Role of GPI-anchored Enzyme in Liposome Detergent-Resistance

In this work, we investigated the role of a glycosylphosphatidylinositol (GPI)-anchored protein, the alkaline phosphatase, on the solubilization of detergent-resistant liposomes. In vivo, GPI-anchored proteins are clustered into sphingolipid- and cholesterol-rich membrane domains and this peculiar composition provides cold-detergent-insolubility. To better understand the mechanisms involved in the clustering of these subdomain components, we built a model, namely sphingolipid- and cholesterol-rich liposomes. We show the cold-Triton X-100 resistance of liposomes before and after insertion of GPI-anchored enzyme. When the amount of incorporated enzyme varied, significant changes in membrane stability occurred. Low protein contents into liposomes increased detergent insolubility, whereas high amounts decreased it. Furthermore, significant differences in the detergent-resistance of each lipid were exhibited between liposomes and proteoliposomes. Thus, the enzyme insertion led to a dramatic decrease of cholesterol solubilization, in line with the existence of cholesterol/GPI interactions. Effect of temperature on detergent resistance was also investigated. Liposome solubilization increased with temperature up to a threshold value of 40/45 degrees C. This was also the temperature at which a phase transition of liposome membrane occurred, as evidenced by Laurdan fluorescence. Although the GPI-anchored enzyme insertion modified membrane stability, no change was observed on phase transition. Our work highlights the importance of GPI-anchored proteins in the structure of sphingolipid- and cholesterol-rich membrane domains, in the detergent-insolubility of these peculiar domains, as well as in interaction of GPI proteins with cholesterol.     

Behavior of a GPI-anchored protein in phospholipid monolayers at the air-water interface

The interaction between alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein (AP-GPI), and phospholipids was monitored using Langmuir isotherms and PM-IRRAS spectroscopy. AP-GPI was injected under C16 phospholipid monolayers with either a neutral polar head (1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC)) or an anionic polar head (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS)). The increase in molecular area due to the injection of protein depended on the surface pressure and the type of phospholipid. At all surface pressures, it was highest in the case of DPPS monolayers. The surface elasticity coefficient E, determined from the pi-A diagrams, allowed to deduct that the AP-GPI-phospholipid mixtures presented a molecular arrangement less condensed than the corresponding pure phospholipid films. PM-IRRAS spectra suggested different protein-lipid interactions as a function of the nature of the lipids. AP-GPI modified the organization of the DPPS deuterated chains whereas AP-GPI affected only the polar group of DPPC at low surface pressure (8 mN/m). Different protein hydration layers between the DPPC and DPPS monolayers were suggested to explain these results. PM-IRRAS spectra of AP-GPI in the presence of lipids showed a shape similar to those collected for pure AP-GPI, indicating a similar orientation of AP-GPI in the presence or absence of phospholipids, where the active sites of the enzyme are turned outside of the membrane.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Spontaneous insertion and partitioning of alkaline phosphatase into model lipid rafts

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the C-terminal end that serves as an anchor to the plasma membrane and could be responsible for the presence of GPI proteins in rafts, a type of functionally important membrane microdomain enriched in sphingolipids and cholesterol. In order to understand better how GPI proteins partition into rafts, the insertion of the GPI-anchored alkaline phosphatase (AP) was studied in real-time using atomic force microscopy. Supported phospholipid bilayers made of a mixture of sphingomyelin–dioleoylphosphatidylcholine containing cholesterol (Chl+) or not (Chl–) were used to mimic the fluid-ordered lipid phase separation in biological membranes. Spontaneous insertion of AP through its GPI anchor was observed inside both Chl+ and Chl– lipid ordered domains, but AP insertion was markedly increased by the presence of cholesterol.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper, we standardize a method for construction an alkaline phosphatase liposome system to mimic matrix vesicles and examine a some kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into liposomes constituted from dimyristoylphosphatidylcholine (DMPC), dilaurilphosphatidylcholine (DLPC) or dipalmitoylphosphatidylcholine (DPPC). This process was time-dependent and >95% of the enzyme was incorporated into the liposome after 4h of incubation at 25 degrees C. Although, incorporation was more rapid when vesicles constituted from DPPC were used, the incorporation was more efficient using vesicles constituted from DMPC. The 395nm diameter of the alkaline phosphatase-liposome system was relatively homogeneous and more stable when stored at 4 degrees C.Alkaline phosphatase was completely released from liposome system only using purified phosphatidylinositol-specific phospholipase C (PIPLC). These experiments confirm that the interaction between alkaline phosphatase and lipid bilayer of liposome is via GPI anchor of the enzyme, alone. An important point shown is that an enzyme bound to liposome does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but a liposome environment affects its kinetic properties, specifically for pyrophosphate.The standardization of such system allows the study of the effect of phospholipids and the enzyme in in vitro and in vivo mineralization, since it reproduces many essential features of the matrix vesicle.     

Plasma membrane homing of tissue nonspecific alkaline phosphatase under the influence of 3-hydrogenkwadaphnin, an antiproliferative agent from Dendrostellera lessertii

Several mammalian enzymes are anchored to the outer surface of the plasma membrane by a covalently attached glycosylphosphatidylinositol (GPI) structure. These include acetylcholinesterase, alkaline phosphatase (AP) and 5'-nucleotidase among other enzymes. Recently, it has been reported that these membrane enzymes can be released into the serum by the GPI-dependent phospholipase D under various medical disturbances such as cancer and/or by chemical and physical manipulation of the biological systems. Treatment of MCF-7 cells with two consecutive effective concentrations of 3-hydrogenkwadaphnin (3-HK, 3 nM) for 48 h enhanced membrane AP activity by almost 330% along with a 40% reduction in the AP activity of the cell culture medium. In addition, our data indicate that 3-HK is capable of inducing mainly the tissue-nonspecific alkaline phosphatase (TNAP) isoenzyme, along with enhancing its thermostability. These findings, besides establishing a correlation between the antiproliferative activity of 3-HK and the extent of plasma membrane AP activity, might assist in the development of new diagnostic tools for following cancer medical treatments.     

The Release and Detection of Endotoxin from Liposomes

Incorporation of lipopolysaccharide (LPS) into liposomes dramatically reduces its ability to coagulateLimulusamebocyte lysate (LAL). The coagulation of LAL is commonly used to signal the presence of endotoxinin vitro.This study demonstrates a simple method to release masked endotoxin from liposomal dispersions using moderate amounts of detergent to form mixed micelles containing lipid, detergent, and LPS. Several parameters were found to affect the degree of liposome solubilization and/or the sensitivity of the LAL assay. These included detergent type and concentration, temperature for solubilization, lipid composition, liposome morphology, and time for test incubation. The nonionic detergent polyoxyethylene 10 lauryl ether (C₁₂E₁₀) proved to be unique in its ability to solubilize liposomes and minimally interfere with endotoxin detection. The LAL endotoxin detection limit for samples dispersed in C₁₂E₁₀varied with the phospholipid component; the sensitivity decreased in the order DSPC > DPPC = EPC DMPC. Cholesterol lowered the solubility limit of the liposomes, but did not appear to affect the LAL assay sensitivity once the liposomes were completely solubilized. The presence of negatively charged phospholipids, DSPG and Pops, also lowered the solubility limit. Pops, but not DSPG, at 10 mol% further decreased the LAL endotoxin detection limit. This detergent-solubilization method should be useful in liposomal LPS immunological studies or in other situations where accurate determination of endotoxin concentration is important.     

Reconstitution of 5′-nucleotidase of bull seminal plasma in spin-labeled liposomes

Seminal plasma separated from freshly ejaculated bull semen contains vesicles with a 5-nucleotidase activity incorporated as an ectoenzyme anchored by glycosyl phosphatidylinositol (GPI). After its extraction from bull seminal plasma vesicles, the protein was purified and reconstituted into hen egg yolk lecithin liposomes obtained through prolonged dialysis of buffered n-octylglucoside detergent solutions of lipid, protein and various effectors against detergent-free solutions. Gel filtration experiments showed that the enzyme incorporated into liposomes in a dimeric form with its two subunits linked by disulfide bridges. In the presence of reduced glutathione, the protein dissociated into monomers and failed to incorporate into liposomes. Electron spin resonance (ESR) experiments, performed with liposomes containing electron spin labels localized at the hydrophilic lipid headgroups (5-doxyl stearic acid) or in the hydrophobic lipid hydrocarbon chains (16-doxyl stearic acid), demonstrated that the incorporation of 5-nucleotidase resulted in the immobilization of the spin probes. Furthermore, the spectral parameters obtained before and after treatment of 5-nucleotidase-containing liposomes with phosphatidylinositol-specific phospholipase C (PI-PLC) indicated that the liposome membrane bilayer did not contain protein segments. This supports the well-known ecto-localization of 5-nucleotidase and rules out a previously reported possibility of a proteic transmembrane anchoring of the enzyme.We thank Mr. Marcello Coli and Dr. Maria Grazia Cantelmi, University of Perugia, for their skillful technical assistance; Dr. Paolo Ghinassi and Dr. Franca Farabegoli, Semen Italy, Diegaro, Cesena, Italy and Dr. Augusto Chiacchierini, Centro Tori Perugia, Italy, for kindly supplying the bull semen; Dr. Maria Grazia Rambotti, Department of Experimental Medicine, University of Perugia, Italy, for her skillful assistance in the electron microscopy experiments; Ms. Darlene I. Morosi for her helpful suggestions.     

ACTH stimulates the release of alkaline phosphatase through Gi-mediated activation of a phospholipase C and the release of inositolphosphoglycan

We have previously reported that ACTH activates a phospholipase C that hydrolyzes glycosylphosphatidylinositol (GPI), which would release inositolphosphoglycan (IPG) to the extracellular medium, and that an IPG purified from Trypanosoma cruzi is able to inhibit ACTH-mediated steroid production in adrenocortical cells. In the present paper, it was found that anti-inositolphosphoglycan antibodies (anti-CRD) increased ACTH-mediated corticosterone production, which indicates that an endogenous IPG is a physiological inhibitor of ACTH response. On the other hand, we investigated the release to the extracellular medium of the GPI-anchored enzyme, alkaline phosphatase, by ACTH. We found that: (a) the released enzyme appeared in the aqueous phase after Triton X-114 partitioning, consistent with loss of the GPI, (b) the phospholipase C inhibitor, U73122, impaired the release of the enzyme by the hormone and (c) two inhibitors of IPG uptake, inositol 2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium. These results suggest that ACTH releases alkaline phosphatase by activation of a phospholipase C. Dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) was able to increase the release of alkaline phosphatase from adrenocortical cells and this effect was inhibited by U73122, suggesting that cAMP is involved in the activation of phospholipase C. In addition, it was found that a pertussis-toxin sensitive G-protein is required for ACTH- and db-cAMP-mediated release of alkaline phosphatase and that incorporation of anti-Gi antibodies in adrenocortical cells inhibited the release of alkaline phosphatase by ACTH. Our results suggest that ACTH increases the release of alkaline phosphatase by activation of a phospholipase C through cAMP and Gi which would contribute to produce IPG It was also found that the two inhibitors of IPG uptake, inositol-2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium of ACTH-treated cells more than in control cells, indicating that ACTH also stimulates the uptake of IPG These data support a role of GPI and the involvement of Gi in ACTH action.     

A simple laboratory experiment to demonstrate the interaction of proteins bearing glycosylphosphatidylinositol anchors with liposomes

We have designed a simple laboratory experiment using different forms of alkaline phosphatase (detergent-solubilized, phospholipase-released and protease-released) and liposomes to demonstrate the relevance of the glycosyl-phosphatidyl inositol tail to anchor proteins in membranes. The results show that only the solubilized alkaline phosphatase form binds to liposome membranes since its anchor structure was preserved after detergent treatment.     

Stability of pressure-extruded liposomes made from archaeobacterial ether lipids

Ether lipids were obtained from a wide range of archaeobacteria grown at extremes of pH, temperature, and salt concentration. With the exception ofSulfolobus acidocaldarius, unilamellar and/or multilamellar liposomes could be prepared from emulsions of total polar lipid extracts by pressure extrusion through filters of various pore sizes. Dynamic light scattering, and electron microscopy revealed homogeneous liposome populations with sizes varying from 40 to 230 nm, depending on both the lipid source and the pore size of the filters. Leakage rates of entrapped fluorescent or radioactive compounds established that those archaeobacterial liposomes that contained tetraether lipids were the most stable to high temperatures, alkaline pH, and serum proteins. Most ether liposomes were stable to phospholipase A2, phospholipase B and pancreatic lipase. These properties of archaeobacterial liposomes make them attractive for applications in biotechnology.     

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

Cell‐free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis‐based Escherichia coli cell‐free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light‐driven proton pump bacteriorhodopsin, consisting of seven transmembrane α‐helices. The cell‐free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3–0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent‐lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.     

Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 x slower in liposomes and 100 x slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.     

Determination and physiological roles of the glycosylphosphatidylinositol lipid remodelling pathway in yeast

In the yeast Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)‐anchored proteins play important roles in cell wall biogenesis/assembly and the formation of lipid microdomains. The lipid moieties of mature GPI‐anchored proteins in yeast typically contain either ceramide moieties or diacylglycerol. Recent studies have identified that the GPI phospholipase A₂ Per1p and O‐acyltransferase Gup1p play essential roles in diacylglycerol‐type lipid remodelling of GPI‐anchored proteins, while Cwh43p is involved in the remodelling of lipid moieties to ceramide. It has been generally proposed that phosphatidylinositol with diacylglycerol containing a C26 saturated fatty acid, which is generated by the sequential activity of Per1p and Gup1p, is converted to inositolphosphorylceramide by Cwh43p. In this report, we constructed double‐mutant strains defective in lipid remodelling and investigated their growth phenotypes and the lipid moieties of GPI‐anchored proteins. Based on our analyses of single‐ and double‐mutants of proteins involved in lipid remodelling, we demonstrate that an alternative pathway, in which lyso‐phosphatidylinositol generated by Per1p is used as a substrate for Cwh43p, is involved in the remodelling of GPI lipid moieties to ceramide when the normal sequential pathway is inhibited. In addition, mass spectrometric analysis of lipid species of Flag‐tagged Gas1p revealed that Gas1p contains ceramide moieties in its GPI anchor.     

Early events in glycosylphosphatidylinositol anchor addition. substrate proteins associate with the transamidase subunit gpi8p

The addition of glycosylphosphatidylinositol (GPI) anchors to proteins occurs by a transamidase-catalyzed reaction mechanism soon after completion of polypeptide synthesis and translocation. We show that placental alkaline phosphatase becomes efficiently GPI-anchored when translated in the presence of semipermeabilized K562 cells but is not GPI-anchored in cell lines defective in the transamidase subunit hGpi8p. By studying the synthesis of placental alkaline phosphatase, we demonstrate that folding of the protein is not influenced by the addition of a GPI anchor and conversely that GPI anchor addition does not require protein folding. These results demonstrate that folding of the ectodomain and GPI addition are two distinct processes and can be mutually exclusive. When GPI addition is prevented, either by synthesis of the protein in the presence of cell lines defective in GPI addition or by mutation of the GPI carboxyl-terminal signal sequence cleavage site, the substrate forms a prolonged association with the transamidase subunit hGpi8p. The ability of the transamidase to recognize and associate with GPI anchor signal sequences provides an explanation for the retention of GPI-anchored protein within the ER in the absence of GPI anchor addition.     

The glycophosphatidylinositol anchor oppositely affects unfolding and refolding of alkaline phosphatase

Regarding the world wide success of artificial chaperone-assisted protein refolding technique and based on its well worked-out mechanism, it is anticipated that the lipid moieties of glycosylphosphatidylinositol (GPI) group, which is present in some membrane proteins, might interfere with the capturing step of the technique. To find an answer, we evaluated the chemical denaturation and also the refolding behavior of insoluble and soluble alkaline phosohatase (ALP), with or without GPI group, respectively. The results indicated that the presence of GPI in the enzyme increased the stability of the protein against chemical denaturation while it decreased its refolding yield by the artificial chaperone refolding technique. The lower refolding yield, compared to soluble ALP (sALP), might be due to a less efficient stripping step caused by new interactions imparted to the refolding elements of the system especially those among the hydrophobic tails of GPI and the capturing agent of the technique. These new interactions will interrupt the kinetics of detergent stripping from the captured molecules by the stripping agent (i.e., cyclodextrins). This situation will lead to higher intermolecular hydrophobic interactions among the refolding protein intermediates leading to their higher misfolding and aggregation.     

Subcellular distribution and cytokine- and chemokine-regulated secretion of leukolysin/MT6-MMP/MMP-25 in neutrophils

Leukolysin, originally isolated from human leukocytes, is the sixth member of the membrane-type matrix metalloproteinase (MT-MMP) subfamily with a potential glycosylphosphatidylinositol (GPI) anchor. To understand its biological functions, we screened subpopulations of leukocytes and localized the expression of leukolysin at the mRNA level to neutrophils. Polyclonal and mono-specific antisera raised against a synthetic peptide from its hinge region recognized a major protein species at 56 kDa and several minor forms between 38 and 45 kDa in neutrophil lysates. In resting neutrophils, leukolysin is distributed among specific granules ( approximately 10%), gelatinase granules ( approximately 40%), secretory vesicles ( approximately 30%), and the plasma membrane ( approximately 20%), a pattern distinct from that of neutrophil MMP-8 and MMP-9. Consistent with its membrane localization and its reported GPI anchor, leukolysin partitions into the detergent phase of Triton X-114 and can be released from intact resting neutrophils by glycosylphosphatidylinositol-specific phospholipase C. Phorbol myristate acetate stimulates neutrophils to discharge 100% of leukolysin from specific and gelatinase granules and approximately 50% from the secretory vesicles and plasma membrane, suggesting that leukolysin can be mobilized by physiological signals to the extracellular milieu as a soluble enzyme. Indeed, interleukin 8, a neutrophil chemoattractant, triggered a release of approximately 85% of cellular leukolysins by a process resistant to a mixture of proteinase inhibitors, including aprotinin, BB-94, pepstatin, and E64. Finally, purified recombinant leukolysin can degrade components of the extracellular matrix. These results not only establish leukolysin as the first neutrophil-specific MT-MMP but also implicate it as a cytokine/chemokine-regulated effector during innate immune responses or tissue injury.     

Glycosylphosphatidylinositol-specific phospholipase D of human serum--activity modulation by naturally occurring amphiphiles

The enzymatic properties of glycosylphosphatidylinositol-specific phospholipase D (EC 3.1.4.50) were characterized using a 6,000-fold purified enzyme. This was obtained in 100 microg amounts from human serum with a recovery of 35%. Pure alkaline phosphatase containing one anchor moiety per molecule was used as substrate. The enzyme is stimulated by n-butanol, but in contrast to other phospholipases this activation is not produced by a transphosphatidylation reaction. The previously reported non-linearity of the specific activity with respect to phospholipase concentration in the test was no longer observed upon purification, indicating inhibitor removal. The serum inhibitor(s) co-chromatograph with serum proteins and lipoproteins. The main part of the inhibitory activity was found in the lipid fraction after protein denaturation and can be subfractionated into acid phospholipids, cholesteryl esters and triacylglycerides. Added phosphatidyl-serine, phosphatidylinositol, phosphatidylglycerol, gangliosides, cholesteryl esters, and sphingomyelins turned out to be strong inhibitors, as well as phosphatidic acid. Phosphatidylethanolamine and various monoacylglycerols were found to be activators. The low glycosylphosphatidylinositol-specific phospholipase activity found in native serum did not increase significantly upon 90% removal of phospholipids by n-butanol. High serum concentrations of strongly inhibiting compounds, complex kinetic interactions among aggregates of these substances, and compartmentalization effects are discussed as possible reasons for the observed inactivity.     

The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.