Subcellular distribution of GTP-binding proteins, Go and Gi2, in rat cerebral cortex

The localization of GTP-binding protein (G-protein) subunits, Go alpha, Gi2 alpha and beta, in subcellular fractions of rat cerebral cortex was determined by means of immunoassays specific for the respective subunits. High concentrations of all three subunits were observed in both crude mitochondrial and microsomal fractions. Muscarinic cholinergic receptors were also densely localized in these fractions. Then the crude mitochondrial and microsomal fractions were subfractionated by sucrose density gradient centrifugation. Each fraction obtained was evaluated morphologically by electron microscopy and biochemically by determination of membrane markers. The crude mitochondrial fraction was subfractionated into myelin, synaptic plasma membrane, and mitochondrial fractions. All the G-protein subunits examined and muscarinic receptors were exclusively localized in the synaptic plasma membrane fraction. Among the submicrosomal fractions, the heavy smooth-surfaced microsomal fraction showed the highest concentrations of all G-protein subunits and receptors, while the rough-surfaced microsomal fraction contained low amounts of them. The heavy smooth-surfaced microsomal fraction also contained high specific activity of (Na(+)-K+)-ATPase, a marker of the plasma membrane. These results indicated that the Go alpha, Gi2 alpha and beta subunits are mainly localized in the plasma membrane in the brain.     

The use of p-toluene sulfonate to dissolve synaptosomal membrane proteins into organic solvents

A method is described by which a large proportion of membrane proteins may be dissolved in organic solvents by the use of p-toluene sulfonate. Synaptosomal membrane fractions from rat cerebral cortex were used as test material. Chloroform-methanol extraction dissolved 7% of proteins, 89% of lipid phosphorus, and 35% of reducing sugars. Further extraction of the residue with 2.5 mm p-toluene sulfonate in chloroform-methanol allowed the dissolution of 45% of the proteins. The final residue contained the rest of the protein and 60% of reducing sugars. The polypeptides in the three fractions showed marked differences in molecular weight and several glycoprotein bands were found in the final residue. The amino acid content of these three protein fractions was also different. It is concluded that p-toluene sulfonate, by lowering the pH and binding to the positive charges in the protein, is able to transfer about half of the synaptosomal membrane proteins into a hydrophobic medium.     

In situ incorporation of Fatty acids into lipids of the outer and inner envelope membranes of pea chloroplasts

When incubated with [1-¹⁴C]acetate and cofactors (ATP, Coenzyme A, sn-glycerol-3-phosphate, UDPgalactose, and NADH), intact chloroplasts synthesized fatty acids that were subsequently incorporated into most of the lipid classes. To study lipid synthesis at the chloroplast envelope membrane level, ¹⁴C-labeled pea (Pisum sativum) chloroplasts were subfractionated using a single flotation gradient. The different envelope membrane fractions were characterized by their density, lipid and polypeptide composition, and the localization of enzymic activities (UDPgalactose-1,2 diacylglycerol galactosyltransferase, Mg²⁺-dependent ATPase). They were identified as very pure outer membranes (light fraction) and strongly enriched inner membranes (heavy fraction). A fraction of intermediate density, which probably contained double membranes, was also isolated. Labeled glycerolipids recovered in the inner envelope membrane were phosphatidic acid, phosphatidyl-glycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol. Their ¹⁴C-fatty acid composition indicated that a biosynthetic pathway similar to the prokaryotic pathway present in cyanobacteria occurred in the inner membrane. In the outer membrane, phosphatidylcholine was the most labeled glycerolipid. Phosphatidic acid, phosphatidylglycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol were also labeled. The ¹⁴C-fatty acid composition of these lipids showed a higher proportion of oleate than palmitate. This labeling, different from that of the inner membrane, could result either from transacylation activities or from a biosynthetic pathway not yet described in pea and occurring partly in the outer chloroplast envelope membrane. This metabolism would work on an oleate-rich pool of fatty acids, possibly due to the export of oleate from chloroplast toward the extrachloroplastic medium. The respective roles of each membrane for chloroplast lipid synthesis are emphasized.     

Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80–90% was found in the intermembrane fraction, while the rest was associated with mitoplast. The intermembrane prostimulated kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplast. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinase, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.     

Subcellular fractionation of porcine neutrophils by nitrogen cavitation and sucrose-density-gradient centrifugation

Neutrophils, isolated in large quantities from porcine blood were disrupted by nitrogen cavitation and separated by differential centrifugation into a nuclear fraction and a post-nuclear supernatant. The latter was subfractionated by sucrose density gradient centrifugation into cytosol, a fraction consisting of membrane vesicles and two granule-rich fractions. The membrane fraction accounted for 1.9% of the protein in the post-nuclear supernatant, the light granule fraction for 2.2% and the dense granule fraction for 4.2%. Catalase, lactate dehydrogenase and malate dehydrogenase were largely confined to the cytosol. The dense granule fraction contained the highest quantities of the hydrolytic enzymes, although the membrane fraction was also rich in alkaline and acid phosphatase and gamma-glutamyl transpeptidase activities. Electron microscopy of the membrane fraction showed intact membrane vesicles, whereas the granular fractions consisted of electron-dense, membrane-bound granules. Two granular fractions were isolated which contained granules of differing size and density. 3H-labeled wheat germ agglutinin bound to the surface of intact neutrophils and when these were disrupted and fractionated the membrane fraction showed a specific binding activity 16-times greater than that of the cavitated sample. The membrane fraction interacted with the detergent digitonin and as a result underwent density perturbation increasing from 1.13 g X cm-3 to 1.18 g X cm-3. Dodecylsulphate-polyacrylamide gel electrophoresis showed the membrane fraction to consist of at least 40 protein bands, with relative molecular masses ranging from 200 000-16 000. The granule fractions contained less protein bands, with a protein composition quite distinct from that of the membrane fraction.     

Lipid Characterization of an Enriched Plasma Membrane Fraction of Dunaliella salina Grown in Media of Varying Salinity

We have developed a rapid procedure for isolating a fraction enriched in plasma membrane from Dunaliella salina using an aqueous two-phase system (dextran/polyethylene glycol, 6.7%/6.7%). An enriched plasma membrane fraction, free of chloroplast and mitochondrial contamination, could be obtained in 2.5 hours. Plasma membrane proteins, which accounted for approximately 1% of the total membrane protein, contained a number of unique proteins compared with the other cell fractions, as shown by gel electrophoresis. The lipids of the plasma membrane fraction from 1.7 molar NaCl-grown cells were extracted and characterized. Phosphatidylethanolamine and phosphatidylcholine were the two most prevalent phospholipids, at 20.6% and 6.0% of the total lipid, respectively. In addition, inositol phospholipids were a significant component of the D. salina plasma membrane fraction. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate accounted for 5.2% and 1.5% of the plasma membrane phospholipid, respectively. Diacylglyceryltrimethylhomoserine accounted for 7.9% of the plasma membrane total lipid. Free sterols were the major component of the plasma membrane fraction, at 55% of the total lipid, and consisted of ergosterol and 7-dehydroporiferasterol. Sterol peroxides were not present in the plasma membrane fraction. The lipid composition of enriched plasma membrane fractions from cells grown at 0.85 molar NaCl and 3.4 molar NaCl were compared with those grown at 1.7 molar NaCl. The concentration of diacylglyceryltrimethylhomoserine and the degree of plasma membrane fatty acid saturation increased in 3.4 molar plasma membranes. The relative concentration of sterols in the plasma membrane fraction was similar in all three NaCl concentrations tested.     

Cell-free analysis of Golgi apparatus membrane traffic in rat liver

 Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway. Accepted: 26 January 1998     

Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80-90% was found in the intermembrane fraction, while the rest was associated with mitoplasts. The intermembrane protein kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplasts. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinases, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.     

ISOLATION OF AN INSULIN SECRETION GRANULE FRACTION

Goosefish islets were homogenized in 0.25 M sucrose and separated into nuclear, mitochondrial + secretion granule, microsomal, and supernatant fractions. Eighty per cent of the cytochrome oxidase activity and 75 per cent of the bioassayed insulin activity were found in the mitochondrial + secretion granule fraction (6000 g for 10 minutes). The mitochondrial + secretion granule fraction was further subfractionated by centrifugation (2 hours at 100,000 g and 0°C) using a continuous linear density gradient 1.0–2.0 M sucrose). Eighteen to 20 subfractions were collected by piercing the bottom of the tube and collecting drops. The total protein was distributed into a bimodal curve consisting of a high density component, which contained 90 per cent of the insulin (secretion granules), and a lower density component, which contained the cytochrome oxidase activity (mitochondria).     

THE IN VIVO PROTEIN SYNTHETIC ACTIVITIES OF FREE VERSUS MEMBRANE-BOUND RIBONUCLEOPROTEIN IN A PLASMA-CELL TUMOR OF THE MOUSE

Cytoplasmic extracts of the transplantable RPC-20 plasma-cell tumor were fractionated by sucrose density gradient centrifugation. Four major fractions were distinguished: (a) microsomes and mitochondria; (b) membrane-free polyribosomes; (c) free monomeric ribosomes; and (d) soluble fraction. The fractions were analyzed for RNA and lipid phosphorus, and their particulate components were characterized by electron microscopy. Particular attention was paid to the problem of membrane contamination of the free polyribosome fraction. It was shown that this contamination was small in relation with the total content of ribosomes in the fraction, and that it consisted primarily of smooth-surfaced membranes which were not physically associated with the polyribosomes themselves. In vivo incorporation studies were carried out by injecting tumor-bearing animals intravenously with leucine-C¹⁴, removing the tumors at various times thereafter, and determining the distribution of protein radioactivity among the gradient-separated cytoplasmic fractions. The free polyribosome and the microsome-mitochondria fractions constituted active centers for protein synthesis. It was shown that nascent protein of the free polyribosome fractions was not associated significantly with the contaminating membranes. The kinetics of labeling during incorporation times up to 11 min suggested that protein synthesized on the free polyribosomes was rapidly transferred in vivo to the soluble fraction of the cell, while protein synthesized by the microsomes and mitochondria remained localized within these elements. It was estimated that the free polyribosome fraction and the microsome-mitochondria fraction accounted for approximately equal proportions of the total cytoplasmic protein synthesis in vivo.     

Glycoproteins and glycolipids of oxyntic cell microsomes I. Glycoproteins: Carbohydrate composition,analytical and preparative fractionation

Isotopically-labeled sugars were incorporated into glycoproteins of isolated bullfrog gastric mucosa. The majority of the label was found in gastric microsomal fractions which were shown to contain membranes derived from the oxyntic cell tubular membrane system and were not significantly contaminated with mucus. The tubular membranes contained exceptionally large quantities of carbohydrate (approx. 260 μg/mg protein). Most of the sugar (73%) was associated with protein in the following molar ratios: hexose, 1.0: fucose, 0.42; hexosamine, 0.62; sialic acid, <0.02. The remaining sugar, predominantly hexose, could be extracted into lipid solvents and was presumably glycolipid.Gastric microsomes were dissolves in sodium dodecyl sulfate and subjected to acrylamide gel electrophoresis and Sephadex G-200 fractionation. The latter preparative procedure yielded several molecular weight classes, each of which contained different sets of proteins and/or glycoproteins; however, the molar ratios of the sugars found in the two carbohydrate containing classes were quite similar.Significant quantities of carbohydrate were also found in gastric microsomal fractions from other species, e.g. pig and rabbit. Furthermore, characteristic proteins and glycoproteins were not present in tadpole gastric microsomes until the later stages of metamorphosis when HCl secretory capability had been established. The above findings suggest that glycoproteins may play an important role in oxyntic cell functions; the possibility of a membrane protective role is discussed.     

ISOLATION AND CHARACTERIZATION OF PLASMA MEMBRANE FRACTIONS FROM GARFISH LEPISOSTEUS OSSEUS OLFACTORY NERVE

Garfish Lepisosteus osseus olfactory nerve, because of its large size and the unusually high concentration of axonal membrane, is an excellent source of axonal membrane. A procedure is described for the isolation of two types of plasma membranes from the nerve which are obtained in yields of about 20 mg (fraction I) and 1.5 mg (fraction II) per g of wet nerve. Both membrane fractions consist mostly of rounded membrane vesicles, with a unit membrane thickness of ~7.5 nm. The two membrane fractions are different in their lipid to protein ratios, Na-K ATPase activities, polypeptide patterns on sodium dodecyl sulfate (SDS) gel electrophoresis, and fatty acid compositions. They have similar phospholipid composition. On the basis of the relative concentration of axonal and Schwann cell plasma membranes in the nerve, the Na-K ATPase activities of the two membrane fractions and a comparison of the properties of the membrane fractions to those of squid and lobster nerve membrane preparations, fraction I seems to be the axonal membrane and fraction II the Schwann cell plasma membrane. Fraction I has a low protein to lipid ratio. Its polypeptide pattern on SDS gel appears to be much more complex as compared to that of fraction II membrane.     

Characterization of two cell-envelope fractions from chemotrophically grownRhodospirillum rubrum

Two cell-envelope fractions were isolated from chemotrophically grown cells ofRhodospirillum rubrum. On the basis of electron-microscopic investigations, chemical analysis, distribution of components involved in respiration, and polyacrylamide gel electrophoresis, the heavy fraction (ρ²⁰=1.246 g per cm³) was identified as cell-wall, and the light fraction (ρ²⁰=1.145 g per cm³) as cytoplasmic-membrane fragments. Electron micrographs showed cell-wall fragments as open structures while cytoplasmic-membrane preparations were composed of closed membrane vesicles. With respect to the main classes of chemical compounds, cell wall could be distinguished from cytoplasmic membranes by a rather low ratio of phospholipids per protein and a high ratio of carbohydrates per protein. The relative proportion of individual neutral sugars as well as phospholipids (except for lysophosphatidyl ethanolamine) revealed no significant differences between both envelope fractions. Fatty acid analysis demonstrated a higher proportion of saturated fatty acids in cell-wall than in cytoplasmic-membrane fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions showed distinct protein compositions. While in cell-wall preparations polypeptides of 43000 and 14000 daltons predominated, 56000- and 52000-dalton polypeptides were the main protein subunits of cytoplasmic membranes. Cross contaminations of both cell-envelope fractions were defined.     

Isolation and characterization of the plasma membrane from Yoshida hepatoma cells

Summary Plasma membranes isolated from Yoshida ascites hepatoma AH-130 by a modification of the method of T. K. Ray (Biochim. Biophys. Acta 196: 1, 1970), were subfractionated into three fractions having densities (d) 1.12, 1.14 and 1.16 by discontinuous sucrose density-gradient. Membrane subfractions were characterized by electron-microscopy, by assay of marker enzymes and by lipid composition. All subfractions appeared to be essentially free from whole mitochondria, lysosomes and nuclei. Subfraction d 1.16 had, the highest 5-nucleotidase, Mg⁺⁺-ATPase and (Na⁺+K⁺)-ATPase activities; cytochromec oxidase was undetectable in any fraction and glucose-6-phosphatase was measurable only in fraction d 1.14. Adenylate cyclase had the highest activity in fractions d 1.14 and 1.16. Cyclic AMP phosphodiesterase was nearly equally distributed in the fractions. Adenylate, cyclase, 5-nucleotidase and Mg⁺⁺-ATPase activities of tumor membrane were lower with respect to liver plasma membrane, while cyclic AMP phosphodiesterase and (Na⁺+K⁺)-ATPase were found to have similar activities in the two membrane preparations. With respect to liver membrane, hepatoma membrane contained a higher amount of glycolipids and a higher amount of phospholipids accounted for mainly, by sphingomyelin, phosphatidylserine and phosphatidic acid. The possible significance of the decrease of adenylate activity in the hepatoma membrane is briefly discussed.     

Further characterization of particulate fractions from lysed cell envelopes of Halobacterium halobium and isolation of gas vacuole membranes

Lysates of cell envelopes from Halobacterium halobium have been separated into four fractions. A soluble, colorless fraction (I) containing protein, hexosamines, and no lipid is apparently derived from the cell wall. A red fraction (II), containing approximately 40 per cent lipid, 60 per cent protein, and a small amount of hexosamines consists of cell membrane disaggregated into fragments of small size. A third fraction (III) of purple color consists of large membrane sheets and has a very similar composition to II, containing the same classes of lipids but no hexosamines; its buoyant density is 1.18 g/ml. The fourth fraction (IV) has a buoyant density of 1.23 g/ml and contains the "intracytoplasmic membranes." These consist mainly of protein, and no lipid can be extracted with chloroform-methanol. Fractions I and II, which result from disaggregation of cell wall and cell membrane during lysis, contain a high proportion of dicarboxyl amino acids; this is in good agreement with the assumption that disruption of the cell envelope upon removal of salt is due to the high charge density. The intracytoplasmic membranes (IV) represent the gas vacuole membranes in the collapsed state. In a number of mutants that have lost the ability to form gas vacuoles, no vacuole membranes or any structure that could be related to them has been found.     

A Comparison between Prolamellar Bodies and Prothylakoid Membranes of Etioplasts of Dark-Grown Wheat Concerning Lipid and Polypeptide Composition

The aim of the present investigation was to find factors critical for the co-existence of prolamellar bodies and prothylakoids in etioplasts of wheat (Triticum aestivum L. cv Starke II). The lipid composition of the prolamellar body and prothylakoid fractions was qualitatively similar. However, the molar ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was higher in the prolamellar body fraction (1.6 ± 0.1), as was the lipid content on a protein basis. Protochlorophyllide was present in both fractions. The dominating protein of the prolamellar body fraction was protochlorophyllide oxidoreductase. This protein was present also in prothylakoid fractions. The other major protein of the prothylakoid fraction was the coupling factor 1, subunit of the chloroplast ATPase. From the lipid and protein data, we conclude that prolamellar bodies are formed when monogalactosyl diacylglycerol is present in larger amounts than can be stabilized into planar bilayer prothylakoid membranes by lamellar lipids or proteins.     

The isolation and characterisation of the plasma membrane fromHalobacterium salinarium

The plasma membrane ofHalobacterium salinarium, strain 1, has been isolated and characterised. A fraction containing cell envelope vesicles was isolated from a cell homogenate by centrifuging. A crude membrane fraction was obtained from the envelope fraction by dialysing it against distilled water, incubating with nucleases and centrifuging. A nucleotide-free purified membrane fraction, identified with the plasma membrane, was obtained by gel-filtration chromatography of the crude membrane fraction on Agarose. The nucleotide-free membrane-rich fraction contained all the cell lipid, including menaquinone and carotenoid, and cytochrome. No amino sugars could be detected. The action of the detergent, sodium dodecyl sulphate, on the nucleotide-free membrane-rich fraction broke up the membrane into smaller particles. The disaggregation occurred in at least two distinct steps. The disaggregated particles could be reaggregated to a fraction which resembled the original membrane by removing the detergent by dialysis or gel-filtration. A fraction which may be analogous to mitochondrial structural protein was isolated by ammonium sulphate fractionation of a preparation of the nucleotide-free membrane-rich fraction dissolved in a mixture of sodium deoxycholate, sodium cholate and sodium dodecyl sulphate. Protein fractions were separated from the nucleotide-free membrane-rich fraction during gel-filtration chromatography on Agarose in the presence of 6m urea. The authors would like to acknowledge the technical assistance of Miss C. Goode and Mrs. J. Wicks. We are indebted to Mrs. A. Flo of the Department of Biochemistry, The Technical University of Norway, Trondheim, for technical assistance in the preparation of samples for electron microscopy.     

SUBCELLUAR SITES FOR SYNTHESIS OF CHONDROMUCOPROTEIN OF CARTILAGE

Microsomes from embryonic cartilage have been subfractionated to yield smooth microsomes and rough microsomes. The in vitro enzymic activities involved in chondroitin sulfate biosynthesis have been assayed in these subfractions. The results demonstrate that all of the activities necessary for linkage to protein as well as for completion of the polysaccharide chain are present in both the rough and smooth fractions. Only in the case of the polymerization of N-acetylgalactosamine and glucuronic acid could enzyme assays be done independent of endogenous acceptor. This enzyme(s) was equally distributed between the rough and smooth fractions. The activities for the addition of xylose and galactose to protein were highest in the rough fraction while that for sulfation was highest in the smooth fraction. These findings suggest that polysaccharide chain-initiation occurs in the rough endoplasmic reticulum and that chain completion occurs in the smooth reticulum. This pattern is consistent with modern theories of synthesis, transfer, and export of extracellular macromolecules.     

Substructure of the Cytoplasmic Membrane of Bacillus megaterium I. Method for the Fractionation of “Ghosts”

A method of fractionation of "ghosts" was devised to identify the chemical components of the cytoplasmic membrane. The method consists of dialyzing the "ghosts" against distilled water, and then dissolving the ghosts in dilute alkali. The ghosts were fractionated into four fractions by use of differential centrifugation. The components of each fraction were analyzed in detail. The ratio of lipid to protein and content of carbohydrate were found to be different for the four fractions. The main two fractions (fractions 2 and 3) contained several types of materials. Fraction 2, which is soluble in alkali and sedimentable at 105,000 x g, contained protein and lipid in the ratio of 2:1; ribonucleic acid was not detectable. Under the electron microscope, the ghosts appeared to have released the cell's cytoplasmic contents, but many small dense particles (about 100 A in diameter) remained adherent to the membrane surface. On the other hand, fraction 2 appeared to be made up only of a membrane structure. No 100 A particles were visible in this fraction. From these results, fraction 2 seemed to be pure membrane material.