Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold-shock

The cholesterol content of spermatozoa with highly unsaturated phospholipids, i.e., ram and bull, which are very susceptible to cold-shock was compared with that of rabbit and human spermatozoa which have more saturated phospholipids and a greater resistance to cold-shock. The level of cholesterol in the seminal plasma was also estimated.Cholesterol was present in ram and bull spermatozoa in comparable amounts (280–346 μg) and at approximately half the level (in micrograms per 10⁹ spermatozoa) in rabbit and human spermatozoa (545–556 μg). The molar ratio of cholesterol: phospholipid in rabbit and human spermatozoa was 0.88 and 0.99, respectively, which is similar to the ratio of these lipids in erythrocytes and higher than the ratio of 0.38 and 0.45 for ram and bull spermatozoa, respectively. This ratio is of some importance in determining the nature and degree of packing in the spermatozoan membrane; higher levels of cholesterol result in a more cohesive, rigid, and impermeable structure. A definite relationship is apparent between the ratio of cholesterol: phospholipid, the ratio of polyunsaturated: saturated phospholipid-bound fatty acids, and the susceptibility of the spermatozoa to cold-shock.     

Levels and distributions of phospholipids and cholesterol in the plasma membrane of neuroblastoma cells.

Murine neuroblastoma cells (clone N-2A) grown in suspension (spinner cells) or attached on a plastic surface (monolayer cells) were used in studies of the phospholipid and cholesterol composition of whole cells, primary plasma membranes, plasma membranes internalized during phagocytosis of polystyrene latex beads, mitochondria and microsomes. Monolayer cells contained higher concentrations of total phospholipid, phosphatidylserine and phosphatidylcholine, and lower concentration of phosphatidylethanolamine than spinner cells. The cholesterol levels and the relative proportions of the various phospholipids were similar in both cell types except phosphatidylethanolamine and sphingomyelin whose proportions were lower in monolayer cells. The primary plasma membranes of the two cell types differed significantly in the relative proportions of all phospholipids, except sphingomyelin, and the phospholipid to protein and the cholesterol to protein ratios were all higher in the membranes of spinner cells. In contrast to these results, all the phospholipid to protein and the cholesterol to protein ratios of the internalized plasma membranes were higher in monolayer than in spinner cells, and the proportions of all phospholipids, except phosphatidylethanolamine, were similar in both cell types. The membrane distributions of individual phospholipids and cholesterol were inferred from comparison of the phospholipid and cholesterol compositions of primary plasma membranes and plasma membranes internalized during phagocytosis of polystyrene beads. The results are consistent with a non-random distribution of most phospholipids in both spinner and monolayer cells, but the patterns of these distributions were different in the two cell types. With regard to cholesterol the results are compatible with a random or a heterogeneous distribution. All the phospholipid to protein ratios of the mitochondrial fraction of both cell types were lower than those of the plasma membranes. However, these ratios of the microsomal fraction were higher than those of the plasma membranes of monolayer cells, whereas they were comparable, with a few exceptions, to those of spinner cell membranes. The cholesterol to phospholipid molar ratios of plasma membranes were 6.4 and 4.3 fold greater than those of the mitochondrial and microsomal fractions, respectively.     

Phospholipid composition modifications influence phospholipase A2 activity in rat liver plasma membranes

The influence of the phospholipid composition and the physico-chemical properties of rat liver plasma membranes on the activity of membrane-bound phospholipase A2 has been investigated. The plasma membrane composition was modified by the aid of exogenous phospholipases A2, C and D, and by butanol treatment. The partially delipidated membranes thus obtained were enriched with different phospholipids. The steady-state fluorescent anisotropy of 1,6-diphenyl-1,3,5-hexatriene and the lipid order parameter-SDPH in the modified membranes were calculated. It was established that the activity of the membrane-bound phospholipase A2 was higher in rigid membranes and was decreased when the membrane lipid bilayer was fluidized.     

Modulation of the lipid composition of boar sperm plasma membranes during an acrosome reaction in vitro

An in vitro acrosome-like reaction was induced in spermatozoa from the boar cauda epididymis by incubation in Tyrode's solution containing 1 mg/ml fatty acid-free bovine serum albumin. Plasma membranes were isolated from the spermatozoa at different times during the incubation and analyzed for their lipid composition. The total lipid, phospholipid, and glycolipid content of the membranes did not change during the acrosome-like reaction, whereas the amount of diacylglycerols and free fatty acids increased. Within the phospholipid class, a decrease of the inositol phospholipid and and sphingomyelin content was observed, whereas the other phospholipids of the plasma membranes did not decrease significantly after 2 h of incubation. Changes in the sterol composition of the membranes were also observed. The onset of the lipid changes was correlated with the uptake of extracellular calcium by the spermatozoa. These results for the lipid changes in isolated sperm plasma membranes during an in vitro acrosome reaction provide the first direct evidence that a modulation of the plasma membrane lipid composition is involved in an acrosome-like reaction of mammalian spermatozoa.     

Influence of the bovine seminal plasma protein PDC-109 on the physical state of membranes.

PDC-109 is the main component of bovine seminal plasma and has been suggested to play an important role in the genesis of bovine sperm cells. Here, the effect of binding of PDC-109 to membranes on the structure and physical properties of the lipid phase was investigated. For that, ESR measurements were undertaken on model membranes (lipid vesicles) and on biological membranes (epididymal spermatozoa) by employing various spin-labeled phospholipids. We found that PDC-109 alters the membrane structure of lipid vesicles as well as of bovine epididymal spermatozoa in that the mobility of spin-labeled phospholipids was reduced in the presence of the protein. This immobilizing effect of the protein was not restricted to analogues of phosphatidylcholine but was also detected with spin-labeled phosphatidylethanolamine. However, the extent of immobilization was lower for phosphatidylethanolamine compared with phosphatidylcholine, supporting the lipid headgroup specificity of the protein. Besides phospholipid headgroups, the physical state of membrane lipids is also important for the interaction of PDC-109 with membranes, in that, e.g., the immobilizing effect of the protein on labeled lipids was larger in membranes above the phase transition temperature compared with the effect below this temperature. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.     

Metabolism of phospholipids by spermatozoa and seminal plasma

1. The hydrolysis of added (32)P-labelled phospholipids by whole ram and bull semen and the separated spermatozoal and plasma components was examined. 2. The ethanolamine phosphoglycerides were rapidly attacked by washed spermatozoa, forming predominantly glycerylphosphorylethanolamine, but with whole semen and seminal plasma a lysophosphatidylethanolamine was also detected. 3. The hydrolysis of lecithin by spermatozoa and plasma was very slow, and glycerylphosphorylcholine was the sole product detected. 4. Ram testicular spermatozoa were comparatively inactive in metabolizing both phospholipids, but ampulla contents showed the same activity as ejaculated semen. 5. Phosphatidylinositol was metabolized by spermatozoa obtained from any portion of the ram reproductive tract and also by seminal plasma. With testicular components, ampulla contents and washed ejaculated spermatozoa, inositol monophosphate, an unidentified phosphate ester and inorganic phosphate were the main products. In contrast, with whole semen and seminal plasma, glycerylphosphorylinositol was the predominant water-soluble phosphate ester. 6. Accessory-gland secretion obtained from vasectomized rams showed a pronounced phospholipase A activity towards ethanolamine phosphoglyceride. 7. On aerobic incubation of whole ram semen there was a decrease in the concentration of all phospholipid classes, although cardiolipin showed the greatest percentage decrease. In the choline phosphoglyceride fraction, this loss was confined to the plasmalogen component. This breakdown of phospholipids was decreased considerably when the spermatozoa were washed, and was not observed when whole bull semen was incubated under similar conditions.     

Translocation of phospholipids between the outer and inner membranes of Salmonella typhimurium.

The reversibility and specificity of phospholipid translocation between the inner and outer membrane of Salmonella typhimurium has been investigated by incorporating exogenous lipids from phospholipid vesicles into the outer membrane of intact cells. Translocation of newly incorporated phospholipids to the inner membrane was demonstrated by decarboxylation of vesicle-derived phosphatidylserine and by recovery of vesicle constituents in both inner and outer membrane fractions. All Salmonella phospholipids tested, as well as phosphatidylcholine and cholesteryl oleate were effectively translocated to the inner membrane. However, no translocation of vesicle-derived lipopolysaccharide or an incomplete biosynthetic precursor of lipid A could be detected. Translocation of phospholipids and cholesteryl ester was rapid and extensive, and appeared to lead to equilibration of the lipids between the two membranes. The mechanism of intermembrane translocation has not been established, but the results are suggestive of diffusional flow across zones of adhesion between the inner and outer membranes.     

Changes in the phospholipid composition and phospholipid asymmetry of ram sperm plasma membranes after cryopreservation

The changes in the phospholipid composition of spermatozoa plasma membranes after freezing were determined by thin-layer chromatography. The results showed an augmentation of the diphosphatidylglycerol and a diminution of phosphatidylglycerol, phosphatidylserine, and phosphatidylethanolamine in sperm plasma membranes after freezing. In intact sperm cells we observed an elevation of the sphingomyelin and phosphatidylinositol levels and a diminution of the phosphatidylethanolamine and diphosphatidylglycerol levels. The effect of freezing on the phospholipid distribution between the inner and outer monolayers of the plasma membrane was also studied using exogenous phospholipases and trinitrobenzene sulfonate. The most important change we observed after freezing, was the translocation of diphosphatidylglycerol from the inner to the outer monolayer of the plasma membrane.     

Comparison of phospholipid effects on insulin-sensitive low Km cyclic AMP phosphodiesterase in adipocyte plasma membranes and microsomes

Both adipocyte plasma membranes and microsomes possess insulin-sensitive low Km cyclic AMP phosphodiesterase activity. The activity of the enzyme from both sources was susceptible to activation by several anionic phospholipids. Activators of the plasma membrane enzyme were lysophosphatidylglycerol greater than lysophosphatidylcholine greater than lysophosphatidylserine greater than phosphatidylserine greater than phosphatidylglycerol. These same phospholipids activated the microsomal enzyme but the extent of activation by each phospholipid was reversed. Neutral phospholipids and other anionic phospholipids were without effect. The phospholipids had no effect on high Km cAMP phosphodiesterase in either membrane. The results suggest that the phospholipid headgroup was an important determinant for enzyme activation by phospholipid. The increased susceptibility of the plasma membrane enzyme to lysophospholipid may be attributed to a difference in the plasma membrane enzyme compared to the microsomal membrane enzyme or to differences in plasma membrane and microsomal membrane phospholipid composition and their ability to regulate low Km cAMP phosphodiesterase activity.     

Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol

Most in vitro studies use 2-dimensional (2D) monolayer cultures, where cells are forced to adjust to unnatural substrates that differ significantly from the natural 3-dimensional (3D) extracellular matrix that surrounds cells in living organisms. Our analysis demonstrates significant differences in the cholesterol and sphingomyelin content, structural organization and cholesterol susceptibility to oxidation of plasma membranes isolated from cells cultured in 3D cultures compared with conventional 2D cultures. Differences occurred in the asymmetry of cholesterol molecules and the physico-chemical properties of the 2 separate leaflets of plasma membranes in 2D and 3D cultured fibroblasts. Transmembrane distribution of other membrane phospholipids was not different, implying that the cholesterol asymmetry could not be attributed to alterations in the scramblase transport system. Differences were also established in the chemical activity of cholesterol, assessed by its susceptibility to cholesterol oxidase in conventional and “matrix” cell cultures. The influence of plasma membrane sphingomyelin and phospholipid content on cholesterol susceptibility to oxidation in 2D and 3D cells was investigated with exogenous sphingomyelinase (SMase) and phospholipase C (PLC) treatment. Sphingomyelin was more effective than membrane phospholipids in protecting cholesterol from oxidation. We presume that the higher cholesterol/sphingomyelin molar ratio is the reason for the higher rate of cholesterol oxidation in plasma membranes of 3D cells.     

Evaluation of plasma membrane stability by detergent-induced rupture of osmotically swollen sperm.

A general assay for plasma membrane stability was developed and tested. Osmotically swollen spermatozoa were ruptured with detergents and their volume distribution was monitored with resistance pulse spectroscopy. The extent of cell breakage was determined and expressed as [D]50, the concentration of detergent necessary to lyse 50% of the initially intact cells. Preliminary experiments established the degree to which spermatozoa could be swollen without lysis (no detergent) and the ability of the method to detect known mixtures of intact and membrane disrupted spermatozoa. [D]50 values were determined for caput (immature) and cauda (mature) ram epididymal spermatozoa with four detergents (cetyltrimethylammonium bromide, sodium dodecylsulfate, Zwittergent 3-14, and sodium deoxycholate). [D]50 values for caput spermatozoa were higher than those for cauda spermatozoa (P less than 0.05) for all detergents but cetyltrimethylammonium bromide. These changes are consistent with a qualitative model of membrane structure and stability based on lipid shape and composition and with the compositional changes known to occur during epididymal maturation. Additional studies using rooster spermatozoa established that a typical cryopreservation protocol leaves the surviving spermatozoa with membranes with greater sensitivity to detergent-induced stress. Since osmotic swelling has been microscopically localized to the tail plasma membrane, the changes in membrane stability can be assigned specifically to that region.     

Regulation of aminophospholipid asymmetry in murine fibroblast plasma membranes by choline and ethanolamine analogues

The regulation of the asymmetric distribution of aminophospholipids in mammalian cell plasma membranes is not understood at this time. One approach to determine the nature of such regulatory mechanisms is to attempt alteration of the plasma membrane phospholipid composition. Choline analogues such as N,N'-dimethylethanolamine and N-monomethylethanolamine lowered the quantity of phosphatidylethanolamine in the plasma membrane of LM fibroblasts grown in defined medium without serum. Ethanolamine supplementation increased the phosphatidylethanolamine content while ethanolamine analogues such as 2-amino-2-methyl-1-propanol, 2-amino-1-butanol, 1-aminopropanol, and 3-aminopropanol did not alter the aminophospholipid content significantly. The transverse distribution of aminophospholipids in the plasma membrane was determined by use of a chemical labelling reagent trinitrobenzenesulfonic acid. The percent phosphatidylethanolamine trinitrophenylated by trinitrobenzenesulfonate in the outer plasma membrane monolayer of LM cells supplemented with choline analogues was not altered. In contrast, ethanolamine analogue supplementation increased the percentage of aminophospholipid in the outer monolayer 2--3-fold. Ethanolamine analogue-containing phospholipids were distributed asymmetrically across the plasma membrane with 85 to 91% being located in the inner monolayer of the plasma membrane, a distribution similar to that of phosphatidylethanolamine. The fatty acyl composition of aminophospholipids in the outer monolayer was in all cases more saturated than in the corresponding phospholipids of the inner monolayer. However, choline analogues and especially the ethanolamine analogues reduced this difference. Thus, base analogues of choline and ethanolamine may alter the aminophospholipid asymmetry, the surface charge, and the acyl chain asymmetry of LM cell plasma membranes.     

Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture,model of the Escherichia coli cytoplasmic membrane

Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes.     

Role of membrane-associated cytoskeleton in maintenance of membrane structure

Various structural components of biological membranes are asymmetrically localized in the two surfaces of the membrane bilayer. This asymmetry is absolute for membrane (glyco) proteins, but only a partial asymmetry has been observed for membrane phospholipids. In the red cell membrane, choline-phospholipids are localized mainly in the outer monolayer whereas aminophospholipids are distributed almost exclusively in the inner monolayer. Several evidences are now available to suggest that this distribution of membrane phospholipids in red cells is directly or indirectly maintained by the membrane-associated cytoskeleton (membrane skeleton). This belief is well supported by the previous as well as recent studies carried out in the authors laboratory. Previously, it has been shown that lipid-lipid interactions play no major role in maintaining the transmembrane phospholipid asymmetry in erythrocytes, and that the asymmetry is lost upon covalent crosslinking of the major membrane skeletal protein, spectrin. The recent data presented here further shows that degradation or denaturation of spectrin indices rapid transbilayer movement of membrane phospholipids in the cells which, in turn, leads to more random phospholipid distributions across the membrane. These studies taken together strongly suggest that the skeleton-membrane associations are the major determinants of the transmembrane phospholipid asymmetry in erythrocytes, and that the dissociation of the skeleton from the membrane bilayer probably results in generation of new reorientation sites for phospholipids in the membrane. Communication No 3648 from C.D.R.I., Lucknow.     

Gonadotropin receptors in plasma membranes of bovine corpus luteum. II. Role of membrane phospholipids.

The role of phospholipids in the binding of 125I-choriogonadotropin to bovine corpus luteum plasma membranes has been investigated with the use of purified phospholipase A and phospholipase C to alter membrane phospholipids. The phospholipase C-digested plasma membrane preparation showed 85 to 90% inhibition of 125I-choriogonadotropin binding activity when 70% of the membrane phospholipid was hydrolyzed. Similarly treatment of plasma membranes with phospholipase A resulted in 45 to 55% hydrolysis of membrane phospholipid and almost 75% inhibition of receptor activity. Both these enzymes hydrolyzed membrane-associated phosphatidylcholine to a greater extent than phosphatidylethanolamine and phosphatidylserine. Phosphorylaminoalcohols of phospholiphase C end products were completely released into the medium, while phospholipase A by-products remained associated with plasma membranes. Addition of a phospholipids suspension or liposomes to plasma membranes pretreated with phospholipase A and C did not restore gonadotropin binding activity. Soluble phosphorylcholine, phosphorylethanolamine, and phosphorylserine and insoluble diglyceride products of phospholipase C action had no effect on receptor activity. In contrast, end products of the phospholipase A action, such as lysophosphatides and fatty acids, inhibited both on the membrane-associated and solubilized receptor activity. Lysophosphatidylcholine was the most effective end product inhibiting the binding of gonadotropin to the receptor, followed by lysophosphatidylethanolamine and lysophosphatidylserine. The inhibitory effects of phospholipase A or lysophosphatides were completely reversed upon removal of membrane-bound phospholipid end products by washing the membranes with defatted bovine serum albumin. However, phospholipase C inhibition could not be overcome by defatted albumin washings. Solubilization of plasma membranes with detergents which had been pretreated with phospholipase C partially restored the inhibited activity. It is concluded that the phospholipase-mediated inhibition of gonadotropin binding activity was due to hydrolysis and alterations of the phospholipid environment in the case of phospholipase C and by direct inhibition by end products in the case of phospholipase A.     

Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage

Cryopreservation causes sub-lethal damage which limits the fertility of frozen thawed spermatozoa. Seminal plasma has been investigated as a cryoprotectant, but has yielded inconsistent results due to considerable variation in its constituents. Individual seminal plasma proteins offer an ideal alternative to whole seminal plasma, and several have been correlated with freezing success. Binder of Sperm Proteins (BSPs) are abundant ram seminal plasma proteins which have been suggested to have significant protective effects on ram spermatozoa during cold shock. This is in direct opposition to bull spermatozoa, where BSPs cause sperm deterioration during in vitro handling. We investigated the potential of BSP1 and BSP5 to prevent freezing associated damage to important functional parameters of ram spermatozoa. BSPs purified by size exclusion chromatography improved post thaw motility and penetration through artificial mucus. Highly purified BSP1 and BSP5, isolated by gelatin affinity and RP-HPLC, improved motility and membrane integrity, and reduced post thaw protein tyrosine phosphorylation. Exposure to BSP5 before freezing increased the amount of phosphatidylethanolamine on the sperm surface after thawing. Neither BSP1 nor BSP5 prevented freezing associated changes in membrane lipid disorder. These results suggest that BSPs may significantly improve freezing outcomes of ram spermatozoa.     

Effect of membrane sterol content on the susceptibility of phospholipids to phospholipase A2

The effects of membrane sterol level on the susceptibility of LM cell plasma membranes to exogenous phospholipases A2 has been investigated. Isolated plasma membranes, containing normal or decreased sterol content, were prepared from mutant LM cell sterol auxotrophs. beta-Bungarotoxin-catalyzed hydrolysis of both endogenous phospholipids and phospholipids introduced into the membranes with beef liver phospholipid exchange proteins was monitored. In both cases, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were degraded at similar rates in normal membranes, while PC hydrolysis was specifically accelerated in sterol-depleted membranes. Additional data suggest that this preferential hydrolysis of PC is not a consequence of the phospholipid head group specificity of the phospholipase, nor of a difference in the accessibility of PC versus PE to the enzyme. Analysis of the reaction products formed during treatment of isolated membranes with phospholipase A2 showed almost no accumulation of lysophospholipids. This was found to be due to highly active lysophospholipase(s), present in LM cell plasma membranes, acting on the lysophospholipids formed by phospholipase A2 action. A soluble phospholipase A2 was partially purified from LM cells and found to behave as beta-bungarotoxin with regard to membrane sterol content. These results demonstrate that the nature of phospholipid hydrolysis, catalyzed by phospholipase A2, can be significantly affected by membrane lipid composition.     

Lipid dynamics in the plasma membrane of ram and bull spermatozoa after washing and exposure to macromolecules BSA and PVP.

Seminal plasma proteins and macromolecules in the external medium have a major influence on the functionality of sperm plasma membranes. In this investigation we have examined their effects on lipid diffusion in the surface membrane of ram and bull spermatozoa as measured by fluorescence recovery after photobleaching (FRAP). Results show that progressive removal of seminal plasma from ram spermatozoa by repeated centrifugation and resuspension in media +/- 4% bovine serum albumin (BSA) or 0.4% polyvinlypyrrolidone (PVP) causes a reduction in lipid diffusion in all regions of the membrane. By contrast, bull sperm membranes respond with an increase in diffusion in all regions. Repeated washing of bull spermatozoa whose membranes were previously immobile (i.e., showed no recovery after FRAP) restored lipid diffusion suggesting an inhibitory effect of seminal plasma proteins. Further analysis by atomic force microscopy revealed a close association between BSA and the plasma membrane. It is concluded that diffusion of lipids in the plasma membrane of ejaculated ram and bull spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium. Mol. Reprod. Dev. 59:306-313, 2001.     

Phospholipid flip-out controls the cell cycle of Escherichia coli

Phospholipids are the principal constituents of biological membranes. In Escherichia coli, phospholipids are involved in the metabolism of other envelope constituents such as lipoprotein, lipopolysaccharide, certain envelope proteins and peptidoglycan. They are also involved in the regulation of the cell cycle. DNAA, the key protein in the initiation of chromosome replication, is activated by acidic phospholipids only when these are in fluid bilayers, whilst interruptions of phospholipid synthesis inhibit both the initiation of chromosome replication and cell division. The transmembrane movement or flip-flop of phospholipids from one monolayer to the other requires the passage of the polar head group through the hydrophobic core of the bilayer. Hence, in many systems, flip-flop is a slow process with half-time of days. Flip-flop accompanies the formation of non-bilayer structure. Such structures form under certain conditions of packing density and composition and have been observed both in vitro and in vivo. In bacteria, flip-flop appears to be extremely rapid, with half-times as fast as 3 min being observed. However, such rapid flip-flop may not be characteristic of all phospholipids. The asymmetrical distribution of phosphatidylethanolamine in the plasma membrane of Bacillus megaterium has been attributed to the existence of two classes of this phospholipid. In E. coli, studies of the metabolic turnover of phosphatidylserine, phosphatidylglycerol and phosphatidic acid also reveal the existence of distinct classes of these phospholipids. In this article I propose that, in E. coli, a class of phospholipids does indeed escape the rapid flip-flop mechanism; this class probably includes a subpopulation of the acidic phospholipids. Therefore during the cell cycle these phospholipids accumulate in the inner monolayer of the cytoplasmic membrane and so cause an increase in its packing density; at a critical density, phospholipids "flip out" from the inner to the outer monolayer. This flip-out occurs once per cycle and initiates cell cycle events.     

Comparison of phospholipid effects on insulin-sensitive low Km cyclic AMP phosphodiesterase in adipocyte plasma membranes and microsomes

Both adipocyte plasma membranes and microsomes possess insulin-sensitive low K_m cyclic AMP phosphodiesterase activity. The activity of the enzyme from both sources was susceptible to activation by several anionic phospholipids. Activators of the plasma membrane enzyme were lysophosphatidylglycerol > lysophosphatidylcholine > lysophosphatidylserine > phosphatidylserine > phosphatidylglycerol. These same phospholipids activated the microsomal enzyme but the extent of activation by each phospholipid was reversed. Neutral phospholipids and other anionic phospholipids were without effect. The phospholipids had no effect on high K_m cAMP phosphodiesterase in either membrane. The results suggest that the phospholipid headgroup was an important determinant for enzyme activation by phospholipid. The increased susceptibility of the plasma membrane enzyme to lysophospholipid may be attributed to a difference in the plasma membrane enzyme compared to the microsomal membrane enzyme or to differences in plasma membrane and microsomal membrane phospholipid composition and their ability to regulate low K_m cAMP phosphodiesterase activity.