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Conventional methods of isolating and reconstituting ryanodine receptors (RyRs) from native membranes into proteoliposomes take a minimum of 2 days to complete. We have developed an alternative strategy that can be used to isolate and reconstitute functional RyRs in just 3 h with a similar degree of purification. RyRs isolated by this method display characteristic functional behaviour as assessed by radioligand binding and single channel analyses., ((c) 2002 Elsevier Science (USA).)

Pulmonary surfactant protein SP-C has been isolated from porcine lungs and treated with dansyl isothiocyanate in chloroform:methanol 2:1 (v/v) solutions,under conditions optimized to introduce a single dansyl group covalently attached to the N-terminalamine group of the protein without loss of its native thioesther-linked palmitic chains. The resulting derivative Dans-SP-C conserves the secondary structure of native SP-C as well as the ability to promote interfacial adsorption of DPPC suspensions and to affect the thermotropic behavior of DPPC bilayers. This derivative can be used to characterize lipid-protein and protein-protein interactions of a native-like SP-C in lipid/protein complexes., (Copyright 2001 Academic Press.)

Unlabelled: Labelling of the hydrophobic surfactant protein B (SP-B) under non-reducing conditions was achieved with [(99m)Tc(CO)(3)(H2O)(3)](+) prepared according to Alberto et al. (JACS, 1998). The binding of radioactivity was protein-specific, with an overall radiochemical yield of 50%. Gel electrophoresis and Westernblot analyses showed no structural changes of SP-B. Spreading properties and surface activity of (99m)Tc-labelled SP-B in an air/water interface coincided with those of unlabelled SP-B. (99m)Tc-SP-B seems to be a promising agent to observe surfactant spreading under clinical conditions., Background: Therapeutic results for surfactant instillation in clinical trials are conflicting. The (99m)Tc-labelling of surfactant would allow to observe its spreading in the lung under clinical conditions., Methods: [(99m)Tc(CO)(3)(H2O)(3)](+) was prepared as described by Alberto et al. (JACS, 1998). This carbonyl complex was used for the direct labelling of surfactant protein B (SP-B) under non-reductive conditions by direct incubation with SP-B at elevated temperature followed by extraction into CHCl(3)/MeOH., Results: The hydrophobic protein SP-B was labelled with [(99m)Tc(CO)(3)(H2O)(3)](+). An overall radiochemical yield of about 50% was achieved. HPLC-analysis revealed a single radiolabelled species according to UV elution profile of SP-B, supported by paper and size exclusion chromatography. Gel electrophoresis confirmed that the dimer structure of SP-B was preserved. Spreading properties of (99m)Tc-labelled SP-B in an air/water interface coincided with those of unlabelled SP-B. Spreading of radioactivity observed in a glass trough of 26 cm x 27 cm with a gamma camera was completed during the first 7-9 sec after application of (99m)Tc-labelled SP-B. The corresponding decrease of surface tension to 45 mN/m at the peripheral surface tension sensors took 7 sec +/- 2 sec (MEAN +/- STD; n = 3)., Conclusions: Direct and specific (99m)Tc-labelling of the hydrophobic surfactant protein B was achieved using the [(99m)Tc(CO)(3)(H2O)(3)](+) precursor. This procedure can easily be used to prepare specifically labelled surfactant mixtures with spreading properties that coincide with those of unlabelled surfactant.

Eosinophils are believed to be one of the important sources of cytokines such as IL-8 at the site of allergic inflammation. It has been demonstrated that pulmonary surfactant protein A (SP-A) plays a potential role in modifying inflammation and the immune function. To verify the regulating effect of SP-A on eosinophil cytokine generation, we studied the effect of SP-A by determining of IL-8 production and expression stimulated with sIgA or PMA. SP-A purified from surfactant recovered from patients with alveolar proteinosis was added to eosinophils isolated by the negative selection method with immunomagnetic beads, and cultured for 24 h. The concentrations of IL-8 in the cell-free supernatants and cell lysates were then measured by ELISA. We also used a semiquantitative reverse transcriptase-polymerase chain reaction assay to detect the effect of SP-A on IL-8 mRNA expression. SP-A inhibited the secretion of IL-8 in a dose-dependent fashion. Suppression of IL-8 production by SP-A was significantly inhibited by SP-A antibody (PE10). SP-A also attenuated expression of IL-8 mRNA in eosinophils. These results indicate that SP-A might have the potential role to modify allergic inflammation by inhibiting IL-8 expression and production from eosinophils., (Copyright 2000 Academic Press.)

Background: Acute respiratory distress syndrome (ARDS) develops in association with many serious medical disorders. Mortality is at least 40%, and there is no specific therapy. A massive influx of activated neutrophils, which damage pulmonary vascular endothelium and alveolar epithelium, leads to alveolar oedema and pulmonary surfactant dysfunction. In-vitro studies show that neutrophil elastase can cleave surfactant-specific proteins and impair surfactant function. If this happens in vivo in ARDS, the response to surfactant therapy will be limited., Methods: Samples of pulmonary surfactant were obtained from the lungs of 18 patients with ARDS and six healthy controls by bronchoalveolar lavage. We separated proteins in these samples according to molecular weight by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). We then used western blotting with monoclonal antibody E8 to detect the major surfactant-specific protein A (SP-A)., Findings: By contrast with controls, 14 of 18 patients had evidence of in-vivo damage to SP-A that resembled damage caused to SP-A when it is cleaved by neutrophil elastase. Controls showed a single band of normal dimers at 66 kDa, whereas 14 of 18 patients showed multiple bands at 66 kDa, 55 kDA, and 30-36 kDa, and six showed additional bands at 36-40 kDa., Interpretation: Direct damage to surfactant-specific proteins occurs in lungs of patients with ARDS, probably by proteolysis. Trials of protein-containing therapeutic surfactant are in progress in ARDS, and our results indicate that the frequent failure to maintain response may result from continuing damage to surfactant by products of activated neutrophils. A combination of surfactant and antiprotease therapy may improve therapeutic prospects.

A simple procedure has been developed for the purification of the surfactant proteins SP-A and SP-D from lung lavage of patients with alveolar proteinosis. The SP-D is purified by fractionation of the supernatant obtained after spinning the lavage at 10000 X g for 40 min, while the bulk of the SP-A is purified by fractionation of the pellet. The supernatant is applied to a maltosyl-agarose column and the bound SP-D is specifically eluted using MnCl2. The pellet is solubilised in 6 M urea and, following renaturation, the solubilised proteins are applied to maltosyl-agarose and SP-A eluted using a gradient of EDTA. Both SP-A and SP-D are further purified by gel-filtration on Superose-6. This procedure has also been used to prepare successfully human SP-A and SP-D from amniotic fluid and may be generally applicable to the isolation of these surfactant proteins from lung washings obtained from other species.

The MAL gene encodes a proteolipid protein displaying a cell type-specific pattern of expression, including T lymphocytes, myelin-forming cells, and epithelial renal MDCK cells, which has been recently identified as a component of detergent-insoluble membranes known to be enriched in glycolipids and cholesterol. Sequence alignment revealed a high degree of conservation of the MAL protein across species and evidenced the existence of a significant level of overall identity between MAL and two other proteins, BENE and the plasmolipin proteolipid. Moreover, using subcellular fractionation of transiently transfected COS-7 cells, both MAL and BENE were identified in detergent-resistant membranes. These results suggest the existence of a novel family of MAL-related proteins (including MAL, BENE, and plasmolipin) with primary structure homologies and with the distinctive features of having unusual biochemical properties such as lipid-like behaviour and/or the ability to reside in glycolipid-enriched membrane microdomains.

The effects of surfactant-associated protein A (SP-A) on lipid adsorption to the air-water interface and accumulation of dipalmitoylphosphatidylcholine (DPPC) in the surface region were investigated at 37 degrees C. Dispersions used were bovine pulmonary lipid extract surfactant with or without neutral lipid (NL). Lipid adsorption was examined with the Wilhelmy plate technique and DPPC accumulation by monitoring surface radioactivity from [14C]DPPC with a scintillation probe. SP-A enhanced the rate of lipid adsorption, while both SP-A and NL increased the extent of DPPC accumulation. At the specific radioactivity used [14C]DPPC monolayers were undetectable: the surface radioactivity arose from surface-associated DPPC beneath the monolayer. At the highest concentration studied (0.3 mg lipid/ml), NL greatly enhanced DPPC accumulation and SP-A attenuated this effect. Langmuir-Blodgett (L-B) films were prepared from [14C]DPPC-labeled dispersions (0.3 mg lipid/ml) at equilibrium surface tension. Autoradiographs of L-B films from lipid extract surfactant exhibited a diffuse misty appearance, while NL promoted formation of heterogeneous DPPC aggregates. Addition of SP-A to lipid extracts without NL generated DPPC aggregates; more uniform larger aggregates appeared in the presence of SP-A and NL. Radiation measurements confirmed that the L-B films were composed of more than monolayers. SP-A did not increase DPPC levels in films deposited from lipid extracts unless NL was present. These results indicate that neutral lipid cooperates with surfactant-associated protein A to organize dipalmitoylphosphatidylcholine in the surface films and enhance formation of a DPPC-rich reservoir below the air-water interface.

Surfactant protein A (SP-A) is an abundant lipoprotein component of pulmonary surfactant that plays multiple roles in surfactant homeostasis within the lung. A simple and rapid purification procedure for SP-A is described. Purified surfactant is washed by centrifugation with Ca2+ containing buffer to remove residual soluble proteins. Following the Ca2+ buffer wash, the surfactant pellet is washed in buffer containing EGTA and Mg2+ which releases the bound SP-A in almost pure form. Subsequent chromatography of the SP-A on Sephacryl S-500 yields homogeneous preparations of the protein. The SP-A purified using this procedure requires no exposure to either detergents or organic solvents to remove lipid. SP-A prepared by this new method inhibits lipid secretion from alveolar type II cells as effectively as SP-A prepared by other methods. In addition, the SP-A depleted surfactant produced in the first step of this procedure is capable of binding exogenous SP-A in a time dependent, saturable and Ca2+ dependent manner.

This study reports the biochemical separation of the hydrophobic constituents of calf lung surfactant into separate fractions from which specific components are excluded. Gel permeation chromatography on LH-20 with acidified chloroform-methanol separated the constituents of calf lung surfactant extract (CLSE) into fractions containing purified phospholipids (PPL), the neutral lipids and phospholipids (N&PL), or the hydrophobic surfactant proteins (SP)-B and -C together with the phospholipids (SP&PL). Extraction of acid to prevent phospholipid degradation after separation reduced recovery of the apoproteins in SP&PL. This fraction was therefore supplemented with protein purified separately to attain the initial levels present in CLSE. Biochemical analyses confirmed that the resulting preparations had the expected composition not only of protein, neutral lipids and phospholipids, but also of the phospholipid head groups. In addition to these fractions obtained with acidified solvent, elution of CLSE with chloroform-methanol without acid yielded the zwitterionic phospholipids substantially depleted of anionic phosphatidylglycerol and phosphatidylinositol. Limited interfacial measurements also demonstrated that the process of separation did not alter the fundamental surface characteristics of the surfactant constituents. Recombined CLSE (rCLSE) reconstituted from all of the separated components had surface activity indistinguishable from the original CLSE. The individual fractions of surfactant components also had average molecular areas at the air-liquid interface which agreed with predictions based on their biochemical composition. These well defined preparations of the hydrophobic constituents of pulmonary surfactant provide the basis for future studies to establish the role of individual components in the function of this complex surface active material.

Two capillary electrophoretic (CE) separation techniques with either simultaneous solvent flow induced by hydrostatic pressure or CE followed by low pressurization with helium were developed for the analysis of extremely hydrophobic proteins, such as the lung surfactant protein SP-C. For both related procedures, buffer solutions containing up to 70% of 2-propanol were used for the capillary electrophoretic separation. This high concentration of organic co-solvent, needed to solubilize the protein, dramatically reduces the electroosmotic flow (EOF) in aminopropyltrimethoxysilane-treated fused-silica capillaries. Because the EOF was insufficient to elute the separated analytes from the capillary, two "pressure-assisted" CE techniques were developed. An additional flow to elute the separated analytes was produced either by raising the inlet of the capillary or by helium pressure. Using the pressurization procedure a baseline separation of the SP-C protein and its dimeric complex was obtained in a 55-minute electrophoretic run, followed by pressure elution of the analyte to the detector. The present combination of pressurization and capillary electrophoresis does not require any detergents or involatile buffer additives, which are usually needed to solubilize extremely hydrophobic lipoproteins. It is therefore applicable to on-line coupling with electrospray mass spectrometry for the direct structural characterization of hydrophobic proteins.

We describe a chemical method for the detection and quantification of surfactant protein C (SP-C), a palmitoylated 3.5-kDa protein, based on the release of free-sulfhydryl groups upon depalmitoylation. SP-C was purified from calf lung surfactant extract by gel filtration on LH-20 and LH-60 columns in chloroform:methanol:0.1 N HCl (19:19:2). SP-C was allowed to react with [14C]iodoacetamide in the presence or absence of triethylamine, a deacylating agent. Unreacted iodoacetamide was removed by extraction with 1% KCl. Polyacrylamide gel electrophoresis followed by fluorography demonstrated that the intensity of the 14C-labeled SP-C bands correlated with the chloroform-soluble radioactivity. Under conditions designed to allow maximal incorporation of radioactivity, the concentration of SP-C was calculated and found to be approximately twice that indicated by the Lowry protein assay. The present assay is sensitive to nanogram levels (approximately 100 ng) of SP-C and is not affected by surfactant protein B or components of the organic solvent system. However, it is slightly inhibited by high concentrations of phospholipids (approximately 30% at a phospholipid:SP-C ratio of 700:1). The low interference of other surfactant components makes this method useful for the determination of SP-C in crude organic extracts and partially purified and purified preparations. It can also be used to detect other hydrophobic palmitoylated proteins, such as myelin proteolipid protein.

Proteolipid protein, the major protein component of central nervous system (CNS) myelin, is known to contain approximately 2% by weight of covalently bound fatty acids. Recently, this protein was also found, though at a lower concentration, in the peripheral nervous system (PNS). The present study was undertaken to determine whether PNS proteolipid protein, like its CNS counterpart, was fatty acylated. Myelin PLP and DM-20, the two proteins that constitute the proteolipid protein fraction, were isolated from bovine sciatic nerves by chromatography on a methylated Sephadex G-100 column. The identity of the isolated proteins was determined by (a) SDS-PAGE, (b) Western blot analysis, and (c) amino acid analysis, and fractions containing only PLP and DM-20 were subjected to acid-methanolysis. Gas-liquid chromatography of the released methyl esters revealed the presence of fatty acids (2.9% w/w) distributed as approximately 47% palmitic, 22% stearic, and 19% oleic acid. Similar results were obtained for bovine white matter proteolipid protein when isolated and analyzed in parallel with the peripheral nerve protein. These data unequivocally demonstrate that both PNS and CNS proteolipid protein are equally acylated.

Neonatal respiratory distress syndrome (RDS) caused by surfactant deficiency is a common disorder in premature infants. Exogenous surfactant therapy improves survival in infants with RDS. However, the phospholipid component of the surfactant has been suggested to be inactivated by a phospholipid hydrolyzing enzyme, phospholipase A2 (PLA2). Although alveolar type II cells produce the surfactant, it is not known whether these cells have any mechanism to protect surfactant from PLA2 hydrolysis. Since alveolar Clara cells express uteroglobin (UG), a PLA2 inhibitory and antiinflammatory protein, and since it has been suggested that alveolar type II cells are derived from Clara cells, we sought to elucidate whether type II cells are also capable of expressing UG gene. By using radioimmunoassay, immunoprecipitation and Western blotting techniques we demonstrate for the first time that type II cells, isolated from mature rabbit lungs, synthesize and secrete UG. The transcription of the UG gene was detected by in situ hybridization using rabbit UG cDNA probe. These results imply that UG, synthesized by type II cells, may protect both endogenous and exogenous surfactant from PLA2 hydrolysis. Moreover, the antiinflammatory properties of UG may prevent the development of chronic inflammatory lung disease, a frequent complication of RDS.

The sarcoplasmic reticulum of rabbit skeletal muscle contains a small "proteolipid," i.e., a protein which is soluble in acidic CHCl3/CH3OH. We propose the name sarcolipin for this small protein, to signify its lipid-like solubility and association with the sarcoplasmic reticulum. We have determined the following amino acid sequence for sarcolipin, using protein chemistry methods: M E R S T R E L C L N F T V V L I T V I L I W L L V R S Y Q Y. This 31-residue sequence includes a 19-residue hydrophobic segment which probably spans the sarcoplasmic reticulum membrane. The molecular weight calculated from the sequence, 3733, agrees with that measured by fast atom bombardment mass spectrometry, showing that sarcolipin contains no attached fatty acyl or other prosthetic groups.

In vitro experiments with granular pneumocytes suggest that surfactant protein-A (SP-A) inhibits secretion of pulmonary surfactant. We examined whether SP-A inhibits surfactant secretion induced by lung distention during lung lavage. Human SP-A was obtained by lung lavage in a patient with pulmonary alveolar proteinosis. After centrifugation of the lavagate, the pellet was repeatedly washed with saline and then extracted with chloroform:methanol. The methanol:saline phase was separated and lyophilized to yield the SP-A product. SDS-PAGE and immunoblot analysis indicated that our preparation of SP-A had only minor contamination with human plasma proteins. To examine secretion, we used freshly killed newborn rabbit pups of 29.5 days gestation and lavaged the lungs by 10 sequential, fresh saline washes. Littermate neighbor pairs were lavaged with SP-A or human plasma protein at concentrations of 1, 6, 10, and 50 micrograms/ml, and disaturated phosphatidylcholine (DSPC) was analyzed as a marker for surfactant. The inhibition of surfactant secretion was maximal at a concentration of 10 micrograms/ml; the average yield by the last two pairs of washes, an index of surfactant secretion, was 286 +/- 41 micrograms/g dry lung weight for SP-A, compared to 405 +/- 37 micrograms/g dry lung weight for controls, an inhibition of 30% (p < 0.005). There were no changes in the volumes of returned lavage or in the concentrations of lactate dehydrogenase or DNA. To test whether SP-A increased cellular uptake of DSPC in the lungs, we prepared radioactive exogenous surfactant, lavaged it into the lungs, and monitored recovery of radioactivity by continued lavage. Recovery was the same in treated and control lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the role of glycation of lens putative gap junctional protein, MIP26, on the permeability as well as on calmodulin mediated gating activity in reconstituted liposomes. Calf lens membranes were incubated with 0-100 mM glucose for 3 days and MIP26 was isolated. There was a glucose concentration dependent increase in the glycation of MIP26 which reached to 2.48 moles/mole of protein with 100 mM glucose. Gel electrophoresis showed that there was no degradation of MIP26 to MIP22 during incubation. Channel permeability was determined by reconstituting MIP26 into asolectin liposomes. There was a MIP26 glycation dependent decrease in the permeability to sucrose. Furthermore, proteoliposomes containing nonglycated MIP26 showed complete uncoupling of the channels with calmodulin whereas the channels containing glycated MIP26 were only partially uncoupled. These results suggest that glycation of MIP26 does interfere with the gating activity in reconstituted liposomes.

The effect of streptozotocin-induced diabetes on the levels of functional mitochondrial anion transport proteins has been determined. The experimental approach utilized for these studies consisted of the extraction of each of four mitochondrial anion transport proteins from rat liver mitoplasts (isolated from diabetic and control animals) with the nonionic detergent Triton X-114, followed by the functional reconstitution of each transporter in a liposomal system via the freeze-thaw-sonication technique. This approach permitted the quantification of transporter function without the complications that occur when such measurements are carried out with intact mitochondria (or mitoplasts). We found that experimental diabetes caused an increase in the extractable and reconstitutable specific (and total) transport activities of the pyruvate and dicarboxylate transporters, a decrease in the activity of the citrate transporter, and no significant change in the activity of the phosphate transporter relative to control values. An examination of the time course of the appearance of changes in the reconstitutable activities of the pyruvate and citrate transporters following the injection of streptozotocin revealed differences. Thus, whereas the activity of the pyruvate transporter displayed the most pronounced increase (193%) 1 week following streptozotocin injection and then subsequently declined from this peak and plateaued at later times (99% and 96% increases at 3 and 8 weeks, respectively), the activity of the citrate transporter progressively decreased with time (31-51% decreases at 1-8 weeks). We suggest that the observed diabetes-induced changes in mitochondrial anion transporter function are predictable on the basis of diabetes-induced alterations in the activities of enzymes that constitute metabolic pathways to which these transporters either supply substrate or remove product. Furthermore, we speculate that mitochondrial anion transport proteins may be regulated in coordination with the enzymes of such associated metabolic pathways.

The chemical and biophysical properties of the proteins in the lipid extracts of lung surfactant have not clearly been determined. These proteins were isolated from lung surfactant lipids by Sephadex LH-20 chromatography and purified with silicic acid chromatography followed by dialysis against organic solvents. The proteolipid thus obtained had a protein to phospholipid ratio of 3 to 1 (w/w). The proteolipid apoprotein had a nominal molecular weight of ca. 5 kDa. We evaluated the functional role of this proteolipid by combining it with proteolipid-depleted surfactant lipids or synthetic dipalmitoylphosphatidylcholine (DPPC) and then measuring with a pulsating bubble surfactometer. The proteolipid and DPPC recombinant reproduced the surface activity of natural lung surfactant. We conclude that this 5 kDa proteolipid apoprotein is a functionally important constituent of lung surfactant.

Bovine cardiac muscle was extracted by an acidic chloroform/methanol mixture. A combination of gel permeation and ion-exchange chromatographies in organic solvents and HPLC allowed the purification of subunits VIIIa (Mr 5400) and VIIIb (Mr 4900) of cytochrome c oxidase and of A6L protein (Mr 7900) of ATP synthase. The identification of the proteins was made possible by measurement of their molecular weight by fast atom bombardment-mass spectrometry (FAB-MS) in conjunction with conventional Edman degradation. The determination by FAB-MS of the molecular weight of A6L protein confirmed its supposed formylated N-terminal methionine.

Since the first description of organic-soluble proteins (i.e., proteolipids), much attention has focused on the isolation, purification, characterization, localization, and function of these intrinsic membrane proteins in a variety of different tissues. Using a rapid purification scheme, which allowed the transfer of organic-soluble proteolipids to aqueous phases, we have isolated proteolipids from cultured human keratinocytes and human epidermis for the first time. A partial characterization of these proteolipids, including molecular-weight determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid composition, and an N-terminal sequencing revealed a preponderance of hydrophobic amino acids (greater than 60% overall and greater than 78% in N-terminal sequence), typical of other proteolipids. The composition of fatty acids, covalently bound to whole purified apoprotein fractions, displayed a predominance of palmitic greater than oleic greater than stearic acids. Comparison of the molecular species of proteolipids isolated from whole epidermis with those obtained from keratinocyte cultures by SDS-PAGE revealed a comparable spectrum of apoprotein species. Finally, subcellular fractionation of cultured keratinocytes, used to localize proteolipids to specific cellular compartments, suggested that one of the major apoprotein species (30 kD) is present in mitochondria, whereas the lower molecular weight species are localized in plasma membrane-enriched fractions. Although evidence is lacking for a specific function(s) of this class of molecules in the epidermis, the hypothesis that it plays a role in epidermal differentiation, for example, as constituents of calcium and/or proton pumps, is discussed.

Alkyl-Sepharose 4B with octyl, decyl, or dodecyl groups as an alkyl chain was a good adsorbent for any type of detergents and a variety of proteins, but not for phospholipids in a vesicle form. When these gels were added to the mixtures of reconstituted proteoliposomes prepared by using bovine band 3 and the protein unincorporated into liposomes, free band 3 in solution was adsorbed onto the gels and the proteoliposomes could be recovered by filtration, suggesting that this procedure, when applicable, permits a rapid isolation of proteoliposomes without loss and dilution of the sample. In addition, the results indicated that Bio-Beads SM-2 resin, which is virtually nonadsorbing for most proteins, can be used in removing any kind of detergents from those protein-detergent mixtures.

Proteolipids from adult rat brain subcellular fractions were purified by a one-step procedure involving chromatography through Sephadex LH-60 eluted with an acidified chloroform-methanol mixture. The protein peak was eluted with the void volume and was free of adventitious lipids. The degree of purification was similar to that attained with the neutral-acidified chloroform-methanol dialysis method with the advantage that this new procedure can be carried out in only 3 h, with a recovery of proteins of 95-100%. Samples containing different lipid/protein ratios passed through the gel gave similar elution profiles. When labeled amino acids or palmitic acid were added to myelin total lipid extracts, no radioactivity was eluted with the protein, indicating that the proteolipid apoproteins purified by this method do not adsorb hydrophobic low-molecular-weight compounds.

An improved preparation of proteolipid from bovine brain white matter is described. The product obtained by repeated acetone precipitation is completely soluble in chloroform-methanol and has a fairly constant composition: 35% protein, 40% galactocerebroside, and about 25% phospholipid.