An apolipoprotein preferentially enriched in cholesteryl ester-rich very low density lipoproteins

Phosphatidyl glycerol is present in lamellar bodies and in the material obtained by alveolar wash representing 12.3 and 11.5%, respectively, of the total phospholipid phosphorus. Lung microsomes catalyze the formation of phosphatidyl glycerol from the known precursors, L-glycerol 3-phosphate and CDP-diglyceride. The rate of [¹⁴C]L-glycerol 3-phosphate incorporation into phosphatidyl glycerol was 30% higher in microsomes as compared to mitochondria. The addition of mercuric chloride inhibited the synthesis of phosphatidyl glycerol, and stimulated the incorporation into another as yet incompletely identified lipid. After pulse labeling of microsomal phosphatidyl glycerol $\text{in vitro}$, further incubation of microsomes with lamellar bodies or alveolar wash resulted in nearly quantitative appearance of label in surfactant.     

Chloroform-soluble nucleotides in Escherichia coli. Role of CDP-diglyceride in the enzymatic cytidylylation of phosphomonoester acceptors

CDP-diglyceride, the precursor of all the phospholipids in Escherichia coli, is cleaved in vitro to phosphatidic acid and CMP by a membrane-bound hydrolase. Since the physiological function of CDP-diglyceride hydrolase is unknown, we have explored the possibility that this enzyme acts in vivo as either a phosphatidyl- or cytidylyltransferase. To distinguish between these two alternatives, partially purified hydrolase was incubated with CDP-diglyceride in the presence of 50% H218O. Analysis of the reaction products by 31P NMR showed that 18O is incorporated exclusively into CMP, suggesting that the enzyme is a cytidylyltransferase. This conclusion is further supported by the following experimental results: (i) the hydrolase catalyzes the transfer of CMP from CDP-diglyceride to Pi; (ii) numerous phosphomonoesters, such as glycerol 3-phosphate, phosphoserine, and glucose 1-phosphate also function as CMP acceptors, but the corresponding compounds lacking the phosphate residues are not substrates for the enzyme; and (iii) CDP-diglyceride hydrolase exchanges [32P]phosphatidic acid for the phosphatidyl moiety of CDP-diglyceride and 32Pi for the beta-phosphate residue of CDP, indicating the involvement of a novel CMP-enzyme complex. These data suggest a biosynthetic role for CDP-diglyceride hydrolase, and extend the possible functions of CDP-diglyceride in the E. coli envelope.     

Phosphatidyl glycerophosphate phosphatase

An enzyme (phosphatidyl glycerophosphate phosphatase) that catalyzes the formation of phosphatidyl glycerol from phosphatidyl glycerophosphate has been rendered soluble by treatment of the particulate fraction of E. coli with Triton X-100 in the presence of EDTA, and has been partially purified. The enzyme is specific for phosphatidyl glycerophosphate and does not catalyze the hydrolysis of other simple phosphomonoesters. It requires Mg(++) for activity and is inhibited by sulfhydryl agents. Some other properties of the enzyme are also described.     

A spectrophotometric method for the assay of cytidine 5'-diphospho-1,2-diacyl-sn-glycerol-dependent enzymes of phospholipid metabolism.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diglyceride) hydrolase, CDP-diglyceride:L-serine O-phosphatidyltransferase, and CDP-diglyceride:sn-glycero-3-phosphate phosphatidyltransferase all release CMP from their liponucleotide substrate, CDP-diglyceride. We have developed a spectrophotometric assay for these enzymes using CMP kinase, pyruvate kinase, and lactate dehydrogenase to couple the release of CMP with the oxidation of NADH. The assay for each of the phospholipid-dependent enzymes was found to be linear both with time and with enzyme concentration. The assay should prove useful for continuous monitoring of enzymatic activity, determination of initial rates of reaction, and detailed kinetic analysis of these enzymes. Since several enzymes and substrates are used in the coupled assay system, the method is limited to analysis of partially purified preparations lacking competing activities.     

Phosphatidylglycerol biosynthesis in Bacillus licheniformis Resolution of membrane-bound enzymes by affinity chromatography on cytidinediphospho-sn-1,2-diacylglycerol Sepharose.

Cytidinediphospho-sn-1,2-diaclglycerol (CDP-diglyceride) has been covalently linked to Sephrose 4B via adipic acid dihydrazide spacer arm forming an effective affinity chromatography column. This liponucleo-tide ligand and sn-glycero-3-phosphate are subtracts for the formation of 3-sn-phoshatidyl-1'-sn-glycero-3'-phosphate (PGP) catalyzed in both eukaryotic and prokaryotic organisms by sn-glycero-3-phosphate: CMP phosphatidlytranferase (PGP synthetase). Using this CDP-diglyceride Sephrose affinity column we were able to resolve the membrane associated 3-sn-phosphatidyl'1-sn-glycerol (PG) synthesizing system present in Bacillus licheniformis into two activities. A PGP synthetase activity was adsorbed to the affinity column and was eluted using buffer containg CDP-diglyceride; a PGP phosphatease acactivity had no affinity for the column. Both PGP synthase and PGP phosphatase of B. licheniformis were associated with a membrane component of the cell as evidenced by sucrose gradient centrifugation, differential centrifugation, and solubilization by buffers containing detergent...     

Kinetics of glycerol uptake by the perfused rat liver. Membrane transport, phosphorylation and effect on NAD redox level.

The kinetics of glycerol uptake by the perfused rat liver were determined according to a model which includes membrane transport, intracellular phosphorylation and competitive inhibition of glycerol phosphorylation by L-glycerol 3-phosphate. The membrane transport obeys first-order kinetics at concentrations below 10 mM in the affluent medium. The K-m of the glycerol phosphorylation was 10 muM and the K-i of the L-glycerol 3-phosphate inhibition was 50 muM. The maximum activity (V) was 3.70 mumoles/min per g liver wet wt. These results are similar to in vitro kinetics of the glycerol kinase, except that K-i was found to be somewhat lower in the intact organ. At low glycerol concentrations, a steep concentration gradient exists across the liver cell membrane. The increase in the lactate to pyruvate concentration ratio during glycerol metabolism is related to the actual concentration of L-glycerol 3-phosphate, not to the rate of glycerol uptake.     

An improved procedure for the synthesis of 14C-labeled phosphatidylserine from cerebral phosphatidic acid

A complete procedure to prepare a highly labeled phosphatidyl-L-[U-14C]serine possessing the same fatty acid composition of brain phospholipids is reported. CDP-diglyceride was synthesized by reaction between phosphatidic acid and CMP-morpholidate as the dicyclohexylcarboxamidium salt. The reaction between CDP-diglyceride and L-[U-14C]serine to produce the labeled phosphatidylserine was catalyzed by the CDP-diglyceride: L-serine phosphatidyl transferase (EC 2.7.8.8) from E. coli. A selective inhibition of phosphatidylserine decarboxylase activity, present as contaminant in the enzyme extract, was introduced in order to avoid a low yield of product. Traces of phosphatidylethanolamine (about 1%) were easily removed by preparative thin-layer chromatography. The yield of the labeled product was as high as 87% and it specific radioactivity was 170 mCi/mmol.     

Synthesis of biologically active spin-labelled radioactive cytidine diphosphodiglyceride, a novel probe for biological membranes.

A versatile synthesis of spin-labelled radioactive cytidine diphospho-sn-1,2-diacylglycerol (CDP-diglyceride) has been developed based on the combination of the enzymatic acylation of radioactive sn-glycero-3-phosphate with 12-doxyl stearic acid and the chemical conversion of the thus obtained spin-labelled radioactive phosphatidic acid with cytidine monophosphomorpholi-date into spin-labelled radioactive CDP-diglyceride. The method for the isolation and purification of the latter compound was described. This obtained CDP-[2-3H]diglyceride contained 10% of fatty acids of paramagnetic nature, presumably present as a covalently bound 12-doxyl stearic acid esters. The biological activity was tested by using the synthesized compound as a substrate in the mitochondrial biosynthesis of phosphatidylglycerol. It was found that spin-labelled CDP-[2-3H]diglyceride prepared as described can be converted in the presence of sn-[2-14C]-glycero-3-phosphate into a spin-labelled [2-3H, 2'-14C]phosphatidylglycerol with isolated rat liver mitochondria, establishing therefore that the site of its utilization is identical with the site of phosphatidylglycerol synthesis in isolated mitochondria, i.e. inner mitochondrial membrane. Results described demonstrate that the synthesized spin-labelled CDP-diglyceride can be used as a specific probe for the spin- and radioactive covalent labelling of polyglycerophosphatides of mitochondrial membranes. Some implications and further possibilities in the study of biological membranes using the spin-labelled radioactive CDP-diglyceride are discussed.     

Fructose-2,6-bisphosphatase and 6-phosphofructo-2-kinase are separable in yeast.

Fructose-2,6-bisphosphatase was purified from yeast and separated from 6-phosphofructo-2-kinase and alkaline phosphatase. The enzyme released Pi from the 2-position of fructose 2,6-bisphosphate and formed fructose 6-phosphate in stoichiometric amounts. The enzyme displays hyperbolic kinetics towards fructose 2,6-bisphosphate, with a Km value of 0.3 microM. It is strongly inhibited by fructose 6-phosphate. The inhibition is counteracted by L-glycerol 3-phosphate. Phosphorylation of the enzyme by cyclic-AMP-dependent protein kinase causes inactivation, which is reversible by the action of protein phosphatase 2A.     

Incorporation of mitochondrial L-glycerol-3-phosphate dehydrogenase into liposomes; effect of sodium oleate and calcium ions

FAD-linked L-glycerol-3-phosphate dehydrogenase purified from liver mitochondria of hyperthyroid rats was incorporated into unilamellar phospholipid liposomes. The incorporation influenced both Vmax,app and Km,app values of the enzyme for its substrate, L-glycerol 3-phosphate. The Km,app for the electron acceptor remained unchanged with a simultaneous slight enhancement of the corresponding Vmax,app value. The steady-state fluorescence anisotropies of the fluorescein isothiocyanate and trimethylammoniumdiphenylhexatriene labels were affected by sodium oleate and calcium ions in the case of both solubilized and liposome-incorporated L-glycerol-3-phosphate dehydrogenase. These results indicate that calcium ions cause a significant alteration of the enzyme conformation. Sodium oleate (used as a model of free fatty acids), besides its direct action on the enzyme itself, affects the enzyme indirectly as well, via alteration of the physical properties of the membrane.     

Identification of cytidine diphosphate-diglyceride in the pineal gland of the rat and its accumulation in the presence of DL-propranolol.

CDP-diglyceride, an important metabolic intermediate in the biosynthesis of phospholipids, has been isolated for the first time from a mammalian tissue. The isolated material, labeled in incubations of intact rat pineal glands with 32P, [3H]cytidine, or [3H]CTP in the presence of DL-propranolol, was chromatographically identical with authentic CDP-diglyceride and was able to serve as phosphatidyl donor in the enzymatic synthesis of phosphatidylinositol and phosphatidyglycerol. It yielded the expected products upon enzymatic and chemical degradation. No dCDP-diglyceride was detected No radioactive CDP-diglyceride was detected following incubations in the absence of propranolol. Stimulation of CDP-diglyceride labeling from 32P1 occurred at propranolol concentrations between 0.03 and 1.0 mM. Net synthesis of the liponucleotide was shown. At 0.1 mM, propranolol incrased the incorporation of radioactivity into phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. When inositol (10 mM) and propranolol (0.1 mM) were both present, phosphatidylinositol labeling was further increased, wheas stimulation of phosphatidylglycerol and CPD-diglyceride labeling was abolished. Since CDP-diglyceride did not accumulate in the absence of the drug, its availability may normally be the limiting factor in phosphatidylinositol and phosphatidylglycerol biosynthesis. When propranol is present, inositol may become limiting and thus may lead to the observed labeling pattern.     

Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDPdiglyceride):L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase) is bound tightly to the ribosomes in crude extracts of Escherichia coli. After separation of the enzyme from the ribosomes by the method of Raetz and Kennedy (Raetz, C.R.H., and Kennedy, E.P. (1974), J. Biol. Chem. 249, 5038), we have purified the enzyme to 97% of homogenekty. The major portion of the overall 5500-fold purification was attained by substrate-specific elution from phosphocellulose using CDP-diglyceride in the presence of detergent. The purified enzyme migrated as a single band with an apparent minimum molecular weight of 54 000 when subjected to electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. The purified enzyme catalyzed exchange reactions between cytidine 5'- monophosphate (CMP) and CDP-diglyceride and between serine and phosphatidylserine. The enzyme also catalyzed the hydrolysis of CDP-diglyceride to form CMP and phosphatidic acid. dCDP-diglyceride was equivalent to CDP-diglyceride in all reactions catalyzed by the enzyme. In addition, the purified enzyme catalyzed the formation of phosphatidylglycerol or phosphatidylglycerophosphate at a very slow rate when serine was replaced as substrate by glycerol or sn-glycero-3-phosphate, respectively. These results suggest catalysis occurs via a ping-pong mechanism through the formation of a phosphatidyl-enzyme intermediate.     

Distribution and properties of CDP-diglyceride:inositol transferase from brain

CDP-diglyceride is converted to phosphatidyl inositol by several particulate subcellular fractions of guinea pig brain, with highest specific activity in the microsomal fraction. Optimal conditions with respect to pH, metal ion concentration, and substrate concentrations have been determined. The reaction was stimulated by the addition of bovine serum albumin and by Tween 80. Of several dl -CDP-diglycerides synthesized and used as substrates in a spectrophoto-metric assay for the enzyme, dl -CDP-didecanoin was the most active. The enzyme showed a high selectivity for myo-inositol. Of a number of compounds tested, only scyllo-inosose and epi-inosose served as substrates. Three inositol isomers and three myo-inositol monophosphates inhibited the reaction slightly. The most potent inhibitor found was galactinol, a myo-inositol galactoside.     

Phospholipid metabolism of wheat grains: Enzymes of the CDP-diglyceride phospholipid biosynthetic pathway

Summary Enzymes of the CDP-diglyceride pathway of phospholipid synthesis, CDP-diacylglycerol synthetase, CDP-diacylglycerol: glycerol 3-phosphate phosphatidyl-transferase and enzymes of phosphatidylserine formation were initially of relatively high specific activities in aleurone cells of wheat and declined upon imbibition. Enzyme activity of phosphatidylinositol synthesis was not detected in dry grains but was present upon imbibition. CDP-diacylglycerol: glycerol 3-phosphate phosphatidyltransferase shifted during imbibition from 85% of the activity in the supernatant of aleurone layers from dry seeds to 98% associated with large particle fractions after 36 hours of imbibition. Phosphatidylserine formation shifted from a dominant location in the 1,500 x g fraction in the dry seed to a predominantly mitochondrial location after 36 hours of imbibition. The subcellular distribution of CDP-diacylglycerol synthetase did not change appreciably upon imbibition from that of the dry seed, 75 to 80% of the activity was found in the supernatant. Only CDP-diacylglycerol: glycerol 3-phosphate phosphatidyltransferase showed increased specific activity late in the imbibition period. GA₃ accelerated the decrease of already declining activities of the CDP-diglyceride enzymes and the changes in their patterns of distribution, augmented the activities of the phosphatidylinositol synthesizing enzyme, and both accelerated and augmented the increase in the activity of the enzyme of phosphatidylglycerol synthesis which occurred late in imbibition.Committee on Institutional Cooperation Travelling Scholar from the University of Chicago.     

Properties of a revertant of Escherichia coli viable in the presence of spermidine accumulation: increase in L-glycerol 3-phosphate.

Escherichia coli CAG2242 cells are deficient in the speG gene encoding spermidine acetyltransferase. When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through the inhibition of protein synthesis by overaccumulation of spermidine. When the cells were cultured with a high concentration of spermidine (4 mM), a revertant strain was obtained. We found that a 55-kDa protein, glycerol kinase, was overexpressed in the revertant and that synthesis of a ribosome modulation factor and the RNA polymerase sigma(38) subunit, factors important for cell viability, was increased in the revertant. Levels of L-glycerol 3-phosphate also increased in the revertant. Transformation of glpFK, which encodes a glycerol diffusion facilitator (glpF) and glycerol kinase (glpK), to E. coli CAG2242 partially prevented the cell death caused by accumulation of spermidine. It was also found that L-glycerol 3-phosphate inhibited spermidine binding to ribosomes and attenuated the inhibition of protein synthesis caused by high concentrations of spermidine. These results indicate that L-glycerol 3-phosphate reduces the binding of excess amounts of spermidine to ribosomes so that protein synthesis is recovered.     

Characterization of phospholipase B of Culex pipiens fatigans

Phospholipase B has been found in the mosquito Culex pipiens fatigans, and some of its properties have been studied. The enzyme had a high optimum temperature (45 degrees C) and broad alkaline pH optimum (8-9). It was inactive toward diacylphospholipids, and hydrolyzed lysolecithin at a higher rate than lysophosphatidyl ethanolamine. The enzyme was heat labile, but lysolecithin protected it against heat inactivation. Phosphatidyl ethanolamine, phosphatidyl choline, deoxycholate, Fe(+++), and Hg(++) inhibited the enzyme markedly. The enzyme was present mainly in larvae; little enzyme activity was detected in pupae or adults. The total and specific activities were highest in IV instar (6 day) and I instar (1st day) larvae, respectively. It was localized in the microsomal fraction and was distributed mainly in the abdomen and thorax of the insect. The enzyme was present at much higher levels of activity in larvae of the mosquitoes Anopheles stephensi and Aedes aegypti.     

Phosphatidyl glycerolphosphate serves as glycerolphosphate donor in polymer synthesis

Phosphatidyl glycerolphosphate was found to serve as the glycerolphosphate donor for polymer synthesis. When CDP-diglyceride and radiolabeled glycerolphosphate were incubated with the membrane enzyme prepared from Streptococcus sanguis, active syntheses of radiolabeled lipids and polymers were observed. The synthesis of polymer was not inhibited by low concentration of unlabeled phosphatidylglycerol. When [3H, 32P]glycerolphosphate was used, the polymer synthesized contained both 3H and 32P. The lipids formed were characterized as phosphatidylglycerol and phosphatidyl glycerolphosphate. The polymers formed from the latter were characterized as lipoteichoic acid like compounds by sodium dodecylsulfate-polyacrylamide gel electrophoresis.     

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.     

Phosphatidyl inositol inhibition of a sperm cyclic AMP-independent protein kinase

Phosphatidyl inositol has been found to inhibit strongly the activity of a cyclic AMP-independent protein kinase located on the external surface of goat epididymal intact spermatozoa. Phosphatidyl inositol at a concentration as low as 10 micrograms/ml inhibited nearly 50% of the ecto-kinase activity for the phosphorylation of the exogenous protein substrate: casein. Phosphatidyl ethanolamine at a relatively high concentration (125 micrograms/ml) inhibited slightly (approx 25%) the activity of the enzyme whereas other phospholipids: phosphatidyl serine and choline, diacyl glycerol, phosphatidic acid and myo-inositol-2-phosphate had no appreciable effect on the kinase activity. Phosphatidyl inositol has also served as a potent inhibitor of the phosphorylation of sperm ecto-phosphoproteins by the endogenous kinase activity of intact spermatozoa. By thin layer chromatography it has been shown that the observed inhibitory effect of the phospholipid was not due to any impurities or degraded products of phosphatidyl inositol. Phosphatidyl inositol inhibited the kinase activity noncompetitively with respect to casein and Mg2+ but uncompetitively with respect to ATP. The results raised the possibility that phosphatidyl inositol-mediated high affinity inhibition of protein kinase(s), may constitute a novel mechanism for the regulatory actins of the phospholipid in mammalian cells.     

Fermentative production of L-glycerol 3-phosphate utilizing a Saccharomyces cerevisiae strain with an engineered glycerol biosynthetic pathway

Interest in L-glycerol 3-phosphate (L-G3P) production via microbial fermentation is due to the compound's potential to replace the unstable substrate dihydroxyacetone phosphate (DHAP) in one-pot enzymatic carbohydrate syntheses. A Saccharomyces cerevisiae strain with deletions in both genes encoding specific L-G3Pases (GPP1 and GPP2) and multicopy overexpression of L-glycerol 3-phosphate dehydrogenase (GPD1) was studied via small-scale (100 mL) batch fermentations under quasi-anaerobic conditions. Intracellular accumulation of L-G3P reached extremely high levels (roughly 200 mM) but thereafter declined. Extracellular L-G3P was also detected and its concentration continuously increased throughout the fermentation, such that most of the total L-G3P was found outside the cells as fermentation concluded. Moreover, in spite of the complete elimination of specific L-G3Pase activity, the strain showed considerable glycerol formation suggesting unspecific dephosphorylation as a mechanism to relieve cells of intracellular L-G3P accumulation. Up-scaling the process employed fed-batch fermentation with repeated glucose feeding, plus an aerobic growth phase followed by an anaerobic product accumulation phase. This produced a final product titer of about 325 mg total L-G3P per liter of fermentation broth.