Phospholipid Phosphorus Research Articles

Cuticular lipids of two resistant and a susceptible strain of houseflies

AbstractA study was made of the composition of the cuticular lipids of two resistant strains of houseflies (Rutgers and Fc), both of which show a reduced rate of absorption of insecticides as a partial mechanism of resistance and a susceptible strain (CSMA). Total lipids, monoglycerides, diglycerides and sterol esters (except in the Fc strain), sterols, fatty acids and phospholipid phosphorus were higher in resistant strains than in the susceptible strain. Phosphatidyl‐ethanolamine and phosphatidyl‐choline were major constituents of the phospholipid fractions and were appreciably higher in the resistant strains. Cuticular wax contents did not differ among strains. Incorporation of lipid precursors, [U‐14]acetate and [32P]orthophosphate, was greater in the cuticle of one or both resistant strains, depending on the lipid component examined.

Distribution of arachidonic acid and other fatty acids in glycerolipids of the rabbit iris

The phospholipid composition, inclusive of higher inositides, fatty acid composition, and in vivo and in vitro incorporation of [ 14C]arachidonate into the rabbit iris glycerolipids were determined. (1) The total phospholipid phosphorus was found to range between 13–14 μmol/g of iris. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin constituted about 85% of the total lipid phosphorus. Ethanolamine plasmalogen, choline plasmalogen and inositol-containing phospholipids constituted about 11,4 and 7% of total lipid phosphorus respectively. (2) Analysis of the fatty acids of the various glycerolipids of the iris revealed the major saturates to be 16 : 0 and 18 : 0 and the major unsaturates to be 18 : 1, 18 : 2 and 20 : 4. Ethanolamine-containing phospholipids contained the highest percentage of 20 : 4 followed by choline-containing phospholipids, diacylglycerol and phosphatidylinositol respectively. Sphingomyelin contained only trace amounts of 20 : 4. (3) It was found that the iris can incorporate, both in vivo and in vitro, [ 14C]arachidonate into its own glycerolipids. In general glycerolipids isolated from the in vitro experiment contained significantly higher radioactivities than those obtained from the in vivo experiment. In vitro about 41% of the [ 14C]radioactivity was recovered in the neutral lipids and phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol contained only 10,2 and 2·8% of the total radioactivity respectively; and in vivo about 25% of the total radioactivity was found in each of phosphatidylcholine and the neutral lipids and 8·4% in phosphatidylethanolamine. (4) No intraocular signs of an inflammatory response were observed 0·5, 1 and 7 hr after intracameral injection of [ 14C]arachidonic acid (2 μg) -bound albumin (0·1 mg).

Lipid composition of Staphylococcus aureus and its derived L-forms.

Two strains of Staphylococcus aureus (Newman and Tazaki) and their derived L-forms were cultured in serum-containing broth and the differences in their lipid compositions were analyzed. Cardiolipin accounted for more than 50% of the total phospholipid phosphorus in L-forms, but for less than 25% in parent bacteria. The cardiolipin content of L-forms was very high through all growth phases, although it increased gradually as growth proceeded. Significant amounts of cholesterol and its esters were present in parent strains and L-forms, all of which incorporated serum cholesterol into the cell membrane. On the other hand, they could be detected in the L-forms but not in the parent strains when they were cultured in serum-free broth. To examine the ability of L-forms to synthesize cholesterol, the cholesterol content of L-forms cultured in serum-free broth was compared with that of the medium. The results indicated that staphylococcal L-forms could synthesize cholesterol and its esters. These differences in lipid composition suggested that modification of membrane lipids may occur as an adaptational change in response to the disappearance of the cell wall.

A rapid sensitive method for determining phospholipid phosphorus involving digestion with magnesium nitrate

AbstractA method is described for the rapid determination of phospholipid phosphorus in samples containing less than 0.5 μg phosphorus. Phospholipid phosphorus is first converted to inorganic phosphate by heating a dried lipid extract briefly over a Bunsen flame in the presence of magnesium nitrate, then dissolving the resulting residue in dilute hydrochloric acid at 95 C. The determination of the inorganic phosphate content of the sample then requires only the addition of Triton X‐100 and an acidic ammonium molybdate‐malachite green reagent. Absorbance is measured at 650 nm after 5 min at room temperature.

Mitochondrial and microsomal phospholipid phosphorus metabolism during postnatal growth in rat heart and liver.

AbstractThe relative specific activities of phospholipids of hearts and livers of newborn and older rats were measured 2 hr after intraperitoneal32P‐orthophosphate. There was a close linear correlation between the specific activity of cardiolipin and the net increase in the amount of this phospholipid. In one‐day‐old animals, the relative specific activities in the heart and liver were highest and exceeded by 7.3‐ and 3‐fold, respectively, the corresponding activities in the adult. The apparent half‐lives of cardiolipins were calculated on the basis of the linear correlation that was found between the net increase in cardiolipin and the rate of incorporation. These half‐lives were 4.7 days in liver and 6.4 days in heart. Though the changes in the phospholipid composition of the organelles during neonatal development were small, the relative specific activities of the individual phospholipids varied considerably. In addition to cardiolipin, there was a good correlation between the specific activity and the net increase in mitochondrial phosphatidylinositol. At birth, the specific activity of mitochondrial phosphatidylcholine in liver was 2.9 times that in microsomes. During the 12 neonatal hours, the specific activity of microsomal phosphatidylcholine increased 6.3‐fold and exceeded the corresponding mitochondrial activity. The relation between the activation of microsomal phosphatidylcholine synthesis and the induction of serum lecithin synthesis in newborn liver is discussed. The finding that at birth the specific activity of mitochondrial phosphatidylcholine unexpectedly was higher than that of microsomal phosphatidylcholine points out the difficulties in interpreting the in vivo evidence for precursor‐product relationship.

A potential role for phospholipids in the regulation of 3-hydroxy-3-methylglutaryl-coenzyme a reductase in cultured C-6 glial cells effects of N, N-dimethylethanolamine

The relation of the activity of the microsomal enzyme, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, to cellular phospholipid composition was studied in C-6 glial cells. Phospholipid composition was perturbed by growth of cells in the naturally occurring amino alcohol, N, N-dimethylethanolamine. After growth of C-6 glia in 5 mM N, N-dimethylethanolamine for 24 h, reductase activity was diminished by 50%. A similar diminution in cholesterol synthesis was observed. This effect was not accompanied by any parallel change in cell growth, DNA synthesis, protein synthesis, fatty acid synthetase activity, or microsomal NADPH-cytochrome c reductase activity. The inhibition of reductase activity by N, N-dimethylethanolamine was prevented by the addition of equimolar concentrations of choline to the culture medium and, also, could be reversed completely by removal of N, N-dimethylethanolamine from the culture medium. The effect of N, N-dimethylethanolamine on reductase was associated with the formation of phosphatidyl- N, N-dimethylethanolamine which accumulated primarily at the expense of phosphatidylcholine and, after 24 h, accounted for 27% of total phospholipid phosphorus. The data demonstrate that incorporation of N, N- dimethylethanolamine into the polar head group of cellular phospholipids has a major impact on the regulation of the reductase. These observations may have particular relevance for the mechanisms of regulation of this enzyme, the cellular adaptation to alterations in membrane lipid composition, and the regulation of cholesterol synthesis in the developing nervous system.

Seasonal changes in the blood composition of captive and free-living White-crowned Sparrows

Seasonal changes in several blood components (cholesterol, phospholipid phosphorus, glyceride glycerol, free fatty acids, and calcium), hematocrit, and body mass were studied in captive and free-living groups of White-crowned Sparrows (Zonotrichia leucophrys gambelii) over a 2-year period. Cholesterol, phospholipid phosphorus, and glyceride glycerol levels were correlated with body mass and all of them changed in parallel during the year (Table 2 and Figs. 1–3). These lipids were elevated during premigratory and migratory periods, minimal during the summer breeding period, and reduced during periods of body molt. Concentrations of them were consistently higher in captive birds than in free-living ones. Free fatty acid levels were highly variable and not correlated with levels of other blood lipids or with body mass (Fig. 4). However, they too increased during premigratory periods. On the other hand, they were consistently higher in free-living sparrows than in captives. Plasma calcium was relatively constant at 3–5 mEq/l between July and the following March (Fig. 5). It increased during the spring, but earlier than preparations for migration by 2–3 weeks. It was also elevated in egg-laying females. The hematocrit rose during the vernal migratory period, but not during the autumnal one; was minimal in breeding birds; and declined during periods of body molt (Fig. 6). Calcium and hematocrit levels were similar in captive and free-living birds. It appears that captive populations of White-crowned Sparrows generally provide relibale information concerning changes in blood composition that are taking place concurrently in the field.

A semi-automated and manual method for determining the total phospholipid phosphorus in amniotic fluid

A rapid method for estimating the total phospholipid content of amniotic fluid has been described that requires 1 ml of sample. Perchloric acid containing a trace of molybdate is the acid digestant for inorganic phosphate release. After digestion at 225 °C for 30 min, dilution with water yields a solution with a hydrogen ion concentration of about 2.5 mol/liter that allows the use of stannous chloride-hydrazine sulfate as the reducing agent for phosphate color development. The color development and measurement may be carried out manually or with use of the AutoAnalyzer at an analysis rate of 40/hr. A coefficient of variation of about 7% and a standard deviation of 0.02 will be obtained with either the manual or semi-automated procedure.

Pyridinolcarbamate and experimental atherosclerosis correlation of hypocholesterolemic and antiatherogenic effects

Rabbits were maintained for 12 weeks on either a control or hypercholesteremic dietary regime, or on comparable diets supplemented with pyridinolcarbamate (PDC) at a level of 30 mg/kg body weight/day. Blood was obtained from all rabbits prior to study and at two-week intervals for analysis of serum cholesterol, phospholipid phosphorus and triglycerides. Animals from each group were sacrificed at 4-week intervals for quantitative assessment of the degree of atherosclerotic involvement of the aorta. All animals in the four groups consumed their entire daily allowance (100 g) of their respective diets, and weight gains throughout the feeding period were comparable in the 4 groups. PDC given with the control chow diet had no effect on serum cholesterol levels but did result in persistent decrease in serum triglycerides and a variable decrease in serum phospholipids during the 12-week feeding period. None of the rabbits on the chow diet, with or without PDC, had any evidence of aortic lesions during the experimental period. Rabbits fed 1% cholesterol administered with chow exhibited markedly elevated levels of serum cholesterol and phospholipids, while serum triglycerides were not significantly different than in the control group. In these animals there was a rapid and progressive increase in aortic atherosclerosis throughout the study, and at 12 weeks plaque involvement was 74 +- 8% of the aortic surface. Addition of PDC to the 1% cholesterol-chow diet resulted in significantly lowered levels of serum cholesterol and phospholipids, but these remained elevated compared to the control levels. There was also a dramatic reduction in the rate and extent of aortic plaque formation. Thus, after 12 weeks on diet, only 27 ± 6% of the aortic surface showed evidence of atheroma. The data suggest that PDC significantly decreases the hypercholesteremia resulting from feeding 1% cholesterol to rabbits, and that this may be largely responsible for the antiatherogenic effect of this drug.

1248 INTERACTION EFFECTS BETWEEN TRACHEAL LIGATION ANDI CERVICAL CORD SECTION ON LUNG DEVELOPMENT IN THE FETAL RABBIT

We have reported that cervical cord section at C1 - C3 level in 22 - 26 day rabbit fetuses results in a 43% reduction in lung wet weight and a 16% reduction in lung DNA when compared to sham operated littermates at harvesting on day 28 - 29. This effect could be due to impaired lung liquid secretion in the cord-sectioned (CS) fetuses. To investigate this aspect, tracheal ligation (TL) with or without high cervical CS was performed in rabbit fetuses at 24 - 26 days which were harvested 2 - 4 days later. Lung weight, lung DNA, lung phospholipid phosphorus, lung lecithin phosphorus and lung lecithin palmitate were compared between fetuses subjected to tracheal ligation alone, those with TL + CS and intact littermate controls. TL alone and TL + CS groups had a mean increase in lung weight/gram body weight of 80% and in lung DNA/gram body weight of 15% four days after operation. No consistent change was demonstrated in total lung phospholipid phosphorus, lecithin and lecithin palmitate. Our results indicate: isolated fetal cervical CS results in lung collapse and impaired growth; cervical CS does not impair lung liquid secretion; retained lung liquid secretion has a powerful growth-promoting effect which completely reverses the growth retarding effect of cord section. We hypothesize that the CNS influences fetal lung growth by controlling the retention rather than the formation of lung liquid.

The effect of rete testis fluid on the metabolism of testicular spermatozoa.

The O2 uptake of spermatozoa collected from the rete testis of conscious rams and boars has been measured polarographically either in the rete testis fluid (RTF) in which they were collected or in a synthetic saline medium (S-RTF) made to resemble the ionic composition of RTF as closely as possible. In both species, O2 uptake of the testicular spermatozoa was higher in RTF than in S-RTF. The addition of some of the organic constituents of RTF was without effect on O2 uptake. Pyruvate, lactate, acetate or alanine increased the O2 consumption of ram spermatozoa in S-RTF. Lactate had no effect on but acetate increased the O2 consumption of pig testicular spermatozoa. Phospholipid phosphorus and fatty acids decreased during incubation of ram spermatozoa in RTF and S-RTF. The addition of substances which increased the O2 consumption of the spermatozoa in S-RTF was without effect on the lipid composition. The fatty acid composition of the phospholipids and the proportions of the various phospholipids were unaffected by incubation. These results indicate that rete testis fluid has an appreciable effect on the metabolism of the spermatozoa it contains, and that this effect cannot be mimicked by any of the known organic constituents of the fluid.

Lectin-mediated agglutination of erythrocyte ghost membranes following depletion of membrane protein and intramembrane particles

Profound digestion of unsealed human erythrocyte ghosts with high concentrations of Pronase results in a near complete loss of intramembrane particles while trypsin digestion is less effective. The small vesicles formed by proteolysis are agglutinable by soybean agglutinin (SBA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA), but not concanavalin A (ConA). Densitometer tracings of Pronase-treated vesicles analyzed on SDS-polyacrylamide gels demonstrated no detectable protein or glycoprotein migrating slower than the marking dye. The vesicles showed a loss of 90% Lowry positive material (the remainder may be non-protein chromogens), near depletion of sialyl residues, no significant change in lipid composition, and equal amounts of phospholipid phosphorus compared to an equal volume of ghosts. The lipid material extracted from Pronase-derived vesicles or intact ghosts inhibited hemagglutination with SBA and WGA but not ConA. SBA but not ConA was found to specifically bind to Pronase-derived vesicles while both lectins bound to native ghosts. These observations suggest that neither the integrity of the intramembrane particles nor the presence of membrane glycoprotein appears essential for SBA-, WGA-, and PHA-mediated agglutination. Furthermore, it appears that native membrane glycolipids (and perhaps glycopeptides) can bind SBA, WGA and PHA. The membrane glycolipids may play a larger role than heretofore realized in lectin-mediated agglutination of cells.

Membrane cholesterol and cell fusion of hen and guinea-pig erythrocytes

1. The cholesterol content of hen erythrocytes was modified by treating the cells with phospholipid liposomes. 2. Depletion of cellular cholesterol, by using liposomes of dipalmitoylglycerophosphocholine or phosphatidylcholine from hen erythrocytes, had no effect on the susceptibility of the cells to fusion induced by oleoylglycerol, but markedly decreased fusion induced by Sendai virus. 3. By contrast, enrichment of cellular cholesterol by using liposomes of dipalmitoylglycerophosphocholine and cholesterol increased cell fusion induced by oleoylglycerol, poly(ethylene glycol) and Sendai virus. 4. Virus-induced cell fusion of guinea-pig erythrocytes, which were enriched in cholesterol by feeding a cholesterol-rich diet to the animals, was also enhanced. 5. Hen erythrocytes that were treated with liposomes prepared from egg phosphatidylcholine contained increased quantities of phospholipid phosphorus and fused readily on incubation with retinol, independently of their cholesterol content. 6. It is suggested that cholesterol may enhance cell fusion by acting to facilitate a phase separation of protein-free areas of lipid bilayer, which subsequently provide the sites for cell fusion.

Fetal lung maturity, as assessed by gas-liquid chromatographic determination of phospholipid palmitic acid in amniotic fluid.

We describe a new and specific method for measurement of lecithin palmitic acid in amniotic fluid. Dipalmitoyl lecithin, the major alveolar surfactant, has previously been estimated by measuring the lecithin-sphingomyelin ratio, total lecithin, total phospholipid phosphorus, and (or) total palmitic acid. Our method is more specific for estimation of dipalmitoyl lecithin, because nonphospholipid sources of palmitic acid are removed by solvent extraction. Using a hexane/2-propanol/sulfuric acid system, we obviated the major interferences from triglycerides and free fatty acids. The palmitic acid derived from the phospholipid fraction is measured by gas-liquid chromatography of its methyl ester. No contribution appears to be made by sphingomyelin palmitic acid--probably owing to the mild hydrolysis conditions. The measured palmitic acid therefore appears to be derived from lecithins, principally dipalmitoyl lecithin. The value for palmitic acid determined by this method correlates well with the lecithin-sphingomyelin ratio and total phospholipid phosphorus. Infants are unlikely to develop respiratory distress syndrome when the measured palmitic acid in amniotic fluid exceeds 8.0 mg/liter, which corresponds to an lecithin-sphingomyelin ratio of 2.0.

Myo-Inositol Metabolism during Lactation and Development in the Rat. The Prevention of Lactation-Induced Fatty Liver by Dietary myo-Inositol

Effects of dietary myo-inositol deprivation were examined during prenatal and postnatal development and during lactation in the rat. The deficient diet contained no detectable myo-inositol while the supplemented diet contained 0.5% (w/w) myo-inositol while the supplemented diet ct contained 0.5% (w/w) myo-inositol at the expense of sucrose. Both diets contained 25% casein, adequate amounts of all known vitamins, choline, and essential fatty acids as well as 0.5% (w/w) phthalylsulfathiazole to depress myo-inositol contribution to the diet by microorganisms. Pregnant rats of the Holtzman strain were fed the respective diets during gestation and lactation, and pups were fed the corresponding diet after weaning until 3 months of age. There were no significant differen-es in body weight between experimental groups. Supplementation of the diet with myo-inositol significanly increased the levels of myo-inositol in plasma, liver, kidney, and intestine of pups at all ages examined, and significantly increased the levels of myo-inositol in the milk and mammary tissue during lactation. During lactation, the myo-inositol deprived dams developed severe fatty livers (31% w/w) characterized by diminished phosphatidyl-inositol (50%) and total phospholipid phosphorus (57%) levels as compared with controls. After weaning, the liver lipid content of the myo-inositol deprived dams returned to normal (4.5%). The data suggest that a possible threshold level of free myo-inositol (approximately 0.15 mumoles/g lipid-free tissue) was required to prevent fatty liver in lactating dams under these dietary conditions. Effects of the deficient diet on fertility were also examined. Based on sperm count and production of offspring, there were no differnences between the experimental and control males. Females of both groups showed equal ability to produce offspring.

Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes

A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. 97 ± 2% of the total membrane phosphorus is accounted for as phospholipid phosphorus. Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been identified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present' in the 49 000–60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences. Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight. Membrane phosphorus was distributed between two chromatographic fractions: one containing the membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.

Glycoprotein-enriched vesicles from sheep erythrocyte ghosts obtained by spontaneous vesiculation.

Sheep erythrocyte membranes have been shown in this laboratory to undergo spontaneous vesiculation when incubated at 4 degrees, fractionating into two bands in dextran gradients (R. McGuire and R. Barber, submitted for publication). While vesicles were observed to be formed in several solvent systems, incubation in the presence of complexors to remove divalent cations was found to be the most efficient method for both vesicle formation and their detachment from the residual membrane. We report here on the characterization of these vesicles formed by spontaneous vesiculation. In the presence of a hypotnoic buffer containing 1 mM EDTA, vesicle production proceeds linearly up to 50 hours and declines, reaching its maximum at 72 hours with up to 20% of the total membrane protein found in the upper band. This upper band is shown in electron micrographs to be composed chiefly of closed vesicles, while the particles in the lower band appear morphologically similar to the original ghosts. Total phospholipid phosphorus and cholesterol in the vesicles are enriched to the same extent, giving a lipid to protein ratio of 2 times that found for whole ghosts. The vesicles contain the same individual phospholipids as the ghosts. The protein composition of these vesicles is unique, in that they are almost depleted in the known extrinsic membrane proteins, while containing practically all types of the various glycoproteins of the original membrane. The two main intrinsic membrane proteins (with apparent molecular weights of 160,000 and 100,000) are found almost exclusively in the vesicles, virtually depleted in the residual ghost-like particles. The protein with 160,000 molecular weight is shown here to be a glycoprotein, giving an anomalous molecular weight on sodium dodecyl sulfate gels and having a molecular weight of approximately 50,000 after lipid extraction. This same glycoprotein appears to fractionate with acetylcholinesterase. From the accessibilities of the substrates to the membrane acetylcholinesterase and NADH-diaphorase, it is concluded that the vesicles are right-side-out and sealed to small molecules. There are more membrane sialic acid residues accessible to neuraminidase in the vesicles (in terms of number of residues/mg og membrane protein) than in ghosts, further supporting the conclustion that these vesicles have a normal orientation and are enriched in glycoproteins. The specific activity of acetylcholinesterase in the vesicles is increased 5- to 6-fold over that found in the original ghosts and almost 20-fold over that in the residual ghost-like particles. Consequently, spontaneous vesiculation occurs simultaneously with the enrichement of specific membrane proteins in certain regions of the lipid bilayer. It is postulated that these domains in the membrane, containing clusters of specific intrinsic membrane proteins, bud out and subsequently release glycoprotein-enriched lipid vesicles.

Phospholipids and creatinine in amniotic fluid in relation to gestational age. II. Complicated pregnancy.

176 samples of amniotic fluid were obtained by abdominal amniocentesis from 69 women with complicated pregnancies (toxemia, diabetes mellitus or rhesus isoimmunization) in the 27th-43rd week of pregnancy. The concentration of creatinine (172 cases), the ratio of lecithin/sphingomyelin (L/S-ratio, 155 cases) and phospholipid phosphorus (155 cases) were determined and related to gestational age. The results were compared with normal pregnancy. The mean level of creatinine in toxemia was significantly higher from the 32nd week till term (p less than 0.05) and the L/S-ratio was significantly higher (p less than 0.05) in the 34th-35th week compared with normal pregnancy. Creatinine concentration was significantly elevated in diabetic pregnancy in the 36th-37th week (p less than 0.05). Creatinine concentration tended to be low in Rh-isoimmunization. A creatinine concentration greater than or equal to 1.8 mg% and a L/S-ration greater than or equal to 2.25 always corresponded to a gestational age greater than or equal to 35th week. To get increased precision in estimating gestational age in complicated as well as in normal pregnancy the determination of both creatinine concentration and L/S-ratio in amniotic fluid is recommended.

Proportions et renouvellement des différents phospholipides des fibroblastes normaux et transformés par le SV 40: Effet de la densité cellulaire

The phospholipid composition and turnover in normal and in SV 40 transformed hamster fibroblasts were studied. The amount of phospholipid phosphorus relative to protein is lower in transformed hamster fibroblasts than in normal fibroblasts. This amount decreases with increasing cell density until stationary growth is reached. The decrease is largest for the normal fibroblasts. In transformed cells, less sphingomyelin and more diphosphatidyl glycerol are found than in normal cells. The turnover of 32P in sphingomyelin is slower in transformed cells than in normal cells ; the contrary is observed with diphosphatidyl glycerol. On the other hand, in transformed cells, phosphatidyl ethanolamine has a faster turnover than phosphatidyl choline, whereas the contrary is observed in normal cells. Finally, the change to stationary growth slows down the turnover of 32P of all phospholipids, this decrease being more important in transformed cells.

Reactivation of the lipid-depleted pyruvate oxidase system from Escherichia coli with cell envelope neutral lipids.

The pyruvate oxidase system of Escherichia coli is composed of a soluble flavoprotein, pyruvate oxidase (EC 1.2.2.2, pyruvate:ferricytochrome b1 oxidoreductase), and an electron transport system associated with the cell envelope-membrane fraction. The membrane particles contain 15% lipid by weight. Fractionation of the lipids revealed that abut one-third are neutral lipids and two-thirds are phospholipids. The relative ratio of ubiquinone to menaquinone within the neutral lipid fraction is 15:1 on a molar basis. Removal of the lipids from the membrane particles by extraction with aqueous acetone or hydrolysis of the phospholipids by treatment with Bacillus cereus phospholipase C results in a complete loss of electron transport activity. Analysis of the particles extracted with aqueous acetone revealed that practically all the neutral lipids and 65% of the phospholipids are removed by this treatment. Phospholipase treatment results in a loss of 75% of the membrane phospholipid phosphorus; however, the diglycerides and the neutral lipids produced by phospholipase hydrolysis remain associated with the particles. Addition of neutral lipid and a detergent, hepta-DL-alanyl dodecylamide to the acetone-extracted material results in a restoration of 37% of the original particle activity. Addition of neutral lipid and hepta-DL-alanyl dodecylamide to phospholipase-treated particles completely restores the original electron transport activity. Furthermore, addition of ubiquinone from either yeast (UQ6) or E. coli (UQ8) will restore pyruvate oxidase activity when the quinones are supplemented with photoinactivated neutral lipid. No restoration of activity to phospholipase-treated particles is noted upon the addition of either menaquinone 6 or menaquinone 8 to the reconstitution system. In fact, these compounds appear to suppress restoration of activity when they are added to reaction mixtures containing neutral lipid and phospholipase-treated particles.