Plant dihydroxyacetone phosphate reductases : purification, characterization, and localization

A cytosolic form of dihydroxyacetone phosphate (DHAP) reductase was purified 200,000-fold from spinach (Spinacia oleracea L.) leaves to apparent electrophoretic homogeneity. The purification procedure included anion-exchange chromatography, gel filtration, hydrophobic chromatography, and dye-ligand chromatography on Green-A and Red-A agaroses. The enzyme, prepared in an overall yield of 14%, had a final specific activity of about 500 μmol of DHAP reduced min⁻¹ mg⁻¹ protein, a subunit molecular mass of 38 kD, and a native molecular mass of 75 kD. A chloroplastic isoform of DHAP reductase was separated from the cytosolic form by anion-exchange chromatography and partially purified 56,000-fold to a specific activity of 135 μmol min⁻¹ mg⁻¹ protein. Antibodies generated in rabbits against the cytosolic form did not cross-react with the chloroplastic isoform. The two reductases were specific for NADH and DHAP. Although they exhibited some dissimilarities, both isoforms were severely inhibited by higher molecular weight fatty acyl coenzyme A esters and phosphohydroxypyruvate and moderately inhibited by nucleotides. In contrast to previous reports, the partially purified chloroplastic enzyme was not stimulated by dithiothreitol or thioredoxin, nor was the purified cytosolic enzyme stimulated by fructose 2,6-bisphosphate. A third DHAP reductase isoform was isolated from spinach leaf peroxisomes that had been prepared by isopycnic sucrose density gradient centrifugation. The peroxisomal DHAP reductase was sensitive to antibodies raised against the cytosolic enzyme and had a slightly smaller subunit molecular weight than the cytosolic isoform.     

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.     

Acyl coenzyme A: cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at −40°C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

NAD+-specific glycerol 3-phosphate dehydrogenase from developing castor bean endosperm

l-Glycerol 3-phosphate dehydrogenase has been isolated and partially purified from the endosperm of developing castor beans. The enzyme is entirely cytosolic and is not found in the plastid fraction. No activity was found in germinating castor beans. The pH optimum for the reduction of dihydroxyacetone phosphate is 8.1 and is 9.6 for the reverse reaction. The molecular weight determined by gel filtration chromatography is between 71,000 and 83,000. Both substrates show substrate inhibition at concentrations about 13 μm for NADH and 400 μm for dihydroxyacetone phosphate. Substrate interaction kinetics gave limiting K_m values of 2.7 and 35.5 μm for NADH and dihydroxyacetone phosphate, respectively. Substrate interaction and product inhibition kinetics were consistent with an ordered sequential mechanism with NADH being the first substrate to bind and NAD⁺ being the last product to dissociate.     

Comparison of the carnitine acyltransferase activities from rat liver peroxisomes and microsomes

Carnitine acyltransferase activities for acetyl- and octanoyl-CoA (coenzyme A) occur in isolated peroxisomal, mitochondrial, and microsomal fractions from rat and pig liver. Solubility studies indicated that both peroxisomal carnitine acyltransferases were in the soluble matrix. In contrast, the microsomal carnitine acyltransferases were tightly associated with their membrane. The microsomal short-chain transferase, carnitine acetyltransferase, was solubilized and stabilized by extensive treatment of the membrane with 0.4 m KCl or 0.3 m sucrose in 0.1 m pyrophosphate at pH 7.5. The same treatment only partially solubilized the microsomal medium-chain transferase, carnitine octanoyltransferase.Although half of the total carnitine acetyltransferase activity in rat liver resides in peroxisomes and microsomes, previous reports have only investigated the mitochondrial activity. Transferase activity for acetyl- and octanoyl-CoA were about equal in peroxisomal and in microsomal fractions. A 200-fold purification of peroxisomal and microsomal carnitine acetyltransferases was achieved using O-(diethylaminoethyl)-cellulose and cellulose phosphate chromatography. This short-chain transferase preparation contained less than 5% as much carnitine octanoyltransferase and acyl-CoA deacylase activities. This fact, plus differences in solubility and stability of the microsomal transferase system for acetyl- and octanoyl-CoA indicate the existence of two separate enzymes: a carnitine acetyltransferase and a carnitine octanoyltransferase in peroxisomes and in microsomes.Peroxisomal and microsomal carnitine acetyltransferases had similar properties and could be the same protein. They showed identical chromatographic behavior and had the same pH activity profiles and major isoelectric points. They also had the same apparent molecular weight by gel filtration (59,000) and the same relative velocities and K_m values for several short-chain acyl-CoA substrates. Both were active with propionyl-, acetyl-, malonyl-, and acetyacetyl-CoA, but not with succinyl- and β-hydroxy-β-methylglutaryl-CoA as substrates.     

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.     

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K_m values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9 ± 2.1 μM and 13.9 ± 0.3 μM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits ～85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl–oleate formation without influencing the retinyl–palmitate formation. Using this inhibitor, we estimate that ～64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.     

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.     

Characterization of Methylglyoxal Synthase from Clostridium acetobutylicum ATCC 824 and Its Use in the Formation of 1,2-Propanediol

A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The K_m and V_max values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min⁻¹ μg⁻¹, respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.     

Lysophosphatidate Acyltransferase in the Microsomes from Maturing Seeds of Meadowfoam (Limnanthes alba)

Lysophosphatidate (LPA) acyltransferase (EC 2.3. 1.51) in the microsomes from the maturing seeds of meadowfoam (Limnanthes alba), nasturtium (Tropaeolum majus), palm (Syagrus cocoides), castor bean (Ricinus communis), soybean (Glycine max), maize (Zea mays), and rapeseed (Brassica napus) were tested for their specificities toward 1-oleoyl-LPA or 1-erucoyl-LPA, and oleoyl coenzyme A (CoA) or erucoyl CoA. All the enzymes could use either of the two acyl acceptors and oleoyl CoA, but only the meadowfoam enzyme could use erucoyl CoA as the acyl donor to produce dierucoyl phosphatidic acid (PA). The meadowfoam enzyme was studied further. It had an optimal activity at pH 7 to 8, and its activity was inhibited by 1 millimolar MnCl₂, ZnCl₂, or p-chloromercuribenzoate. In a test of substrate specificity using increasing concentrations of either 1-oleoyl-LPA or 1-erucoyl-LPA, and either oleoyl CoA or erucoyl CoA, the enzyme activity in producing PA was highest for dioleoyl-PA, followed successively by 1-oleoyl-2-erucoyl-PA, dierucoyl-PA, and 1-erucoyl-2-oleoyl-PA. In a test of substrate selectivity using a fixed combined concentration, but varying proportions, of 1-oleoyl-LPA and 1-erucoyl-LPA, and of oleoyl CoA and erucoyl CoA, the enzyme showed a pattern of acyl preference similar to that observed in the test of substrate specificity, but the preference toward oleoyl moiety in the substrates was slightly stronger. The meadowfoam microsomes could convert [¹⁴C]glycerol-3-phosphate to diacylglycerols and triacylglycerols in the presence of erucoyl CoA. The meadowfoam LPA acyltransferase is unique in its ability to produce dierucoyl-PA, and should be a prime candidate for use in the production of trierucin oils in rapeseed via genetic engineering.     

Regulation of ADP-Glucose Pyrophosphorylase from Chlorella vulgaris

ADP-glucose pyrophosphorylase was partially purified from Chlorella vulgaris 11h. 3-Phosphoglycerate activated the enzyme by lowering the Michaelis constant for glucose-1-phosphate (from 0.97 to 0.36 millimolar in the presence of 2 millimolar phosphoglycerate) and ATP (from 0.23 to 0.10 millimolar), as well as increasing the V_max. Saturation curves for 3-phosphoglycerate were hyperbolic and the activator concentration at half V_max value for 3-phosphoglycerate was 0.41 millimolar either in the presence or absence of phosphate. Phosphate inhibited the enzyme in a competitive manner with respect to glucose-1-phosphate, but did not affect the Michaelis constant value for ATP. 3-Phosphoglycerate changed neither the inhibitor concentration at half V_max value of 1.0 millimolar for phosphate nor the hyperbolic inhibition kinetics for phosphate. The enzyme required divalent cations for its activity. The activation curves for Mn²⁺ and Mg²⁺ were highly sigmoidal. The activator concentration at half V_max values for Mn²⁺ and Mg²⁺ were 2.8 and 3.7 millimolar, respectively. With optimal cations, the Michaelis constant values for ATP-Mn and ATP-Mg were 0.1 and 0.4 millimolar, respectively.     

Partial purification and specificity of triacylglycerol: Sterol acyltransferase from Sinapis alba

Triacylglycerol: sterol acyltransferase is present in roots of Sinapis alba seedlings. The enzyme is located predominantly in the cell membrane structures sedimenting at 300–16 000 g but can be solubilized by acetone treatment and buffer extraction. During gel filtration on Sephadex G-100 the acyltransferase activity was separated into two peaks corresponding to MW 1.8 × 10¹⁴ and MW ? 10⁵, respectively. A number of natural 3β-hydroxysterols can be esterified by the solubilized acyltransferase. The rate of esterification is much higher for sterols containing a planar ring system. The number and position of double bonds, as well as the structure of the side chain at C- 17 of the sterol molecule, are of secondary importance. Triacylglycerols containing fatty acids C, C₆-C₂₂ can be utilized as acyl donors. Among triacylglycerols containing saturated fatty acids, tripalmitoylglycerol (C_16:0) is the best acyl donor. For triacylglycerols containing C₁₈-fatty acids the following sequence was observed: trioleoylglycerol (C_18:1) > trilinoleoylglycerol (C_18:2) > trilinolenoylglycerol (C_18:3) > tristearoylglycerol (C_18:0).     

Glycerolipid synthetic capacity of rat liver peroxisomes

Investigations on rat liver peroxisomal glycerolipid synthetic capability were performed. Highly purified peroxisomal preparations contained dihydroxyacetone-phosphate acyltransferase, acyldihydroxyacetone-phosphate reductase, and fatty acid-CoA ligase activities. Glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, diacylglycerol cholinephosphotransferase, diacylglycerol ethanolaminephosphotransferase and ethanol acyltransferase activities were low in activity or not detected. These results suggest that the peroxisomes are specialized to contribute to the synthesis of ether-linked glycerolipids. If peroxisomes contribute towards the synthesis of non-ether-linked glycerolipids (i.e., ester-linked) then translocation of acyl glycerophosphatide (acyl dihydroxyacetone phosphatide) from peroxisomes to endoplasmic reticulum would be expected to occur.     

Purification and properties of dihydroxyacetone kinase from Gluconobacter suboxydans

Different carbon and nitrogen sources had little effect on the level of dihydroxyacetone kinase formed in the cells of Gluconobacter suboxydans. The enzyme was purified to homogeneity from cell-free extract of the organism by ammonium sulfate fractionation and chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200 (60-fold purification, 6% yield). Its molecular weight was 260,000; it was stabilized by addition of ATP, dithiothreitol, 2-mercaptoethanol or EDTA, and it reacted optimally at pH 6.5. d-Glyceraldehyde was equally as effective as DHA as a phosphate acceptor (K_m: 0.30 mM each). UTP showed 15% of the reactivity of ATP as a phosphate donor. K_m values for ATP were 0.33 mM in phosphorylation of dihydroxyacetone and 0.39 mM with d-glyceraldehyde. The enzyme activity was dependent on Mg²⁺ but not on Mn²⁺. The reaction with dihydroxyacetone as an acceptor was inhibited by d-glyceraldehyde. The inhibition was competitive with respect to dihydroxyacetone 3K_i=0.09 mM) and noncompetitive with respective to ATP (K_i=5.7 mM).     

Purification and characterization of a secreted purple phosphatase from soybean suspension cultures

We purified and partially sequenced a purple (λ_max = 556 nanometers) acid phosphatase (APase; EC 3.1.3.2) secreted by soybean (Glycine max) suspension-culture cells. The enzyme is a metalloprotein with a Mn²⁺ cofactor. This APase appears to be a glycoprotein with a monomer subunit molecular weight of 58,000 and an active dimer molecular weight of approximately 130,000. The protein has an isoelectric point of about 5.0 and a broad pH optimum centered near 5.5. The purified enzyme, assayed with p-nitrophenyl phosphate as the substrate, has a specific activity of 512 units per milligram protein and a K_m of approximately 0.3 millimolar; phosphate is a competitive inhibitor with a K_i of 0.7 millimolar. This APase is similar to one found in soybean seed meal but dissimilar to that found in soybean seedlings.     

Isolation of a sn-glycerol 3-phosphate: NAD oxidoreductase from spinach leaves.

An NAD-dependent glycerol 3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD oxidoreductase; EC 1.1.1.8) has been purified from spinach leaves by a three-step procedure involving ion-exchange, gel filtration, and affinity chromatography. The enzyme has been purified over 10,000-fold to a specific activity of 38. It has a molecular weight of approximately 63,500. The pH optimum for the reduction of dihydroxyacetone phosphate is 6.8 and for glycerol 3-phosphate oxidation it is 9.5. During dihydroxyacetone phosphate reduction hyperbolic kinetics were observed when either NADH or dihydroxyacetone phosphate was the variable substrate, but concentrations of NADH greater than 150 μm were inhibitory. Michaelis constants were 0.30–0.35 mm for dihydroxyacetone phosphate and 0.01 mm for NADH. Glycerol 3-phosphate oxidation obeyed Michaelis-Menten kinetics with a K_m of 0.19 mm for NAD and 1.6 mm for glycerol 3-phosphate. The enzyme was specific for those substrates, and dihydroxyacetone, glyceraldehyde, glyceraldehyde 3-phosphate, NADPH, NADP, and glycerol were not utilized. The spinach leaf enzyme appears to be in the cytoplasm and probably functions for the production of glycerol 3-phosphate from dihydroxyacetone phosphate.     

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.     

Changes in the activities of dihydroxyacetone phosphate and glycerol-3-phosphate acyltransferases in rat liver under various conditions

Activities of enzymes relating to the acyl dihydroxyacetone phosphate (acyl DHAP) pathway were determined in rat liver under conditions known to elevate the peroxisomal β-oxidation activity. In fasted and streptozotocin-induced diabetic rats, DHAP acyltransferase activity showed a small but significant increase, though the activities of glycerol-3-phosphate (GP) acyltransferase and alkyl DHAP synthase were not changed. After 2 weeks, feeding of 20% partially hydrogenated marine oil, the activity of DHAP acyltransferase also increased to 140% of the control. The feeding of 0.25% clofibrate and 2% di(2-ethylhexyl)phthalate (DEHP) increased the activities of both DHAP and GP acyltransferases by 2- to 3-fold, whereas alkyl DHAP synthase activity decreased under the same conditions. A fractionation study showed that the increases in the activities of DHAP acyltransferase and acyl /alkyl DHAP reductase in the liver of rats treated with DEHP occurred mainly in peroxisomes and microsomes, respectively. The phospholipid contents per mg protein of the isolated hepatic peroxisomes from rats were as follows (percent of the control): fasting, 62%; diabetic, 69%; high fat-diet, 89%; clofibrate-treated, 126%; DEHP-treated, 119%. These results suggest that glycerophospholipid metabolism might also be controlled by peroxisomal enzymes under physiological and pathological conditions.     

Purification and characterization of an acid phosphatase from Trichoderma harzianum

An acid phosphatase from Trichoderma harzianum was purified in a single step using a phenyl-Sepharose chromatography column. A typical procedure showed 22-fold purification with 56% yield. The purified enzyme showed as a single band on SDS-PAGE with an apparent molecular weight of 57.8 kDa. The pH optimum was 4.8 and maximum activity was obtained at 55°C. The enzyme retained 60% of its activity after incubation at 55°C for 60 min. The K _m and V _max values for p-nitrophenyl phosphate (p-NPP) as a substrate were 165 nM and 237 nM min^?1, respectively. The enzyme was partially inhibited by inorganic phosphate and strongly inhibited by tungstate. Broad substrate specificity was observed with significant activities for p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate and phenyl phosphate.