

Complementation in Zellweger syndrome: biochemical analysis of newly generated peroxisomes.

The Zellweger syndrome is characterized by a defect which results in the abnormal biogenesis of peroxisomes. As a consequence, metabolic activities associated with peroxisomes such as the oxidation of very long chain fatty acids, the synthesis of plasmalogens, and the catabolism of phytanic and pipecolic acids are impaired. Since this disorder is genetically heterogeneous and several complementation groups are known, we were able to study the normalization of peroxisomal activity during the process of complementation. The restoration of catalase and dihydroxyacetone phosphate acyltransferase activities peaked within 3-4 days postfusion while the oxidation of lignoceric acid was much delayed (7-8 days). Electron microscopy indicated that by 6 days following hybridization, peroxisome structure and density in heterokaryons was comparable to normal control cells. The heterogenous biochemical response during peroxisome normalization could be due to several factors including a possible requirement for restoration of peroxisomal structural integrity for maximum activation of certain metabolic pathways.     

Properties of the enzymes catalyzing the biosynthesis of lysophosphatidate and its ether analog in cultured fibroblasts from Zellweger syndrome patients and normal controls

The activities, properties, and steady-state kinetics of the five enzymes catalyzing the synthesis of 1-acyl- and 1-alkyl-sn-glycerol 3-phosphate in the cultured skin fibroblasts from Zellweger syndrome patients and normal controls were studied in detail. Judging from their Km and Vmax values, glycerol phosphate acyltransferase (EC 2.3.1.15), acyl/alkyl dihydroxyacetone phosphate reductase (EC 1.1.1.101), and acyl coenzyme A reductase (long-chain alcohol forming), appear to be affected only slightly by the absence of peroxisomes characteristic of the Zellweger syndrome. Glycerophosphate acyltransferase also showed no differences in N-ethylmaleimide sensitivity nor in inhibition by dihydroxyacetone phosphate between these cell types. Dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) and alkyl dihydroxyacetone phosphate synthase (EC 2.5.1.26) have altered activity and kinetic constants in homogenates from Zellweger syndrome fibroblasts. Dihydroxyacetone phosphate acyltransferase has similar Km (DHAP) values in both control and Zellweger syndrome cells; however, the value for the Vmax in Zellweger syndrome cells is only 6% of that found in the controls. This is interpreted as indicating that this enzyme is not defective in this disease but is simply present at a depressed level. Also, this enzyme activity has a maximum rate at pH 7.0-7.5 in the mutant cells as opposed to pH 5.4 in the controls. Acylation of dihydroxyacetone phosphate by control cell homogenate was stimulated by N-ethylmaleimide at both pH 5.7 and 7.5 whereas this activity from Zellweger syndrome cells was slightly inhibited at pH 5.7 and strongly inhibited at pH 7.5. In the absence of detergent, dihydroxyacetone phosphate acyltransferase in the Zellweger syndrome cells was much more labile to trypsin than in the control cells. Alkyl dihydroxyacetone phosphate synthase had a slightly higher Km (33 vs 17 microM) for palmitoyl dihydroxyacetone phosphate and a lower Vmax (0.07 vs 0.24 mU/mg protein) in the Zellweger syndrome cells as compared to controls. Although this is a substantial decrease in activity, it probably contributes little to the decreased rate of ether lipid synthesis in these cells. The major problem in this respect is apparently the loss of dihydroxyacetone phosphate acyltransferase activity. All of these enzymes, in both control and Zellweger syndrome cell homogenates, are sedimentable by centrifugation at 100,000g. Also, with the exception of dihydroxyacetone phosphate acyltransferase they had similar patterns of inactivation by heat in both cell types.     

Leishmania major expresses a single dihydroxyacetone phosphate acyltransferase localized in the glycosome, important for rapid growth and survival at high cell density and essential for virulence

Despite major advances in the understanding of pathogenesis of the human protozoan parasite Leishmania major, little is known about the enzymes and the primary precursors involved in the initial steps of synthesis of its major glycerolipids including those involved in virulence. We have previously demonstrated that the initial step of acylation of the precursor glycerol 3-phosphate is not essential for the synthesis of ester and ether phospholipids in this parasite. Here we show that Leishmania expresses a single acyltransferase with high specificity for the precursor dihydroxyacetone phosphate and shows the best activity in the presence of palmitoyl-CoA. We have identified and characterized the LmDAT gene encoding this activity. LmDAT complements the lethality resulting from the loss of both dihydroxyacetone phosphate and glycerol-3-phosphate acyltransferase activities in yeast. Recombinant LmDAT exhibits biochemical properties similar to those of the native enzyme of the promastigote stage parasites. We show that LmDAT is a glycosomal enzyme and its loss in a delta lmdat/delta lmdat null mutant results in complete abrogation of the parasite dihydroxyacetone phosphate acyltransferase activity. Furthermore, lack of LmDAT causes a major alteration in parasite division during the logarithmic phase of growth, an accelerated cell death during stationary phase, and loss of virulence. Together, our results demonstrate that LmDAT is the only dihydroxyacetone phosphate acyltransferase of the L. major localized in the peroxisome, important for growth and survival and essential for virulence.     

Peroxisomes and ether lipid biosynthesis in rat testis and epididymis

Plasmalogens are a main component of the spermatozoon membrane, playing a crucial role in their maturation. The initial steps in plasmalogen biosynthesis are catalyzed by two peroxisomal enzymes, dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase. The localization of both enzymes in the membrane of peroxisomes implies that plasmalogen-producing cells should contain this organelle. To unravel the putative source of spermatozoan plasmalogens we investigated which cell types in the testis and epididymis are endowed with peroxisomes. To this extent, testicular and epididymal tissue was analyzed at the protein and RNA levels by means of light and electron microscopical immunocytochemistry as well as by Western and Northern blotting. Proteins and mRNAs of peroxisomal enzymes, especially those of dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase, were detected in the testis and epididymis. In the testis, peroxisomes were localized exclusively in Leydig cells and not in cells of the seminiferous tubules, implying that the latter do not contribute to the biosynthesis of plasmalogens of the sperm membrane. In contrast, peroxisomes could be clearly visualized in the epithelial cells of the epididymis. The results suggest that peroxisomes in epithelial cells of the rat epididymis play a pivotal role in the biosynthesis of plasmalogens destined for delivery to the sperm plasma membrane.     

Comparison of the activities of some peroxisomal and extraperoxisomal lipid-metabolizing enzymes in liver and extrahepatic tissues of the rat.

Peroxisomal (acyl-CoA oxidase and peroxisomal dihydroxyacetone-phosphate acyltransferase) and extraperoxisomal (mitochondrial fatty acid oxidation, extraperoxisomal dihydroxyacetone-phosphate acyltransferase, mitochondrial and microsomal glycerophosphate acyltransferases) lipid-metabolizing enzymes were measured in homogenates from rat liver and from seven extrahepatic tissues. Except for jejunal mucosa and kidney, extrahepatic tissues contained very little acyl-CoA oxidase activity. Peroxisomal dihydroxyacetone-phosphate acyltransferase, taken as the activity that was not inhibited by 5 mM-glycerol 3-phosphate, was present in all tissues examined, and its specific activity in liver and extrahepatic tissues was roughly of the same order of magnitude. Clofibrate treatment increased the activity of acyl-CoA oxidase in liver, and to a smaller extent also in kidney, but did not influence the activity of peroxisomal dihydroxyacetone-phosphate acyltransferase. Comparison of the activities of peroxisomal and extraperoxisomal lipid-metabolizing enzymes in extrahepatic tissues and in liver, an organ in which the contribution of peroxisomes to fatty acid oxidation and to glycerolipid synthesis has been estimated previously, suggests that, as in liver, peroxisomal long-chain fatty acid oxidation is of minor quantitative importance in extrahepatic tissues, but that in these tissues (micro)-peroxisomes are responsible for most of the dihydroxyacetone phosphate acylation and, consequently, for initiating ether glycerolipid synthesis.     

Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver

Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25–30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent K_m values (0.1 and 0.5 mm) for dihydroxyacetone phosphate. The V_max was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a K_m = 0.1 mm and no change in V_max. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [³H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [³H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.     

Redundant systems of phosphatidic acid biosynthesis via acylation of glycerol-3-phosphate or dihydroxyacetone phosphate in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611-7616, 1997). Using the gat1 mutant strain TTA1, we show here that Gat1p present in both subcellular fractions accepts glycerol-3-phosphate and dihydroxyacetone phosphate as a substrate. Similarly, the additional acyltransferase(s) present in the endoplasmic reticulum can acylate both precursors. In contrast, yeast mitochondria harbor an enzyme(s) that significantly prefers dihydroxyacetone phosphate as a substrate for acylation, suggesting that at least one additional independent acyltransferase is present in this organelle. Surprisingly, enzymatic activity of 1-acyldihydroxyacetone phosphate reductase, which is required for the conversion of 1-acyldihydroxyacetone phosphate to 1-acylglycerol-3-phosphate (lysophosphatidic acid), is detectable only in lipid particles and the endoplasmic reticulum and not in mitochondria. In vivo labeling of wild-type cells with [2-3H, U-14C]glycerol revealed that both glycerol-3-phosphate and dihydroxyacetone phosphate can be incorporated as a backbone of glycerolipids. In the gat1 mutant and the 1-acylglycerol-3-phosphate acyltransferase slc1 mutant, the dihydroxyacetone phosphate pathway of phosphatidic acid biosynthesis is slightly preferred as compared to the wild type. Thus, mutations of the major acyltransferases Gat1p and Slc1p lead to an increased contribution of mitochondrial acyltransferase(s) to glycerolipid synthesis due to their substrate preference for dihydroxyacetone phosphate.     

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.     

Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids

The variant CHO-K1 cell line, NRel-4, is unable to synthesize plasmalogens because of a severe reduction in dihydroxyacetonephosphate acyltransferase (DHAPAT) activity (Nagan, N., A. K. Hajra, L. K. Larkins, P. Lazarow, P. E. Purdue, W. B. Rizzo, and R. A. Zoeller. 1998. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol. Biochem. J. 332: 273-279). Northern analysis demonstrated that the loss of this activity was attributable to a severe reduction in mRNA levels for DHAPAT. Transfection of NRel-4 cells with a plasmid bearing the human DHAPAT cDNA recovered DHAPAT activity and plasmalogen biosynthesis. Examination of clonal isolates from the transfected population showed that recovery of as little as 10% of wild-type DHAPAT activity restored plasmalogen levels to 55% of normal, whereas in one isolate, NRel-4.15, which overexpressed DHAPAT activity by 6-fold over wild-type cells, plasmalogen levels were returned only to wild-type values. Although the rate of plasmenylethanolamine biosynthesis was restored in NRel-4.15, the biosynthesis of nonether glycerolipids was either decreased or unaffected, suggesting that peroxisomal DHAPAT does not normally contribute to nonether glycerolipid biosynthesis. These data demonstrate that a defect in the gene that codes for peroxisomal DHAPAT is the primary lesion in the NRel-4 cell line and that the peroxisomal DHAPAT is essential for the biosynthesis of plasmalogens in animal cells.     

Genetic and biochemical heterogeneity in patients with the rhizomelic form of chondrodysplasia punctata — a complementation study

Summary 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.     

Induction of the peroxisomal glycerolipid-synthesizing enzymes during differentiation of 3T3-L1 adipocytes. Role in triacylglycerol synthesis

The glycerophosphate backbone for triglyceride synthesis is commonly believed to be created through the conversion of dihydroxyacetone phosphate (DHAP) by glycerophosphate dehydrogenase (GPD) to sn-glycerol 3-phosphate (GP), which is then converted by glycerophosphate acyltransferase (GPAT) to 1-acyl-GP. Consistent with this, GPD and GPAT are highly induced during differentiation of mouse 3T3-L1 preadipocytes. While the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway for glycerolipid synthesis is commonly believed to be involved only in glycerol ether lipid synthesis, we report here that during conversion of 3T3-L1 preadipocytes to adipocytes, the specific activity of peroxisomal DHAP acyltransferase (DHAPAT) is increased by 9-fold in 6 days, while acyl-DHAP:NADPH reductase is induced by 5-fold. A parallel increase in the catalase (the peroxisomal marker enzyme) activity is also seen. In contrast, the specific activity of alkyl-DHAP synthase, the enzyme catalyzing the synthesis of the ether bond, is decreased by 60% during the same period. Unlike microsomal GPAT, the induced DHAPAT is found to have high activity at pH 5.5 and is resistant to inhibition by sulfhydryl agents, heat, and proteolysis. On subcellular fractionation, DHAPAT is found to be associated with microperoxisomes whereas GPAT activity is mainly present in microsomes. Northern blot analyses reveal that induction of DHAPAT can be largely explained through increases in DHAPAT mRNA. A comparison of microsomal and peroxisomal glycerolipid synthetic pathways, using D-[3-(3)H, U-(14)C]glucose as the precursor of the lipid glycerol backbone shows that about 40-50% of triglyceride is synthesized via the acyl-DHAP pathway. These results indicate that the acyl-DHAP pathway is important not only for the synthesis of ether lipids, but also for the synthesis of triacylglycerol and other non-ether glycerolipids.     

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 either phospholipid biosynthesis, we have studied the properties of acyl-CoA:dihydroxyacetone phosphate acyltransferase 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.     

The relative utilization of the acyl dihydroxyacetone phosphate and glycerol phosphate pathways for synthesis of glycerolipids in various tumors and normal tissues.

Rates of phosphatidate synthesis from dihydroxyacetone phosphate via acyl dihydroxyacetone phosphate or glycerol phosphate are compared in homogenates of 13 tissues, most of which are deficient in glycerol phosphate dehydrogenase (EC 1.1.1.8). In all tissues examined, dihydroxyacetone phosphate entered phosphatidate more rapidly via acyl dihydroxyacetone phosphate than via glycerol phosphate. Tissues with a relatively low rate of phosphatidate synthesis via glycerol phosphate, showed no compensating increase in the rate of synthesis via acyl dihydroxyacetone phosphate. The rates at which tissue homogenates synthesize phosphatidate from dihydroxyacetone phosphate via glycerol phosphate increase as glycerol phosphate dehydrongenase increase. Both glycerol phosphate dehydrogenase and glycerol phosphate: acyl CoA acyltransferase (EC 2.3.1.15) are more active than dihydroxyacetone phosphate : acyl CoA acyltransferase (EC 2.3.1.42). Thus, all the tissue homogenates possessed an apparently greater capability to synthesize phosphatidate via glycerol phosphate than via acyl dihydroxyacetone phosphate, but did not express this potential. This result is discussed in relation to in vivo substrate limitations.     

Chinese hamster ovary cell mutants defective in peroxisome biogenesis. Comparison to Zellweger syndrome

We have previously reported the isolation of Chinese hamster ovary (CHO) cell mutants that are defective in the biosynthesis of plasmalogens, deficient in at least two peroxisomal enzymes (dihydroxyacetonephosphate (DHAP) acyltransferase and alkyl-DHAP synthase), and in which catalase is not found within peroxisomes (Zoeller, R. A., and Raetz, C. R. H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5170). We now provide further evidence that three such strains are more generally defective in peroxisome biogenesis. Electron microscopic cytochemistry revealed that the mutants did not contain recognizable peroxisomes. However, immunofluorescence microscopy using an antibody directed against peroxisomal integral membrane proteins revealed the presence of peroxisomal membrane ghosts resembling those seen in cells of patients suffering from one of the human peroxisomal disorders, Zellweger syndrome. Immunoblot analyses, using antibodies specific for peroxisomal matrix proteins, demonstrated deficiencies of peroxisomal proteins in the mutant CHO cells that were similar to those in Zellweger syndrome. Fusion of a CHO mutant with fibroblasts obtained from Zellweger patients resulted in restoration of peroxisomal dihydroxyacetonephosphate acyltransferase and peroxisomal acyl-coenzyme A oxidation activities. The hybrid cells also regained the ability to synthesize plasmenylethanolamine. Moreover, normal peroxisomes were seen by immunofluorescence in the hybrid cells. These results indicate that the hybrid cells have recovered the ability to assemble peroxisomes and that, although the mutant CHO cells are biochemically and morphologically very similar to cells from patients with Zellweger syndrome, the genetic lesions are distinct. Our somatic cell mutants should be useful in identifying factors and genes involved in peroxisome biogenesis and may aid the genetic categorization of the various peroxisomal disorders.