The enzymes of phospholipid synthesis in Clostridium butyricum

We have examined extracts of Clostridium butyricum for several enzymes of phospholipid synthesis. Membrane particles were shown to catalyze the formation of CDP-diglyceride from [3H]CTP and phosphatidic acid. The reaction was dependent on Mg2+ and stimulated by monovalent cations. CDP-diglyceride formed in vitro was found to be a substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. The formation of phosphatidylglycerophosphate from added CDP-diglyceride and [U-14C]sn-glycerol-3-phosphate was dependent on Mg2+ and Triton X-100. The dephosphorylation of endogenously-generated phosphatidylglycerophosphate to yield phosphatidylglycerol was observed to be pH-dependent. The formation of phosphatidylserine from CDP-diglyceride and L-[3-14C]serine was stimulated by Mg2+ and Triton X-100. dCDP-diglyceride was a suitable substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. Phosphatidylserine decarboxylase activity was barely detectable in membrane particles from C. butyricum. The addition of E. coli membrane particles provided efficient phosphatidylserine decarboxylase activity in this system. Although plasmalogens are the principal lipids of C. butyricum, none of the products of phospholipid synthesis formed in vitro contained measurable amounts of plasmalogens. The subcellular distribution of both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase in C. butyricum was also studied. Both were found to be membrane-associated.     

Partial purification and properties of mammalian phosphatidylglycerophosphatase

The phosphatidylglycerophosphatase (EC 3.1.3.27) activity of rat liver mitochondria was investigated by assaying the conversion of 14C-labelled phosphatidylglycerophosphate to phosphatidylglycerol. The activity was associated with a mitochondrial membrane fraction and could not be released into solution employing techniques applicable to a peripheral membrane protein. The enzyme was partially purified by sonication, pH 5.0 precipitation, and gel filtration. Various ionic and nonionic detergents as well as numerous divalent cations inhibited the phosphatase. The enzyme displayed a high affinity for phosphatidylglycerophosphate.     

Affinity of phosphatidylglycerophosphate synthetase for the liponucleotides in Bacillus subtilis

Abstract In Bacillus subtilis , the synthesis of phosphatidylglycerol, the more reactive phospholipid, was investigated in vitro. The phosphatidylglycerophosphate synthetase was exclusively localized in the membrane fraction. Three phospholipids (phosphatidylglycerophosphate, phosphatidylglycerol and diphosphatidylglycerol) were synthesized by this fraction. Exogenous CDP-diglyceride and dCDP-diglyceride were substrates with the same K _m (0.11 mM). In coupled system with CDP-diglyceride synthetase, endogenous dCDP-diglyceride was a less effective substrate than CDP-diglyceride.     

Intracellular distribution of enzymes of phospholipid metabolism in several gram-negative bacteria.

Cell-free extracts of Salmonella typhimurium, Serratia marcescens, Enterobacter aerogenes, and Micrococcus cerificans contained the following enzymatic activities related to phospholipid metabolism: cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diglyceride):l-serine O-phosphatidyltransferase (phosphatidylserine synthase), phosphatidylserine decarboxylase, CDP-diglyceride:sn-glycero-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase), phosphatidylglycerophosphate phosphatase, and CDP-diglyceride hydrolase. The intracellular distribution of these enzymatic activities as determined by sucrose density gradient centrifugation of cell-free extracts was shown to be similar in each species investigated. The phosphatidylserine decarboxylase, phosphatidylglycerophosphate synthase, and CDP-diglyceride hydrolase activities were all associated with the cell envelope fraction, whereas the phosphatidylserine synthase activity was associated mainly with the ribosomal fraction. These enzymatic activities are comparable and have an intracellular distribution similar to those found in Escherichia coli cell-free extracts. Therefore, the pathways established for phospholipid biosynthesis in E. coli can also account for the synthesis of the major phospholipids (phosphatidylethanolamine and phosphatidylglycerol) in several other gram-negative organisms. In addition, the unusual ribosomal association of the phosphatidylserine synthase from E. coli (Raetz and Kennedy, J. Biol. Chem. 247:2008-2014, 1972) appears to be a general property for this activity in several other bacterial species.     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

Purification and characterization of a membrane-associated phosphatidylserine synthase from Bacillus licheniformis

A CDP-diacylglycerol-dependent phosphatidylserine synthase was solubilized from Bacillus licheniformis membranes and purified to near homogeneity. The purification procedure consisted of CDP-diacylglycerol-Sepharose affinity chromatography followed by substrate elution from blue dextran-Sepharose. The purified preparation showed a single band with an apparent relative molecular mass of 53 000 daltons when subjected to sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Proteolytic digestion of the enzyme yielded a smaller (41 000 daltons) active form. The preparation was free of any phosphatidylglycerophosphate synthase, phosphatidylserine decarboxylase, CDP-diacylglycerol hydrolase, and phosphatidylserine hydrolase activities. The utilization of substrates and the formation of products occurred with the expected stoichiometry. Radioisotopic exchange patterns between related substrate and product pairs suggest a sequential Bi-Bi reaction as opposed to the ping-pong mechanism exhibited by the well-studied phosphatidylserine synthase of Escherichia coli [Larson, T. J., & Dowhan, W. (1976) Biochemistry 15, 5212-5218]. The B. licheniformis enzyme was also found to be markedly dissimilar to the E. coli enzyme with regard to association with detergent micelles, affinity for ribosomes, and antigenicity.     

The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase.

To further define the genes and gene products responsible for the in vivo conversion of phosphatidylglycerophosphate to phosphatidylglycerol in Escherichia coli, we disrupted two genes (pgpA and pgpB) which had previously been shown to encode gene products which carried out this reaction in vitro (T. Icho and C. R. H. Raetz, J. Bacteriol. 153:722-730, 1983). Strains with either gene or both genes disrupted had the same properties as the original mutants isolated with mutations in these genes, i.e., reduced in vitro phospholipid phosphatase activities, normal growth properties, and an increase in the level of phosphatidylglycerophosphate (1.6% versus less than 0.1% in wild-type strains). These results demonstrate that these genes are not required for either normal cell growth or the biosynthesis of phosphatidylglycerol in vivo. In addition, the total phosphatidylglycerophosphate phosphatase activity in the doubly disrupted mutant was reduced by only 50%, which indicates that there is at least one other gene that encodes such an activity and thus accounts for the lack of a dramatic effect on the biosynthesis of anionic phospholipids in these mutant strains. The phosphatidic acid and lysophosphatidic acid phosphatase activities of the pgpB gene product were also significantly reduced in gene-interrupted mutants, but the detection of residual phosphatase activities in these mutants indicated that additional genes encoding such phosphatases exist. The lack of a significant phenotype resulting from disruption of the pgpA and pgpB genes indicates that these genes may be required only for nonessential cell function and leaves the biosynthesis of phosphatidylglycerophosphate as the only step in E. coli phospholipid biosynthesis for which a gene locus has not been identified.     

Biosynthesis of phosphatidylglycerol and phosphatidylglycerolphosphate in submitochondrial membranes isolated from guinea pig liver is absolutely dependent on CDP-diglycerides imported from microsomal membranes

The biosynthesis of radioactively labelled phosphatidylglycerol via phosphatidylglycerophosphate in outer and inner mitochondrial membranes isolated from guinea pig liver was found to depend absolutely on CDP-diglycerides, which could not be biosynthesized in these membranes. The requirement for CDP-diglycerides in the biosynthesis of labelled phosphatidylglycerol could be fulfilled by the transfer of biosynthesized [3H]CDP-diglycerides from the microsomal membranes to the outer and inner mitochondrial membranes.     

Suppression of the lethal effect of acidic-phospholipid deficiency by defective formation of the major outer membrane lipoprotein in Escherichia coli.

The Escherichia coli pgsA3 allele encoding a defective phosphatidylglycerophosphate synthase is lethal for all but certain strains. Genetic analysis of such strains has revealed that the lethal effect is fully suppressed by the lack of the major outer membrane lipoprotein that consumes phosphatidylglycerol for its maturation.     

Purification and characterization of the aspartate chemoreceptor

The chemoreceptor for aspartate in Salmonella typhimurium was purified from an Escherichia coli strain containing a plasmid bearing the receptor's structural gene (tar). The receptor was solubilized from salt-washed membranes with the nonionic detergent octyl-beta-D-glucopyranoside and purified by a combination of ion exchange, molecular sieve and hydroxyapatite-agarose chromatography. The inclusion of glycerol and 1,10-phenanthroline in all buffers used prior to ion exchange chromatography prevented scission of the receptor by an endogenous proteolytic activity. The solubilized receptor was estimated to have a molecular weight of 248,000 from its behavior on Sephacryl S-300, suggesting that the receptor may be organized as a multimer containing 4 +/- 1 identical subunits. Circular dichroic measurements of the purified protein indicate that 78% of its residues are arranged in helical secondary structures.     

Localization of phospholipid biosynthetic enzyme activities in cell-free fractions derived from Rhodopseudomonas sphaeroides

The phospholipid biosynthetic enzyme activities: CDP-diglyceride synthetase, phosphatidylglycerophosphate synthetase, PGP phosphatase, phosphatidylserine (PS) synthase, PS decarboxylase, and S-adenosyl-L-methionine:phosphatidylethanolamine (AdoMet:PE) N-methyltransferase were detected in crude cell-free extracts of Rhodopseudomonas sphaeroides. CDP-diglyceride synthetase and phosphatidylglycerophosphate synthetase co-enriched with penicillin-binding protein activity, a known cytoplasmic membrane marker, throughout fractionation of cell-free extracts of both chemoheterotrophically and photoheterotrophically grown cells. PS decarboxylase also co-enriched with the cytoplasmic membranes in fractions derived from chemoheterotrophically and photoheterotrophically grown cells, but substantially greater quantities of PS decarboxylase activity was found in the chromatophores derived from photoheterotrophically grown cells than could be accounted for by cytoplasmic membrane contamination of this sample. PS synthase (60% of the recovered activity) and S-adenosyl-L-methionine:phosphatidylethanolamine N-methyltransferase (90% of the recovered activity) were found in the supernatant fraction after high speed centrifugation of crude cell lysates, suggesting that these enzyme activities were not tightly membrane associated. The localization of phospholipid biosynthetic enzyme activity in R. sphaeroides is discussed in terms of the biosynthesis of the photosynthetic membranes.     

Reconstitution of phosphate-linked antiport from Streptococcus lactis

Membrane protein solubilized by octyl-beta-D-glucopyranoside in the presence of dispersed phospholipid was incubated with bath-sonicated liposomes and additional detergent. The proteoliposomes formed on dilution showed transport and exchange properties consistent with a reconstitution of phosphate:sugar 6-phosphate antiport. Thus, phosphate self-exchange was found only when protein from induced cells was used; this exchange was blocked by a sugar 6-phosphate, not by a sugar 1-phosphate; and proteoliposomes supported an accumulation of 2-deoxyglucose 6-phosphate with no added source of energy. Solubilization and reconstitution of protein was most effective when performed in the presence of gram-positive phospholipids.     

A rapid method for reconstitution of bacterial membrane proteins

We have devised a simple method for the reconstitution of bacterial membrane proteins directly from small (1-20 ml) volumes of cell culture, thus eliminating the preparation of membrane vesicles. Cells are subjected to simultaneous lysozyme digestion and osmotic lysis, and after brief centrifugation ghosts are solubilized in 1.2% octyl-beta-D-glucopyranoside (octylglucoside) in the presence of added carrier lipid and an osmolyte. Aliquots of the clarified supernatant are suitable for reconstitution, as documented by using extracts from three different Gram-negative cells to recover both inorganic phosphate (Pi)-linked antiport and oxalate:formate exchange activities in proteoliposomes. These proteoliposomes are physically stable, non-leaky and can sustain a membrane potential and, because functional porins do not reconstitute, the artificial system has transport characteristics similar to those found when proteoliposomes are obtained using standard methods. This method should become an important tool for the screening and characterization of large numbers of strains, both wild-type and mutant.     

Secondary structure and oligomerization of the E. coli glycerol facilitator

The Major Intrinsic Proteins are found throughout the bacterial, plant, and animal kingdoms and are responsible for the rapid transport of water and other small, polar solutes across membranes. The superfamily includes the aquaporins, the aquaglyceroporins, and the glycerol facilitators. We have overexpressed and purified the Escherichia coli inner membrane glycerol facilitator. Approximately 7.5 mg of 95% pure protein is obtained from 1 L of Escherichia coli cells using immobilized metal affinity chromatography. Well-resolved matrix-assisted laser desorption ionization mass spectra were obtained by solubilization of the protein in octyl-beta-D-glucopyranoside (M(r) = 33 650.3; error approximately 0.4%). The recombinant glycerol facilitator is inserted into the bacterial inner membrane, is functional, and is inhibited by HgCl(2). Polyacrylamide gel electrophoresis suggests that the facilitator is predominantly monomeric when solubilized with dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, and sodium dodecyl sulfate, but that it self-associates, forming soluble oligomers when urea is used during extraction. Similar oligomeric species are demonstrated to exist in the bacterial membrane by chemical cross-linking experiments. Circular dichroism analysis shows that the protein is predominantly alpha-helical. Helix content is significantly higher in protein prepared in the absence of urea (42-55%) than in its presence (32%). A possible role for the facilitator oligomers in interactions with, and regulation of, the glycerol kinase is discussed.     

Participation of the microsomal CDP-diglycerides in the mitochondrial biosynthesis of phosphatidylglycerol

Participation of microsomal CDP-diglycerides in mitochondrial biosynthesis of phosphatidylglycerol was studied by [3H]palmitoyl, [14C]linoleoyl, and [14C]arachidonoyl CDP-diglycerides and [3H]CDP-diglycerides which were bound to microsomal membranes, incubated with unlabelled mitochondrial membranes, and further incubated in the presence of radioactive sn-glycero-3-phosphate under conditions required for mitochondrial phosphatidylglycerol biosynthesis. Ten to 15% of microsomal radioactive CDP-diglycerides was transferred to mitochondrial membranes and incorporated into mitochondrial radioactive lipids identified as phosphatidylglycerol, phosphatidylglycerophosphate, and, when [14C]linoleoyl CDP-diglycerides were used, diphosphatidylglycerol (cardiolipin).     

Solubilization of ligand-stabilized vasopressin receptors from plasma membranes of bovine kidney and rat liver

The solubilization of vasopressin receptors from plasma membranes of bovine kidney and rat liver by different detergents was investigated. A prerequisite for the extraction of vasopressin receptors retaining binding affinity for their ligand was the stabilization of the receptors by the prior formation of the membrane-bound hormone-receptor complexes. The vasopressin-receptor complexes from both kidney and liver membranes were solubilized in a high yield with dodecyl-beta-D-maltoside and 3-laurylamido-N,N'-dimethylpropylaminoxide. Several other nonionic detergents including octyl-beta-D-glucopyranoside effectively extracted the hepatic vasopressin receptor. For the hormone-receptor complex solubilized from bovine kidney with dodecyl-beta-D-maltoside, a Stokes' radius of 5.8 nm was determined.     

Lipid composition of integral purple membrane by 1H and 31P NMR

In the purple membrane (PM) of halobacteria, lipids stabilize the trimeric arrangement of bacteriorhodopsin (BR) molecules and mediate the packing of the trimers in a regular crystalline arrangement. To date, the identification and quantification of these lipids has been based either on lipid extraction procedures or structural models. By directly solubilizing PMs from Halobacterium salinarum in aqueous detergent solutions (SDS or Triton X-100), we avoided any separation or modification steps that might modify the lipid composition or even the lipid molecules themselves. Our analysis of integral PM preparations should resolve partially conflicting literature data on the lipid composition of the PM. Using 31P and 1H NMR of detergent-solubilized but otherwise untreated samples, we found two glycolipids and 6.4 +/- 0.1 phospholipids per BR molecule, 4.4 +/- 0.1 of the latter being the phosphatidylglycerophosphate methyl ester. The only glycolipid detected was S-TGD-1. For an additional glycolipid, glycocardiolipin, that was recently identified in lipid extracts, we show that it was produced mainly during the lipid extraction procedure but also was partially dependent on the preparation of the PM suspensions.     

Regulation of the balanced synthesis of membrane phospholipids. Experimental test of models for regulation in Escherichia coli

In Escherichia coli, highly effective regulation controls the balanced synthesis of membrane phospholipids, important for optimal growth. Regulation is such that normally about 70% of a common pool of cytosine liponucleotide precursor is utilized by phosphatidylserine synthase and eventually converted to phosphatidylethanolamine, while about 30% is utilized by the competing enzyme phosphatidylglycerophosphate synthase and converted to phosphatidylglycerol (25%) plus cardiolipin (5%). Although the ratio of phosphatidylglycerol to cardiolipin may vary with conditions of growth, the sum of these two lipids remains relatively constant at about 30% of the total. Alternative models, postulating coordinate regulation of the two competing enzymes, or independent feedback regulation are proposed. These models were tested in experiments in which phosphatidylglycerol was continuously removed from growing cells treated with arbutin (4-hydroxyphenyl-O-beta-D-glucoside), causing its conversion to arbutinphosphoglycerol (Bohin, J.-P., and Kennedy, E.P. (1984) J. Biol. Chem. 259, 8388-8393.) The synthesis of phosphatidylglycerol was increased by a factor of 7 in cells treated with arbutin, with only small changes in phospholipid composition and with no significant change in the level of phosphatidylglycerophosphate synthase. The synthesis of phosphatidylethanolamine was not significantly increased, decisively eliminating the model that requires coordinate regulation of phosphatidylserine synthase and phosphatidylglycerophosphate synthase, and supporting the model of independent feedback inhibition, sensitive to very small changes in composition of cellular phospholipids.     

Coordinate increases in the enzyme activities responsible for phosphatidylglycerol synthesis and CTP:cholinephosphate cytidylyltransferase activity in developing rat lung

The enzymes responsible for the biosynthesis of phosphatidylglycerol, CTP:phosphatidate cytidylyltransferase, CDP-diacylglycerol: glycerophosphate phosphatidyltransferase and phosphatidylglycerophosphate phosphatase demonstrated a coordinate increase in activity in fetal rat lung at term when the demand for pulmonary surfactant increases. The activity of CTP:cholinephosphate cytidylyltransferase, the enzyme responsible for CDP-choline production also increased in the perinatal period. The activity of cholinephosphate cytidylyltransferase in fetal and neonatal cytosol was stimulated by the addition of phosphatidylglycerol but no effect was noted with cytosol from adult lung. These results are consistent with the suggestion that the activity of cholinephosphate cytidylyltransferase, a potential rate-determining enzyme in pulmonary phosphatidylcholine synthesis, may be regulated in the perinatal period both through an activation by phosphatidylglycerol and by an increase in total enzyme units.