Solubilization of thyroid peroxidase by nonionic detergents.

We have examined the ability of nonionic detergents to solubilize thyroid peroxidase from a porcine thyroid particulate fraction, as measured by the release of peroxidase activity into the supernatant fraction after centrifugation at 105,000 X g for 1 hour and the retardation of the supernatant peroxidase of Sepharose 6B. The parameters of peroxidase solubilization by Triton X-100 have been investigated in detail. Under optimum conditions, 60 to 95% of the thryoid peroxidase and about 50% of the total protein is released into the 105,000 X g, 1-hour supernatant. Under the optimum conditions established with Triton X-100, a series of Brij detergents of different chemical structure were equally effective in releasing peroxidase and protein. The protein patterns of the supernatants obtained with these detergents were similar on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, suggesting that the detergents studied release similar membrane proteins. The Triton X-100 and Brij 58 supernatants were chromatographed separately on Sepharose 6B equilibrated with 0.1% Triton X-100 or Brij 58, respectively. In both cases, 75 to 80% of the peroxidase activity was retarded, thereby indicating that the nonionic detergents effect solubilization of the peroxidase rather than dispersal of nonsedimentable membrane fragments. These studies report the first successful solubilization of thyroid peroxidase by nonionic detergents. Together with previous evidence from our laboratory, these experiments indicate that thyroid peroxidase is an integral membrane protein.     

Binding, activation, and solubilization of the Ca2+-ATPase from sarcoplasmic reticulum by nonionic detergents

Interactions between delipidated Ca2+-ATPase from sarcoplasmic reticulum and four nonionic detergents--dodecyl octaoxyethyleneglycol monoether (C12E8), Triton X-100, Brij 58, and Brij 35--were characterized with respect to activation of ATPase activity, binding, and solubilization. C12E8 and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C12E8. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C12E8 and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C12E8 are more useful when bulk solubilization is the goal.     

Plasma membrane association and preliminary characterization of Drosophila sperm surface glycosidases

Previous studies have identified β‐N‐acetylglucosaminidase (GlcNAc'ase) and α‐mannosidase activities on the Drosophila melanogaster sperm surface which may have a role in fertilization. The aim of this study was to investigate their linkage to the sperm plasma membrane. We verified that glycosidases are not peripherally adsorbed to the cell surface by evaluating their resistance to release by KI, by buffered salt solutions of high ionic strength or alkaline buffers. Glycosidases were released from the sperm surface by detergents and, only to a minor extent, by mild proteolysis. Differential detergent solubilization pointed out that Triton X‐114 was the most effective releasing agent for GlcNAc'ase and CHAPS for mannosidase. No activity was released from the membrane by a phosphatidylinositol‐specific phospholipase C (PI‐PLC). The released forms were quite hydrophilic in phase separation experiments with Triton X‐114. This finding indicates the presence of a hydrophobic domain limited to a single transmembrane helix or/and the presence of an extensive glycosilation. The use of a Con‐A binding assay demonstrated that both the enzymes are glycosilated. The molecular weight of the released glycosidases estimated by gel filtration was 158 kDa for GlcNAc'ase and 317 kDa for mannosidase. These results suggest that Drosophila melanogaster GlcNAc'ase and mannosidase are mannosylated integral membrane proteins that would function as exoenzymes with their active sites accessible in the extracellular space. Mol. Reprod. Dev. 52:166–173, 1999. © 1999 Wiley‐Liss, Inc.     

Solubilization and hydrophobicity test by Triton X-114-partitioning of NADPH-protochlorophyllide oxidoreductase from the unicellular alga Scenedesmus obliquus, mutant C-2A'

NADPH-protochlorophyllide oxidoreductase (PChilde reductase, EC 1.3.1.33), a key enzyme in light-dependent greening and the conversion of etioplasts into chloroplasts was investigated in the the greening mutant C-2A' of the unicellular green alga Scenedesmus obliquus. In the absence of detergent, the solubilization of the enzyme increased with high glycerol concentrations in the buffer. Solubilization capacities of 4 non-ionic or zwitterionic detergents, Triton X-100, CHAPS, octylglucoside and decyl-maltopyranoside, were compared. Due to the addition of these detergents, the enzyme activity in the soluble fraction was increased severalfold. Hydrophobicity of the enzyme was analyzed by Triton X-114 phase partitioning. The protein had a preference for the aqueous phase, but its distribution was strongly influenced by the glycerol concentration of the buffer. These results indicate that the PChlide reductase of the green alga Scenedesmus obliquus is a hydrophobic, membrane-associated enzyme, but not an integral membrane protein.     

Binding,Activation, and Solubilization of the Ca2+-ATPase from Sarcoplasmic Reticulum by Nonionic Detergents

Interactions between delipidated Ca²⁺-ATPase from sarcoplasmic reticulum and four nonionic detergents—dodecyl octaoxyethyleneglycol monoether (C₁₂E₈), Triton X-100, Brij 58, and Brij 35—were characterized with respect to activation of ATPase activity, binding, and solubilization. C₁₂E₈ and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C₁₂E₈. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C₁₂E₈ and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C₁₂E₈ are more useful when bulk solubilization is the goal.     

Ease of solubilization of five marker enzymes in three preparations of rat renal brush border membranes

The ability of eight stripping agents to solubilize five marker enzymes from rat renal brush border membranes isolated by three different preparative methods was examined. Protein and enzyme activities - alkaline phosphatase (APase), L-leucine aminopeptidase (LAPase), gamma-glutamyl transpeptidase (GGTase), gamma-glutamyl hydrolase (GGHase) and maltase - solubilized by the treatments were expressed as percent of total activity recovered in excess of control values. The relative enzyme activity and the solubilization factor were determined for each marker enzyme in every treated sample and the treatments with the eight agents compared. Trypsin treatment released > 80% of LAPase and < 10% of total membrane protein. Papain treatment released only 16--23% of total membrane protein but most of the enzyme activities except APase. Neuraminidase had no solubilizing effect. 4--10% of total membrane protein was solubilized by LiCl treatment but no marker enzyme activities were released. Less total membrane protein was released by treatment with proteolytic enzymes or LiCl than with the detergents Triton X-100, hexadecyltrimethylammonium bromide, sodium deoxycholate, and sodium dodecylsulfate. APase activity was the least readily solubilized. Correlating the degree of solubilization for five marker enzymes with the types of stripping agents used and with the appearance of the membrane surface when examined by electron microscopy led to the suggestion that LAPase, GGTase, GGHase and maltase molecules are part of an interwoven surface layer of membrane proteins which can be disrupted by transamidation and transesterification reactions. APase appears to be more strongly associated with the intact lipid matrix than the bulk of the membrane protein.     

Solubilization and partial purification of the adenosine triphosphatase from a corn root plasma membrane fraction

The K(+)-stimulated ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 x Mo 17) by solubilization with 30 millimolar octyl-beta-d-glucopyranoside followed by precipitation with dilute ammonium sulfate. The specific activity of the enzyme was increased about five times by this procedure. The molecular weight of the detergent-extracted ATPase complex was estimated to be at least 500,000 daltons by chromatography on a Bio-Gel A-5m column. Negative staining electron microscopy indicated that the detergent-extracted material consisted of amorphous particles, while the ammonium sulfate precipitate was composed of uniform vesicles with an average diameter of 100 nanometers. The protein composition of the ammonium sulfate precipitate was significantly different from that of the plasma membrane fraction when compared by sodium dodecyl sulfate gel electrophoresis. The characteristics of the partially purified ATPase resembled those of the plasma membrane associated enzyme. The ATPase required Mg(2+), was further stimulated by K(+), was almost completely inhibited by 0.1 millimolar diethylstilbestrol, and was not affected by 5.0 micrograms per milliliter oligomycin. Although the detergents sodium cholate, deoxycholate, Triton X-100 and Lubrol WX also solubilized some membrane protein, none solubilized the K(+)-stimulated ATPase activity. Low concentrations of each detergent, including octyl-beta-d-glucopyranoside, activated the ATPase and higher concentrations inactivated the enzyme. These results suggest that the plasma membrane ATPase is a large, integral membrane protein or protein complex that requires lipids to maintain its activity.     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

Action of surface-active agents on arylsulfatase-C of human cultured fibroblasts

Arylsulfatase-C is a microsomal membrane-bound enzyme with unusual biochemical and genetic properties. Whether it is a single enzyme hydrolyzing different sterol sulfates or a complex of enzymes, with each enzyme hydrolyzing a specific substrate, has not been resolved. Its locus has been mapped to the human X chromosome but appears to escape inactivation. As a first step to clarify its biochemical properties, a systematic search was undertaken for a suitable detergent that can release this enzyme from human cultured fibroblast membranes in a form that is biologically active and electrophoretically mobile. Four non ionic (Triton X-100, Nonidet P-40, Digitonin, and saponin) and four amphoteric (lysolecithin, Zwittergent, Miranol, and Chaps) detergents were studied. At 1% concentration, they released more than 80% of the activity into a low-speed supernatant fraction, except for Saponin which had no effect. With Triton X-100 and Miranol representing the two groups of detergents, significant release occurred only when the detergent concentrations exceeded their respective critical micelle concentrations, thus indicating that arylsulfatase-C is an integral membrane protein. The apparent molecular weight of the detergent-enzyme complex, ascertained by gel filtration, was 85,000 in the presence of Triton X-100 and 335,000 in the presence of Miranol. However, only the preparation solubilized by Miranol (and Chaps, to a lesser degree) permitted migration of the enzyme in nitrocellulose acetate during electrophoresis at pH 7.0, while the enzyme extracted with all other detergents remained at the origin. Therefore, the amphoteric detergent, Miranol, appears to fulfill the requirements for further characterization of the membrane-bound arylsulfatase-C in human cultured fibroblasts.     

Novel procedure for extraction of a latent grape polyphenoloxidase using temperature-induced phase separation in triton x-114

Polyphenoloxidase from grape berries is extracted only by nonionic detergents with a hydrophilic-lipophilic balance between 12.4 and 13.5. The enzyme was partially purified in latent form, free of phenolics and chlorophylls, by using temperature phase partitioning in a solution of Triton X-114. This method permits the purification of the enzyme with the same fold purification as the commonly used method, but with a yield three times higher and a 90% reduction in time needed. The latent enzyme can be activated by different treatments, including trypsin and cationic and anionic detergents. Cetyltrimethylamonium bromide was found to be the most effective detergent activator, followed by sodium dodecyl sulfate. Polyphenoloxidase in grape berries, in spite of being an integral membrane protein, had an anomalous interaction with Triton X-114, remaining in the detergent-poor phase after phase separation. This could be explained by its having a short hydrophobic tail that anchors it to the membrane.     

Purification of 2,3-oxidosqualene cyclase from rat liver.

A rapid and simple purification of milligram amounts of 2,3-oxidosqualene cyclase, an integral membrane enzyme that catalyzes the cyclization of squalene epoxide to lanosterol, is reported. Several nonionic detergents (Triton X-100, Tween 80, Emulphogene, and lauryl maltoside) were evaluated for solubilization of oxidosqualene cyclase from rat liver microsomes. At a detergent concentration of 5 mg/ml, lauryl maltoside was approximately 10 times more effective than Emulphogene in the solubilization of oxidosqualene cyclase; Triton X-100 and Tween 80 were less effective than Emulphogene as judged by the relative specific activities of the solubilized enzyme. Treatment of microsomes with lauryl maltoside resulted in a selective solubilization of the cyclase with concomitant activation of the enzyme. The solubilized enzyme was purified to homogeneity by fast protein liquid chromatography. The purified enzyme consists of a single subunit that has an apparent molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme obeys saturation kinetics and the apparent Km of (2,3)-oxidosqualene is 15 microM; the apparent kcat/Km is 200 M-1.min-1. An improved assay of the enzyme that utilizes high performance liquid chromatography methods is also described.     

Differential effects of detergents upon the soluble and particulate guanylate cyclases from rat lung.

The enzyme guanylate cyclase is present in both particulate and soluble form in rat lung homogenates. As previously reported, the soluble enzyme can be activated by preincubation in the presence of O₂. The inactive (nonactivated) soluble enzyme is also stimulated by nonionic detergents, in the order Tween 20 > Lubrol PX > Triton X-67 > Triton X-100. The activated enzyme, however, was inhibited by these detergents in the reverse order. Sodium deoxycholate and lysolecithin were potent inhibitors of both inactive and activated enzyme. The activity of the particulate enzyme was stimulated by Lubrol PX > Triton X-100 > Triton X-67 > Tween 20. At a low concentration of lysolecithin or deoxycholate the particulate activity was increased; however, when detergent/protein > 1, inhibition was seen. In the case of deoxycholate, the inhibition could be reversed if excess deoxycholate was removed either by chromatography or by forming mixed micelles with Lubrol PX; however, deoxycholate inhibition of the soluble enzyme was irreversible. The stimulation by detergents of the particulate enzyme was apparently the result of solubilization. The effects upon the activity of the soluble enzyme were interpreted in terms of a model which assumes two hydrophobic regions on the enzyme surface. The two regions differ in hydrophobicity with the more hydrophobic region only being exposed as a result of activation. Interaction of a nonionic detergent with the less hydrophobic region stimulates activity, while interaction with the more hydrophobic region results in inhibition.     

Aminoacylase I is not a glycolipid-anchored ectoenzyme in pig kidney.

Subcellular fractionation of pig kidney cortex revealed that aminoacylase I (EC 3.5.1.14, N-acyl-L-amino-acid aminohydrolase) is predominantly a soluble enzyme with only 0.5% of the total activity being recovered in the membrane fraction. The aminoacylase I activity associated with the membrane preparations displayed neither rapid release following incubation with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis nor the distinctive differential pattern of detergent solubilization which was seen with glycosyl-phosphatidylinositol-anchored proteins (renal dipeptidase, alkaline phosphatase). When fractionated by phase separation in Triton X-114, integral membrane proteins of kidney microvillar membranes partitioned predominantly (greater than 90%) into the detergent-rich phase. In contrast, only 3.7% of aminoacylase I activity associated with microvillar membranes partitioned into the detergent-rich phase. Aminoacylase I activity of pig kidney would therefore appear to be a hydrophilic protein in nature and is not, as suggested previously, a G-PI-anchored integral membrane protein.     

Kinetic properties and solubilization of microsomal cholesterol ester hydrolase from rat liver

Some kinetic properties of the microsomal cholesterol ester hydrolase (CEH) have been examined in rat liver. The reaction was linear with time up to 60 min and with enzyme concentration up to 0.3 mg/mL, and a pH optimum of 6.7 for enzyme activity was observed. Cholesterol esterase exhibited the following apparent kinetic constants: Km, 68.88 microM and Vmax, 33 Units/mg protein. Cholesteryl palmitate was hydrolyzed to a much greater extent than cholesteryl oleate by the enzyme. Product inhibition with cholesterol and palmitic acid was not apparent; however, oleic acid added to the system reduced markedly microsomal CEH activity. The present paper also reports the solubilization of cholesteryl palmitate hydrolase from the microsomal fraction by pretreating it with Triton X-100, sodium deoxycholate, and sodium dodecylsulfate. All ionic and non-ionic detergents tested are capable of making the enzyme soluble, and maximal effects were found at higher concentrations of detergents although the esterase activity was strongly inhibited. Triton X-100 was found to be more effective than sodium deoxycholate and sodium dodecylsulfate in enzyme and protein solubilization. When the direct effects of detergents on CEH activity were studied, progressive concentration-dependent inhibitions were observed.     

Plasma membrane association, purification, and partial characterization of mouse sperm beta 1,4-galactosyltransferase

Gamete recognition in the mouse is mediated, in part, by the binding of sperm surface galactosyltransferase (GalTase) to appropriate substrates in the egg zona pellucida. In this paper, sperm GalTase is shown to be an externally oriented, integral plasma membrane component. GalTase is not peripherally adsorbed to the cell surface, nor is it bound to cell surface glycoside substrates. GalTase can be released from the surface of intact sperm by either mild proteolysis or by detergent under conditions in which the sperm membranes remain intact as judged by double-label indirect immunofluorescence. Detergent-solubilized sperm GalTase has been purified to apparent homogeneity by affinity chromatography and characterized as a beta 1,4-GlcNAc:GalTase by substrate and kinetic analyses. Purified and membrane-bound GalTase both show an unusual thermal inactivation above 39-40 degrees C, whereas other sperm enzyme activities as well as GalTase activity from other cell types are temperature-dependent. Purified sperm GalTase inhibits sperm binding to the egg zona pellucida, consistent with its proposed role during gamete recognition.     

Effect of the hydrophile-lipophile balance of non-ionic detergents (Triton X-series) on the solubilization of biological membranes and their integral b-type cytochromes.

The solubilization of four integral membrane proteins (i.e. cytochrome b-561 of the chromaffin granule membrane, cytochrome b5 of the endoplasmic reticulum and the mitochondrial b-type cytochrome(s) as well as cytochrome c oxidase) has been studied at 0 degrees C using the non-ionic detergents of the Triton X-series having the common hydrophobic 4(1,1,3,3-tetramethylbutyl)phenoxy (t-octyl-phenoxy) group and a variable average number (n) of polar ethylene oxide units added. Following a pre-extraction of peripheral membrane and matrix proteins with low and high salt concentration and a weak non-ionic detergent (Tween 20, average hydrophile-lipophile balance (HLB) = 16.7), the amount of heme proteins solubilized by subsequent Triton X-solutions was measured. With the detergents tested the degree of solubilization decreased in the sequence cytochrome b-561 greater than cytochrome b5 greater than mitochondrial cytochrome(s) b and parallelled the effect of the detergents on light scattering and the phospholipid to protein ratio of the three membranes. For all the b-cytochromes, the solubilizing power of the detergent increased with decreasing average length of the polar ethylene oxide chain and the hydrophile-lipophile balance as long as clouding did not occur (e.g. Triton X-114,n = 7.5 and HLB = 12.4). Thus, the greatest difference in the degree os solubilization of the three cytochromes was observed with Triton X-405 (n = 40 and HLB = 17.9). All the cytochromes were most efficiently solubilized (i.e. approx. 90%) by Triton X-100 (n = 9.5 and HLB = 13.5).     

The major protein of pancreatic zymogen granule membranes (GP-2) is anchored via covalent bonds to phosphatidylinositol

GP-2, the major integral protein characteristic of the pancreatic zymogen granule membrane can be released from the membrane by the action of a phosphatidylinositol specific phospholipase C (PI-PLC). In a hydrophobic/hydrophilic phase separation system using the non-ionic detergent Triton X-114, the membrane-bound form of the protein went from the detergent phase into the hydrophilic phase upon action of the phospholipase. PI-PLC solubilization of GP-2 unmasked an antigenic determinant similar to the cross-reacting determinant of the trypanosome variant surface glycoproteins. This determinant being a distinctive feature of the glycan moiety of phosphatidyl-inositol anchored membrane proteins, it established the glycosyl-phosphatidyl-inositol nature of the GP-2 membrane anchor. Since soluble GP-2 is also found in the contents of the granule and is secreted intact into the pancreatic juice, it is likely that one of the mechanisms responsible for its release could be a specific phospholipase. GP-2 is the first glycosyl-phosphatidyl-inositol-anchored protein that is integral to the membrane of an organelle and not located at the surface of the cell.     

The Sperm‐surface glycoprotein,SGP, is necessary for fertilization in the frog,Xenopus laevis

To identify a molecule involved in sperm‐egg plasma membrane binding at fertilization, a monoclonal antibody against a sperm‐surface glycoprotein (SGP) was obtained by immunizing mice with a sperm membrane fraction of the frog, Xenopus laevis, followed by screening of the culture supernatants based on their inhibitory activity against fertilization. The fertilization of both jellied and denuded eggs was effectively inhibited by pretreatment of sperm with intact anti‐SGP antibody as well as its Fab fragment, indicating that the antibody recognizes a molecule on the sperm's surface that is necessary for fertilization. On Western blots, the anti‐SGP antibody recognized large molecules, with molecular masses of 65–150 kDa and minor smaller molecules with masses of 20–28 kDa in the sperm membrane vesicles. SGP was distributed over nearly the entire surface of the sperm, probably as an integral membrane protein in close association with microfilaments. More membrane vesicles containing SGP bound to the surface were found in the animal hemisphere compared with the vegetal hemisphere in unfertilized eggs, but the vesicle‐binding was not observed in fertilized eggs. These results indicate that SGP mediates sperm‐egg membrane binding and is responsible for the establishment of fertilization in Xenopus.     

The microvillus 110K cytoskeletal protein is an integral membrane protein

A protein of 110,000 MW connects actin filaments to the plasma membrane in microvilli of intestinal epithelial cells. In the present study four independent lines of evidence suggest that the 110K protein is directly bound to the lipid bilayer. The solubilization of the 110K protein requires detergents and removal of detergent after solubilization results in aggregation. The 110K protein partitions into the detergent phase in Triton X-114 solutions. It is selectively incorporated into liposomes. It is specifically labeled with the hydrophobic probe ¹⁴C-phenylisothiocyanate. In addition we present a purification scheme for the 110K protein in milligram amounts. This represents the simplest system of membrane to filament attachment, in which an integral membrane protein is also a cytoskeletal protein.     

Characterization and partial purification of squalene-2,3-oxide cyclase from Saccharomyces cerevisiae.

The membrane nature of squalene oxide cyclase from Saccharomyces cerevisiae was investigated by comparing properties of the enzyme recovered from both microsomes and the soluble fraction of the yeast homogenate. The "apparent soluble" form and microsomal form of the enzyme were both stimulated by the presence of mammalian soluble cytoplasm and corresponded to one another in response to detergents Triton X-100 and Triton X-114. The observed strong dependence of the enzyme activity on the presence of detergents and the behavior of the enzyme after Triton X-114 phase separation were peculiar to a lipophilic membrane-bound enzyme. A study of the conditions required to extract the enzyme from microsomes confirmed the lipophilic character of the enzyme. Microsomes, exposed to ipotonic conditions to remove peripheral membrane proteins, retained most of the enzyme activity within the integral protein fraction. Quantitative dissociation of the enzyme from membranes occurred only if microsomes were treated with detergents (Triton X-100 or octylglucoside) at concentrations which alter membrane integrity. The squalene oxide cyclase was purified 140 times from yeast microsomes by (a) removal of peripheral proteins, (b) extraction of the enzyme from the integral protein fraction with octylglucoside, and (c) separation of the solubilized proteins by DEAE Bio-Gel A chromatography. Removal of the peripheral proteins seemed to be a key step necessary for obtaining high yields.