Regulation of mammalian cell membrane biosynthesis.

This review explores current information on the interrelationship between phospholipid biochemistry and cell biology. Phosphatidylcholine is the most abundant phospholipid and it biosynthesis has been studied extensively. The choline cytidylyltransferase regulates phosphatidylcholine production, and recent advances in our understanding of the mechanisms that govern cytidylyltransferase include the discovery of multiple isoforms and a more complete understanding of the lipid regulation of enzyme activity. Similarities between phosphatidylcholine formation and the phosphatidylethanolamine and phosphatidylinositol biosynthetic pathways are discussed, together with current insight into control mechanisms. Membrane phospholipid doubling during cell cycle progression is a function of periodic biosynthesis and degradation. Membrane homeostasis is maintained by a phospholipase A-mediated degradation of excess phospholipid, whereas insufficient phosphatidylcholine triggers apoptosis in cells.

Complete Genome Sequence of Anaeromyxobacter sp. Fw109-5, an Anaerobic, Metal-Reducing Bacterium Isolated from a Contaminated Subsurface Environment.

We report the genome sequence of Anaeromyxobacter sp. Fw109-5, isolated from nitrate- and uranium-contaminated subsurface sediment of the Oak Ridge Integrated Field-Scale Subsurface Research Challenge (IFC) site, Oak Ridge Reservation, TN. The bacterium's genome sequence will elucidate its physiological potential in subsurface sediments undergoing in situ uranium bioremediation and natural attenuation.

The Rhodobacter capsulatus genome.

The genome of Rhodobacter capsulatus has been completely sequenced. It consists of a single chromosome containing 3.5 Mb and a circular plasmid of 134 kb. This effort, started in 1992, began with a fine-structure restriction map of an overlapping set of cosmids that covered the genome. Cosmid sequencing led to a gapped genome that was filled by primer walking on the chromosome and by using lambda clones. Methods had to be developed to handle strong stops in the high GC (68%) inserts. Annotation was done with the ERGO system at Integrated Genomics, as was the reconstruction of the cell's metabolism. It was possible to recognize 3709 orfs of which functional assignments could be made with high confidence to 2392 (65%). Unusual features include the presence of numerous cryptic phage genomes embedded in the chromosome.

Combined effect of a lecithin and a bile salt on pancreatic lipase activity.

Lipolysis is regulated by the presence of amphiphilic compounds such as bile salts and lecithin, which are adsorbed at the triglyceride-water interface and therefore influence the approach of water-soluble pancreatic lipase to its insoluble substrate (emulsified triglycerides). The partition of bile salts between the lipid and the aqueous phase is of prime importance in the expression of lipase activity. Lipase activity was determined as a function of different combinations of concentrations of deoxycholate and dipalmitoylphosphatidyl choline. From zeta-potential measurements, it is evident that lecithin affects the partition of bile salts, most probably by displacing deoxycholate molecules. Our results indicate that lecithin cannot be classified a priori as inhibitor or activator of pancreatic lipase. As an amphiphilic compound, lecithin exerts a synergistic effect with bile salts via the formation of mixed micelles. The final effect on lipolysis depends on the ratio of lecithin to bile salt: low ratios enhance enzyme activity, whereas high ratios lead to inhibition.

The genome of the obligately intracellular bacterium Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies.

Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, alpha-proteobacterium, is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, 17 putative pseudogenes, and a substantial proportion of noncoding sequence (27%). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences and a unique serine-threonine bias associated with the potential for O glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly(G-C) tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Genes associated with pathogen-host interactions were identified, including a small group encoding proteins (n = 12) with tandem repeats and another group encoding proteins with eukaryote-like ankyrin domains (n = 7).

Pancreatic lipase assays with triglycerides as substrate: contribution of each sequential reaction to product formation.

Human pancreatic lipase assays are usually performed in the presence of either emulsified triglycerides or diglycerides within the limits of their solubility. Two reactions are catalyzed in the presence of triglycerides: hydrolysis of triglycerides to diglycerides, and diglycerides to monoglycerides. The contribution of each reaction to the final result was determined after extensive kinetic studies on the appearance and/(or) accumulation of intermediates and/(or) products. Acylated glycerides were analyzed after extraction from the reaction mixture, separation of lipid classes by thin-layer chromatography, and quantification by capillary gas chromatography. The results show that after 10 min of reaction in the presence of high concentrations of triolein, 75% of the released fatty acids arise from the first reaction. Relative merits and disadvantages of each substrate (triglyceride or diglyceride) are discussed in terms of practicability.

Kinetics of the two-step hydrolysis of triacylglycerol by pancreatic lipases.

Pancreatic lipases catalyze the hydrolysis of triacylglycerol in a sequential manner. First, triacylglycerol is hydrolyzed to 1,2-diacylglycerol, which is subsequently converted to 2-monoacylglycerol. We studied the kinetics of trioleoylglycerol hydrolysis by rabbit and human pancreatic lipases. The products (acylglycerols and fatty acid) were analyzed by extraction from the reaction mixture, separation by thin-layer chromatography, and quantification by capillary gas chromatography. The first-order rate constants of trioleoylglycerol and dioleoylglycerol hydrolysis were calculated showing that both enzymes hydrolyze dioleoylglycerol faster than trioleoylglycerol. Using rabbit pancreatic lipase, we found that deoxycholate enhanced dioleoylglycerol hydrolysis to a higher degree than trioleoylglycerol hydrolysis. Colipase increased both rate constants similarly at high deoxycholate concentrations (35 mM), while at low concentrations (5 mM) a selectivity toward trioleoylglycerol was observed. From the variation of the rate constants with respect to temperature, we calculated the apparent activation energies of trioleoylglycerol and dioleoylglycerol hydrolysis to be 59.8 kJ.mol-1 and 53.5 kJ.mol-1, respectively. Upon storage, both rabbit and human pancreatic lipases showed a greater loss of activity toward dioleoylglycerol as compared to trioleoylglycerol, suggesting that different conformational elements of the enzyme molecule are responsible for the interaction with each substrate.

Distribution of CTP:phosphocholine cytidylyltransferase (CCT) isoforms. Identification of a new CCTbeta splice variant.

CTP:phosphocholine cytidylyltransferase is a major regulator of phosphatidylcholine biosynthesis. A single isoform, CCTalpha, has been studied extensively and a second isoform, CCTbeta, was recently identified. We identify and characterize a third cDNA, CCTbeta2, that differs from CCTbeta1 at the carboxyl-terminal end and is predicted to arise as a splice variant of the CCTbeta gene. Like CCTalpha, CCTbeta2 is heavily phosphorylated in vivo, in contrast to CCTbeta1. CCTbeta1 and CCTbeta2 mRNAs were differentially expressed by the human tissues examined, whereas CCTalpha was more uniformly represented. Using isoform-specific antibodies, both CCTbeta1 and CCTbeta2 localized to the endoplasmic reticulum of cells, in contrast to CCTalpha which resided in the nucleus in addition to associating with the endoplasmic reticulum. CCTbeta2 protein has enzymatic activity in vitro and was able to complement the temperature-sensitive cytidylyltransferase defect in CHO58 cells, just as CCTalpha and CCTbeta1 supporting proliferation at the nonpermissive conditions. Overexpression experiments did not reveal discrete physiological functions for the three isoforms that catalyze the same biochemical reaction; however, the differential cellular localization and tissue-specific distribution suggest that CCTbeta1 and CCTbeta2 may play a role that is distinct from ubiquitously expressed CCTalpha.

Lipid activation of CTP: phosphocholine cytidylyltransferase alpha: characterization and identification of a second activation domain.

The CTP:phosphocholine cytidylyltransferase (CCT) governs the rate of phosphatidylcholine (PtdCho) biosynthesis, and its activity is governed by interaction with membrane lipids. The carboxy-terminus was dissected to delineate the minimum sequences required for lipid responsiveness. The helical domain is recognized as a site of lipid interaction, and all three tandem alpha-helical repeats from residues 257 through 290 were found to be required for regulation of enzymatic activity by this domain. Truncation of the carboxy-terminus to remove one or more of the alpha-helical repeats yielded catalytically compromised proteins that were not responsive to lipids but retained sufficient activity to accelerate PtdCho biosynthesis when overexpressed in vivo. The role of the helical region in lipid-activation was tested further by excising residues 257 through 309 to yield a protein that retained a 57-residue carboxy terminal domain fused to the catalytic core. This construct tested the hypothesis that the helical region inhibits activity in the absence of lipid rather than activates the enzyme in the presence of lipid. This hypothesis predicts constitutive activity for CCTalpha[Delta257-309]; however, this protein was tightly regulated by lipid with activities comparable to the full-length CCTalpha, in both the absence and presence of lipid. Activation of CCTalpha[Delta257-309] was dependent exclusively on anionic lipids, whereas full-length CCTalpha responded to either anionic or neutral lipids. Phosphatidic acid delivered in Triton X-100 micelles was the preferred activator of the second lipid-activation domain. These data demonstrate that CCTalpha can be regulated by lipids by two independent domains: (i) the three amphipathic alpha-helical repeats that interact with both neutral and anionic lipid mixtures and (ii) the last 57 residues that interact with anionic lipids. The results show that both domains are inhibitory in the absence of lipid and activating in the presence of lipid. Removal of both domains results in a nonresponsive, dysregulated enzyme with reduced activity. The data also demonstrate for the first time that the 57-residue carboxy-terminal domain in CCTalpha participates in lipid-mediated regulation and is sufficient for maximum activation of enzyme activity.

Overexpression of a mammalian ethanolamine-specific kinase accelerates the CDP-ethanolamine pathway.

Ethanolamine kinase (EKI) is the first committed step in phosphatidylethanolamine (PtdEtn) biosynthesis via the CDP-ethanolamine pathway. We identify a human cDNA encoding an ethanolamine-specific kinase EKI1 and the structure of the EKI1 gene located on chromosome 12. EKI1 overexpression in COS-7 cells results in a 170-fold increase in ethanolamine kinase-specific activity and accelerates the rate of [3H]ethanolamine incorporation into PtdEtn as a function of the ethanolamine concentration in the culture medium. Acceleration of the CDP-ethanolamine pathway does not result in elevated cellular PtdEtn levels, but rather the excess PtdEtn is degraded to glycerophosphoethanolamine. EKI1 has negligible choline kinase activity in vitro and does not influence phosphatidylcholine biosynthesis. Acceleration of the CDP-ethanolamine pathway also does not change the rate of PtdEtn formation via the decarboxylation of phosphatidylserine. The data demonstrate the existence of separate ethanolamine and choline kinases in mammals and show that ethanolamine kinase can be a rate-controlling step in PtdEtn biosynthesis.

Cloning and characterization of a second human CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CCT) is a key regulator of phosphatidylcholine biosynthesis, and only a single isoform of this enzyme, CCTalpha, is known. We identified and sequenced a human cDNA that encoded a distinct CCT isoform, called CCTbeta, that is derived from a gene different from that encoding CCTalpha. CCTbeta transcripts were detected in human adult and fetal tissues, and very high transcript levels were found in placenta and testis. CCTbeta and CCTalpha proteins share highly related, but not identical, catalytic domains followed by three amphipathic helical repeats. Like CCTalpha, CCTbeta required the presence of lipid regulators for maximum catalytic activity. The amino terminus of CCTbeta bears no resemblance to the amino terminus of CCTalpha, and CCTbeta protein was localized to the cytoplasm as detected by indirect immunofluorescent microscopy. Whereas CCTalpha activity is regulated by reversible phosphorylation, CCTbeta lacks most of the corresponding carboxyl-terminal domain and contained only 3 potential phosphorylation sites of the 16 identified in CCTalpha. Transfection of COS-7 cells with a CCTbeta expression construct led to the overexpression of CCT activity, the accumulation of cellular CDP-choline, and enhanced radiolabeling of phosphatidylcholine. CCTbeta protein was posttranslationally modified in COS-7 cells, resulting in slower migration during polyacrylamide gel electrophoresis. Expression of CCTbeta/CCTalpha chimeric proteins showed that the amino-terminal portion of CCTbeta was required for posttranslational modification. These data demonstrate that a second, distinct CCT enzyme is expressed in human tissues and provides another mechanism by which cells regulate phosphatidylcholine production.

The role of CDP-diacylglycerol synthetase and phosphatidylinositol synthase activity levels in the regulation of cellular phosphatidylinositol content.

The regulation of phosphatidylinositol synthesis was examined by cloning and expressing in COS-7 cells the human cDNAs encoding the two enzymes in the biosynthetic pathway. Human CDP-diacylglycerol synthetase (cds1) and phosphatidylinositol synthase (pis1) clones were identified in the human expressed sequence-tagged (EST) data base, and full-length cDNAs were obtained by library screening. The cds1 cDNA did not possess a recognizable mitochondrial import signal, and the activity of the expressed Cds1 protein was stimulated by nucleoside triphosphates in vitro, indicating that cds1 did not encode the mitochondrial-specific isozyme. There were two mRNA species (3.9 and 5.6 kilobases) detected on Northern blots hybridized with the cds1 probe that were expressed at distinctly different levels in various human tissues. Consistent with the presence of the two mRNAs, a cDNA predicted to encode a second human CDP-diacylglycerol synthetase (cds2) was also uncovered in the EST data base. In contrast to the two cds mRNAs, a single, 2.1-kilobase pis1 mRNA was uniformly expressed in all human tissues examined. Expression of the pis1 gene led to the overproduction of both phosphatidylinositol synthase and phosphatidylinositol:inositol exchange reactions, indicating that the Pis1 polypeptide catalyzed both of these activities. Phosphatase treatment of cell extracts abolished the CMP-independent phosphatidylinositol:inositol exchange reaction, and exchange activity was completely restored by the addition of CMP. Overexpression of cds1 or pis1 alone or in combination did not enhance the rate of phosphatidylinositol biosynthesis. Also, overexpression did not result in a significant proportional increase in the cellular levels of CDP-diacylglycerol or phosphatidylinositol. These data illustrate that the levels of Cds1 and Pis1 protein expression are not critical determinants of cellular PtdIns content and argue against a determining role for the activity of either of these enzymes in the regulation of PtdIns biosynthesis.