Isolation of the C3 complement component and its C3d subunit from IY-1 fraction of Cohn's fractionation of human plasma

C3 complement component and its C3d subunit were isolated from the IY-1 Cohn's fraction, which is the waste of industrially produced albumin and immunoglobulins. The first step was the fractionation of precipitate IY-1 by polyethylene glycol (PEG) 4000 to a final concentration of 16% PEG. The precipitate formed was separated by centrifugation. The supernatant contained the C3d subunit of C3, and the redissolved 16% PEG precipitate contained the C3 component. Then the supernatant and the dissolved precipitate were subjected to anion-exchange chromatography on DEAE-Toyopearl 650 M. In the last step fractions containing C3 and C3d concentrated by ultrafiltration were chromatographed on Sephacryl S-200.     

A comparison of methods for the isolation and fractionation of reticulocyte ribosomes

1. Polysomes, ribosomes and pH5 enzymes were isolated from rabbit reticulocytes by acidifying the post-mitochondrial supernatant to pH6.0 to precipitate all ribonucleoprotein particles and about half the pH5 enzymes; the precipitate was redissolved in buffer, pH7.6, and fractionated by zone centrifuging. 2. The isolation of polysome-rich and ribosome-rich fractions from the post-mitochondrial supernatant was also examined. 3. Studies of the stability of polysomes revealed that dissociation into sub-units occurred when both bound and free Mg(2+) was chelated by EDTA or when the pH was increased above pH8.8.     

Effect of temperature on the separation of soybean 11 S and 7 S protein fractions during bipolar membrane electroacidification

The purpose of this study was to evaluate the effect of temperature (10 and 27 degrees C) on the efficiency of bipolar membrane electroacidification (BMEA) to fractionate soybean proteins. BMEA is a technology derived from electrodialysis, based on the isoelectric precipitation of proteins. It appears that temperature has a significant effect on the selective precipitation of the soybean protein fractions, mainly 11 S and 7 S, during BMEA. At 27 degrees C, the precipitation profile of the four protein fractions is situated in a pH range from 6.6 to 4.4, with no possibility of separating any of theses fractions. However, at 10 degrees C, the 11 S globulin precipitates at a higher pH than at 27 degrees C, pH 6.7 vs 5.9, allowing the fractionation of 11 S from the other fractions. Using electroacidification it is possible to obtain a precipitate solution enriched in the 11 S fraction (71.8% of 11 S and 10.8% of 7 S) and a supernatant solution enriched in the 7 S fraction (46.6% of 7 S and 4.6% of 11S).     

Partial purification and characterization of gizzard erosion-inducing substance in heated casein-histidine mixture

A heated casein-histidine mixture, or the model compound of gizzard erosion-inducing fish meal, was prepared, and a gizzard erosion-inducing substance in this mixture was partially purified. The mixture was refluxed with hydrochloric acid, and the supernatant was obtained. The supernatant was then adjusted to the pH of the isoelectric point of casein and the resulting precipitate collected. The precipitate was further hydrolyzed with papain at a neutral pH. After acidification of the reaction mixture, the supernatant and the precipitate were separated. The supernatant fraction, which was toxic to induce gizzard erosion, was submitted to a gel filtration of Sephadex G-25, and resolved into five fractions. Gizzard erosion-inducing toxicity was found only in the second fraction. The non-toxic second fraction was also prepared by the same treatment from the non-toxic material of heated casein. The characters of these toxic and non-toxic second fractions were compared by using several analytical methods. Thin layer chromatography, as well as gel permeation chromatography, revealed that more than eight components having widely distributed molecular weights existed in both second fractions. By reversed phase chromatography, these fractions were found to consist of various peptides (or peptide-like compounds). Some characterizations, however, were performed. It failed to detect any difference in the components between the toxic and non-toxic second fractions by any analytical method used.     

Preparation of immunoglobulins G,A,M (IgGAM) for therapeutic use. Conditions for enrichment in IgA or in IgM]

Antibodies directed against viruses and bacteria are not equally distributed among the main classes of immunoglobulins, e.g. IgG, IgA and IgM. It has been found that IgM is mostly concerned with certain antibacterial activities (Salmonella, Escherichia coli and Pseudomonas) and IgA with high antibody titers for poliomyelitis virus I whereas antibody activities against many viruses such as influenza and measles virus occur preferentially in the IgG population. Furthermore, isolated immunoglobulin deficiency syndromes are actually well known. In the light of these findings, new concepts of immunotherapy have developed. Massive i.v. IgG-therapy is already widely used in congenital and acquired severe hypogammaglobulinemia. Preparations enriched in IgA and IgM are needed to complete the immunotherapeutical possibilities. Such a fraction called IgGAM has already been prepared in our Institute. Fraction III obtained during large scale fractionation is used as starting material and caprylic acid for the precipitation of most proteins other than the immunoglobulins present in fraction III. The immunoglobulin concentrate is finally obtained by ethanol precipitation of the caprylic acid supernatant. The present study is concerned with various modifications of the initial technique in order to obtain fractions more specially enriched in IgA or in IgM. In some cases the standard IgGAM fraction has been submitted to a further fractionation step, such as adsorption of IgG on DEAE-cellulose or precipitation of certain immunoglobulins achieved by Rivanol or by lowering the salt concentration. In other trials the fractionation procedure starting from fraction III has been modified. Rivanol has been used as a precipitating agent for the subfractionation of fraction III. It is well known that IgG is soluble in the presence of Rivanol. This technique was thus used in order to obtain preparations enriched mainly in IgM and IgA. The precipitate obtained after the addition of Rivanol was dissociated by NaCl and the solution further subfractionated by caprylic acid. In a similar way PEG was associated with the caprylic acid precipitation step. PEG precipitates proteins mainly in function of their molecular weight. However, the enrichment of IgM of the final fraction did not exceed 32% and much IgM was lost under the experimental conditions. It proved easiest to suspend fraction III in distilled water leaving IgM in the precipitate; it is dissolved and the solution submitted to a slightly modified caprylic acid precipitation step. This fraction contains 35-40% IgM, few (2-6%) IgA and about 50% IgG whereas an IgA (35%) enriched fraction is obtained when fraction III is solubilized with acetate at pH 6.2 and then submitted to precipitation by caprylic acid under slightly modified conditions as compared with our standard IgGAM. Thus, simple modifications of the standard procedure allow to prepare fractions enriched more specially in IgM or IgA. Fractions poor or almost devoid of IgG can also be obtained...     

SUBCELLULAR LOCALIZATION OF TRYPTOPHAN-5-MONO-OXYGENASE IN BOVINE PINEAL GLANDS AND RAPHE NUCLEI

Abstract— Tryptophan-5-mono-oxygenase from both bovine raphe nuclei and pineal glands was activated by preincubation with dithiothreitol and ferrous ion at pH 8.5. The optimum pH for the enzyme activity lies between pH 6.4 and 7.3. Preincubation increased the activity of the enzyme from raphe nuclei by about 20 times in both the homogenate and 105,000 g precipitate prepared from it. Activity in the 105,000 g supernatant fraction was about trebled. Corresponding increases in pineal gland enzyme activity were noted: 100 times in homogenate and 105,000 g precipitate and 15 times in 105,000 g supernatant fluid. Total recoveries of activated enzyme from the homogenate prepared in hypo-osmotic medium, in the 105,000 g supernatant and precipitate, were 87.1% and 79.0% for raphe nuclei and pineal glands respectively. Of this, 89.5-91.3% in the case of the raphe nuclei and 76.0-82.0% in the case of the pineal glands, was found in the precipitate. In contrast, 85-90% of the lactate dehydrogenase activity was found in the supernatant fraction. The results of subcellular fractionation revealed that the raphe nuclear enzyme was located in both 'mitochondrial' and 'microsomal' fraction while the pineal gland enzyme was effectively restricted to the 'mitochondrial' fraction. The structural characteristics of the fraction were confirmed by electron microscopy.     

The Isolation of Soluble Proteins,Glycoproteins, and Proteoglycans from Bone

Procedures are described for the isolation from bone of fractions containing proteins, glycoproteins and proteoglycans. Extraction of powdered bone with solutions of the sodium salts of ethylendiaminetetra-acetic acid (EDTA) at pH 7.5 solubilised about 7% of the organic material. These extracts contained about 1.8% of the total collagen and at least 60% of the total non-collagenous protein of bone. The extracts were dialysed against water to remove EDTA and then against a pH 5 buffer. At this stage a precipitate (Cl) formed which was removed by centrifugation. The supernatant was applied to a column of the carboxylio ion-exchange resin, Amberlite CG-50. The effluent at pH 5 contained the proteoglycans and more-acidic glycoproteins and was therefore named the Acidic Fraction (AF). The material adsorbed to the resin (Fraction G2) was eluted by equilibration to pH 8. AF was further fractionated by cetylpyridinium chloride (CPC) precipitation into three relatively pure components:(i) CP-S, a glycoprotein soluble in CPC, (ii) bone sialoprotein (BSP) which formed a CPC precipitate soluble in 0.2M-MgCl₂; and (iii) a proteoglycan fraction which formed a CPC precipitate insoluble in 0.2M-MgCl₂. The G2 fraction contained most of the soluble collagen together with glycoproteins and other non-collagenous proteins. These were fractionated by chromatography on DEAE-cellulose at pH 7.2 using stepwise elution with increasing concentrations of NaCl. Some resolution of the mixture was obtained, though most of the fractions contained more than one component. These procedures have been used on an analytical scale to assess the yields and recoveries of total protein, hydroxyproline and sialic acid in the fractions described above. This has been compared with the large scale procedure for the preparation of the fractions, which have been studied in previous work.     

The subcellular localization of cerebral phosphoproteins sensitive to electrical stimulation

1. On incubating cerebral-cortex slices at 37° in an oxygenated medium marked changes resulted in the subcellular distribution of proteins and phosphoproteins in the tissue. The protein content of the nuclear fraction more than doubled, whereas the yields of microsomal and supernatant proteins were both markedly decreased. The amount of phosphoprotein/mg. of protein decreased in the microsomal and supernatant fractions, but showed little change in the nuclear and mitochondrial fractions. The loss of microsomal protein could be partly prevented by rinsing the slices briefly in cold sucrose solution before dispersion; the altered subcellular distribution was apparently related to contamination of the dispersing solution with traces of salts from the medium. 2. The subcellular location of the phosphoprotein sensitive to the effects of electrical pulses applied to cerebral slices in vitro has been reinvestigated by two different procedures. Comparison between unstimulated and stimulated slices after incubation in the presence of [³²P]orthophosphate showed that phosphoprotein radioactivity increased on stimulation to a greater extent in a membrane-rich fraction than in a mitochondria-rich fraction, these being obtained by immediate density-gradient fractionation of the tissue dispersion. With fractions isolated by differential centrifuging the percentage increase in a combined mitochondrial and nuclear fraction was 5% as compared with 24% (P<0·02) in the microsomal fraction and 30% in the original dispersion before fractionation. The sensitive phosphoprotein therefore appears to be located in structures sedimenting with the microsomal fraction, rather than with the nuclear fraction as previously claimed.     

Isoelectric precipitation and gel chromatography for purification of monoclonal IgM

A preparative scale purification procedure of monoclonal IgM from hybridoma culture supernatant with high protein content is described. The procedure consists of three steps starting with ultrafiltration followed by isoelectric precipitation and gel chromatography. Cells and debris from culture supernatant were removed by microfiltration. The clarified supernatant was concentrated 400-fold in a hollow fibre ultrafiltration apparatus (cut off 100 000 daltons). The concentrate was titrated with dilute histidine/HCl buffer close to the isoelectric point of the IgM. Precipitated proteins were harvested by centrifugation, washed and redissolved. The protein fraction containing the IgM was further purified by gel chromatography on a Sephacryl S300 column. This procedure leads to product recovery of 40% and purity of 99% related to total protein.     

ENZYME ACTIVITIES IN CHICK EMBRYONIC CARTILAGE : Their Subcellular Distribution in Isolated Chondrocytes

Some characteristic enzymatic activities were determined in chick embryonic cartilage and compared with the analogous activities in bone and liver. Chondrocytes were isolated, broken by sonication, and subjected to subcellular fractionation to yield a nuclear pellet, the mitochondrial, lysosomal, and microsomal fractions, and the high speed supernatant solution. It was established that these fractions are characterized by enzymatic activities usually associated with similar fractions in other tissues, but with some quantitative differences. Lysozyme, a particulate-associated enzyme in other tissues, was not detected in any subcellular fraction even by the sensitive technique of microzone electrophoresis and is therefore considered to be primarily extracellular in cartilage.     

Determination of the Optimal Ammonium Sulfate Concentration for the Fractionation of Rabbit, Sheep, Horse, and Goat Antisera

Various ammonium sulfate concentrations and reaction conditions were employed in the fractionation of sera from rabbits, sheep, horses, and goats. Precipitates and supernatant fluids were analyzed by electrophoresis to study the effects of the controlled variables. At room temperature, the third precipitate in 35% saturated (NH(4))(2)SO(4) was the best fraction from both rabbit and sheep sera; 80 to 90% of the gamma globulins were recovered. The second and third precipitates of horse sera proteins in 30% saturated (NH(4))(2)SO(4) were both satisfactory, but only 44% of the gamma globulin was recovered after three precipitations. Goat sera yielded a very satisfactory fraction; 80% of the gamma globulin was recovered after two precipitations-the first in 30% and the second in 45% saturated (NH(4))(2)SO(4). The composition of these fractions was not influenced by the pH of the sulfate solutions (pH 5.8 and 7.2), by a range of normal room temperatures (20 to 30 C), or by diluting the sera before fractionation. Crude globulins and fluorescein isothiocyanate-labeled globulins were successfully refractionated by one precipitation in the optimal sulfate concentration for the appropriate animal species. The refractionated products contained considerably less beta and alpha globulins than did the original crude fractions and little or no albumin.     

Studies of the distribution of vitamin A as ester and alcohol and of carotenoids in plasma proteins of several species

The distribution of vitamin A ester, alcohol, and lutein was studied in fractions of chicken plasma proteins obtained by ammonium sulfate fractionation or dialysis, 6–8 hr. after oral administration of vitamin A and lutein in an oily medium. The losses of vitamin A and carotenoids during fractionation were considerable in some cases, losses of vitamin A being as large as 50% at times. Although this made the interpretation of the distribution between precipitate and supernatant difficult, the following conclusions seem to be possible. Vitamin A ester was found to be associated with the least soluble, and vitamin A alcohol and lutein with the more soluble protein fractions. No mono- or diesters of lutein could be demonstrated in the plasma.     

Cellular site and state combination of the adenosine 3':5'-cyclic monophosphate persisting after excitation of cerebral tissues.

1. The subcellular distribution of cyclic AMP in guinea-pig cerebral-cortical tissue was determined before and after electrical stimulation, and after a period of continued incubation after electrical stimulation, with and without the presence of histamine. 2. Electrical stimulation and histamine increased the cyclic AMP content of all fractions, the greatest increase occurring in the supernatant fraction. 3. Continued incubation after cessation of electrical stimulation diminished the cyclic AMP of the total homogenate and supernatant fractions, but increased that of the synaptosomal fraction. 4. Further fractionation of the synaptosomal fractions from stimulated tissues suggested that most of their soluble cyclic AMP was in a higher-molecular-weight form than was that of the tissue supernatant. 5. It is suggested that protein binding and cytoplasmic transport of cyclic AMP are involved in the changes observed.     

Rapid preparation of samples for compositional sugar analysis of the "degraded polysaccharide" fraction of lipopolysaccharides from Vibrio cholerae

A simple and rapid method was devised for direct isolation and fractionation of the "degraded polysaccharide" (DPS) fraction of O-antigenic (or endotoxic) lipopolysaccharides (LPS) directly from heat-killed Vibrio cholerae (O1 and non-O1) cells without separating the LPS. Neither phenol-water extraction nor ultracentrifuge is needed in this method. V. cholerae NIH 41 was used as standard. The cells (3-5 g wet weight) were heated in 5% acetic acid at 100 C for 1.5 hr. The acetic acid extract obtained as the supernatant by centrifugation was evaporated to dryness in vacuo, and the resultant residue was dissolved in 10 ml of distilled water. The solution was mixed with 2 volumes of acetone, and the supernatant obtained by centrifugation was mixed with 5 volumes of acetone and centrifuged. Fraction Sed. II was recovered as the precipitate, while the supernatant was evaporated to dryness in vacuo, yielding fraction Sup. III. Sed. II had a sugar composition that was identical, at least qualitatively, to that of DPS isolated from LPS of the corresponding strain except for the absence of a fructose component in the case of V. cholerae NIH 41, while instead Sup. III from V. cholerae NIH 41 contained fructose.     

Effects of ATP on regulation of galactosyltransferase-I activity responsible for synthesis of the linkage region between the core protein and glycosaminoglycan chains of proteoglycans.

We report that ATP enhances the activity of galactosyltransferase-I, which synthesizes the linkage region between glycosaminoglycan chains and the core proteins of proteoglycans. The enzyme activity in cell-free fractions prepared from cultured human skin fibroblasts was measured by high-performance liquid chromatographic detection of galactosyl-xylosyl-(4-methylumbelliferone) produced from 4-methylumbelliferyl-beta-D-xyloside used as an acceptor. ATP at 2 mM increased the enzyme activity by about 60% in the 110 x g supernatant of the cell homogenate, but not in the supernatant or precipitate fractions obtained by 100,000 x g centrifugation. When both fractions (the 100,000 x g supernatant and precipitate) were mixed, the additional ATP increased the enzyme activity. This increase was canceled by heat treatment or trypsin digestion of the 100,000 x g supernatant. In addition, the 100,000 x g precipitate, which was prepared from the 110 x g supernatant preincubated with ATP, exhibited increased activity, and this increase was abolished by alkaline phosphatase treatment. These results suggest that a protein kinase in the 100,000 x g supernatant activates galactosyltransferase-I activity.     

Comparison of soluble and membrane polypeptides ofSynechocystis 6308 by two-dimensional electrophoresis

Two-dimensional electrophoresis was carried out on fractions of the cyanobacteriumSynechocystis 6308 (ATCC 27150). Phycobilisomes isolated fromSynechocystis in 0.75M potassium phosphate buffer, pH 6.8, (KPi) plus Triton X-100 showed 5 prominent polypeptides when examined by two-dimensional electrophoresis. Ultracentrifugation of cells broken in KPi lacking Triton yielded three fractions, a membrane-containing pellet, a green supernatant, and a less dense yellow supernatant. The three fractions yielded a total of 272 polypeptides visualized by silver staining of two-dimensional gels. Fourteen polypeptides were found only in the yellow fraction, 14 polypeptides were found only in the green fraction, and 16 polypeptides were found only in the membrane fraction; 23 polypeptides were found in all three fractions. The crude Triton-containing KPi extract contained 62, and a 50 mM HEPES extract contained 55, of the 272 polypeptides visualized in these fractions. Two-dimensional electrophoresis combined with subcellular fractionation may be useful tools for examining changes in polypeptide composition caused by nitrogen starvation.     

MORPHOLOGICAL AND CHEMICAL STUDIES OF COLLAGEN FORMATION : II. Metabolic Activity of Collagen Associated with Subcellular Fractions of Guinea Pig Granulomata

Electron micrographs of thin sections of nuclear, microsomal, and mitochondrial fractions obtained from a carrageenin-induced granuloma showed considerable contamination of the heavier by the lighter fractions. Striated collagen fibrils could be identified in the nuclei + debris fraction. Only a few striated fibrils occurred in the mitochondrial fraction; very fine filaments (diameter 50 A) could be seen in this fraction, but could not be distinguished with certainty from fibrillar material derived from broken nuclei. 35 per cent of the mitochondrial and 80 per cent of the microsomal collagen was extractable by 0.2 M NaCl and could be purified by the standard methods of solution and reprecipitation. The amino acid composition of these collagen fractions determined by ion exchange chromatography was within the range normally found for collagen and gelatin from other mammalian species, allowing for 10 to 20 per cent of some non-collagenous contaminant of the microsomal collagen. Hydroxyproline and proline were isolated by chromatography on paper from hydrolysates of the nuclear, mitochondrial, and microsomal collagen fractions, after incubation of tissue slices with L-¹⁴C-proline. The specific activities of the hydroxyproline from these collagens were in the approximate ratio 1:2:6, while that of bound hydroxyproline derived from the supernatant was only 1, indicating primary synthesis of collagen in the microsomes. Attempts to demonstrate incorporation of L-¹⁴C-proline into collagen or into free hydroxyproline in cell free systems were unsuccessful, nor was it possible to demonstrate non-specific incorporation of L-¹⁴C-valine into TCA-insoluble material by various combinations of subcellular fractions.     

Regulation of insulin secretion by energy metabolism in pancreatic B-cell mitochondria. Studies with a non-metabolizable leucine analogue.

This study compares the collagen types present in rabbit ear cartilage with those synthesized by dissociated chondrocytes in cell culture. The cartilage was first extracted with 4M-guanidinium chloride to remove proteoglycans. This step also extracted type I collagen. After pepsin solubilization of the residue, three additional, genetically distinct collagen types could be separated by fractional salt precipitation. On SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis they were identified as type II collagen, (1 alpha, 2 alpha, 3 alpha) collagen and M-collagen fragments, a collagen pattern identical with that found in hyaline cartilage. Types I, II, (1 alpha, 2 alpha, 3 alpha) and M-collagen fragments represent 20, 75, 3.5, and 1% respectively of the total collagen. In frozen sections of ear cartilage, type II collagen was located by immunofluorescence staining in the extracellular matrix, whereas type I collagen was closely associated with the chondrocytes. Within 24h after release from elastic cartilage by enzymic digestion, auricular chondrocytes began to synthesize type III collagen, in addition to the above-mentioned collagens. This was shown after labelling of freshly dissociated chondrocytes with [3H]proline 1 day after plating, fractionation of the pepsin-treated collagens from medium and cell layer by NaCl precipitation, and analysis of the fractions by CM(carboxymethyl)-cellulose chromatography and SDS/polyacrylamide-gel electrophoresis. The 0.8 M-NaCl precipitate of cell-layer extracts consisted predominantly of type II collagen. The 0.8 M-NaCl precipitate obtained from the medium contained type I, II, and III collagen. In the supernatant of the 0.8 M-NaCl precipitation remained, both in the cell extract and medium, predominantly 1 alpha-, 2 alpha-, and 3 alpha-chains and M-collagen fragments. These results indicate that auricular chondrocytes are similar to chondrocytes from hyaline cartilage in that they produce, with the exception of type I collagen, the same collagen types in vivo, but change their cellular phenotype more rapidly after transfer to monolayer culture, as indicated by the prompt onset of type III collagen synthesis.     

Inhibition by various steroids on dihydrotestosterone binding to androphilic protein in human benign prostatic hypertrophy.

The effect of various steroidal compounds on the binding of the androphilic protein to dihydrotestosterone in the cytosol of tissues of human benign prostatic hypertrophy was examined. Androgens, as well as estrogens and gestagens showed an inhibitory effect on the binding. Non-steroidal anti-androgens were revealed to be weak inhibitors on the binding. Two androphilic proteins were observed in Sephadex G-200 chromatography in fractions eluted at the void volume and in fractions appearing at the site of IgG, and the rate of inhibition on the binding of both fractions by various steroids was compared. The rate of inhibition by various compounds was generally higher in the IgG fraction than in the void volume fraction. When the ligand and inhibitors were incubated with the cytosol prior to fractionation by Sephadex chromatography, rate of inhibition was lower than that obtained when the ligand and inhibitors were reacted with fractions after chromatography. Implications of the difference observed in these two experiments are not clear at this moment. The results obtained by the protamine precipitation experiment were almost the same as those by the experiment using the extract of the unfractionated acetone-dried cytosol, therefore, the protamine procedure does not seem to precipitate the tissue specific androphilic protein specifically.     

Separation of the primary dehydrogenase from the cytochromes of the nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium

A selective extraction procedure was developed for sequentially extracting a fraction containing the primary dehydrogenase and a fraction containing the cytochromes of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase of Bacillus megaterium KM membranes. The primary dehydrogenase (NADH-2,6-dichlorophenolindophenol oxidoreductase) activity was extracted from sonically treated membranes with 0.4% sodium deoxycholate for 30 min at 4 C. The insoluble residue was extracted with 0.4% sodium deoxycholate in 1 m KCl for 30 min at 25 C. A combination of the two extracts and dilution in Mg(2+) gave good recovery of the original membrane NADH oxidase activity. The primary dehydrogenase fraction contained 41% of the membrane protein, no cytochromes, flavine adenine dinucleotide as the sole acid-extractable flavine, and most of the membrane ribonucleic acid (RNA). The cytochrome-containing fraction had 16% of the membrane protein, 61% of the membrane cytochrome with the same relative amounts of cytochromes a and b as the original membrane, no acid-extractable flavine, little RNA, and no oxidoreductase activity. The oxidoreductase fraction remained soluble after removal of deoxycholate whereas the cytochrome fraction became insoluble after removal of deoxycholate-KCl, but the precipitated fraction could be redissolved in 0.4% sodium deoxycholate. Treatment of both fractions with ribonuclease to destroy all of the RNA present did not affect the ability of the fractions to recombine into a functional oxidase unit. Treatment of either fraction with phospholipase A prevented restoration of a functional oxidase when the oxidoreductase and cytochrome fractions were treated in solution, but no affect on restoration of oxidase was observed when the phospholipase A treatment was carried out with the soluble oxidoreductase fraction and the insoluble cytochrome fraction.