Expression of Cds4/5 of Arabidopsis chloroplasts in E. coli reveals the membrane topology of the C-terminal region of CDP-diacylglycerol synthases.

CDP-diacylglycerol synthases (Cds) are conserved from bacteria to eukaryotes. Bacterial CdsA is involved not only in phospholipid biosynthesis but also in biosynthesis of glycolipid MPIase, an essential glycolipid that catalyzes membrane protein integration. We found that both Cds4 and Cds5 of Arabidopsis chloroplasts complement cdsA knockout by supporting both phospholipid and MPIase biosyntheses. Comparison of the sequences of CdsA and Cds4/5 suggests a difference in membrane topology at the C-termini, since the region assigned as the last transmembrane region of CdsA, which follows the conserved cytoplasmic domain, is missing in Cds4/5. Deletion of the C-terminal region abolished the function, indicating the importance of the region. Both 6 × His tag attachment to CdsA and substitution of the C-terminal 6 residues with 6 × His did not affect the function. These 6 × His tags were sensitive to protease added from the cytosolic side in vitro, indicating that this region is not a transmembrane one but forms a membrane-embedded reentrant loop. Thus, the C-terminal region of Cds homologues forms a reentrant loop, of which structure is important for the Cds function.

Structures of the Mitochondrial CDP-DAG Synthase Tam41 Suggest a Potential Lipid Substrate Pathway from Membrane to the Active Site.

In mitochondria, CDP-diacylglycerol (CDP-DAG) is a crucial precursor for cardiolipin biosynthesis. Mitochondrial CDP-DAG is synthesized by the translocator assembly and maintenance protein 41 (Tam41) through an elusive process. Here we show that Tam41 adopts sequential catalytic mechanism, and report crystal structures of the bulk N-terminal region of Tam41 from Schizosaccharomyces pombe in the apo and CTP-bound state. The structure reveals that Tam41 contains a nucleotidyltransferase (NTase) domain and a winged helix domain. CTP binds to an "L"-shaped pocket sandwiched between the two domains. Rearrangement of a loop region near the active site is essential for opening the CTP-binding pocket. Docking of phosphatidic acid/CDP-DAG in the structure suggests a lipid entry/exit pathway connected to the "L"-shaped pocket. The C-terminal region of SpTam41 contains a positively charged amphipathic helix crucial for membrane association and participates in binding phospholipids. These results provide detailed insights into the mechanism of CDP-DAG biosynthesis in mitochondria.

Legionella Effector AnkX Disrupts Host Cell Endocytic Recycling in a Phosphocholination-Dependent Manner.

The facultative intracellular bacterium Legionella pneumophila proliferates within amoebae and human alveolar macrophages, and it is the causative agent of Legionnaires' disease, a life-threatening pneumonia. Within host cells, L. pneumophila establishes a replicative haven by delivering numerous effector proteins into the host cytosol, many of which target membrane trafficking by manipulating the function of Rab GTPases. The Legionella effector AnkX is a phosphocholine transferase that covalently modifies host Rab1 and Rab35. However, a detailed understanding of the biological consequence of Rab GTPase phosphocholination remains elusive. Here, we broaden the understanding of AnkX function by presenting three lines of evidence that it interferes with host endocytic recycling. First, using immunogold transmission electron microscopy, we determined that GFP-tagged AnkX ectopically produced in mammalian cells localizes at the plasma membrane and tubular membrane compartments, sites consistent with targeting the endocytic recycling pathway. Furthermore, the C-terminal region of AnkX was responsible for association with the plasma membrane, and we determined that this region was also able to bind the phosphoinositide lipids PI(3)P and PI(4)P in vitro. Second, we observed that mCherry-AnkX co-localized with Rab35, a regulator of recycling endocytosis and with major histocompatibility class I protein (MHC-I), a key immunoregulatory protein whose recycling from and back to the plasma membrane is Rab35-dependent. Third, we report that during infection of macrophages, AnkX is responsible for the disruption of endocytic recycling of transferrin, and AnkX's phosphocholination activity is critical for this function. These results support the hypothesis that AnkX targets endocytic recycling during host cell infection. Finally, we have demonstrated that the phosphocholination activity of AnkX is also critical for inhibiting fusion of the Legionella-containing vacuole (LCV) with lysosomes.

In silico characterization of 1,2-diacylglycerol cholinephosphotransferase and lysophospha-tidylcholine acyltransferase genes in Glycine max L. Merrill.

The enzymes 1,2-diacylglycerol cholinephosphotrans-ferase (CPT) and lysophosphatidylcholine acyltransferase (LPCAT) are important in lipid metabolism in soybean seeds. Thus, understand-ing the genes that encode these enzymes may enable their modification and aid the improvement of soybean oil quality. In soybean, the genes encoding these enzymes have not been completely described; there-fore, this study aimed to identify, characterize, and analyze the in silico expression of these genes in soybean. We identified two gene models encoding CPT and two gene models encoding LPCAT, one of which presented an alternative transcript. The sequences were positioned on the physical map of soybean and the promoter regions were analyzed. Cis-elements responsible for seed-specific expression and responses to biotic and abiotic stresses were identified. Virtual expression analysis of the gene models for CPT and LPCAT indicated that these genes are expressed under different stress conditions, in somatic embryos during differentiation, in immature seeds, root tissues, and calli. Putative ami-no acid sequences revealed the presence of transmembrane domains, and analysis of the cellular localization of these enzymes revealed they are located in the endoplasmic reticulum.

Unraveling the Phosphocholination Mechanism of the Legionella pneumophila Enzyme AnkX.

The intracellular pathogen Legionella pneumophila infects lung macrophages and injects numerous effector proteins into the host cell to establish a vacuole for proliferation. The necessary interference with vesicular trafficking of the host is achieved by modulation of the function of Rab GTPases. The effector protein AnkX chemically modifies Rab1b and Rab35 by covalent phosphocholination of serine or threonine residues using CDP-choline as a donor. So far, the phosphoryl transfer mechanism and the relevance of observed autophosphocholination of AnkX remained disputable. We designed tailored caged compounds to make this type of enzymatic reaction accessible for time-resolved Fourier transform infrared difference spectroscopy. By combining spectroscopic and biochemical methods, we determined that full length AnkX is autophosphocholinated at Ser521, Thr620, and Thr943. However, autophosphocholination loses specificity for these sites in shortened constructs and does not appear to be relevant for the catalysis of the phosphoryl transfer. In contrast, transient phosphocholination of His229 in the conserved catalytic motif might exist as a short-lived reaction intermediate. Upon substrate binding, His229 is deprotonated and locked in this state, being rendered capable of a nucleophilic attack on the pyrophosphate moiety of the substrate. The proton that originated from His229 is transferred to a nearby carboxylic acid residue. Thus, our combined findings support a ping-pong mechanism involving phosphocholination of His229 and subsequent transfer of phosphocholine to the Rab GTPase. Our approach can be extended to the investigation of further nucleotidyl transfer reactions, which are currently of reemerging interest in regulatory pathways of host-pathogen interactions.

Platelet Activating Factor (PAF) biosynthesis is inhibited by phenolic compounds in U-937 cells under inflammatory conditions.

Interleukin 1 beta (IL-1ß) induced platelet activating factor (PAF) synthesis in U-937 cells through stimulation of acetyl-CoA:lysoPAF-acetyltransferase (lyso PAF-AT) at 3 h and DTT-independentCDP-choline-1-alkyl-2-acetyl-sn-glycerol cholinophosphotransferase (PAF-CPT) at 0.5 h. The aim of this study was to investigate the effect of tyrosol (T), resveratrol (R) and their acetylated derivatives(AcDs) which exhibit enhanced bioavailability, on PAF synthesis in U-937 after IL-1ß stimulation. The specific activity of PAF enzymes and intracellular levels were measured in cell homogenates. T and R concentration capable of inducing 50% inhibition in IL-1ß effect on lyso PAF-AT was 48 µΜ ± 11 and 157 µΜ ± 77, for PAF-CPT 246 µΜ ± 61 and 294 µΜ ± 102, respectively. The same order of concentration was also observed on inhibiting PAF levels produced by IL-1ß. T was more potent inhibitor than R (p<0.05). AcDs of T retain parent compound inhibitory activity, while in the case of R only two AcDs retain the activity. The observed inhibitory effect by T,R and their AcDs, may partly explain their already reported beneficial role.

A highly dynamic ER-derived phosphatidylinositol-synthesizing organelle supplies phosphoinositides to cellular membranes.

Polyphosphoinositides are lipid signaling molecules generated from phosphatidylinositol (PtdIns) with critical roles in vesicular trafficking and signaling. It is poorly understood where PtdIns is located within cells and how it moves around between membranes. Here we identify a hitherto-unrecognized highly mobile membrane compartment as the site of PtdIns synthesis and a likely source of PtdIns of all membranes. We show that the PtdIns-synthesizing enzyme PIS associates with a rapidly moving compartment of ER origin that makes ample contacts with other membranes. In contrast, CDP-diacylglycerol synthases that provide PIS with its substrate reside in the tubular ER. Expression of a PtdIns-specific bacterial PLC generates diacylglycerol also in rapidly moving cytoplasmic objects. We propose a model in which PtdIns is synthesized in a highly mobile lipid distribution platform and is delivered to other membranes during multiple contacts by yet-to-be-defined lipid transfer mechanisms.

Plasmodium CDP-DAG synthase: an atypical gene with an essential N-terminal extension.

Cytidine diphosphate diacylglycerol synthase (CDS) diverts phosphatidic acid towards the biosynthesis of CDP-DAG, an obligatory liponucleotide intermediate in anionic phospholipid biosynthesis. The 78kDa predicted Plasmodium falciparum CDS (PfCDS) is recovered as a 50 kDa conserved C-terminal cytidylyltransferase domain (C-PfCDS) and a 28kDa fragment that corresponds to the unusually long hydrophilic asparagine-rich N-terminal extension (N-PfCDS). Here, we show that the two fragments of PfCDS are the processed forms of the 78 kDa pro-form that is encoded from a single transcript with no alternate translation start site for C-PfCDS. PfCDS, which shares 54% sequence identity with Plasmodium knowlesi CDS (PkCDS), could substitute for PkCDS in P. knowlesi. Experiments to disrupt either the full-length or the N-terminal extension of PkCDS indicate that not only the C-terminal cytidylyltransferase domain but also the N-terminal extension is essential to Plasmodium spp. PkCDS and PfCDS introduced in P. knowlesi were processed in the parasite, suggesting a conserved parasite-dependent mechanism. The N-PfCDS appears to be a peripheral membrane protein and is trafficked outside the parasite to the parasitophorous vacuole. Although the function of this unusual N-PfCDS remains enigmatic, the study here highlights features of this essential gene and its biological importance during the intra-erythrocytic cycle of the parasite.

Molecular cloning and expression of a novel cholinephosphotransferase involved in glycoglycerophospholipid biosynthesis of Mycoplasma fermentans.

A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using alpha-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5'-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.

Molecular cloning and characterization of the guinea pig cholinephosphotransferase gene.

Cholinephosphotransferase (CPT), the terminal enzyme in the de novo synthesis of phosphatidylcholine (PC), has an important role in regulating the acyl group of PC in mammalian cells. A 593bp cDNA coding for the 3(')-end of the CPT gene has been cloned from guinea pig liver using degenerative oligos based on the human CPT gene. It has 85% amino acid homology with the human CPT enzyme and amino acid variations were found to cluster at few points. Restriction enzyme polymorphisms were found particularly with respect to BamHI and NcoI. Hydrophobic and helix plot analysis of the sequence shows a similar pattern to human counterpart except for amino acid residues 142-179 and 173-179. PCR analysis suggested that a predominant pseudogene may be present in guinea pig and also the intronic sequences were much shorter when compared to the human CPT gene. We are the first to report on the C-terminal 195 amino acid residues of the CPT gene from any animal species alike in many aspects of cellular metabolism. The probable differences in genomic organization and its expression in different cancer cells have been discussed here having CPT as an important target for cancer drug development.

A 24 bp cis-acting element essential for the transcriptional activity of Plasmodium falciparum CDP-diacylglycerol synthase gene promoter.

CDP-diacylglycerol synthase (CDS) is a key rate-limiting enzyme in the phospholipid metabolism of Plasmodium falciparum, converting phosphatidic acid to CDP-diacylglycerol. The CDS gene is predominantly expressed in the mature intraerythrocytic stages. Consequently, we physically and functionally characterized the CDS gene promoter. The mRNA transcription initiation site was mapped 121 bp upstream of the CDS gene translation start site. A 1909 bp 5' upstream sequence was isolated and found to be transcriptionally active thus constituting a functional CDS promoter. Mapping of this promoter identified a 44 bp cis-acting sequence, located between -1640 and -1596 bp upstream of the ATG codon, essential for efficient transcriptional activity. This 44 bp sequence binds specifically to nuclear factors from trophozoite stage parasites. We further showed that a 24 bp element, lying within the 44 bp sequence, mediates the specific binding to nuclear proteins and shows no significant homology to known eukaryotic DNA consensus sequence elements that bind transcription factors. The deletion of the 24 bp element abrogated promoter activity, indicating that this cis-acting sequence element is essential for efficient transcription of the CDS gene.

Scanning alanine mutagenesis of the CDP-alcohol phosphotransferase motif of Saccharomyces cerevisiae cholinephosphotransferase.

Cholinephosphotransferase (EC 2.7.8.2) catalyzes the formation of a phosphoester bond via the transfer of a phosphocholine moiety from CDP-choline to diacylglycerol forming phosphatidylcholine and releasing CMP. A motif, Asp113-Gly114-(X)2-Ala117-Arg118-(X)8-Gly127+ ++-(X)3-Asp131-(X)3-Asp135, located within the CDP-choline binding region of Saccharomyces cerevisiae cholinephosphotransferase (CPT1 ?/Author: Please confirm that a gene is meant here.) is also found in several other phospholipid synthesizing enzymes that catalyze the formation of a phosphoester bond utilizing a CDP-alcohol and a second alcohol as substrates. To determine if this motif is diagnostic of the above reaction type scanning alanine mutagenesis of the conserved residues within S. cerevisiae cholinephosphotransferase was performed. Enzyme activity was assessed in vitro using a mixed micelle enzyme assay and in vivo by determining the ability of the mutant enzymes to restore phosphatidylcholine synthesis from radiolabeled choline in an S. cerevisiae strain devoid of endogenous cholinephosphotransferase activity. Alanine mutants of Gly114, Gly127, Asp131, and Asp135 were inactive; mutants, Ala117 and Arg118 displayed reduced enzyme activity, and Asp113 displayed wild type activity. The analysis described is the first molecular characterization of a CDP-alcohol phosphotransferase motif and results predict a catalytic role utilizing a general base reaction proceeding through Asp131 or Asp135 via a direct nucleophilic attack of the hydroxyl of diacylglyerol on the phosphoester bond of CDP-choline that does not proceed via an enzyme bound intermediate. Residues Ala117 and Arg118 do not participate directly in catalysis but are likely involved in substrate binding or positioning with Arg118 predicted to associate with a phosphate moiety of CDP-choline.

CDP-choline:1,2-diacylglycerol cholinephosphotransferase.

Cholinephosphotransferase transfers a phosphocholine moiety from CDP-choline to diacylglycerol thus forming phosphatidylcholine (PtdCho) and CMP. This reaction defines the ultimate step in the Kennedy pathway for the genesis of de novo synthesized PtdCho. Hence, the intracellular location of cholinephosphotransferase identifies both the site from which de novo synthesized PtdCho is transported to other organelles and the site from which it is assembled with proteins and other lipids for secretion from the cell during the generation of lung surfactant, lipoproteins, and bile. Most subcellular fractionation studies observed the majority of cholinephosphotransferase activity in the endoplasmic reticulum, although the method of subcellular fractionation was found to grossly affect these results with activity alternately dispersed within Golgi, nuclear, and mitochondrial fractions. Coupling subcellular fractionation results with immunofluorescence or electron microscopy studies would resolve the issue of the site of PtdCho synthesis. However, antibodies have yet to be generated to cholinephosphotransferase since its integral membrane-bound nature has prevented its purification from any source and a mammalian cholinephosphotransferase cDNA has also yet to be isolated. However, cholinephosphotransferase genes have recently been isolated from the yeast Saccharomyces cerevisiae. Structure/function analysis of the S. cerevisiae cholinephosphotransferase has allowed for an in depth molecular examination resulting in the identification of the catalytic site. In addition, this analysis has generated the predicted amino acid data necessary to produce antibodies to pursue the site of PtdCho synthesis in this organism, as well as to provide information that should allow for the isolation of mammalian cholinephosphotransferase cDNA(s).

CDP-diacylglycerol synthase from mammalian tissues.

CDP-diacylglycerol resides at the branch point of glycerolipid biosynthesis as precursor of both the phosphoinositides and phosphatidylglycerol. The discovery of the phosphoinositide signal transduction pathway and the recognition of its prominent role in intracellular communication has focused new attention on CDP-diacylglycerol synthase. As a rate-limiting step in this pathway, it is a likely target for regulation. Exploration of this possibility will be facilitated by the recent cloning of mammalian CDP-DAG synthase.

Gene cloning and characterization of CDP-diacylglycerol synthase from rat brain.

A cDNA encoded a 462-amino acid protein, which showed CDP-diacylglycerol synthase (CDS) activity was cloned for the first time as the vertebrate enzyme molecule from rat brain cDNA library. The deduced molecular mass of this rat CDS was 53 kDa, and putative primary structure included several possible membrane- spanning regions. At the amino acid sequence level, rat CDS shared 55.5%, 31. 7%, and 20.9% identity with already known Drosophila, Saccharomyces cerevisiae, and Escherichia coli CDS, respectively. This rat CDS preferred 1-stearoyl-2-arachidonoyl phosphatidic acid as a substrate, and its activity was strongly inhibited by phosphatidylglycerol 4, 5-bisphosphate. By immunoblotting analysis of COS cells overexpressed with the epitope-tagged for rat CDS, a 60-kDa band was detected. By epitope-tag immunocytochemistry, the CDS protein was mainly localized in close association with the membrane of the endoplasmic reticulum of the transfected cells. The intense mRNA expression of CDS was localized in the cerebellar Purkinje cells, the pineal body, and the inner segment of photoreceptor cells. Additionally, very intense expression was detected in postmitotic spermatocytes and spermatids.

Cloning of CDP-diacylglycerol synthase from a human neuronal cell line.

A critical step in the supply of substrate for the phosphoinositide signal transduction pathway is the formation of the liponucleotide intermediate, CDP-diacylglycerol, catalyzed by CDP-diacylglycerol synthase. Further insight into the regulation of phosphoinositide biosynthesis was sought by cloning of the gene for the vertebrate enzyme. Sequence of the corresponding gene from Drosophila was used to prepare a probe for screening of a human neuronal cell cDNA library. A cDNA was isolated with a predicted open reading frame of 1,332 bases, encoding a protein of 51 kDa. The amino acid sequence showed 50% identity (75% similarity) to that of Drosophila eye CDP-diacylglycerol synthase and substantial similarity to the Saccharomyces cerevisiae and Escherichia coli homologues. Northern blot analysis, with human cDNA riboprobes, suggested that the corresponding mRNA was expressed in all human tissues examined. Expression of the human cDNA in COS cells resulted in a more than fourfold increase in CDP-diacylglycerol synthase activity. Knowledge of the sequence of vertebrate CDP-diacylglycerol synthase should facilitate further investigations into its regulation and the possible existence of distinct isoforms.

Evolutionary comparison of enzyme activities of phosphatidylcholine metabolism in the nervous system of an invertebrate (Loligo pealei), lower vertebrate (Mustelus canis) and the rat.

While steady-state kinetic parameters (metabolite pools, Km and activation energies) are partially known for the enzymes involved in phosphatidylcholine synthesis and degradation in mammalian brain, they are not available for the nervous system of lower vertebrates or invertebrates. Since the extent of evolutionary development of an enzyme is not known a priori, we evaluated the kinetic and thermodynamic parameters of choline kinase, CTP:phosphocholine cytidylyltransferase, choline phosphotransferase and glycerophosphorylcholine phosphodiesterase in squid (Loligo pealei) optic lobe, dogfish (Mustelus canis) and rat brain. For all these enzyme activities, basic similarities in Km and inhibitor effect were found. The same was true for the activation energies Ea, with the exception of squid choline kinase and dogfish cytidylyltransferase. Treatment of microsomal membranes with phospholipase C sharply inhibited cytidylyltransferase activity in all three animal species. In dogfish brain, glycerophosphorylcholine phosphodiesterase activity was undetectable. Our results are consistent with the notion that the kinetic properties of the enzyme activities leading to the preservation of the phosphatidylcholine membranous pool may have appeared early in metazoan evolution and been fully conserved in mammals.

The potential for platelet-activating factor synthesis in brain: properties of cholinephosphotransferase and 1-alkyl-sn-glycero-3-phosphate acetyltransferase in microsomal fractions of immature rabbit cerebral cortex.

The synthesis of platelet-activating factor (PAF) was studied in microsomal fractions of cerebral cortices of 15-day-old rabbits. These included: a total microsomal fraction P3, rough and smooth microsomes, R and S, and microsomal fraction P derived from isolated nerve cell bodies. Cholinephosphotransferase (CPT) generating PAF from alkylacetylglycerol had the highest specific activities in fractions R and P (24 and 6 times the homogenate values, based on membrane phospholipid content). This CPT activity differed from that which synthesized phosphatidylcholine as the latter was sensitive to dithiothreitol inhibition and was more readily inhibited by Triton X-100. As the CPT activity for PAF synthesis relies on the production of alkylacetylglycerol we studied the acetyltransferase which forms 1-alkyl-2-acetyl-sn-glycero-3-phosphate (AAGP). This enzyme had the highest specific activity in fraction R, followed by fractions P3 and P. There was evidence that the acetyltransferase was more active in a phosphorylated form. NaF maximized the recovery of AAGP products in the assays. The pH optimum for acetylation was in a range of 8.0-9.0. Lyso PAF did not inhibit the formation of AAGP and the rates of formation of PAF by acetylation were less than 5% of values for AAGP synthesis. During AAGP formation there was no evidence for subsequent alkylacetylglycerol formation in the absence of NaF, but a small formation of radioactive PAF could be demonstrated from AAGP under the CPT assay conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

CDPcholine:1,2-diacylglycerol cholinephosphotransferase from rat liver microsomes. I. Solubilization and characterization of the partially purified enzyme and the possible existence of an endogenous inhibitor.

The solubilization and partial purification of cholinephosphotransferase (CDPcholine:1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2) from rat liver microsomes were examined in the presence of ionic (sodium deoxycholate), nonionic (Triton X-100, n-octylglycoside), or zwitter ionic (CHAPS) detergents. Among the four detergents tested, only sodium deoxycholate was found to be an efficient solubilizer of cholinephosphotransferase activity from microsomal membranes, whereas the other three detergents caused irreversible inactivation of the enzyme at the solubilization step. Addition of phospholipids at the solubilization step, or after solubilization of the membrane proteins, could not preserve or reconstitute activity to any extent. The sodium deoxycholate-solubilized activity was partially purified by gel permeation chromatography (Superose 12HR). The partially purified preparation appeared to consist of a large aggregate containing phospholipids; further dissociation of the protein-phospholipid complex caused complete inactivation of the enzyme. The partially purified cholinephosphotransferase showed a specific activity of 100-130 nmol/min/mg protein, which is the highest activity reported to date from any tissue source; this amounts to a 4-fold enrichment of cholinephosphotransferase activity from the original KCl-washed rat liver microsomes. Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) activity was copurified and 6-fold enriched with a total recovery of 60%. During the purification of cholinephosphotransferase activity, a putative endogenous inhibitor of cholinephosphotransferase was also solubilized and was isolated from the microsomal membranes. This heat-labile, nondialyzable inhibitor was shown to act specifically on cholinephosphotransferase and not on ethanolaminephosphotransferase. Further characterization of the inhibitory activity revealed that it may act at the binding step of the cholinephosphotransferase to its lipid substrate, diacylglycerol.

Study of properties of cholinephosphotransferase from fetal guinea pig lung mitochondria and microsomes.

We have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, Km values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on Km and Vmax indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID50 values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.