Low Concentrations Of Deoxycholate Research Articles

Role of calcium in adaptive cytoprotection and cell injury induced by deoxycholate in human gastric cells.

We have developed an in vitro model of adaptive cytoprotection induced by deoxycholate (DC) in human gastric cells and have shown that pretreatment with a low concentration of DC (mild irritant, 50 microM) significantly attenuates injury induced by a damaging concentration of DC (250 microM). This study was undertaken to assess the effect of the mild irritant on changes in intracellular Ca2+ and to determine if these perturbations account for its protective action. Protection conferred by the mild irritant was lost when any of its effects on intracellular Ca2+ were prevented: internal Ca2+ store release via phospholipase C and inositol 1,4, 5-trisphosphate sustained Ca2+ influx through store-operated Ca2+ channels or eventual Ca2+ efflux. We also investigated the relationship between Ca2+ accumulation and cellular injury induced by damaging concentrations of DC. In cells exposed to high concentrations of DC, sustained Ca2+ accumulation as a result of extracellular Ca2+ influx, but not transient changes in intracellular Ca2+ content, appeared to precede and induce cellular injury. We propose that the mild irritant disrupts normal Ca2+ homeostasis and that this perturbation elicits a cellular response (involving active Ca2+ efflux) that subsequently provides a protective action by limiting the magnitude of intracellular Ca2+ accumulation.

Temperature-Induced Differential Kinetic Properties between an Initial Burst and the Following Steady State in Membrane-Bound Enzymes: Studies on Lathosterol 5-Desaturase

The NADH-dependent lathosterol 5-desaturation reaction that forms 7-dehydrocholesterol is biphasic, an initial burst followed by steady state. The steady-state phase is slower than the burst phase, because the latter diffusion of the lathosterol substrate within the microsomal membrane must occur before the next reaction can take place [Y. Takakuwa, H. Nishino, Y. Ishibe, and T. Ishibashi (1994)J. Biol. Chem.269, 27889–27893]. In the present study, changes in the structure and function of the membrane were examined by measurement of the Arrhenius activation energy of lathosterol 5-desaturase at various temperatures between 2 and 45°C. At the burst phase, there was a lack of discontinuity in the Arrhenius plots at the presumed phase transition temperature for the microsomal membrane. However, the plots of the activities of the steady state showed breaks at around 17 and 32°C. It was concluded that phospholipid phase transition affects the steady-state phase but not the burst phase. Furthermore, treatment of microsomes with low concentrations of deoxycholate, known to perturb the membrane integrity, resulted in a break of the activation energy of the burst phase. These results have revealed further evidence for our previous model suggesting interaction between the substrate and enzyme within the microsomal membrane via lateral diffusion.

In situ two-dimensional crystallization of a polytopic membrane protein: the cardiac gap junction channel.

In situ crystallization of rat ventricular gap junctions was accomplished by sequential dialysis of membranes against low concentrations of deoxycholate and dodecyl-beta-D-maltoside. Lipids are removed without solubilizing the protein, and the increased protein concentration in the membrane plane facilitates two-dimensional crystallization in the native membrane environment. The two-dimensional crystals have a nominal resolution of 16 A and display plane group symmetry p6 with a = b = 85 A and gamma = 120 degrees. Electron crystallography reveals that the cardiac gap junction membrane channel is formed by a hexameric cluster of protein subunits, and this hexameric design appears to be a recurring quaternary motif for the multigene family of gap junction proteins. Exposure of membranes to low concentrations of detergents may provide an approach for in situ two-dimensional crystallization of other connexins as well as other membrane proteins, especially those that are labile when solubilized as protein-detergent micelles.

Trapping of the beta-adrenergic receptor in the hormone-induced state.

Isoproterenol and other agonists readily dissociate from the beta-adrenergic receptor in turkey erythrocyte membranes. However, when a low concentration of deoxycholate is added, the receptor locks the prebound agonist; i.e., the rate of dissociation of the prebound agonist decreases drastically. The dissociation of prebound antagonists is slightly increased by deoxycholate. Locking, which is thus agonist specific, occurs in the cold, is reversed when detergent is removed from the membranes, and appears not to require the guanyl nucleotide binding protein of the adenylate cyclase system. It is suggested that this induced fit of a receptor to an agonist represents the specific conformational response that normally propagates in the receptor molecule in its interaction with the next component along the pathway of signal transmission.

Transmembrane orientation of an early biosynthetic form of acetylcholine receptor delta subunit determined by proteolytic dissection in conjunction with monoclonal antibodies

The transmembrane topology of acetylcholine receptor (AChR) delta subunit, synthesized in vitro and co-translationally integrated into dog pancreas rough microsomal membranes, was studied using limited proteolysis and domain-specific immunoprecipitation. Forty-four kilodaltons (kd) of the 65-kd delta subunit comprise a single fragment that is inaccessible to exhaustive proteolytic digestion from the cytoplasmic surface of the membrane by trypsin, chymotrypsin, thermolysin, and pronase. Previously, we have shown that this 44-kd "protected" fragment contains the amino terminus of the intact molecule and all of the core oligosaccharides (Anderson, D.J., P. Walter, and G. Blobel (1982) J. Cell Biol. 93: 501-506). Here we demonstrate that this domain can be further dissected into a 26-kd fragment, together with low molecular weight material, when the membranes are rendered permeable to trypsin by low concentrations of deoxycholate (Kreibich, G., P. Debey, and D. D. Sabatini (1973) J. Cell Biol. 58: 436-462). This 26-kd fragment contains all of the core oligosaccharides present on the intact subunit and therefore constitutes at least part, if not all, of the extracellular domain. The remaining low molecular weight material may derive from the membrane-embedded domain; our data imply that as much as 18 kd may be internal to the lipid bilayer. On the other hand, part of the cytoplasmic pole of AChR-delta can be recovered as a discrete, 12-kd fragment upon mild trypsinization of intact vesicles. We have used this 12-kd fragment to identify anti-AChR-delta monoclonal antibodies (mAbs) that react with the cytoplasmic domain of this subunit. Partial proteolytic fragmentation of the AChR in vitro translation products, in topologically well defined rough microsomes, may be used as a general assay to characterize the domain specificity of anti-AChR mAbs. For example, in the case of AChR-beta, we were able to identify two mAbs that recognize extracellular and cytoplasmic fragments, respectively.

Disposition of gangliosides and sialosylglycoproteins in neuronal membranes.

Labeled gangliosides and glycoproteins were obtained by incubation of homogenized neuronal perikarya from rat brain with CMP-[3H]N-acetyl neuraminic acid. The highest degree of labelling was observed in a subcellular fraction that also showed the highest specific activities for several ganglioside glycosyltransferases. The [3H]sialosylglycoconjugates of this fraction remained associated with the membranes after treatment with 1 M-KCl, 125 mM-EDTA, repeated freezing and thawing, or controlled sonication, but were solubilized by sodium deoxycholate (DOC) at a concentration high enough to solubilize the choline phospholipids. About 75% of th neuraminidase-labile sialosyl residues of these labeled endogenous gangliosides and glycoproteins were protected from the action of added neuraminidase or pronase or both enzymes added together. The protection was not abolished by pretreatment of the membranes with high ionic strength or with EDTA but was abolished by sonication or low concentration of DOC. Between 50 and 80% of the neuraminidase-labile sialosyl residues of the gangliosides of the neuronal perikaryon membrane fraction labelled in vivo by an intracerebral injection of N-[3H]acetylmannosamine were, at 3 h after the injection, also protected from the action of added neuraminidase. The protection was abolished by the addition of DOC. In contrast with behaviour of the labeled glycoconjugates of this neuronal perikaryon fraction, the gangliosides and sialosylglycoproteins from intact synaptosomes were accessible to neuraminidase. It is suggested that most gangliosides and sialosylglycoproteins are sialosylated as intrinsic components of the neuronal perikaryon membrane fraction and that at some stage of the process of transport through the axon and incorporation into the synaptic plasma membrane they change their accessibility to added enzymes.

Purification and characterization of the 53,000-dalton glycoprotein from the sarcoplasmic reticulum.

A 53,000-dalton intrinsic glycoprotein of the sarcoplasmic reticulum was separated from the Ca2+ + Mg2+-ATPase by dissolution with low concentrations of deoxycholate in the presence of 1 M KCl and purified in two successive gel filtration steps. It was aggregated and eluted at the void volume when subjected to gel filtration in the presence or absence of deoxycholate. When subsequently chromatographed in the presence of sodium dodecyl sulfate, the glycoprotein eluted in pure form as a monomer. The glycoprotein contained 48% nonpolar amino acids. It also contained 4 mol of glucosamine and 18 mol of mannose per mol of protein, suggesting that it contained two chains of (GlcNAc)2, (Man)9. The 53,000-dalton glycoprotein was completely removed from deoxycholate extracts of sarcoplasmic reticulum by affinity chromatography on concanavalin A Sepharose. Elution of glycoproteins with alpha-methyl-D-mannoside and deoxycholate resulted in co-purification of the 53,000-dalton glycoprotein and 160,000-dalton glycoprotein previously observed in sarcoplasmic reticulum. The apparent molecular weight of the glycoprotein was reduced from 53,000 to 49,000 after digestion with endo-beta-N-acetylglucosaminidase H (Endo H) and its reactivity with concanavalin A (Con A) was lost. There was no change in molecular weight of the glycoprotein and no diminution of its reactivity with Con A when sealed vesicles of sarcoplasmic reticulum were treated with Endo H. Endo H reduced the molecular weight and the Con A reactivity of the protein when the vesicles were made permeable by detergents. These observations, together with our previous demonstration that the glycoprotein reacts with a cycloheptaamylose-fluorescamine complex in sealed vesicles (Michalak, M., Campbell, K. P., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 1317-1326), show that the glycoprotein is a transmembrane protein. A protein of approximately 53,000 daltons was labeled when the sarcoplasmic reticulum was reacted with the photoaffinity label [32P]8-N3-cAMP. The labeled protein was neither the glycoprotein nor the high affinity calcium-binding protein since it was not sensitive to Endo H and was sensitive to trypsin digestion.

Resolution of microsomal membranes into fractions differing in polypeptide composition

Microsomes from rat liver, prepared by gel filtration, were subjected to centrifugation in a continuous sucrose density gradient containing a low concentration of deoxycholate. The membranes were subfractionated into five bands differing in appearance and equilibrium density. Each band, when analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, displayed a characteristic population of membrane proteins.

Adenosine 3',5'-monophosphate-dependent phosphorylation of a 6000 and a 22,000 dalton protein from cardiac sarcoplasmic reticulum.

In canine cardiac sarcoplasmic reticulum, adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22,000 and 24,000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640-2647). The other protein that the 32P label incorporates into has a molecular weight of approximately 6000. Like the 22,000 dalton protein, the 6000 dalton protein has characteristics of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the 32P label is incorporated more rapidly into the 22,000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22,000 and the 6000 dalton protein are still phosphorylated, but only to about a quarter of the activity found when cyclic AMP and protein kinases are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22,000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins. Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation the phosphorylated 22,000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22,000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.

Effects of a supernatant protein activator on microsomal squalene-2,3-oxide-lanosterol cyclase.

A soluble protein termed "supernatant protein factor" (SPF) that stimulates microsomal squalene epoxidase has been isolated in this laboratory (Ferguson, J.B., and Bloch, K. (1977) J. Biol. Chem. 252, 5381-5385). We now show that the purified protein also stimulates microsomal squalene-2,3-oxide leads to lanosterol cyclase but has no effect on the subsequent conversion of lanosterol to cholesterol. Phospholipid, specifically phosphatidylglycerol or phosphatidylethanolamine, is required for maximal stimulation of the cyclase by purified SPF. The response of microsomal squalene epoxide-lanosterol cyclase to SPF was abolished by pretreatment of the membranes with phospholipase A2 or by low concentrations of deoxycholate, indicating that an intact membrane system is required. Digestion of intact microsomes with trypsin had no effect on the SPF-stimulated cyclase activity. However, in the presence of 0.4% deoxycholate, trypsin completely inhibited microsomal squalene epoxide-lanosterol cyclase. We conclude that the cyclase is located on the luminal side of the microsomal membrane. SPF also significantly enhances the formation of lanosterol from squalene-2,3-oxide already bound to microsomes. This finding is constant with the proposal that SPF influences intramembrane events.

The role of components of the endoplasmic reticulum in the biosynthesis of cytochrome P-450.

1. Antibodies have been prepared to rat hepatic cytochrome P-450 and their specificity demonstrated. These antibodies have been used to investigate the biosynthesis of cytochrome P-450 in vitro and in situ in various components of the endoplasmic reticulum. 2. A preparation of heavy rough endoplasmic reticulum translocates proteins newly biosynthesized in vitro vectorially into the luminal space and these are released by low concentrations of deoxycholate. A significant proportion of the radioactivity found in this released fraction is incorporated into cytochrome P-450. 3. Following incorporation of [14C]leucine by perfused rat liver, radioactively labelled cytochrome P-450 can be found in the intrascisternal content of heavy rough, light rough and smooth endopalsmic reticulum and also in a solublized Golgi preparation. 4. We suggest that at least part of the newly biosynthesized cytochrome P-450 is translocated into the intracisternal space of the rough endoplasmic and then passes through the other components of the endoplasmic reticulum before insertion at its ultimate membrane locus.

Preparation of membrane-bound and of solubilized [formula omitted] from rectal glands of Squalus acanthias. The effect of preparative procedures on purity, specific and molar activity

A simple method is described for the routine preparation of larger quantities of purified (Na + + K +)-ATPase from the rectal glands from Squalus acanthias and for solubilization of the purified enzyme in a highly active form. Microsomes are prepared by homogenization of the glands in a Waring Blendor followed by differential centrifugation. They keep their activity for years when stored at −70°C. Based on the earlier method (Jørgensen, P.L. and Skou, J.C. (1971) Biochim. Biophys. Acta 233, 366–380), enzyme with a specific activity of 1500 μmol P i · mg −1 protein · h −1 was prepared by treating the microsomes with low concentrations of deoxycholate followed by differential centrifugation, and with a yield of 70% of the activity in the deoxycholate-treated microsomes. The purified enzyme can be dissolved in deoxycholate in the presence of cholesterol, and after a single centrifugation to remove undissolved enzyme, the specific activity of the solubilized enzyme is increased to 2400–2600 μmol P i · mg −1 protein · h −1. Precipitation of the solubilized enzyme leads to a decrease in specific activity to 1500 μmol P i · mg −1 protein · h −1 and to a decrease in molar activity.

In vitro synthesis of the Ca 2+ transport ATPase by ribosomes bound to sarcoplasmic reticulum membranes

The calcium transport ATPase (M(r) 100,000) from sarcoplasmic reticulum membranes was synthesized in a cell-free translation system containing rough microsomes or detergent-treated bound polysomes from 14- to 16-day old chicken embryo muscles. Immunoprecipitates obtained from total translation mixtures treated with anti-ATPase antiserum contained 1.5% of the total radioactivity incorporated in vitro. A polypeptide with the electrophoretic mobility, isoelectric point, and [(35)S]methionine-labeled tryptic peptide pattern of the mature ATPase was a major component of these immunoprecipitates. By contrast, free polysomes from the same source, which were capable of high levels of in vitro protein synthesis, did not yield immunoprecipitable ATPase. ATPase synthesized in rough microsomes was not released by treatment with 10 mM EDTA in a high-salt medium (0.5 M KCl) which removes ribosomes and peripheral membrane proteins. Furthermore, labeled ATPase remained associated with the microsomes after these were treated with low concentrations of deoxycholate (0.1 mg/mg of protein in 0.3 ml) which release the luminal content of the vesicles. Only with higher deoxycholate concentrations (0.5 mg/mg of protein in 0.3 ml), which cause membrane dissolution, was the labeled ATPase found on the detergent extracts. These observations indicate that newly synthesized ATPase discharged from bound ribosomes is transferred directly to the sarcoplasmic reticulum membranes where it is incorporated as an integral membrane protein.

Purification of phospholamban, a 22,000-dalton protein from cardiac sarcoplasmic reticulum that is specifically phosphorylated by cyclic AMP-dependent protein kinase.

Very low concentrations deoxycholate (DOC) were used to isolate two proteins from canine cardiac sarcoplasmic reticulum. These two proteins are phospholamban, a 22,000 dalton protein, and the Ca/sup 2 +/ + Mg/sup 2 +/-ATPase, the major protein of the sarcoplasmic reticulum, responsible for the active transport of calcium. The 22,000 dalton protein is first solubilized in a very low concentration of DOC and then subjected to column chromatography. After molecular weight sieving on a Sephadex G-75 column, the 22,000 dalton protein appears as a purified protein on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The purified protein is specifically phosphorylated by cyclic AMP-dependent protein kinase. Phospholipids are still bound to the isolated protein. The Ca/sup 2 +/ + Mg/sup 2 +/-ATPase is purified by first solubilizing all the extrinsic proteins with a low concentration of DOC. An increasing amount of DOC is then added to yield the purified Ca/sup 2 +/ + Mg/sup 2 +/-ATPase. This protein is at least 95% pure. Adding additional DOC to the purified enzyme enhances the enzyme's ability to hydrolyze ATP. (ERB)

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins.

Rat liver microsomal glycoproteins were purified by affinity chromatography on concanavalin A Sepharose columns from membrane and content fractions, separated from rough microsomes (RM) treated with low concentrations of deoxycholate (DOC). All periodic acid-Schiff (PAS)-positive glycoproteins of RM showed affinity for concanavalin A Sepharose; even after sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, most of the microsomal glycoproteins bound [125I]concanavalin A added to the gels, as detected by autoradiography. Two distinct sets of glycoproteins are present in the membrane and content fractions derived from RM. SDS acrylamide gel electrophoresis showed that RM membranes contain 15--20 glycoproteins (15--22% of the total microsomal protein) which range in apparent mol wt from 23,000 to 240,000 daltons. A smaller set of glycoproteins (five to seven polypeptides), with apparent mol wt between 60,000 and 200,000 daltons, was present in the microsomal content fraction. The disposition of the membrane glycoproteins with respect to the membrane plane was determined by selective iodination with the lactoperoxidase (LPO) technique. Intact RM were labeled on their outer face with 131I and, after opening of the vesicles with 0.05% DOC, in both faces with 125I. An analysis of iodination ratios for individual proteins separated electrophoretically showed that in most membrane glycoproteins, tyrosine residues are predominantly exposed on the luminal face of the vesicles, which is the same face on which the carbohydrate moieties are exposed. Several membrane glycoproteins are also exposed on the cytoplasmic surface and therefore have a transmembrane disposition. In this study, ribophorins I and II, two integral membrane proteins (mol wt 65,000 and 63,000) characteristic of RM, were found to be transmembrane glycoproteins. It is suggested that the transmembrane disposition of the ribophorins may be related to their possible role in ribosome binding and in the vectorial transfer of nascent polypeptides into the microsomal lumen.

Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol.

The glycoproteins of microsomes and cytosol were studied. Various washing procedures did not release the proteins from the microsomes, and immunological tests demonstrated that the sialoproteins are not serum components. Low concentrations of deoxycholate and incubation in 0.25 M sucrose solution liberated a small amount of microsomal sialoprotein and this fraction exhibited a high degree of labeling of protein-bound N-acetylneuraminic acid. A part of the glycoprotein fraction could not be solubilized, even with a high concentration of the detergent. Thoroughly perfused rat liver contained sialoproteins in the particle-free supernate. The level of sialoprotein present could not be due to contamination with serum or broken organelles. The high in vivo incorporation of [3H]glucosamine into protein-bound sialic acid of Golgi membranes and cytosol was paralleled by a delayed and lesser rate of incorporation into the rough and smooth microsomal membranes. This incorporation pattern suggests the possibility that the glycoproteins of cytosol and Golgi may later be incorporated into the membrane of the endoplasmic reticulum.

Lipoteichoic acid: a specific inhibitor of autolysin activity in Pneumococcus.

The choline-containing pneumococcal lipoteichoic acid (Forssman antigen) is a powerful inhibitor of the homologous autolytic enzyme, an N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28, MUCOPEPTIDE AMIDOHYDROLASE). Low concentrations of deoxycholate can reverse the inhibition. Wall teichoic acid preparations are inactive at several hundred-fold higher concentrations. Activation of an inactive form of autolysin by in vitro incubation with choline-containing cell walls is also inhibited by lipoteichoic acid. Addition of lipoteichoic acid to the growth medium of pneumococcal cultures causes chain formation, resistance to stationary phase lysis, and penicillin tolerance. It is suggested that a physiological role of lipoteichoic acids may be in the in vivo control of autolysin activity.

Membranes of mammary gland: X. Adenosine triphosphate dependent calcium accumulation by golgi apparatus rich fractions from bovine mammary gland

Golgi apparatus rich fractions from lactating bovine mammary gland had an Mg 2+-dependent, Ca 2+-stimulated adenosine triphosphatase. These Golgi apparatus fractions also accumulated Ca 2+ in vitro. Accumulation of Ca 2+ required ATP and could be abolished by treatment either with low concentrations of deoxycholate followed by ultrasound, or by heating at 100 °C for 10 min. The adenosine triphosphatase activity of Golgi apparatus was strongly stimulated by low concentrations of Ca 2+ and moderately stimulated by high concentrations of K +. This activity was unaffected by Na + and was not inhibited by ouabain. The pH optimum for the Mg 2+-dependent hydrolysis of ATP was 7.5, the K m was 5 × 10 −5 M and the activation energy was 6 000 calories/mole. This Mg 2+-dependent adenosine triphosphatase activity was also found in rough endoplasmic reticulum, smooth microsomes and milk fat globule membrane, the latter membrane being derived directly from the apical plasma membrane. All of these membrane fractions had the ability to specifically accumulate Ca 2+. Specific accumulation was highest with smooth microsomes and lowest with milk fat globule membrane with Golgi apparatus and rough endoplasmic reticulum being intermediate. These observations provide one plausible explanation for intracellular Ca 2+ accumulation and secretion into milk. Further, these results help explain the ultrastructural observations of casein micelle formation in secretory vesicles elaborated by Golgi apparatus.

The effect of delipidation on the adenosine triphosphatase of sarcoplasmic reticulum. Electron microscopy and physical properties.

Electron micrographs of negatively stained vesicles regenerated from the purified ATPase of sarcoplasmic reticulum show projections from the membrane which are similar to those previously observed on the intact parent membrane. They were also seen on sarcoplasmic vesicles from which all proteins other than the ATPase had been extracted with low concentrations of deoxycholate. These projections consist of a knob joined to the membrane by a narrow stalk, which together account for about for about half of the ATPase molecule. The overall length of the molecule is probably 10–12 nm. Inactivation of the enzyme in 1% deoxycholate followed by removal of the detergent gave non-vesicular aggregates which lacked projections and remained inactive. If lipid was removed from the protein before the detergent, optically clear solutions were obtained which contained much smaller particles, including single molecules of ATPase protein. These were approximately equidimensional (about 6.0 nm across) and could not be clearly related to the projections of the parent membrane. The cireular dichroism spectrum showed only a small decrease in content of α-helix relative to the active enzyme (35% to 28%), but no more than 5% of the original activity could be regenerated by recombination with phospholipids. The disappearance of the projections and the limited aggregation of the lipid-free molecule suggested an irreversible loss of hydrophobic surface following the replacement of lipid by deoxycholate. A structural model which would account for this behaviour is discussed.