Exchange of phospholipids between brain membranes in vitro

1. When unlabelled mitochondria from guinea-pig brain were incubated with a (32)P-labelled microsomal fraction from brain there was a transfer of phospholipid to the mitochondria, which could not be accounted for by an aggregation of microsomes and mitochondria or an exchange with microsomes contaminating the mitochondria. Under similar circumstances there was a transfer of phospholipid from (32)P-labelled mitochondria to microsomes, indicating that the process was one of exchange. 2. The transfer from microsomes was greatly stimulated by a non-dialysable heat-labile macromolecular component in the brain supernatant fraction but not by the concentration of the particulate fractions. 3. Phospholipid-exchange processes occurred most readily between pH7 and 7.5 and were inhibited by the presence of myelin and on the addition of lysophosphatidylcholine. 4. The rates of transfer of individual phospholipids from brain microsomes to mitochondria were similar. 5. (32)P-labelled microsomes could slowly donate phospholipid to the isolated synaptosomal (nerve-ending) fraction but the phospholipids of the myelin fraction did not exchange. 6. Subfractionation of the synaptosomal fraction after [(32)P]phospholipid transfer showed that the mitochondria were most actively labelled during the incubation. All of the isolated individual synaptosomal membranes were capable of acquiring phospholipid on incubation with a (32)P-labelled brain supernatant fraction although a greater percentage was again exchanged by the mitochondrial fraction.     

Ageing-Induced Alterations in Lipid/Phospholipid Profiles of Rat Brain and Liver Mitochondria: Implications for Mitochondrial Energy-Linked Functions

Effects of ageing on the lipid/phospholipid profile of brain and liver mitochondria from rats were examined. In the brain mitochondria the contents of total phospholipid (TPL) and cholesterol (CHL) increased with simultaneous increase in the TPL/CHL (mole:mole) ratio. The proportion and contents of lysophospholipid (Lyso), sphingomyelin (SPM), phosphatidylinositol (PI), phosphatidylserine (PS) and diphosphatidylglycerol (DPG) components increased, with maximal increases seen for PS and PI; phosphatidylcholine (PC) and phosphatidylethanolamine (PE) components registered decrease. In the liver mitochondria contents of TPL and CHL increased. However, the TPL/CHL (mole:mole) ratio was not altered. Lyso, PI and PS increased. However, the magnitude of increase was competitively lower; PE and DPG decreased. SPM and PC did not change as a consequence of ageing. These changes altered the contents of individual phospholipids in the two membrane systems. Respiration with glutamate, pyruvate + malate, succinate and ascorbate + N,N,N’,N’-tetramethyl-p-phenylenediamine was significantly impaired in brain mitochondria from old animals. For liver mitochondria the respiratory activity declined with glutamate and succinate. Correlation studies by regression analysis revealed that the lipid/phospholipid classes regulate respiratory function differently in the mitochondria from the two tissues. The respiration-related parameters in the brain mitochondria were dependent on multiple lipid/phospholipid components, and the process of regulation was complex compared to the liver mitochondrial functions.     

Effect of N-ethylmaleimide on rat liver phosphatidylcholine-specific and non-specific transfer protein activities: its dependence on donor liposome composition

Phosphatidylcholine exchange between liposomes and mitochondria catalyzed by rat liver phosphatidylcholine transfer protein is strongly stimulated by N-ethylmaleimide (NEM) when PC/PI (molar ratio, 4:1) donor liposomes are used. In the presence of PC/PE or PC liposomes the exchange activity by this protein is unaffected. In the same experimental conditions, the activity of rat liver non-specific transfer protein is always stimulated by N-ethylmaleimide with all the types of liposomes tested in the order PC/PI greater than PC/PE greater than PC. Since the effect of NEM depends on the type of liposomes used and appears to be similar for both phospholipid transfer proteins, the possibility that their mode of action implies the formation of a ternary complex should be considered. As far as non-specific transfer protein is concerned, its interaction could vary depending on the nature of the exchanging membranes. Data are also presented indicating that when the two transfer proteins are together their activity is additive, therefore suggesting a specific role in phospholipid biomembrane assembly for each of them.     

Phospholipid exchange reactions within the liver cell

1. Isolated rat liver mitochondria do not synthesize labelled phosphatidylcholine from CDP-[(14)C]choline or any phospholipid other than phosphatidic acid from [(32)P]phosphate. The minimal labelling of phosphatidylcholine and other phosphoglycerides can be attributed to microsomal contamination. However, when mitochondria and microsomes are incubated together with [(32)P]phosphate, the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of the reisolated mitochondria become labelled, suggesting a transfer of phospholipids between the two fractions. 2. When liver microsomes or mitochondria containing labelled phosphatidylcholine are independently incubated with the opposite un-labelled fraction, there is a substantial and rapid exchange of the phospholipid between the two membranes. Exchange of phosphatidylinositol also occurs rapidly, whereas phosphatidylethanolamine and phosphatidic acid exchange only slowly. There is no corresponding transfer of marker enzymes. The transfer of phosphatidylcholine does not occur at 0 degrees , and there is no requirement for added substrate, ATP or Mg(2+), but the omission of a heat-labile supernatant fraction markedly decreases the exchange. 3. After intravenous injection of [(32)P]phosphate, short-period labelling experiments of the individual phospholipids of rat liver microsomes and mitochondria in vivo give no evidence for a similar exchange process. However, the incubation of isolated microsomes and mitochondria with [(32)P]phosphate also fails on reisolation of the fractions to demonstrate a precursor-product relationship between the individual phospholipids of the two membranes. 4. The intraperitoneal injection of [(32)P]phosphate results in a far greater proportion of the dose entering the liver than does intravenous administration. After intraperitoneal administration of [(32)P]phosphate the specific radioactivities of the individual phospholipids are in the order microsomes > outer mitochondrial membrane > inner mitochondrial membrane. 5. The incorporation of (32)P into cardiolipin is very slow both in vivo and in vitro. After labelling in vivo the radioactivity in the cardiolipin persists compared with that of the other phospholipids, whose specific radioactivities in the microsomes and mitochondrial fragments decay at a similar rate to that of the acid-soluble phosphate pool. 6. The possibility of phospholipid exchange processes occurring in the liver cell in vivo is discussed, and it is suggested that only a small but highly labelled part of the endoplasmic-reticulum lipoprotein pool is involved in the transfer.     

THE EXCHANGE OF PHOSPHATIDYLCHOLINE AND OF PHOSPHATIDYLINOSITOL IN SHEEP BRAIN

—The exchange of phospholipids between liposomes and brain mitochondria has been studied in the presence of pH 5·1 supernatant fluids derived from rat, guinea pig, sheep and ox brains. The exchange phenomenon was similar to that observed in liver and heart, but phosphatidylinositol and not phosphatidylcholine was the most rapidly exchanging phospholipid. The phosphatidylcholine exchange activity was purified 186-fold from sheep brain and the protein fraction contained two major and several minor protein species. The phosphatidylcholine and phosphatidylinositol exchange activities have been shown to have very similar molecular weights and isoelectric points. However, their behaviour in response to changes in liposomal surface charge suggested that separate proteins might be involved in stimulating the exchange of the two phospholipid classes.     

Effect of environmental temperature on the phospholipid metabolism of gill mitochondria of Carcinus maenas

The different types of phospholipids extracted from gill mitochondria of crab Carcinus maenas have been analysed and it was found that a significant increase of the phosphatidylethanolamine (PE) content and a concomitant decrease of the phosphatidylcholine (PC) amount are present in animals living in low temperatures. The incorporation of [³H]ethanolamine in total phospholipids, PE and PC, was demonstrated in gill mitochondria and a thermal alteration of the in vivo exchange of PE between mitochondria and 10,000 g supernatant is suggested by the kinetics of the incorporation. It is suggested that the conversion of PE to PC by N-methylation is very low in crab gills. There is a marked action of acclimation temperature on the gills-hemolymph exchange of PC and PE. It is postulated that the changes reported at the level of the PE → PC conversion by N-methylation and in phospholipid exchange between hemolymph and gills could be implicated in adapting the organism to seasonal fluctuations of environmental temperatures.     

Investigation of lipid-exchange lipoproteins from rat liver and hepatoma 27]

A phospholipid exchange lipoprotein from the postmicrosomal supernatant of rat hepatoma 27, which stimulated in vitro the exchange of sphingomyelin between mitochondria and microsomes, was found. Sphingomyelin is incorporated into the mitochondria under incubation of this complex with rat liver mitochondria (in which sphingomyelin is absent) an microsomes. Under the same conditions the phospholipid exchange lipoproteins of rat liver do not transfer sphingomyelin form microsomes to mitochrondria.     

Topographical Distribution of Base Exchange Activities in Rat Brain Subcellular Fractions

Abstract: Enrichment in the base-exchange activities was found in the micro-somal fraction of rat brain, with less activity being associated with nuclei, mitochondria and synaptosomes. The distribution of the choline base exchange in microsomal subfractions differed from that for serine and ethanolamine and these three activities seemed asymmetrically distributed in the microsomes. Choline exchange activity was trypsin-sensitive and presumably was located on the cytoplasmic side of the microsomes, while serine and ethanolamine exchange activities were trypsin-insensitive and were assumed to be located on the luminal side of the microsomes. Treatment of rat brain microsomes with phospholipases A, C and D produced significant losses of membrane-bound base exchange activities. Some activity was restored in phospholipase C-treated microsomes by exogenous phospholipid, but significant restoration was not observed in phospholipase A-treated microsomes by such additions. Exogenous phospholipid stimulated choline and ethanolamine exchange activities, but not serine exchange activity of phospholipase D-treated microsomes. The exchange activities of rat brain microsomes differed in their responses to treatment with phospholipases, choline exchange activity in general being more sensitive than either serine or ethanolamine activities.     

A universal lipid exchange protein from rat hepatoma.

The postmicrosomal protein fraction from rat hepatoma 27 adjusted to pH 5.1 stimulates phospholipid exchange between rat liver microsomes and mitochondria with higher rates and in a less specific way than the corresponding fraction from rat liver. A phospholipid exchange protein has been purified to homogeneity from the hepatoma pH-5.1 supernatant by gel filtration on Sephadex G-75 and ion-exchange chromatography on carboxymethylcellulose. The isolated protein had a molecular weight of 11200 as determined by electrophoresis on polyacrylamide in the presence of dodecyl sulfate and of 11168 as calculated from the amino acid composition. Isoelectric focusing showed a single band at pH 5.2. in the assay system rat liver microsomes leads to mitochondria the protein exhibits a complete lack of substrate specificity transferring all the major microsomal phospholipids to about the same extent. The possible role of the isolated phospholipid exchange protein in the chemical dedifferentiation of hepatoma cell membranes is discussed.     

Intracellular phospholipid movement and the role of phospholipid transfer proteins in animal cells

The mechanism of the intracellular movement of phospholipids from their site of synthesis in the endoplasmic reticulum to mitochondria and other cell membranes is a major unsolved problem of cell biology. Phospholipid transfer proteins of varying specificity found in the soluble supernatant fractions of many tissues catalyze the transfer of phospholipids from microsomes to mitochondria in vitro. They are postulated to play a similar role in vivo, but evidence for their function in living cells is lacking. We have now used an analogue of choline, N-propyl-N,N-dimethylethanolamine [PDME, (2-hydroxyethyl)dimethylpropylammonium hydroxide], to devise a test for the function of the transfer proteins in living cells. The rates of translocation of newly synthesized phosphatidylcholine and the analogue phosphatidyl-PDME in living cells were compared with the rates of transfer in vitro catalyzed by soluble transfer proteins extracted from the same cells. Labeled PDME, choline, and ethanolamine were found to be rapidly incorporated into the lipids of isolated rat hepatocytes and of baby hamster kidney (BHK-21) cells in culture. The translocation of newly synthesized phosphatidylcholine and phosphatidyl-PDME was very rapid in both types of cells with a half-time for equilibration of a few minutes, while the translocation of phosphatidylethanolamine was much slower, with a half-time 20-80 fold longer than those of the other two phospholipids. We then compared these relative rates of movement with the activities of the phospholipid transfer proteins of the respective cells. Partially purified phosphatidylcholine transfer protein from rat liver transfers phosphatidylcholine and phosphatidyl-PDME at identical rates but transfers phosphatidylethanolamine at a rate too low to be detected. This result is consistent with an essential function of this transfer protein in vivo. In contrast, partially purified phosphatidylcholine phospholipid transfer protein from BHK cells transfers phosphatidylcholine rapidly, while no transfer of phosphatidyl-PDME and phosphatidylethanolamine was detected. We further found that the specific phosphatidylcholine transfer protein of BHK cells accounts for nearly all of the transfer activity detected in the crude soluble fraction. The rapid translocation of phosphatidyl-PDME in vivo in BHK cells is therefore inconsistent with the postulate that soluble phospholipid transfer proteins are responsible for the rapid movement of phospholipids from microsomes to mitochondria in living cells.     

Phospholipids of rat tissues after feeding pure phosphatidyl ethanolamine and lecithin

Pure phosphatidyl ethanolamine and lecithin from egg yolks were fed to rats in saline or in olive oil and the changes in individual phospholipids in the intestinal wall, liver, and plasma of the animals were studied. Ingestion of olive oil alone produced increased levels of all phospholipid fractions in each of the three tissues. Feeding phosphatidyl ethanolamine in saline resulted in slightly increased plasma phospholipids, but levels of liver total phospholipids were greatly reduced; when phosphatidyl ethanolamine was fed with olive oil, liver phospholipids were again reduced but this reduction was confined to the phosphatidyl ethanolamine and phosphatidic acid fractions. Feeding lecithin alone did not produce significant changes in levels of plasma or tissue phospholipids. The results suggest that liver phospholipid synthesis is depressed by feeding phosphatidyl ethanolamine; in the presence of olive oil, hepatic synthesis of phosphatidyl ethanolamine seems to be more selectively inhibited.     

Adaptive changes in lipid composition of rat liver plasma membrane during postnatal development following maternal ethanol ingestion

The fatty acid composition of constituent phospholipids and the cholesterol content of rat liver plasma membranes were determined subsequent to maternal alcohol ingestion during pregnancy and lactation. The alcoholic group was given a liquid Metrecal diet containing 37% ethanol-derived calories. The control group was pair-fed an isocaloric sucrose/Metrecal diet. Litters were killed for lipid analyses at days 5, 15 and 25 after birth. These studies revealed that the total phospholipid phosphorus was similar and increased significantly with age in both groups. Cholesterol also increased significantly with age in both groups but was greater in the alcoholic pups, resulting in a higher cholesterol/phospholipid molar ratio. While the phosphatidylethanolamine (PE) content increased with age in both groups, that of sphingomyelin decreased. Phosphatidylserine + phosphatidylinositol (PS + PI) was significantly higher in the control group at all ages studied. A consistent increase of C22:6 in phosphatidylcholine (PC), sphingomyelin, PS + PI and in the total phospholipid fraction from alcoholic pups was observed. Although other fatty acid changes were found in PC, PS + PI and sphingomyelin, PE was not affected. These results suggest that specific adaptive changes were induced in the liver plasma membrane lipids of the progeny from alcoholic rats.     

Activity and properties of CTP: cholinephosphate cytidylyltransferase in adult and fetal rat lung.

Cholinephosphate cytidylyltransferase (CTP : cholinephosphate cytidylyltransferase, EC 2.7.7.15) is located in both the microsomal and supernatant fractions of adult lung when the tissue is homogenized in 0.145 M NaCl. The activity is located predominantly in the supernatant fraction in fetal lung. Cholinephosphate cytidylyltransferase in the supernatant from fetal lung is stimulated 4- to 6-fold by the additions of total lung lipid. Serine phosphoglycerides and inositol phosphoglycerides specifically caused stimulation whereas choline phosphoglycerides and ethanolamine phosphoglycerides produced no stimulation. Lysophosphatidylcholine cause some stimulation, but only at high concentrations. A number of detergents were investigated. All produced inhibition except for the ampholytic detergent, miranol H2M which was not inhibitory. None of the detergents produced any stimulation of activity. Cytidylyltransferase activity in fetal lung when assayed in the absence of lipid is about 25% of the adult. The activity when assayed in the presence of lipid is equal or slightly higher than adult levels. The activity, measured without added phospholipid, increases 5- to 6-fold within 12 h after birth, to values higher than in the adult. The activity, measured in the presence of phospholipid, increased almost linearly from -2 day until +1 day. There is an inverse relationship between the concentration of phospholipid in the fetal lung supernatant and the degree of lipid stimulation. Chromatographic experiments with Biogel A 1.5 columns have shown that cytidylyltransferase can exist in two molecular sizes, a small molecular size that requires phospholipid for activity, and a larger molecular weight species which does not require the addition of phospholipid for activity. Fetal lung has a higher proportion of the low molecular weight form than adult lung. The small molecular weight species can be converted to the larger molecular weight form by the addition of phospholipids.     

Nonspecific lipid transfer protein (sterol carrier protein 2) is bound to rat liver mitochondria: its role in spontaneous intermembrane phospholipid transfer

In the present study we have investigated the transfer of phospholipids between vesicles and rat liver mitochondria. Transfer was measured by electron paramagnetic resonance spectroscopy using vesicles that contained spin-labeled phospholipids. A spontaneous transfer was observed which could be strongly inhibited by treating the mitochondria with the thiol reagent mersalyl. Transfer was also greatly reduced after a saline wash of the mitochondria; the transfer activity was then recovered in the wash. This activity was inhibited by tryptic digestion and mersalyl. By gel chromatography, enzyme immunoassay and immunoblotting it was demonstrated that the activity in the wash was due to the nonspecific lipid transfer protein (sterol carrier protein 2). We could estimate that up to 85% of the spontaneous phospholipid transfer between vesicles and rat liver mitochondria was mediated by this transfer protein.     

N-acylethanolamine phospholipid metabolism in normal and ischemic rat brain

N-Acylethanolamine phospholipids accumulate in rat brain during post-decapitative ischemia. Small amounts of these phospholipids consisting primarily of diacyl and alkenylacyl species can be detected within 15 min of ischemia and they increase linearly for 60 min. This ischemia-induced synthesis is more pronounced in developing rat brain (approx. 5.0 nmol/h per mumol lipid P) than in adult brain (0.4 nmol). Pulse labeling experiments with subcellular preparations of 10-day-old rat brain indicate a precursor-product relationship between ethanolamine phospholipids and their N-acyl analogs. N-Acylation of endogenous substrates occurs with both microsomes and mitochondria, exhibits a pH optimum of 10 and requires 1 mM Ca2+ for maximal (0.2 mM Ca2+ for half maximal) activity. Cell-free preparations of both developing and adult rat brain contain a phosphodiesterase which hydrolyzes N-acylphosphatidylethanolamine to phosphatidic acid and N-acylethanolamine. The latter is further hydrolyzed to fatty acid and ethanolamine by an amidohydrolase. [1-3H]Ethanolamine, injected intracerebrally or intraperitoneally into 13- and 18-day-old rats, is incorporated into brain ethanolamine phospholipids. Since small amounts of radioactivity are also associated with N-acylethanolamine phospholipids 5 and 24 h after injection of the substrate, it appears that these phospholipids may occur at a very low level as a natural lipid constituent of rat brain.     

Purification, characterization and substrate specificity of rabbit lung phospholipid transfer proteins.

Three phospholipid transfer proteins, namely proteins I, II and III, were purified from the rabbit lung cytosolic fraction. The molecular masses of phospholipid transfer proteins I, II and III are 32 kilodaltons (kDa), 22 kDa and 32 kDa, respectively; their isoelectric point values are 6.5, 7.0 and 6.8, respectively. Phospholipid transfer proteins I and III transferred phosphatidylcholine (PC) and phosphatidylinositol (PI) from donor unilamellar liposomes to acceptor multilamellar liposomes; protein II transferred PC but not PI. All the three phospholipid transfer proteins transferred phosphatidylethanolamine poorly and showed no tendency to transfer triolein. The transfer of [14C]PC from unilamellar liposomes to multilamellar liposomes facilitated by each protein was affected differently by the presence of acidic phospholipids in the PC unilamellar liposomes. In an equal molar ratio of acidic phospholipid and PC, phosphatidylglycerol (PG) reduced the activities of proteins I and III by 70% (P = 0.0004 and 0.0032, respectively) whereas PI and phosphatidylserine (PS) had an insignificant effect. In contrast, the protein II activity was stimulated 2-3-times more by either PG (P = 0.0024), PI (P = 0.0006) or PS (P = 0.0038). In addition, protein II transferred dioleoylPC (DOPC) about 2-times more effectively than dipalmitoylPC (DPPC) (P = 0.0002), whereas proteins I and III transferred DPPC 20-40% more effectively than DOPC but this was statistically insignificant. The markedly different substrate specificities of the three lung phospholipid transfer proteins suggest that these proteins may play an important role in sorting intracellular membrane phospholipids, possibly including lung surfactant phospholipids.     

Phospholipid-transfer proteins in human liver and primary liver carcinoma

Investigations have been carried out on phospholipid-transfer activity of the cytosol and the phospholipid composition of subcellular membranes from human liver and primary liver carcinoma. In both human liver and primary liver carcinoma cytosolic fractions, the transfer activity for phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin has been observed for the first time. The transfer rate of PC and PE in normal human liver was almost equal, whereas sphingomyelin-transfer activity was much slower. In carcinoma cells, the transfer activity for PE and PC was significantly enhanced, while sphingomyelin transfer remained unchanged. Comparative investigations with HepG2 cultured cells have revealed a high PE-transfer activity in this cell line. Parallel with the phospholipid-transfer activity modifications in neoplasic cells, changes in the phospholipid composition of microsomes and mitochondria have been observed. The content of PC and PE in hepatocarcinoma cells was decreased in microsomes, while in the mitochondria it was increased. The possible role of the phospholipid-transfer proteins in the maintenance of membrane composition and structure is discussed.     

Inhibitory effect of glucose on the maturation of rat liver mitochondria at birth. Phospholipid and oxidative metabolism

(1) The rate of ATP synthesis coupled with succinate oxidation in rat liver mitochondria is low at birth and increases rapidly during the first postnatal hours (Nakazawa, T., Asami, K., Suzuki, H. and Yakawa, O. (1973) J. Biochem. 73, 397-406). A glucose injection given to newborn rats immediately after birth seemed to delay this maturation process. (2) Glucose administration specifically diminished the rate of 32Pi incorporation into phosphatidylcholine both in microsomes and in mitochondria while other phospholipids remained unaffected. (3) In newborn rat liver, 32Pi incorporation into phospholipids can be explained by de novo synthesis of phospholipids in microsomes followed by transfer to mitochondria with two exceptions phosphatidylserine and sphingomyelin. Indeed, after a 20-min incorporation of 32Pi into phospholipids, the specific radioactivity of phosphatidylserine and sphingomyelin was higher in mitochondria than in microsomes. (4) As far as phospholipid synthesis is concerned, no precursor-product relationship could be observed between light and heavy mitochondria.     

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane Ca2(+)-ATPase

The role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane (SPM) Ca2(+)-ATPase was investigated by its reconstitution into different phospholipids. Retention of activity of the solubilized Ca2(+)-ATPase depended on addition of exogenous phospholipids. As the cholate concentration used for solubilization of native SPM increased, a larger excess of exogeneous phospholipids, relative to membrane protein, had to be added to maintain optimal activity. Highest ATP-dependent Ca2+ transport activity was obtained when reconstitution was carried out in calf brain phospholipids (BPLs) followed by soybean phospholipids (SPLs) and the lowest in egg PC; reconstitution at a 40:1 weight ratio of exogenous phospholipids to native SPM protein resulted in ATP-dependent Ca2+ transport of 40.0 +/- 4.16, 23.4 +/- 8.48, and 11.54 +/- 2.31 nmol of Ca2+ (mg of protein)-1 (5 min)-1, respectively. Partial substitution of egg PC with BPLs led to an increase in the activity of the reconstituted Ca2+ pump. The highest ATP-dependent Ca2+ uptake was obtained when ratios of 15:25 or 10:30 egg PC to BPLs were used. Testing the individual phospholipids participating in the BPL mixture showed that addition of PS to egg PC led to a consistent increase in Ca2+ pump activity. Substitution of 50% of the PC with PS resulted in a 3.8-fold higher ATP-dependent Ca2+ uptake than that obtained in egg PC alone. No other phospholipid tested--PE, SM, or PI--had a similar effect. Increasing the proportion of PS within the BPL mixture above its original content led to a gradual decrease in the reconstituted SPM Ca2+ pump activity. Enrichment of asolectin with PS led first to increased Ca2+ pump activity; then, as the proportion of PS increased, Ca2+ transport of the reconstituted pump decreased. An increased proportion of PE, SM, or PI within the BPLs or asolectin, above their original contents, resulted in decreased Ca2+ transport. These results indicate that optimal SPM Ca2+ pump activity requires the combined presence of a critical amount of PC and PS within the reconstituted membrane.