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.     

Glycolipid transfer protein from bovine brain

Glycolipid transfer protein from bovine brain has been purified partially by ammonium sulfate precipitation, CM-52 ion-exchange, and Sephadex G-75 column chromatography. Both pyrene-labeled and tritium-labeled glucocerebrosides have been used to study the kinetics of protein-mediated transfer between donor and acceptor vesicles. Protein accelerates glucocerebroside transfer but does not accelerate phospholipid transfer. In colyophilized small sonicated vesicles (10% glucocerebroside, 90% 1-palmitoyl-2-oleoyl-phosphatidylcholine) about two-thirds of the glycolipid is transferred in 2 h and the remaining one-third does not transfer (up to 5 h). For donor and acceptor vesicles made of dipalmitoylphosphatidylcholine or 1-palmitoyl-2-oleoyl-phosphatidylcholine, glucocerebroside (10% in donors) is transferred rapidly only when both the donor and acceptor matrix phospholipids are in the liquid-crystalline state. If either donor or acceptor vesicles are in the gel state, transfer protein mediated transfer is much reduced. The amount of transfer protein bound specifically to glucocerebroside-containing vesicles is nearly equal above and below the matrix phospholipid phase transition temperature. Bound protein transfers glucocerebroside upon addition of acceptor vesicles.     

Membrane properties modulate the activity of a phosphatidylinositol transfer protein from the yeast, Saccharomyces cerevisiae

A phospholipid transfer protein from yeast (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391) was 2800-fold enriched by an improved procedure. The specificity of this transfer protein and the influence of membrane properties of acceptor vesicles (lipid composition, charge, fluidity) on the transfer activity were determined in vitro using pyrene-labeled phospholipids. The yeast transfer protein forms a complex with phosphatidylinositol or phosphatidylcholine, respectively, and transfers these two phospholipids between biological and/or artificial membranes. The transfer rate for phosphatidylinositol is 19-fold higher than for phosphatidylcholine as determined with 1:8 mixtures of phosphatidylinositol and phosphatidylcholine in donor and acceptor membrane vesicles. If acceptor membranes consist only of non-transferable phospholipids, e.g., phosphatidylethanolamine, a moderate but significant net transfer of phosphatidylcholine occurs. Phosphatidylcholine transfer is inhibited to a variable extent by negatively charged phospholipids and by fatty acids. Differences in the accessibility of the charged groups of lipids to the transfer protein might account for the different inhibitory effects, which occur in the order phosphatidylserine which is greater than phosphatidylglycerol which is greater than phosphatidylinositol which is greater than cardiolipin which is greater than phosphatidic acid which is greater than fatty acids. Although mitochondrial membranes contain high amounts of negatively charged phospholipids, they serve effectively as acceptor membranes, whereas transfer to vesicles prepared from total mitochondrial lipids is essentially zero. Ergosterol reduces the transfer rate, probably by decreasing membrane fluidity. This notion is supported by data obtained with dipalmitoyl phosphatidylcholine as acceptor vesicle component; in this case the transfer rate is significantly reduced below the phase transition temperature of the phospholipid.     

Use of spin-labeled and fluorescent lipids to study the activity of the phospholipid transfer protein from maize seedlings.

The transfer of spin-labeled and fluorescent lipids between sonicated vesicles and different host membranes has been measured in the presence or absence of a phospholipid transfer protein purified from maize seedlings. It was found that the protein has little specificity towards the phospholipid head group and allows the transfer of hydrophobic long chain phospholipids. By contrast, no transfer of a cholesterol analogue could be detected. By EPR spectroscopy, evidence is presented that shows that the protein catalyzes the incorporation of labeled phospholipids in the outer monolayer of the acceptor membranes. The efficiency of the transfer depends largely on the nature of the acceptor: erythrocytes are more difficult to label than chromaffin granules or liposomes made with unsaturated lipids. Thus, consistent with the high activation energy observed, the transfer is facilitated when it involves fluid membranes. These results are in favor of a process involving the exchange of phospholipids, facilitated by a shuttle protein rather than a fusion mechanism.     

Phospholipid transfer between vesicles. Dependence on presence of cytochrome P-450 and phosphatidylcholine-phosphatidylethanolamine ratio

The rate of transfer of spin-labeled phospholipid from donor vesicles of sonicated 1-acyl-2-(10-doxylstearoyl)-sn-glycero-3-phosphocholine to other vesicle was determined as a function of content of cytochrome P-450 and the phosphatidylcholine/phosphatidylethanolamine ratio in the acceptor vesicles. The transfer rate was measured as an increase in intensity that resulted from a decrease in the line width in the EPR spectrum of the spin-labeled phospholipids as they was transferred to the nonspin-labeled acceptor vesicles. A lower transfer rate was observed for acceptor vesicles of pure egg phosphatidylcholine vesicles than for vesicles for a mixture of phosphatidylcholine and phosphatidylethanolamine. The presence of cytochrome P-450 in the acceptor vesicles further increased the transfer rate. Those alterations in the mole ratios of the protein and the two phospholipids that made the bilayer of the reconstituted vesicles more like the membrane of the endoplasmic reticulum resulted in an increase in phospholipid-transfer rate. The mole ratios of components that produce high phospholipid-transfer rates were similar to those that in an earlier study produced a 31P-NMR spectrum characteristic of a nonbilayer phase. These findings suggest that, in the membrane of the endoplasmic reticulum, phospholipid exchange may be an important element in function and interaction with other intracellular organelles.     

Phospholipid transfer proteins in microorganisms

Phospholipid transfer activity has been demonstrated in cell lysates of Saccharomyces cerevisiae, Rhodopseudomonas sphaeroides and Bacillus subtilis, and proteins facilitating phospholipid transfer from the first two organisms have recently been purified. The phospholipid transfer protein from S. cerevisiae has mol. wt. 35 000 with a specificity of transfer for phosphatidylinositol and phosphatidylcholine. The purified phospholipid transfer protein from R. sphaeroides has mol. wt. 27 000 and, although it has the ability to transfer all phospholipid species tested it displays a preference for phosphatidylglycerol. The cellular levels of phospholipid transfer activity in both S. cerevisiae and R. sphaeroides are not strictly related to the level of subcellular membranes. However, in photosynthetically grown R. sphaeroides, the distribution of the activities between soluble and membrane-associated forms is correlated with the level of intracytoplasmic membrane (a postulated membrane substrate).     

Phospholipid transfer between vesicles: Dependence on presence of cytochrome P-450 and phosphatidylcholine-phosphatidylethanolamine ratio

The rate of transfer of spin-labeled phospholipid from donor vesicles of sonicated 1-acyl-2-(10-doxylstearoyl)-sn-glycero-3-phosphocholine to other vesicles was determined as a function of content of cytochrome P-450 and the phosphatidylcholine/phosphatidylethanolamine ratio in the acceptor vesicles. The transfer rate was measured as an increase in intensity that resulted from a decrease in the line width in the EPR spectrum of the spin-labeled phospholipids as they were transferred to the nonspin-labeled acceptor vesicles. A lowe transfer rate was observed for acceptor vesicles of pure egg phosphatidylcholine vesicles than for vesicles of a mixture of phosphatidylcholine and phosphatidylethanolamine. The presence of cytochrome P-450 in the acceptor vesicles further increased the transfer rate. Those alterations in the mole ratios of the protein and the two phospholipids that made the bilayer of the reconstituted vesicles more like the membrane of the endoplasmic reticulum resulted in an increase in phospholipid-transfer rate. The mole ratios of components that produce high phospholipid-transfer rates were similar to those that in an earlier study produced a ³¹P-NMR spectrum characteristic of a nonbilayer phase. These findings suggest that, in the membrane of the endoplasmic reticulum, phospholipid exchange may be an important element in function and interaction with other intracellular organelles.     

A new fluorimetric method to measure protein-catalyzed phospholipid transfer using 1-acyl-2-parinaroylphosphatidylcholine

A new, simple and versatile method to measure phospholipid transfer has been developed, based on the use of a fluorescent phospholipid derivative, 1-acyl-2-parinaroylphosphatidylcholine. Vesicles prepared of this phospholipid show a low level of fluorescence due to interactions between the fluorescent groups. When phospholipid transfer protein and vesicles consisting of non-labeled phosphatidylcholine are added the protein catalyzes an exchange of phosphatidylcholine between the labeled donor and non-labeled acceptor vesicles. The insertion of labeled phosphatidylcholine into the non-labeled vesicles is accompanied by an increase in fluorescence due to abolishment of self-quenching. The initial rate of fluorescence enhancement was found to be proportional to the amount of transfer protein added. This assay was applied to determine the effect of membrane phospholipid composition on the activity of the phosphatidylcholine-, phosphatidylinositol- and non-specific phospholipid transfer proteins. Using acceptor vesicles of egg phosphatidylcholine and various amounts of phosphatidic acid it was observed that the rate of phosphatidylcholine transfer was either stimulated, inhibited or unaffected by increased negative charge depending on the donor to acceptor ratio and the protein used. In another set of experiments acceptor vesicles were prepared of phosphatidylcholine analogues in which the ester bonds were replaced with ether bonds or carbon-carbon bonds. Assuming that only a strictly coupled exchange between phosphatidylcholine and analogues gives rise to the observed fluorescence increase, orders of substrate preference could be established for the phosphatidylcholine- and phosphatidylinositol transfer proteins.     

Asymmetry and transposition rates of phosphatidylcholine in rat erythrocyte ghosts.

Purified phospholipid exchange protein from beef heart cytosol is used to accelerate the exchange of phospholipids between labeled sealed ghosts and phosphatidylcholine/cholesterol liposomes. The purified protein accelerates the transfer of phosphatidylcholine and, to a lesser degree, that of sphingomyelin, phosphatidylinositol, and lysophosphatidylcholine. The presence of exchange protein does not accelerate the exchange of phospholipids between intact red blood cells and liposomes, but 75% of the phosphatidylcholine of sealed ghosts is readily available for exchange. The remaining 25% is also exchangeable but at a slower rate. When the exchange is assayed between inside-out vesicles and liposomes, 37% of the phosphatidylcholine is readily available, and 63% is exchanged at a slower rate. These results are consistent with an asymmetric distribution of phosphatidylcholine in isolated erythrocyte membrane fractions. The sum of the forward and backward transposition of phosphatidylcholine between the inside and outside layers of sealed ghost membranes amounts to 11% per hour, and the half-time for equilibration is 2.3 h. Significatnly lower values are obtained for the inside-out vesicles (half-time for equilibration: 5.3 h). These results suggest that, during the formation of the vesicles, the asymmetry of phosphatidylcholine is partially preserved, but structural changes occur in the membrane that affect the rate of membrane transposition of phosphatidylcholine.     

Localization and characterization of the sn-glycerol-3-phosphate acyltransferase in Rhodopseudomonas sphaeroides

The membrane localization and properties of the Rhodopseudomonas sphaeroides sn-glycerol-3-phosphate acyltransferase have been examined utilizing enzymatically prepared acyl-acyl carrier protein (acyl-ACP) substrates as acyl donors for sn-glycerol-3-phosphate acylation. Studies conducted with membranes prepared from chemotrophically and phototrophically grown cells show that sn-glycerol-3-phosphate acyltransferase activity is predominantly (greater than 80%) associated with the cell's cytoplasmic membrane. Enzyme activity associated with the intracytoplasmic membranes present in phototrophically grown R. sphaeroides was within the range attributable to cytoplasmic membrane contamination of this membrane fraction. Enzyme activity was optimal at 40 degrees C and pH 7.0 to 7.5, and required the presence of magnesium. No enzyme activity was observed with any of the long-chain acyl-CoA substrates examined. Vaccenoyl-ACP was the preferred acyl-ACP substrate and vaccenoyl-ACP and palmitoyl-ACP were independently utilized to produce lysophosphatidic and phosphatidic acids. With either vaccenoyl-ACP or palmitoyl-ACP as sole acyl donor substrate, the lysophosphatidic acid formed was primarily 1-acylglycerol-3-phosphate and the Km(app) for sn-glycerol-3-phosphate utilization was 96 microM. The implications of these results to the mode and regulation of phospholipid synthesis in R. sphaeroides are discussed.     

Concerning the mechanism of action of bovine liver phospholipid exchange protein: Exchange or net transfer

Bovine liver phospholipid exchange protein catalyzes the transfer of phosphatidylcholine between donor and acceptor populations of single bilayer phospholipid vesicles. In comparing egg and dimyristoylphosphatidylcholine vesicles, larger transfer rates are found for the unsaturated phospholipid. The bidirectional transfer rates measured from donor to acceptor and from acceptor to donor, are equivalent, suggesting that the protein facilitates an exchange rather than a net transfer of phosphatidylcholine.     

Unsaturated aminophospholipids are preferentially retained by the fast skeletal muscle CaATPase during detergent solubilization. Evidence for a specific association between aminophospholipids and the calcium pump protein.

When fast twitch skeletal muscle vesicles (SR) and purified calcium pump protein are stripped with the nonionic detergent C12E8 (octaethylene glycol dodecyl ether), not all the membrane phospholipids are removed from the calcium pump protein. Maximal extraction produces a remnant of 6-8 mol of phospholipid/mole of calcium ATPase (CaATPase). In contrast to native SR and the prestripped purified CaATPase, the remaining phospholipid is markedly enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS) in both preparations; the remaining lipid is also enriched in phospholipid that is predominantly unsaturated. In addition, virtually all of the associated PE is plasmalogenic (96% as opposed to 63% in the native SR). The amino-specific cross-linking reagent DFDNB (1,5-difluoro-2,4-dinitrobenzene sulfonic acid) and the amino binding reagent TNBS (2,4,6-trinitrobenzene sulfonic acid) were utilized to identify the monolayer of the native preparation where these phospholipids reside, and to determine which phospholipids are closely associated with the calcium pump protein following detergent treatment. These studies demonstrate that PE and PS are closely associated with the pump protein, PE residing almost exclusively in the outer monolayer of SR, while PS resides in the inner monolayer. Nonspecific phospholipid exchange protein was shown to be capable of exchanging phospholipids from donor vesicles into those phospholipids associated with the CaATPase; stripping of lipid-exchanged vesicles with C12E8 exhibited the same specificity with regard to head-group species (i.e., PE is markedly enriched in the extracted protein associated fraction). The results suggest that specific protein-lipid interactions exist, favoring the association of plasmalogenic aminophospholipids with the calcium pump protein.     

Phosphatidylinositol exchange protein. Effects of membrane structure on activity and evidence for a ping-pong mechanism.

Phosphatidylinositol exchange protein, purified from bovine cerebral cortex, catalyzes the transfer of phosphatidylinositol and, to a lesser extent, phosphatidylcholine between rat liver microsomes and egg phosphatidylcholine liposomes. Transfer activity is sensitive to pH, temperature, and the method of liposome preparation. Variation of the phospholipid composition of the liposomes produces vesicles for which the apparent Michaelis constant decreases with increasing molar proportions of phosphatidylinositol. Interaction of exchange protein with liposomes containing radioactively labeled phosphatidylcholine allows the isolation of a phospholipid-protein complex; dissociation of this complex occurs upon subsequent interaction with unlabeled liposomes. Changes in the concentration of the two membrane species, microsomes and liposomes, yield results which are interpreted in terms of a ping-pong kinetic mechanism for the protein-catalyzed, intermembrane transfer of phospholipids.     

Catalytic properties of phospholipid exchange protein from bovine heart.

A protein which catalyzes the exchange of phosphatidylcholine between membranes has been purified from heart tissue homogenates up to 300-fold by acidic pH precipitation, (NH4)2SO4 precipitation, gel filtration, and ion-exchange chromatography. Binding of the protein to phosphatidylcholine liposomes as measured by Sepharose chromatography was nondetectable. However, isoelectric focusing experiments showed that individual molecules of phosphatidylcholine were transferred from liposomes to the soluble, partially purified protein. Exchange of phospholipid between liposomes and mitochondria was not affected by the presence of moderate amounts of cholesterol in liposomes. A search for competitive inhibitors among moieties similar to phosphatidylcholine failed to show strong binding sites in the hydrophilic part of the substrate. High concentrations of Na+, Ca2+ and Mg2+ impaired the exchange activity.     

Biophysical and biochemical characterization of reconstituted and purified Rhodobacter sphaeroides cytochrome c oxidase in phospholipid vesicles sheds insight into its functional oligomeric structure

Discontinuous sucrose gradient ultracentrifugation was used to separate liposomes containing Rhodobacter sphaeroides cytochrome c oxidase (pCOV) from liposomes devoid of the enzyme, and the biophysical and biochemical properties of pCOV were compared to unpurified liposomes containing cytochrome c oxidase (COV). Isolated and purified R. sphaeroides cytochrome c oxidase (COX) was reconstituted into asolectin phospholipid vesicles by cholate dialysis, and this preparation was purified further on a discontinuous sucrose gradient to isolate only those vesicles which contained the enzyme (pCOV). After centrifugation at 300,000g for 22h, 80% of the enzyme recovered was in a single band. The number of COX molecules per pCOV liposome was estimated by measuring the visible absorbance spectrum of cytochrome c oxidase (for heme aa(3)) and inorganic phosphate concentration (for phospholipid). The number of COX molecules incorporated per pCOV was estimated to be approximately one (0.72+/-0.19-1.09+/-0.28). The pCOV exhibited similar physical properties as COV; respiratory control ratios (indicators of endogenous proton permeability) and maximum enzymatic turnover number at pH 7.4 were comparable (6.0+/-1.3 and 535+/-130s(-1)). Furthermore, proton pumping activities of the pCOV were at least 70% of COV, indicating that discontinuous sucrose gradient centrifugation is a useful technique for functional experiments in R. sphaeroides cytochrome c oxidase. Our results suggest that the monomeric form of R. sphaeroides COX when reconstituted into a phospholipid bilayer is completely functionally active in its ability to perform electron transfer and proton pumping activities of the enzyme.     

Transfer of cholesterol between phospholipid vesicles mediated by the steroidogenic acute regulatory protein (StAR)

The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in steroidogenic cells. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P-450scc converts this cholesterol into pregnenolone. We tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P-450scc to acceptor vesicles containing P-450scc but no cholesterol, using P-450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P-450scc activity in acceptor vesicles 5-10-fold following the addition of donor vesicles. Transfer of cholesterol to acceptor vesicles was rapid and sufficient to maintain a linear rate of pregnenolone synthesis for 10 min. The effect of N-62 StAR in stimulating P-450scc activity was specific for cholesterol transfer and was not due to vesicle fusion or P-450scc exchange between vesicles. Maximum stimulation of P-450scc activity in acceptor vesicles required preincubation of N-62 StAR with phospholipid vesicles prior to adding donor vesicles. The amount of N-62 StAR causing half-maximum stimulation of P-450scc activity in acceptor vesicles was 1.9 microm. Half-maximum stimulation required more than a 10-fold higher concentration of R182L N-62 StAR, a mutant associated with congenital lipoid adrenal hyperplasia. N-62 StAR-mediated transfer of cholesterol between vesicles showed low dependence on the cholesterol concentration in the donor vesicles. Thus StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria.     

Concentration of neutral lipids in the phospholipid surface of substrate particles determines lipid transfer protein activity

To better understand the mechanism of lipid transfer protein (LTP) action and the effects of altered lipoprotein composition on its activity, we evaluated the dependence of LTP activity on the concentrations of cholesteryl ester (CE) and/or triglyceride (TG) in the phospholipid bilayer of substrate particles. Phosphatidylcholine (PC)-cholesterol liposomes containing up to 2 mole% TG and/or CE were prepared by cholate dialysis and used as either the donor of lipids to, or the acceptor of lipids from, low density lipoproteins (LDL). CE or TG transfer from liposomes of varying neutral lipid content to LDL showed saturation kinetics with an apparent Km of less than or equal to 0.2 mole%. Throughout this concentration-dependent response. PC transfer, which depended on the same LTP-donor particle binding interactions as those required for neutral lipid transfer, was essentially unchanged. Lipid transfer in the reverse direction (from LDL to liposomes of varying neutral lipid content) followed the same kinetics showing that transfer between the two particles is tightly coupled and bidirectional. When liposomes contained both TG and CE, these lipids competed for transfer in a manner analogous to that previously noted with lipoprotein substrates. In conclusion, CE and TG transfer activities are determined by the concentration of these lipids in the phospholipid surface of donor and acceptor particles. At low TG and CE concentrations, LTP bound to the liposome surface as indicated by PC transfer, but only a portion of these interactions actually facilitated a neutral lipid transfer event. Thus, the overall rate of neutral lipid transfer, and the competition between TG and CE for transfer, depend on the concentrations of these lipids in the phospholipid layer.     

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P-450scc incorporated into vesicles prepared from these phospholipids

Cytochrome P-450scc, which catalyses the conversion of cholesterol to pregnenolone in steroidogenic tissues, can be incorporated into artificial phospholipid vesicles and cholesterol binding to the cytochrome is affected by the composition of the vesicles. We have purified the phospholipids from the inner mitochondrial membrane fraction of the bovine corpus luteum where the cytochrome is located. The composition in mol % was 49% phosphatidylcholine, 34% phosphatidylethanolamine, 8.7% cardiolipin, 6.4% lysophosphatidylethanolamine and 1.5% phosphatidylinositol. The ratio of cholesterol to phospholipid (mol/mol) in the inner membrane fraction was 0.14 to 1. The Km for cholesterol of purified luteal cytochrome P-450scc incorporated into vesicles prepared from the total inner mitochondrial membrane phospholipids was 0.063 mol of cholesterol per mol of phospholipid. Removal of the cardiolipin component of the inner mitochondrial membrane phospholipids prior to preparation of vesicles caused a four fold increase in the Kd of cytochrome P-450 for cholesterol and a two fold increase in Km. The data suggests that in the inner mitochondrial membrane of the bovine corpus luteum the cholesterol concentration is less than saturating for cytochrome P-450scc.     

A protein purified from pig brain accelerates the intermembranous translocation of mono- and dihexosylceramides,but not the translocation of phospholipids

By the use of an assay that measures the transfer of [³H]galactosylceramide from donor to acceptor liposomes, a protein has been purified 1683-fold from pig brain. The most purified fraction was purified to homogeneity as judged by electrophoresis on 15% polyacrylamide gel in the presence of sodium dodecyl sulfate. The protein has a molecular weight of 23000 as determined by the gel electrophoresis and 18500 as estimated by gel filtration through Sephadex G-75. The protein accelerates the transfer of labeled glycolipids at the following relative rates: 100 for glucosylceramide, 43 for lactosylceramide, 17 for galactosyldiglyceride, and 15 for galactosylceramide. The lipid-transfer stimulated by the protein is specific to glycolipids; the protein does not accelerate the transfer of labeled phosphatidylcholine and phosphatidylethanolamine from donor to acceptor liposomes.     

The occurrence of phosphatidyl choline exchange protein in leaves

The transfer of phosphatidyl choline between liposomes was stimulated by the protein fractions from spinach leaves, etiolated and greening leaves of $\text{Avena}$ seedlings. This is confirmed by the transfer of [¹⁴C]phosphatidyl choline or spin-labeled phosphatidyl choline between donor and acceptor liposomes. ESR spectrum changes also indicated that no spin-labeled phosphatidyl choline was released from donor liposomes by spinach leaf protein unless acceptor liposomes were present. [¹⁴C]phospholipids were transferred from liposomes to both spinach chloroplasts and $\text{Avena}$ etiochloroplasts by phosphatidyl choline exchange protein from germinated castor bean endosperms and further from liposomes to spinach chloroplasts by spinach leaf protein. These results support the view that phosphatidyl choline in the plastid is supplied from the synthesis site, the endoplasmic reticulum, by phospholipid exchange protein.