Identification of odoriferous sulfanylalkanols in human axilla secretions and their formation through cleavage of cysteine precursors by a C-S lyase isolated from axilla bacteria

Human axillary odor is known to be formed upon the action of Corynebacteria sp. on per se odorless axilla secretions. Besides the known odoriferous acids, we report the occurrence in human axilla secretions of four odoriferous sulfanylalkanols, namely 3-sulfanylhexan-1-ol (3), 2-methyl-3-sulfanylbutan-1-ol (4), 3-sulfanylpentan-1-ol (5), and 3-methyl-3-sulfanylhexan-1-ol (6). These compounds have a pungent sweat/kitchen odor, also reminiscent of onions with some fruity connotations, and perception thresholds in the pg/l range. It was postulated that the odorless precursors for these compounds are cysteine conjugates. Bacterial isolates obtained from the human axilla and belonging to the Corynebacteria were, indeed, found to have the enzymatic capacity to release various thiols from cysteine conjugates. The metC gene, which is known to code for a cystathione-beta-lyase, was cloned from the axilla isolate Corynebacterium striatum Ax20 and heterologously expressed in E. coli. The pure recombinant enzyme cleaves various cysteine conjugates and has a similar substrate specificity as the cell homogenates of the wild-type. The recombinant enzyme was finally incubated with odorless axilla secretions and shown to release odoriferous thiols.     

A broad diversity of volatile carboxylic acids, released by a bacterial aminoacylase from axilla secretions, as candidate molecules for the determination of human-body odor type

Human body odor is to a large part determined by secretions of glands in the axillary regions. Two key odoriferous principles, 3-methylhex-2-enoic acid (3MH2; 4/5) and 3-hydroxy-3-methylhexanoic acid (HMHA; 6) have been shown to be released from glutamine conjugates secreted in the axilla by a specific N(alpha)-acyl-glutamine aminoacylase (N-AGA) obtained from axilla isolates of Corynebacteria sp. However, the low number of different odorants reported in humans stands in contrast to the observed high inter-individual variability in body odors. Axilla secretions of individual donors were, therefore, analyzed in detail. The secretions were treated with N-AGA, analyzed by GC/MS, and compared to undigested controls. Over 28 different carboxylic acids were released by this enzyme from odorless axilla secretions (Table 1). Many of these body odorants have not been reported before from a natural source, and they include several aliphatic 3-hydroxy acids with 4-Me branches, 3,4-unsaturated, 4-Et-branched aliphatic acids, and a variety of degradation products of amino acids. The odor threshold of some of the acids was found to be in the range of 1 ng. Most of these compounds were present in all donors tested, but in highly variable relative amounts, and they are, thus, candidate molecules as key components of a 'compound odor' determining the individual types of human body odor.     

The sequential action of a dipeptidase and a beta-lyase is required for the release of the human body odorant 3-methyl-3-sulfanylhexan-1-ol from a secreted Cys-Gly-(S) conjugate by Corynebacteria

Human axillary odor is formed by the action of Corynebacteria on odorless axilla secretions. Sulfanylalkanols, 3-methyl-3-sulfanylhexan-1-ol in particular, form one key class of the odoriferous compounds. A conjugate with the dipeptide Cys-Gly has been reported as the secreted precursor for 3-methyl-3-sulfanylhexan-1-ol. Here, we confirm the Cys-Gly-(S) conjugate as the major precursor of this odorant, with lower levels of the Cys-(S) conjugate being present in axilla secretions. The enzymatic release of 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate by the axilla isolate Corynebacterium Ax20 was thus investigated. Cellular extracts of Ax20 released 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate and from the Cys-(S) conjugate, whereas the previously isolated C-S lyase of this bacterial strain was only able to cleave the Cys-(S) conjugate. o-Phenanthroline blocked the release from the Cys-Gly-(S) conjugate but did not affect cleavage of the Cys-(S) conjugate, indicating that in a first step, a metal-dependent dipeptidase hydrolyzes the Cys-Gly bond. This enzyme was purified by four chromatographic steps and gel electrophoresis, and the partial amino acid sequence was determined. The corresponding gene was cloned and expressed in Escherichia coli. It codes for a novel dipeptidase with a high affinity toward the Cys-Gly-(S) conjugate of 3-methyl-3-sulfanylhexan-1-ol. Co-incubating either the synthetic Cys-Gly-(S) conjugate or fresh axilla secretions with both the C-S lyase and the novel dipeptidase did release 3-methyl-3-sulfanylhexan-1-ol, proving that the sequential action of these two enzymes from the skin bacterium Corynebacterium Ax20 does release the odorant from the key secreted precursor.     

Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements

The x-ray structure of the EcoRI endonuclease-DNA complex (3) suggests that hydrogen bonds between amino acids, glutamic acid 144, arginine 145, and arginine 200, and major groove base moieties are the molecular determinants of specificity. We have investigated residue 144 using aspartate and glutamine substitutions introduced by site-directed mutagenesis. Substitution with glutamine results in a null phenotype (at least a 2000-fold reduction in activity). On the other hand, the aspartic acid mutant (ED144) retained in vivo activity. Substrate binding and catalytic studies were done with purified ED144 enzyme. The affinity of the ED144 enzyme for the canonical sequence 5'-GAATTC-3' is about 340-fold less than the wild-type (WT) enzyme, while its affinity for nonspecific DNA is about 50 times greater. The ED144 enzyme cleaves one strand in the EcoRI site in plasmid pBR322 with a kcat/Km similar to WT. In contrast to the WT enzyme, the ED144 enzyme dissociates after the first strand cleavage. Partitioning between cleavage and dissociation at the first and second cleavage steps for the ED144 enzyme is extremely salt-sensitive. The altered partitioning results largely from a destabilization of the enzyme-DNA complex, particularly the enzyme-nicked DNA complex, with only small changes in the respective cleavage rates. The hydrogen bonds of Glu-144 are critical, they appear to act cooperatively with other specificity contacts to stabilize the enzyme-DNA complex.     

Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from the novel marine isolate, JAMB-A94

A gene, agaA, for a novel beta-agarase from the marine bacterium JAMB-A94 was cloned and sequenced. The 16S rDNA of the isolate had the closest match, of only 94.8% homology, with that from Microbulbifer salipaludis JCM11542(T). The agaA gene encoded a protein with a calculated molecular mass of 48,203 Da. The deduced amino acid sequence showed 37-66% identity to those of known agarases in glycoside hydrolase family 16. A carbohydrate-binding module-like amino acid sequence was found in the C-terminal region. The recombinant enzyme was hyper-produced extracellularly when Bacillus subtilis was used as a host. The purified enzyme was an endo-type beta-agarase, yielding neoagarotetraose as the main final product. It was very thermostable up to 60 degrees C. The optimal pH and temperature for activity were around 7.0 and 55 degrees C respectively. The activity was not inhibited by EDTA (up to 100 mM) and sodium dodecyl sulfate (up to 30 mM).     

Purification and characterization of a bioluminescence-related fatty acyl esterase from Vibrio harveyi

Vibrio harveyi extracts contain three polypeptides (32, 42, and 57 kDa) which are involved in long-chain aldehyde biosynthesis and can be labeled with [3H] tetradecanoic acid (+ATP) and/or [3H]tetradecanoyl-CoA. These proteins have been separated from other labeled bands by ammonium sulfate fractionation, and the 32-kDa polypeptide has been further purified to homogeneity by ion-exchange, gel filtration, and hydroxylapatite chromatography. In aqueous buffers at pH 7, the 32-kDa protein catalyzes the hydrolysis of tetradecanoyl-CoA at a low rate (0.01 mumol/min/mg) to form free fatty acids. The thioesterase rate is slightly increased by phosphate, which also protects the enzyme against inhibition by the sulfhydryl reagent N-ethylmaleimide. Acyl-CoA cleavage is dramatically stimulated (up to 100-fold) by certain organic solvents, in particular glycerol and ethylene glycol, with the fatty acyl group being transferred to the alcohol acceptors. These enzymatic properties may be related to the role of the 32-kDa esterase in generating fatty acids for subsequent use in the V. harveyi bioluminescent system.     

Brain Endo-Oligopeptidase A, a Putative Enkephalin Converting Enzyme

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterized as a thiol endopeptidase. This enzyme, known to hydrolyze the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin, has been shown to produce, by a single cleavage, Leu5-enkephalin or Met5-enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration-dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be eight to 13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin, and dynorphin B are of the same order, a result indicating that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme.     

Ether glycerolipids: novel substrates for studying specificity of enzymes involved in glycerolipid biosynthesis in higher plants

Ether glycerolipids, predominantly alkylacylglycerols and alkylacylglycerophosphocholines, are synthesized in photomixotrophic rape (Brassica napus) suspension cells from various exogenous monoalkylglycerols. The stereospecific distribution of acyl moieties was studied in these ether glycerolipids with regard to chain-length and degree of unsaturation of alkyl moieties and compared with the distribution of acyl moieties in the corresponding endogenous acyl glycerolipids. The results show the following: (1) Alkylacylglycerophosphocholines replaced up to one-half of the corresponding physiological membrane lipids, i.e. diacylglycerophosphocholines, without changing the total amount of cholineglycerophospholipids as compared to untreated cells. (2) The composition of acyl moieties in total lipids of rape cells was practically unaltered by fatty acids derived via oxidative cleavage from the various alkyl moieties of either glycerolipids. (3) In 1-O-alkyl-2-acylglycerols derived from exogenous alkylglycerols and in endogenous 1,2-diacylglycerols compositions of acyl moieties were found to be different indicating that different pathways were operative in the biosynthesis of these two neutral glycerolipids. (4) Enzymes involved in synthesizing molecular species of 1-O-alkyl-2-acylglycerophosphocholines or 2-O-alkyl-1-acylglycerophosphocholines as well as 1,2-diacylglycerophosphocholines showed similar specificities with regard to chain-length and degree of unsaturation of both alkyl and corresponding acyl moieties. Thus, ether glycerolipids formed by plant cells from exogenous alkylglycerols are suitable metabolites for studying the specificity of enzymes involved in the biosynthesis of glyerolipids.     

Uclacyanins,stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: plant-specific mononuclear blue copper proteins.

The cDNAs encoding plantacyanin from spinach were isolated and characterized. In addition, four new cDNA sequences from Arabidopsis ESTs were identified that encode polypeptides resembling phytocyanins, plant-specific proteins constituting a distinct family of mononuclear blue copper proteins. One of them encodes plantacyanin from Arabidopsis, while three others, designated as uclacyanin 1, 2, and 3, encode protein precursors that are closely related to precursors of stellacyanins and a blue copper protein from pea pods. Comparative analyses with known phytocyanins allow further classification of these proteins into three distinct subfamilies designated as uclacyanins, stellacyanins, and plantacyanins. This specification is based on (1) their spectroscopic properties, (2) their glycosylation state, (3) the domain organization of their precursors, and (4) their copper-binding amino acids. The recombinant copper binding domain of Arabidopsis uclacyanin 1 was expressed, purified, and shown to bind a copper atom in a fashion known as "blue" or type 1. The mutant of cucumber stellacyanin in which the glutamine axial ligand was substituted by a methionine (Q99M) was purified and shown to possess spectroscopic properties similar to uclacyanin 1 rather than to plantacyanins. Its redox potential was determined by cyclic voltammetry to be +420 mV, a value that is significantly higher than that determined for the wild-type protein (+260 mV). The available structural data suggest that stellacyanins (and possibly other phytocyanins) might not be diffusible electron-transfer proteins participating in long-range electron-transfer processes. Conceivably, they are involved in redox reactions occurring during primary defense responses in plants and/or in lignin formation.     

The transamination of L-cystathionine, L-cystine and related compounds by a bovine kidney transaminase

An enzyme which actively transaminates L-cystathionine, L-cystine, L-lanthionine and S-aminoethyl-L-cysteine has been purified from bovine kidney. The transaminase appears to be pure up to 90% and probably consists of two subunits of similar molecular mass of about 47 kDa. The enzymatic products arising from the transamination of L-cystathionine and related compounds spontaneously cyclize into ketiminic structures, which are the immediate precursors of unusual imino acids recovered in biological materials. The specificity towards other amino acid and oxo acid acceptors is similar to the specificity exhibited by rat kidney glutamine transaminase. This suggests that the sulfur amino acid transaminations that have been described could be performed by the bovine kidney glutamine transaminase.     

A novel and efficient enzymatic method for the production of peptides from unprotected starting materials

We report herein the development of a novel and efficient enzymatic method for the production of oligopeptides. This newly discovered method is a simple, cost-effective process, using unprotected amino acids as substrates in an aqueous solution and producing peptides in high yield. The target of our initial screen was l-alanyl-L-glutamine, a dipeptide of significant industrial interest by virtue of its widespread use in infusion therapy. By means of the screening of microorganisms that can catalyze the peptide-forming reaction producing l-alanyl-L-glutamine from L-alanine methylester (acyl donor) and L-glutamine (nucleophile), we discovered that Empedobacter brevis ATCC 14234 produced l-alanyl-L-glutamine most efficiently. The newly found enzyme purified from E. brevis ATCC 14234 facilitates significantly high production yields of l-alanyl-L-glutamine from L-alanine methylester and L-glutamine in an aqueous solution--more than 80% yield based on L-alanine methylester. In addition, this enzyme has wide substrate specificity--both for acyl donors and nucleophiles--and can catalyze peptide-forming reactions not only to produce various dipeptides from the corresponding amino acid esters and amino acids but also to produce various oligopeptides from the corresponding amino acid esters and peptides.     

Folate synthesis in plants: purification, kinetic properties, and inhibition of aminodeoxychorismate synthase

pABA (p-aminobenzoate) is a precursor of folates and, besides esterification to glucose, has no other known metabolic fate in plants. It is synthesized in two steps from chorismate and glutamine, the first step being their conversion into glutamate and ADC (4-aminodeoxychorismate). In Escherichia coli, two proteins forming a heterodimeric complex are required for this reaction, but, in plants and lower eukaryotes, a single protein is involved. The Arabidopsis enzyme was expressed in E. coli and was purified to homogeneity. The monomeric enzyme (95 kDa) catalyses two reactions: release of NH3 from glutamine (glutaminase activity) and substitution of NH3 for the hydroxy group at position 4 of chorismate (ADC synthase activity). The kinetic parameters of the plant enzyme are broadly similar to those of the bacterial complex, with K(m) values for glutamine and chorismate of 600 and 1.5 microM respectively. As with the bacterial enzyme, externally added NH3 was a very poor substrate for the plant enzyme, suggesting that NH3 released from glutamine is preferentially channelled to chorismate. The glutaminase activity could operate alone, but the presence of chorismate increased the efficiency of the reaction 10-fold, showing the interdependency of the two domains. The plant enzyme was inhibited by dihydrofolate and its analogue methotrexate, a feature never reported for the prokaryotic system. These molecules were inhibitors of the glutaminase reaction, competitive with respect to glutamine (K(i) values of 10 and 1 microM for dihydrofolate and methotrexate respectively). These findings support the view that the monomeric ADC synthase is a potential target for antifolate drugs.     

Purification and properties of a phospholipase A2/lipase preferring phosphatidic acid,bis(monoacylglycerol) phosphate,and monoacylglycerol from rat testis

Phospholipase A(2) (PLA(2)) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca(2+) ions for activity and exhibited both phosphatidic acid-preferring PLA(2) and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA(2) and lipase activities. The sequence of NH(2)-terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA(2) activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA(2)-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA(2), and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments.     

Vacuolar localization of the enzymatic synthesis of hydroxycinnamic acid esters of malic acid in protoplasts from Raphanus sativus leaves

Protoplasts from leaves of radish ( Raphanus sativus L. var. sativus ) were examined for the subcellular localization of p -coumaric, caffeic, ferulic and sinapic acid esters of malic acid and the enzyme(s) involved in their syntheses. Vacuoles isolated from leaf protoplasts contained all the hydroxycinnamic acid esters as well as all the dependent enzyme activities. Protein from leaf vacuoles was shown to form the hydroxycinnamoylmalic acids, using the corresponding hydroxycinnamic acid glucose esters (1-O-acyl glucosides) as acyl donors. It is proposed that the vacuole is the cell compartment for synthesis and deposition of the hydroxycinnamoylmalic acids.     

The 1986 Borden award lecture. The role of the kidney in amino acid metabolism and nutrition

Measurement of the arteriovenous differences for free amino acids across rat kidney reveals that glycine and citrulline are removed and serine and arginine are added to the circulation. In addition, glutamine is taken up in large quantities by kidneys of animals that need to excrete large quantities of acid (e.g., diabetic animals, NH4Cl-fed animals, and animals fed a high protein diet). Glutamine is the major precursor of urinary ammonia and thus renal glutamine metabolism plays a key role in acid-base homeostasis. This process occurs primarily in the cells of the convoluted proximal tubule. Glutamine carbon is converted to glucose in acidotic rats and is totally oxidized in dogs. Regulation of glutamine metabolism occurs at two levels: acute regulation and chronic regulation. Acute regulation is, in part, mediated through a fall in intracellular [H+]. This activates alpha-ketoglutarate dehydrogenase and, ultimately, glutaminase. Chronic regulation involves induction of key enzymes, including, in the rat, glutaminase, glutamate dehydrogenase, and phosphoenolpyruvate carboxykinase. During the acidosis of prolonged starvation, the kidneys' requirement for glutamine must be met from muscle proteolysis and thus becomes a drain on lean body mass. Serine synthesis occurs by two separate pathways: from glycine by the combined actions of the glycine cleavage enzyme and serine hydroxymethyltransferase and from gluconeogenic precursors using the phosphorylated-intermediate pathway. Both pathways are located in the cells of the proximal tubule. Conversion of glycine to serine is ammoniagenic and the activity of the glycine cleavage enzyme is increased in acidosis. The function of serine synthesis by the phosphorylated-intermediate pathway is not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transacylase formation of bis(monoacylglycerol)phosphate

Recent work within our laboratory has focused on the enzymes we hypothesize are involved in the biosynthesis of bis(monoacylglycerol)phosphate from phosphatidylglycerol. Here we describe a transacylase, active at acidic pH values, isolated from a macrophage-like cell line, RAW 264.7. This enzyme acylates the head group glycerol of sn-3:sn-1' lysophosphatidylglycerol to form sn-3:sn-1' bis(monoacylglycerol)phosphate. Here we demonstrate that this enzyme uses two lysophosphatidylglycerol molecules, one as an acyl donor and another as an acyl acceptor, and that the acyl contributions from all other lipids tested are comparatively minor. This enzyme prefers saturated acyl chains to monounsaturates, 16 and 18 carbon fatty acids over 14 carbon fatty acids, and saturated acyl chains at the sn-1 position to monounsaturated acyl chains on the sn-2 carbon of lysophosphatidylglycerol. We present data which show the transacylase activity depends on the presence of a lipid-water interface and the lipid polymorphic state.     

Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates

Ribonuclease III cleaves double-stranded (ds) structures in bacterial RNAs and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, the biochemical properties of A. aeolicus (Aa)-RNase III and the reactivity epitopes of its substrates are not known. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of ～70-85°C, with either Mg2+ or Mn2+ supporting efficient catalysis. Small hairpins based on the stem structures associated with the Aquifex 16S and 23S rRNA precursors are cleaved at sites that are consistent with production of the immediate precursors to the mature rRNAs. Substrate reactivity is independent of the distal box sequence, but is strongly dependent on the proximal box sequence. Structural studies have shown that a conserved glutamine (Q157) in the Aa-RNase III dsRNA-binding domain (dsRBD) directly interacts with a proximal box base pair. Aa-RNase III cleavage of the pre-16S substrate is blocked by the Q157A mutation, which reflects a loss of substrate binding affinity. Thus, a highly conserved dsRBD-substrate interaction plays an important role in substrate recognition by bacterial RNase III.     

A novel Dps-type protein from insect gut bacteria catalyses hydrolysis and synthesis of N-acyl amino acids

A novel type of a microbial N-acyl amino acid hydrolase (AAH) from insect gut bacteria was purified, cloned and functionally characterized. The enzyme was obtained from Microbacterium arborescens SE14 isolated from the foregut of larvae of the generalist herbivore Spodoptera exigua. The substrates of AAH are N-acyl-glutamines previously reported to elicit plant defence reactions after introduction into the leaf during feeding. The isolated AAH catalyses the hydrolysis of the amide bond (K(m) = 36 micromol l(-1)) and, less efficient, the formation (K(m) = 3 mmol l(-1)) of the elicitor active N-acyl amino acids. The AAH from M. arborescens SE14 shows no homology to known fatty acyl amidases (EC 3.5.1.4) but belongs to the family of Dps proteins (DNA-binding protein from starved cell). In line with other DPS proteins AAH is a homododecamer (monomer 17 181 Da) and contains iron atoms (c. 1-16 iron atoms per subunit). Unlike genuine DPS proteins the enzyme does not significantly bind DNA. Amino acid hydrolase is the first member of the DPS family that catalyses the cleavage or formation of amide bonds. The participation of a microbial enzyme in the homeostasis of N-acyl-glutamines in the insect gut adds further complexity to the interaction between plants and their herbivores.     

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.