Identification, purification, and characterization of a thermally stable lipase from rice bran. A new member of the (phospho) lipase family.

A thermally stable lipase (EC 3.1.1.3.) was first identified in rice (Oryza sativa) bran, and the enzyme was purified to homogeneity using octyl-Sepharose chromatography. The enzyme was purified to 7.6-fold with the final specific activity of 0.38 micromol min(-1) mg(-1) at 80 degrees C using [9,10-(3)H]triolein as a substrate. The purified enzyme was found to be a glycoprotein of 9.4 kD. Enzyme showed a maximum activity at 80 degrees C and at pH 11.0. The protein was biologically active and retained most of its secondary structure even at 90 degrees C as judged by the enzymatic assays and far-ultraviolet circular dichroism spectroscopy, respectively. Differential scanning calorimetric studies indicated that the transition temperature was 76 degrees C and enthalpy 1.3 x 10(5) Calorie mol(-1) at this temperature. The purified lipase also exhibited phospholipase A(2) activity. Colocalization of both the hydrolytic activities in reverse-phase high-performance liquid chromatography and isoelectric focusing showed that the dual activity was associated with a single protein. Further, a direct interaction between both the substrates and the purified protein was demonstrated by photoaffinity labeling, using chemically synthesized analogs of triolein and phosphatidylcholine (PC). Apparent K(m) for triolein (6.71 mM) was higher than that for PC (1.02 mM). The enzyme preferentially hydrolyzed the sn-2 position of PC, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate inhibited both lipase and phospholipase activities of the purified enzyme. This enzyme is a new member from plants in the family of lipases capable of hydrolyzing phospholipids.

Purification and characterization of acyl-acyl carrier protein synthetase from oleaginous yeast and its role in triacylglycerol biosynthesis.

Fatty acids are activated in an ATP-dependent manner before they are utilized. We describe here how the 10 S triacylglycerol biosynthetic multienzyme complex from Rhodotorula glutinis is capable of activating non-esterified fatty acids for the synthesis of triacylglycerol. The photolabelling of the complex with [(32)P]azido-ATP showed labelling of a 35 kDa polypeptide. The labelled polypeptide was identified as acyl-acyl carrier protein (ACP) synthetase, which catalyses the ATP-dependent ligation of fatty acid with ACP to form acyl-ACP. The enzyme was purified by successive PAGE separations to apparent homogeneity from the soluble fraction of oleaginous yeast and its apparent molecular mass was 35 kDa under denaturing and reducing conditions. Acyl-ACP synthetase was specific for ATP. The K(m) values for palmitic, stearic, oleic and linoleic acids were found to be 42.9, 30.4, 25.1 and 22.7 microM, respectively. The antibodies to acyl-ACP synthetase cross-reacted with Escherichia coli acyl-ACP synthetase. Anti-ACP antibodies showed no cross-reactivity with the purified acyl-ACP synthetase, indicating no bound ACP with the enzyme. Immunoprecipitations with antibodies to acyl-ACP synthetase revealed that this enzyme is a part of the 10 S triacylglycerol biosynthetic complex. These results demonstrate that the soluble acyl-ACP synthetase plays a novel role in activating fatty acids for triacylglycerol biosynthesis in oleaginous yeast.

Identification, purification, and characterization of monoacylglycerol acyltransferase from developing peanut cotyledons.

Biosynthesis of diacylglycerols in plants occurs mainly through the acylation of lysophosphatidic acid in the microsomal membranes. Here we describe the first identification of diacylglycerol biosynthetic activity in the soluble fraction of developing oilseeds. This activity was NaF-insensitive and acyl-CoA-dependent. Diacylglycerol formation was catalyzed by monoacylglycerol (MAG) acyltransferase (EC ) that transferred an acyl moiety from acyl-CoA to MAG. The enzyme was purified by successive chromatographic separations on octyl-Sepharose, blue-Sepharose, Superdex-75, and palmitoyl-CoA-agarose to apparent homogeneity from developing peanut (Arachis hypogaea) cotyledons. The enzyme was purified to 6,608-fold with the final specific activity of 15.86 nmol min(-1) mg(-1). The purified enzyme was electrophoretically homogeneous, and its molecular mass was 43,000 daltons. The purified MAG acyltransferase was specific for MAG and did not utilize any other acyl acceptor such as glycerol, glycerol-3-phosphate, lysophosphatidic acid, and lysophosphatidylcholine. The K(m) values for 1-palmitoylglycerol and 1-oleoylglycerol were 16.39 and 5.65 micrometer, respectively. The K(m) values for 2-monoacylglycerols were 2- to 4-fold higher than that of the corresponding 1-monoacylglycerol. The apparent K(m) values for palmitoyl-, stearoyl-, and oleoyl-CoAs were 17.54, 25.66, and 9.35 micrometer, respectively. Fatty acids, phospholipids, and sphingosine at low concentrations stimulated the enzyme activity. The identification of MAG acyltransferase in oilseeds suggests the presence of a regulatory link between signal transduction and synthesis of complex lipids in plants.

Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast.

Triacylglycerol is one of the major storage forms of metabolic energy in eukaryotic cells. Biosynthesis of triacylglycerol is known to occur in membranes. We report here the isolation, purification, and characterization of a catalytically active cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis from Rhodotorula glutinis during exponential growth. The complex was characterized and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S triacylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase from the complex were microsequenced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase sequences. Immunoprecipitation and immunolocalization studies show the presence of a cytosolic multienzyme complex for triacylglycerol biosynthesis. Chemical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate that the cytosol is one of the sites for triacylglycerol biosynthesis in oleaginous yeast.

Synthesis of azidophospholipids and labeling of lysophosphatidylcholine acyltransferase from developing soybean cotyledons.

A photoreactive substrate analog of lysophosphatidylcholine (LPC), 1-([(4-azidosalicyl)-12-amino)]dodecanoyl-sn-glycerol-3-phospho cholin e (azido-LPC) was synthesized. Fast atom bombardment mass spectrometry was employed to confirm the structures of azido-LPC and its intermediates. Azido-LPC was used to label putative acyl-CoA:LPC acyltransferase from microsomal membranes of developing soybean cotyledons. The synthesized substrate analog acts as a substrate for the target acyltransferases and phospholipases in the dark. When the microsomal membranes were incubated with the acyl acceptor analog and immediately photolyzed, LPC acyltransferase was irreversibly inhibited. Photoinactivation of the enzyme by the photoprobe decreased in the presence of LPC. Microsomal membranes were photolyzed with 125I-labeled azido-LPC and analyzed by SDS-PAGE followed by autoradiography. These revealed that the analog preferentially labeled 54- and 114-kDa polypeptides. Substrate protected the labeling of both the polypeptides. In our earlier report, the same polypeptides were also labeled with photoreactive acyl-CoA analogs, suggesting that these polypeptides could be putative LPC acyltransferase(s). These results demonstrated that the photoreactive phospholipid analog could be a powerful tool to label acyltransferases involved in lipid biosynthesis.

Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.

Lipid peroxidation and ethanol-related tumor promotion in Fischer-344 rats treated with tobacco-specific nitrosamines.

Male Fischer-344 rats were treated, by gavage, with a total dose of 40 mmol/kg of N'-nitrosonornicotine (NNN) or 20 mmol/kg of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), three times a week for 4 weeks. One week afterwards the rats were fed an isocaloric liquid diet containing 7% (v/v) ethanol and continued on this diet until killed. Cumulative ethane exhaled by a rat by 180 min was measured at 54 weeks of the start of the study and was found to increase significantly (P < 0.001) with either NNN or NNK treatment but more so when followed by ethanol consumption. Other indices of lipid peroxidation, cholesterol and phospholipids were measured in the lipid extracts from the liver, esophagus and lungs at 55 weeks. Ethanol consumption increased the amount of cholesterol and phospholipids per g of tissue in naïve or NNN- and NNK-treated rats. All peroxidative indices measured, i.e. malondialdehyde (MDA), diene- and triene-conjugates and lipid fluorescence, were significantly increased in the liver, the main metabolic and peroxidative site, with ethanol consumption in rats whether they were treated with NNN or NNK or remained untreated. Overall, the indices of lipid peroxidation also showed an increase in other tissues, but the results differed with different indices. The differences in indices may be due to differences in lipid peroxidation products measured or to differences in their rates of production and degradation or conversion to other products. However, the largest increases in indices were seen with ethanol consumption by either NNN- or NNK-treated rats. Incidence of tumors in the tissues was also assessed and showed about a two-fold increase with ethanol consumption in the tumors of esophagus, oral cavity, lungs and liver induced by either NNN or NNK. Ethanol also caused an increase in the mean frequency and mean size of the tumors induced. The results suggest that ethanol-related promotion of NNN- and NNK-induced tumors may result from increased lipid peroxidation in the target tissue.

Use of photoreactive substrates for characterization of lysophosphatidate acyltransferases from developing soybean cotyledons.

Photoreactive lipid analogs, namely, 1-acyl-2-(12-azidooleoyl)glycero-3-phosphocholine (N3-PC) and 1-acyl-2-(12-azidooleoyl)glycero-3-phosphoethanolamine (N3-PE) have been synthesized as previously described [R. Rajasekharan and J. D. Kemp (1994) J. Lipid Res. 35, 45-51]. Azidophosphatidic acid was produced by hydrolyzing N3-PC with phospholipase D. All of the lysophospholipid analogs, 2-(12-azidooleoyl)glycero-3-phosphate (N3-LPA), 2-(12-azidooleoyl)glycero-3-phosphocholine (N3-LPC), and 2-(12-azidooleoyl)glycero-3-phosphoethanolamine (N3-LPE), were produced from appropriate azidophospholipids by lipase treatment. The photoactive lysophospholipid analogs were recognized as substrates by acyltransferases in the dark and as irreversible inhibitors after photolysis with uv light. The photoinactivation of acyltransferases by azidolysophospholipids was protected by the addition of natural lysophospholipids. Incubation of developing soybean microsomal membranes with N3-LPA followed by photolysis resulted in 69% inhibition of lysophosphatidic acid (LPA) acyltransferase and also had significant inhibitory effects on lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) acyltransferases, indicating that the LPA analog interacts with all the lysophospholipid acyltransferases. When the membranes were photolyzed with N3-LPC or N3-LPE and assayed, the membranes showed approximately 50% inactivation of LPC and LPE acyltransferase activities, whereas LPA acyltransferase was unaffected, suggesting that a single enzyme might acylate both LPC and LPE. The recognition of these photoreactive lipid analogs by acyltransferases will facilitate the identification and purification of these membrane-bound enzymes.

Photolabeling of soybean microsomal membrane proteins with photoreactive acyl-CoA analogs.

Synthesis of 32P-labeled 12-azidooleoyl-CoA and 125I-labeled 12-[(azidosalicyl)amino]dodecanoyl-CoA (ASD-CoA) was achieved. The synthesized radioactive, photoreactive reagents were tested as photoaffinity labels for acyl-CoA:lysophosphatidylcholine acyltransferase from the microsomal membranes of developing soybean cotyledons. When a mixture of microsomal membranes and the azidooleoyl-CoA or ASD-CoA were incubated in the dark, the analogs were recognized as substrate and competitive inhibitor, respectively. The enzyme preferentially utilizes unsaturated acyl-CoAs rather than saturated acyl-CoAs. Incubation of microsomal membranes with acyl-CoA analogs and immediately followed by photolysis resulted in an irreversible inhibition of lysophosphatidylcholine acyltransferase activity. Analysis of photolyzed microsomal membranes by SDS/PAGE and autoradiography revealed that azidooleoyl-CoA preferentially labeled eight acyl-CoA binding proteins, but ASD-CoA labeled only three polypeptides with molecular masses of 110, 90 and 32 kDa that are commonly labeled by both the analogs. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme against photoinactivation by azidooleoyl-CoA and ASD-CoA, respectively. The protection was profound in 110-kDa polypeptide indicating that this protein could be lysophosphatidylcholine acyltransferase. These results demonstrate that the photoaffinity of acyl-CoA analogs makes them potential probes to identify and characterize lipid biosynthetic enzymes.

Synthesis of photoreactive phosphatidylethanolamine and its interaction with phospholipase A2.

A photoreactive derivative of phosphatidylethanolamine, N-(4-azidobenzoyl)phosphatidylethanolamine (AB-PE), was synthesized by acylation of phosphatidylethanolamine with an N-hydroxysuccinimide ester of 4-azidobenzoic acid. The substantial photosensitivity exhibited by AB-PE correlated with a marked decrease in the absorption spectra of the compound. The compound proved sensitive to lipase and phospholipase A2 hydrolysis but resistant to phospholipase C and D activities. Photolysis of a sonicated dispersion of AB-PE containing phospholipase A2 resulted in irreversible inhibition of the enzyme. Addition of natural phosphatidylethanolamine provided protection against photoinactivation.

Photoaffinity labeling of acyl-CoA oxidase with 12-azidooleoyl-CoA and 12-[(4-azidosalicyl)amino]dodecanoyl-CoA.

Synthesis of 32P-labeled CoA of high specific activity was achieved using partially purified dephospho-CoA kinase (EC 2.7.1.24) from pig liver with [gamma-32P]ATP as donor and dephospho-CoA as acceptor. A photoaffinity dodecanoic acid analog, 12-[(4-azidosalicyl)amino]dodecanoic acid was synthesized, as were its CoA derivative (ASD-CoA) and the CoA derivative of 12-azidooleic acid. The CoA derivatives were synthesized from azido fatty acid analogs by acyl-CoA synthetase. The synthesized photolabile reagents were tested as photoaffinity labels for acyl-CoA oxidase (EC 1.3.99.3) from Arthrobacter species. When a mixture of oxidase and the acyl-CoA analogs were incubated in the absence of ultraviolet light, the analogs were recognized as substrate. Acyl-CoA oxidase was incubated in the presence of acyl-CoA analogs and immediately photolyzed, which resulted in irreversible inhibition. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme from photoactivated inhibition by 12-azidooleoyl-CoA and ASD-CoA, respectively. Analysis of photolyzed enzyme preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that both analogs preferentially labeled a 54,000 molecular weight protein. These results demonstrate that the photoaffinity acyl-CoA analogs have potential application as probes to identify and characterize lipid biosynthetic enzymes and to identify the active site of these proteins.

Substrate-mediated purification and characterization of a 3-hydroxybenzoic acid-6-hydroxylase from Micrococcus.

3-Hydroxybenzoic acid-6-hydroxylase from Micrococcus sp. was purified to homogeneity in a single step using the substrate-mediated interaction of the enzyme with blue-Sepharose. The enzyme was bound to the affinity matrix in the presence of 3-hydroxybenzoic acid and was eluted in its absence. The molecular weight of the purified enzyme is 70,000 with no subunit structure. The flavoenzyme required the exogenous addition of FAD for its complete activity and had a strict preference for NADH over NADPH. The activity of the enzyme was drastically inhibited by Cu2+ and Hg2+ and the inhibition was reversed by thiol reagents.

A direct nonchromatographic assay for 1-acyl-sn-glycerol-3-phosphate acyltransferase.

1-Acyl-sn-glycerol-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase) which catalyzes the acylation of 1-acyl-sn-glycerol-3-phosphate to phosphatidic acid is generally assayed by the use of a radioactive substrate followed by a time-consuming chromatographic separation of substrate and product. We report a direct and highly sensitive nonchromatographic assay for this enzyme based on the ability of Escherichia coli alkaline phosphatase to dephosphorylate 1-acyl-sn-glycerol-3-phosphate but not phosphatidic acid. This selective hydrolysis coupled with the use of 32P-labeled 1-acyl-sn-glycerol-3-phosphate as substrate permits measurement of the product, 32P-labeled phosphatidic acid by solvent extraction or precipitation. We also report a series of enzymatic reactions for the efficient conversion of 32Pi to 32P-labeled 1-acyl-sn-glycerol-3-phosphate.