THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.     

HeLa cell plasma membranes. Changes in membrane protein composition during the cell cycle.

HeLa cells grown in suspension culture were synchronized by amethopterin block and thymidine reversal. In some cases an additional Colcemid block was used to obtain mitotic cells. From the various phases of the cell cycle, cells were harvested and the plasma membranes isolated. The membrane proteins were solubilized in sodium dodecyl sulphate and separated by gel electrophoresis in the presence of sodium dodecyl sarcosinate. About 35 protein bands, five of which were stained with periodic acid-Schiff reagent, appeared. Most of the bands were identical in all membrane preparations, but a few minor bands seemed to be associated with limited periods of the cell cycle. In particular, the cells in mitosis apparently contained plasma membrane proteins which did not occur in other phases. Amino acid analyses of the plasma membranes revealed no significant cell cycle-dependent changes in the amino acid composition.     

Antibodies to the most tightly bound proteins in eukaryotic DNA

DNA released from eukaryotic cells by proteases/SDS or by alkali/SDS still contains distinct proteins which are not removed by these cell lysis procedures nor by subsequent phenol treatment. The proteins most tightly bound to DNA can only be isolated by degradation of DNA. In contrast to the protein-DNA complexes, the protein material isolated after degradation of DNA is sensitive to protease treatment. Moreover, the isolated protein material tends to form aggregates which are insoluble in buffers not containing detergents. They are only poorly soluble in buffers containing SDS. The partially solubilized material can be separated by SDS-polyacrylamide gel electrophoresis into two main bands. Antibodies were raised in rabbits against the polypeptides contained in these main bands. Immunofluorescence micrographs are presented of cells treated with the antibodies. The results indicate that the proteins characterized by their involvement in extremely stable protein-DNA complexes also occur independently of DNA in eukaryotic cells.     

Membranes of chromaffin granules. Isolation and partial characterization of two proteins

Membranes of chromaffin granules were isolated from the adrenal glands of four different species. The solubilized membrane proteins could be resolved into several bands by polyacrylamide-gel electrophoresis (alkaline and acid gel systems). Two major protein components appeared to be common to the chromaffin granule membranes of ox, horse, pig and man. The various membrane proteins of bovine chromaffin granules were separated by filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. Two major membrane proteins (A and B) were obtained in purified form. Treatment of protein A with 2-mercaptoethanol before electrophoresis resulted in two more rapidly migrating subunits, whereas protein B was unaffected by mercaptoethanol treatment. The amino acid compositions of the two purified proteins were determined. They are very similar to that of the total membrane proteins but significantly different from that of the chromogranins, the soluble proteins of chromaffin granules.     

Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 and partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor alpha subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-[gamma-32P]triphosphate, a protein of 95 kilodaltons representing the insulin receptor beta subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins.     

Purification and properties of a membrane-bound insulin binding protein, a putative receptor, from Neurospora crassa.

The protein that is responsible for specific, high-affinity binding of insulin to the surface of Neurospora crassa cells has been purified to homogeneity. The insulin binding activity of solubilized plasma membranes resembled that of intact cells with regard to affinity of binding, specificity for mammalian insulins, and amount of insulin bound per cell. Insulin binding activity was purified from Triton X-100 solubilized membranes in two steps: FPLC on a MonoQ HR5/5 column; and affinity chromatography on insulin-agarose. The pure material migrated as a single band of ca. 66 kDa on SDS gels, pI = 7.4 by isoelectric focusing. The protein bound 5.34 pmol of insulin/micrograms, or 35% of that expected for univalent binding. Cross-linking of 125I-insulin to pure protein or to solubilized membranes revealed a single labeled band of 67-70 kDa on SDS gels. In nonreducing native gels, two labeled bands of ca. 55 and 110 kDa were produced after cross-linking, and two bands of similar molecular weight bound iodinated insulin after transfer to nitrocellulose filters. These may correspond to active monomer and dimer forms. The pure protein possessed no protein kinase activity against itself, or against exogenous substrates (histone H2, casein, or the synthetic peptide Glu80-Tyr20), and possessed no detectable phosphorylated amino acids. It is suggested, however, that this 66-kDa protein is the "receptor" that mediates insulin-induced downstream metabolic effects.     

Detection and immunolocalization of glycoproteins of the plasma membrane of maize sperm cells

Summary After purifying plasma membranes from isolated maize sperm cells by aqueous polymer two-phase partition, peripheral and integral proteins were solubilized from the plasma membrane with Triton X-114 and separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Silver staining revealed 10 bands (19–68 kDa) of peripheral membrane proteins and about 40 bands (12–120 kDa) of integral proteins. Peroxidase-conjugated Con A was used to detect the surface glycopeptides. It was found that Con A particularly stained 8 peripheral polypeptide bands, including 68, 66, 55, 51,48, 44, 36, and 32 kDa, and 6 integral polypeptide bands, 68, 51, 48, 44, 38, and 34 kDa. These bands differed from those of somatic samples. Staining specificity was demonstrated by the control in the presence of competing inhibitory sugar. The above result indicates the existence of mannosyl and glucosyl residues in the surface glycoproteins of maize sperm cells. The prominent peripheral 68 kDa polypeptide was further separated into 4 spots by isoelectric focusing and sodium dodecyl sulfate two-dimensional (IEF-SDS 2-D) electrophoresis, showing pI values from 5.5 to 5.8. Three prominent glycopeptides (68, 48, and 32 kDa) were localized on the plasma membrane of maize sperm cells via the fluorescein isothiocyanate (FITC) technique. About 25% of sperm cells showed an intense positive reaction in each immunological labelling. The results agree with our previous labelling of the surface of isolated viable maize sperm cells with Con A-FITC.Abbreviations FITC fluorescein isothiocyanate - Con A Canavalia ensiformis agglutinin - HRP horseradish peroxidase - RCA Ricinus communis agglutinin - WGA Triticum vulgaris agglutinin     

Morphological Phenomena Associated with Penicillinase Induction and Secretion in Bacillus licheniformis

Cells of uninduced Bacillus licheniformis (strain 749) in mid-logarithmic phase have no extensive intracytoplasmic membrane. After induction with cephalosporin C, characteristic organelles that contain tubules and vesicles with single-layered membranes and no visible internal substructure can be seen in thin sections in the periplasm. A magnoconstitutive penicillinase producer (749/C) contains similar structures. It is suggested that they represent a penicillinase secretory apparatus. In the first 15 min after induction, negatively stained preparations of induced 749 show large intracellular vesicles without individual contact with the cell surface. Negatively stained 749/C and fully induced 749 contain invaginations comparable to the structures seen in thin sections. When protoplasts of induced 749 and of 749/C are prepared, vesicles and tubules similar to those seen in thin sections of whole cells are liberated from the cell. Growing protoplasts of induced 749 show massive convolutions of the peripheral membrane, multiple layers of membrane, and characteristic long, slender tubules extending from the protoplast surface. These phenomena are not observed in uninduced 749 except for the production of a relatively small number of tubules. In 749/C, there were fewer convolutions than in induced 749, although tubule production was similar. Multiple layers of membrane were not observed in 749/C. The relation of the penicillinase secretory structures to mesosomes and to secretory structures of other organisms is discussed.     

Isolation and staging of horse seminiferous tubules by transillumination

Stages of the spermatogenic cycle in the horse were determined by trans-illumination of enzymically isolated, seminiferous tubules and were verified by whole-mounted tubules observed by Nomarski optics and by conventional histology. Isolated tubules were obtained from young (less than 2 years) and adult (4-10 years) horses by enzymic digestion. Dispersed tubules were separated into three different groups based on the presence, size, and intensity of a dark region in the centre of the tubules: (1) pale--homogeneously light, (2) spotty--light on the periphery with a wide spotty region in the central two-thirds, or (3) dark--an intensely dark, narrow region through the central one-third. Seminiferous tubules from young stallions separated easily, but were only of the homogeneously light pattern as they lacked mature spermatids. After observation by Nomarski optics and bright-field microscopy, pale tubules under transillumination largely contained Stages I and II, spotty tubules contained Stages V and VI, and dark tubules contained Stages VII and VIII of the spermatogenic cycle. In-vitro incorporation of [3H]thymidine in spermatogonia and preleptotene/leptotene primary spermatocytes of these tubules confirmed the viability of germ cells in isolated tubules, and ultrastructural analysis confirmed excellent preservation of normal structure of seminiferous epithelium in isolated tubules. Hence, segments of seminiferous tubules in specific stages of the spermatogenic cycle can be obtained from enzymically digested horse testes when viewed by transillumination.     

CHLAMYDOMONAS FLAGELLA : I. Isolation and Electrophoretic Analysis of Microtubules, Matrix, Membranes, and Mastigonemes

Methods were developed for the isolation of Chlamydomonas flagella and for their fractionation into membrane, mastigoneme, "matrix," and axoneme components. Each component was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flagellar membrane in situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of ~ 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "9 + 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 + 2 flagella of wild-type cells and of "9 + 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubulins 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively     

Separation and Partial Characterization Of Rat Liver Cell Membrane Components

The objectives of this research were to separate and partially characterize the components of rat liver cell plasma membranes. The plasma membranes of liver cells obtained from normal Wistar male rats were isolated using density gradients. The purified membrane was then solubilized in phenol-urea-acetic acid and gel electrophoresis was performed on the solubilized membrane components. Samples of the eluate collected following preparative gel electrophoresis were phosphorus positive-ninhydrin positive; phosphorus negative-ninhydrin positive; phosphorus po6itive-ninhydrin negative; and phosphorus negative-ninhydrin negative. The amino acid analyses showed that the proteins of the separated components were different and that no one component contained more than 6% of the total membrane protein. Liver cell plasma membranes contain components that differ from those of red blood cell plasma membranes. These components can be further divided into subgroups of proteins, i.e., phospholipoproteins and protein alone. The concept of a group of proteins composing the membrane is extended to one of various subgroups on the basis of their different affinity for lipid.     

Plasmodium lophurae: membrane proteins of erythrocyte-free plasmodia and malaria-infected red cells

Plasma membranes of normal duckling erythrocytes were prepared by blender homogenization and nitro-en decompression. Surface membrane vesicles of red cells infected with the avian malaria Plasmodium lophurae were produced by nitrogen decompression. Membranes of erythrocyte-free malaria parasites were removed from cytoplasmic constituents by Dounce homogenization. These membranes were collected by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Red cell membranes had a buoyant density of 1.159 g/cm3, whereas plasmodial membranes banded at 2 densities: 1.110 g/cm3 and 1.158 g/cm3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the isolated red cell membranes revealed 7 major protein bands with molecular weights (MW) ranging from 230, 000 to 22,000, and 3 glycoprotein bands with MW of 160,000, 88,000 and 37,000. Parasite membranes also had 7 major bands with MW ranging from 100,000 to 22,000. No glycoproteins were identifiable in these membranes. The proteins of the surface membranes from infected red cells had MW similar to those from normal red cells; however, there was some evidence of a reduction in the amount of the high MW polypeptides. The red cell membrane contained 79 nmoles sialic acid/mg membrane protein, whereas plasmodial membranes had 8 nmoles sialic acid/mg membrane protein. The sialic acid content of the surface membranes of infected red cells was significantly smaller than that of normal cells. Lactoperoxidase-glucose oxidase-catalyzed iodination of intact normal and malaria-infected erythrocytes labeled 7 surface components. Although no observable differences in iodinatable proteins were seen in these preparations, there was a striking reduction in the iodinatability of erythrocytic membranes obtained from P. lophurae-infected cells. Erythrocyte-free plasmodia bound very little radioactive iodine; the small amount of radioactivity was distributed among 3 major bands with MW of 42,000, 32,000 and 28,000. It is suggested that the alterations of the surface of the P. lophurae-infected erythrocyte do not occur by a wholesale insertion of plasmodial membrane proteins into the red cell plasma membrane, but rather that there are parasite-mediated modifications of existing membrane polypeptides.     

Lymphocyte plasma membranes. III. Composition of lymphocyte plasma membranes from normal and immunized rats

Isolated plasma membranes of thymic and splenic lymphocytes from unimmunized and immunized rats of the inbred ACI and F344 strains were analyzed for chemical and enzymatic composition, for membrane protein patterns by polyacrylamide gel electrophoresis and for membrane-associated immunoglobulins. After immunization, the thymic and splenic lymphocyte membranes from F344 rat contained less carbohydrate and higher phospholipid contents than control animals. In both ACI and F344 inbred rat strains the membrane phospholipid to cholesterol weight ratio increased significantly after immunization. The electrophoretic patterns of solubilized membrane proteins and of iodinated external membrane proteins were similar in unimmunized and immunized animals.When thymic and splenic lymphocytes of normal or immunized animals were surface radioiodinated, solubilized in Triton X-100, NP-40 or 10 M urea in 1.5 M acetic acid and analyzed by immunoprecipitation, labeled IgM immunoglobulin was recovered from thymic lymphocytes but both labeled IgG and IgM were recovered from splenic lymphocytes. However, when unlabeled isolated plasma membranes were solubilized in 1% Triton X-100 and analyzed by immunodiffusion in agarose gels, both IgG and IgM were identified in thymic and splenic cells.     

Extramembraneous particles and structural variations of tubular myelin figures in rat lung surfactant

Tubular myelin figures of pulmonary surfactant were examined by electron microscopy after fixation in glutaraldehyde and postfixation in an osmium tetroxide-ferrocyanide mixture. Bilayered membranes were seen as parallel arrays or as lattices with spacings varying from about 36 to 50 nm. This method also produced good visualization of drumstick-like particles, 5 nm in diameter and about 15 nm in length. The particles were regularly spaced at intervals of 16 nm in rows along the rectangular angles of myelin membranes. Depending on the size of the tubules the particles contacted each other in the center of the tubules at low diameters (tubular diameter less than 40 nm) and formed a continuous filamentous central core, or they were separated from one another (tubular diameter greater than 40 nm). In the latter case the central core had a hollow appearance. Based on further findings employing tannic acid, lipid extraction with 2,2-dimethoxypropane, and a ruthenium red-osmium tetroxide technique for the demonstration of polyanionic proteins it is suggested that these particles are protein in nature and that they are involved in the formation and maintenance of the structure of tubular myelin. A new concept of the ultrastructure of tubular myelin figures is proposed.     

Vectorial discharge of nascent polypeptides attached to chloroplast thylakoid membranes.

Chloroplast thylakoids with attached ribosomes were isolated from Chlamydomonas reinhardti. They were allowed to incorporate labeled amino acids into polypeptides. Labeled membranes were recovered from the reaction mixture, and a portion was treated with puromycin. The amount of labeled polypeptides released to the medium, and to the membranes by puromycin was determined by comparing radioactivity in soluble protein before, and after untreated, and puromycin-treated membranes were solubilized with the detergent Nonidet P-40. About 20% of the radioactive protein associated with the membranes was in nascent chains which were terminated by puromycin. Essentially all of terminated nascent chains remained with the membranes, and thus, were vectorially released. The results support the hypothesis that polypeptides which are synthesized by thylakoid-bound ribosomes are being incorporated into the membranes as they are synthesized.     

Membrane retrieval in the guinea-pig neurohypophysis. Isolation and characterization of secretory vesicles and coated microvesicles after radiolabel incorporation in vivo.

We have developed small-scale methods for the isolation and biochemical characterization of subcellular fractions from single guinea-pig posterior-pituitary glands. Secretory vesicles and coated microvesicles produced in this way were of similar purity to those isolated from large amounts of tissue by conventional ultracentrifugation. [35S]Cysteine injected into the hypothalamus was found in the soluble contents of secretory vesicles isolated from the neural lobes 24 h later. High-pressure liquid-chromatographic analysis revealed that the radiolabel was incorporated into the expected neurosecretory products (oxytocin, vasopressin and neurophysin) and also into a biosynthetic intermediate in the vasopressin system. The membranes of secretory vesicles were labelled with [3H]choline 24 h after its hypothalamic injection. Little or no [3H]choline could be demonstrated in coated microvesicles at this time, although these structures were labelled 5 days after injection. Stimulating hormone secretion by chronic dehydration produced a significant fall in [3H]choline content of the secretory-vesicle membranes without any transfer of label into coated microvesicles, suggesting that coated microvesicles are not involved in membrane retrieval in the neurohypophysis.     

Transtubular transport of proteins in rabbit proximal tubules

The purpose of the present experiments was to study possible different pathways of intracellular transport of proteins after luminal and basolateral uptake in isolated rabbit proximal tubules. Tubules were exposed to cationized ferritin (CF) in the perfusion fluid and horseradish peroxidase (HRP) in the bath simultaneously or to HRP in the bath alone for 30 min. The peritubular fluid (bath) and perfusion fluid were then exchanged and the tubules either fixed immediately or allowed to function during chase-periods for 10, 20, 30, or 60 min before fixation to follow the migration of the proteins through the cells. The proteins were to a large extent found separated in different vacuoles and lysosomes at all time periods studied, indicating separate pathways after uptake via the luminal and basolateral membranes respectively. About 0.5% of the CF taken up by the cells was transported through the cells and became located in the intercellular spaces. HRP was transported from the peritubular fluid to the apical cytoplasm of the tubules indicated by a gradual accumulation of small HRP-containing vesicles, first in the basal part of the cells and then in the apical cytoplasm. In tubules perfused with both CF and HRP in the perfusate, the CF and HRP were found together in apical vacuoles and lysosomes. After perfusion with HRP alone, this tracer was found in similar large vacuoles and lysosomes in the apical cytoplasm, in contrast to the small HRP-filled vacuoles seen after uptake from the bath.     

Association of Rho(D) polypeptides with the membrane skeleton in Rho(D)-positive human red cells

The Rho(D) antigen was recently identified as a 28,000 to 33,000 m.w. polypeptide expressed on the surface of human Rho(D)+ cells. We now show that 70 to 80% of the Rho(D) polypeptides remain firmly associated with the membrane skeleton (detergent-insoluble matrix) obtained after treatment of isolated membranes with Triton X-100. The same treatment solubilized most of the major sialoglycoprotein, glycophorin A. The membrane skeleton-bound Rho(D) polypeptides were not solubilized by procedures that dissociated spectrin, actin, and glyceraldehyde-3-phosphate dehydrogenase from the membrane. Affinity-purified 125I-labeled anti-Rho(D) antibodies bound to intact Rho(D)+ cells, Rho(D)+ membranes, and isolated membrane skeletons from Rho(D)+ cells, but not to Rho(D)- cells. The binding to Rho(D)+ cells was competitively inhibited efficiently by Rho(D)+ membranes and weakly by Rho(D)- membranes. When isolated unsealed Rho(D)+ and Rho(D)- membranes were labeled by lactoperoxidase-catalyzed iodination and solubilized in Triton X-100, Rho(D) polypeptides were immune precipitated only from Rho(D)+ membranes.     

Isolation of synaptosomal plasma membranes from cholinergic nerve terminals and a comparison of their proteins with those of synaptic vesicles

Plasma membranes were purified from purely cholinergic nerve endings (synaptosomes) isolated from the electric organ of Torpedo marmorata. Synaptosomes were lysed, membranes recovered and further separated by density gradient centrifugation. A fraction was obtained enriched in 5'-nucleotidase, Na+, K+-activated ATPase and acetylcholine esterase. Morphological examination showed abundant membrane fragments of the size range of synaptosomes and few of vesicle size. The fraction has a characteristic protein composition upon gel electrophoresis. Five reproducible major bands with apparent Mr of 100000, 75000, 52000, 42000 and 35000--33000 are found. A gel-electrophoretic comparison with proteins from synaptic vesicles from the same source (major bands Mr 160000, 147000, 34000 and 25000) was made. Comigration of major bands was detected in one-dimensional gel electrophoresis with the 42000-Mr, 35000--33000-Mr and 34000-Mr components. Upon two-dimensional gel electrophoresis the 42000-Mr component comigrates with a similar component in vesicles, recently characterized as actin; the other components are different. The presence of tubulin-like polypeptides is unlikely. Beside actin, all major vesicle proteins are often detected in small amounts in the plasma membrane preparation. It cannot be decided if they result from fused or contaminating vesicle membranes, but since they are essentially absent in some preparations, it seems that the plasma membrane does not contain vesicle proteins.     

In vitro effect of free bile acids on the bile canalicular membrane phospholipids in the rat.

Liver cell plasma membranes of male rats were isolated and separated into two fractions, one rich in bile canalicular membranes (BCM) and the other comprising the rest of the plasma membrane (PM). Aliquots of BCM, PM, and microsomes were incubated with deoxycholic, chenodeoxycholic, or cholic acid at bile acid - membrane phospholipid mole ratios up to 100, and the phospholipid solubilization from the PM and from microsomes was linear and apparently nonselective, while that from BCM was biphasic and distinctly selective. Phosphatidyl choline and phosphatidyl ethanolamine made up 90% of the phospholipids solubilized from the BCM at a bile acid - membrane phospholipid mole ratio sufficient to solubilize about 50% of the total phospholipids of the BCM. Of particular interest was the observation that the molecular species and fatty acid composition of the phospholipids solubilized from the BCM under these experimental conditions were similar to those of bile obtained from the same animal, and were quite unlike those solubilized at higher bile acid - phospholipids mole ratios. The data are discussed in terms of the mechanism of the biliary secretion of phospholipids.