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Peptides have shown great potential in acting as template for developing versatile carrier platforms in nanomedicine, aimed at selective delivery of drugs to only pathological tissues saving its normal neighbors. Cell-penetrating peptides (CPPs) are short oligomeric peptides capable of translocating across the cell membrane while simultaneously employing multiple mechanisms of entry. Most CPPs exist as disordered structures in solution and may adopt a helical conformation on interaction with cell membrane, vital to their penetrative capability. Herein, we report a series of cationic helical amphipathic peptides (CHAPs), which are topologically constrained to be helical. The peptides were tested against cervical and breast cancer cells for their cell penetration and drug delivery potential. The cellular uptake of CHAP peptides is independent of temperature and energy availability. The activity of the peptides is biocompatible in bovine serum. CHAPs delivered functional methotrexate (MTX) inside the cell as CHAP-MTX conjugates. CHAP-MTX conjugates were more toxic to cancer cells than MTX alone. However, the CHAP-MTX conjugates were less toxic to HEK-293 cells compared with the cancer cells suggesting higher affinity towards cancer cells.

Vascular diseases are an increasing health issue, and common alloplastic, allogenic or autologous vascular grafts show frequent complications. The aim of this study is to develop an acellular, xenogenic bypass-graft from a bovine carotid artery (BAC) using detergent-based protocols. We compared decellularization with sodium desoxycholate (DOA), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), sodium dodecyl sulfate (SDS), and Triton X100 and improved suitable methods by variation of concentration, buffer system, incubation time, temperature, rinsing, and flow rate. All processes were evaluated systematically based on cellular residues, biocompatibility, structural and mechanical integrity. Decellularization with SDS and Triton X100 was not sufficient for the removal of cellular components. We optimized protocols using 1% DOA and Chaps by a buffered system at 37 °C with extended decellularization and rinsing. Decellularization with DOA depleted DNA to 0.5 ± 0.1% and soluble proteins to 0.6 ± 0.2%. Using Chaps, DNA was reduced to 0.2 ± 0.2% and proteins to 0.6 ± 0.3%. The improved protocols eliminated RNA completely from the matrix, and no cytotoxic effects were detected. Mechanical and structural integrity of decellularized tissues was comparable to non-decellularized controls. Our method effectively removed cellular components from the extracellular matrix while preserving the structural and mechanical integrity of the tissue. Decellularized BACs could be a promising alternative for vascular replacement therapy.

Biologic scaffolds are derived from mammalian tissues, which must be decellularized to remove cellular antigens that would otherwise incite an adverse immune response. Although widely used clinically, the optimum balance between cell removal and the disruption of matrix architecture and surface ligand landscape remains a considerable challenge. Here we describe the use of time of flight secondary ion mass spectroscopy (ToF-SIMS) to provide sensitive, molecular specific, localized analysis of detergent decellularized biologic scaffolds. We detected residual detergent fragments, specifically from Triton X-100, sodium deoxycholate and sodium dodecyl sulphate (SDS) in decellularized scaffolds; increased SDS concentrations from 0.1% to 1.0% increased both the intensity of SDS fragments and adverse cell outcomes. We also identified cellular remnants, by detecting phosphate and phosphocholine ions in PAA and CHAPS decellularized scaffolds. The present study demonstrates ToF-SIMS is not only a powerful tool for characterization of biologic scaffold surface molecular functionality, but also enables sensitive assessment of decellularization efficacy.We report here on the use of a highly sensitive analytical technique, time of flight secondary ion mass spectroscopy (ToF-SIMS) to characterize detergent decellularized scaffolds. ToF-SIMS detected cellular remnants and residual detergent fragments; increased intensity of the detergent fragments correlated with adverse cell matrix interactions. This study demonstrates the importance of maintaining a balance between cell removal and detergent disruption of matrix architecture and matrix surface ligand landscape. This study also demonstrates the power of ToF-SIMS for the characterization of decellularized scaffolds and capability for assessment of decellularization efficacy. Future use of biologic scaffolds in clinical tissue reconstruction will benefit from the fundamental results described in this work.

Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold’s regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cell-removing detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology. Statement of Significance Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response. Unfortunately, conventional techniques (SEM, SHG) are not conducive to identifying denatured collagen molecules in tissues. We demonstrate the first investigation into the molecular denaturation of collagen in decellularized ECM enabled by a novel Collagen Hybridizing Peptide (CHP) that specifically binds to unfolded collagen chains. We show that SDS and Triton X-100 denature collagen molecules while CHAPS and SD cannot. Such detection has been nearly impossible with other existing techniques. The CHP technique will advance our understanding about the effect of the cell-removing process on ECM, and lead to development of the decellularization technology.

Cholesterol is an important lipid molecule and is needed for all mammalian cells. In various cell types, excess cholesterol is stored as cholesteryl esters; acyl-CoA:cholesterol acyltransferase 1 (ACAT1) plays an essential role in this storage process. ACAT1 is located at the endoplasmic reticulum and has nine transmembrane domains (TMDs). It is a member of the membrane-bound O-acyltransferase (MBOAT) family, in which members contain multiple TMDs and participate in a variety of biological functions. When solubilized in the zwitterionic detergent CHAPS, ACAT1 can be purified to homogeneity with full enzyme activity and behaves as a homotetrameric protein. ACAT1 contains two dimerization motifs. The first motif is located near the N-terminus and is not conserved in MBOATs. Deletion of the N-terminal dimerization domain converts ACAT1 to a dimer with full catalytic activity; therefore, ACAT1 is a two-fold dimer. The second dimerization domain, located near the C-terminus, is conserved in MBOATs; however, it was not known whether the C-terminal dimerization domain is required for enzyme activity. Here we show that treating ACAT1 with non-ionic detergent, Triton X-100 or octyl glucoside, causes the enzyme to become a two-fold monomer without any enzymatic activity. Detergent exchange of Triton X-100 with CHAPS restores ACAT1 to a two-fold dimer but fails to restore its enzymatic activity. These results implicate that ACAT1 requires hydrophobic subunit interactions near the C-terminus in order to remain active as a two-fold dimer. Our results also caution the use of Triton X-100 or octyl glucoside to purify other MBOATs.

Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVβ3 and αVβ5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVβ3 and αVβ5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8.

Lung transplantation is the last option for the treatment of end stage chronic lung disorders. Because the shortage of donor lung organs represents the main hurdle, lung regeneration has been considered to overcome this hurdle. Recellularization of decellularized organ scaffold is a promising option for organ regeneration. Although detergents are ordinarily used for decellularization, other approaches are possible. Here we used high alkaline (pH12) sodium hydroxide (NaOH)-PBS solution without detergents for lung decellularization and compared the efficacy on DNA elimination and ECM preservation with detergent based decellularization solutions CHAPS and SDS. Immunohistochemical image analysis showed that cell components were removed by NaOH solution as well as other detergents. A Collagen and GAG assay showed that the collagen reduction of the NaOH group was comparable to that of the CHAPS and SDS groups. However, DNA reduction was more significant in the NaOH group than in other groups (p < 0.0001). The recellularization of HUVEC revealed cell attachment was not inferior to that of the SDS group. Ex vivo functional analysis showed 100% oxygen ventilation increased oxygen partial pressure as artificial hemoglobin vesicle-PBS solution passed through regenerated lungs in the SDS or NaOH group. It was concluded that the NaOH-PBS based decellularization solution was comparable to ordinal decellularizaton solutions and competitive in cost effectiveness and residues in the decellularized scaffold negligible, thus providing another potential option to detergent for future clinical usage.

Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). The protein extracts of T. solium cysts are complex mixtures including cyst's and host proteins. Little is known about the influence of using different detergents in the efficiency of solubilization-extraction of these proteins, including relevant antigens. Here, we describe the use of CHAPS, ASB-14 and Triton X-100, alone or in combination in the extraction buffers, as a strategy to notably increase the recovery of proteins that are usually left aside in insoluble fractions of cysts. Using buffer with CHAPS alone, 315 protein spots were detected through 2D-PAGE. A total of 255 and 258 spots were detected using buffers with Triton X-100 or ASB-14, respectively. More protein spots were detected when detergents were combined, i.e., 2% CHAPS, 1% Triton X-100 and 1% ASB-14 allowed detection of up to 368 spots. Our results indicated that insoluble fractions of T. solium cysts were rich in antigens, including several glycoproteins that were sensitive to metaperiodate treatment. Host proteins, a common component in protein extracts of cysts, were present in larger amounts in soluble than insoluble fractions of cysts proteins. Finally, antigens present in the insoluble fraction were more appropriate as a source of antigens for diagnostic procedures.

The basement membrane complex (BMC) is a critical component of the extracellular matrix (ECM) that supports and facilitates the growth of cells. This study investigates four detergents commonly used in the process of tissue decellularization and their effect upon the BMC. The BMC of porcine urinary bladder was subjected to 3% Triton-X 100, 8 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 4% sodium deoxycholate or 1% sodium dodecyl sulfate (SDS) for 24 h. The BMC structure for each treatment group was assessed by immunolabeling, scanning electron microscopy (SEM) and second harmonic generation (SHG) imaging of the fiber network. The composition was assessed by quantification of dsDNA, glycosaminoglycans (GAG) and collagen content. The results showed that collagen fibers within samples treated with 1% SDS and 8 mM CHAPS were denatured, and the ECM contained fewer GAG compared with samples treated with 3% Triton X-100 or 4% sodium deoxycholate. Human microvascular endothelial cells (HMEC) were seeded onto each BMC and cultured for 7 days. Cell-ECM interactions were investigated by immunolabeling for integrin β-1, SEM imaging and semi-quantitative assessment of cellular infiltration, phenotype and confluence. HMEC cultured on a BMC treated with 3% Triton X-100 were more confluent and had a normal phenotype compared with HMEC cultured on a BMC treated with 4% sodium deoxycholate, 8 mM CHAPS and 1% SDS. Both 8 mM CHAPS and 1% SDS damaged the BMC to the extent that seeded HMEC were able to infiltrate the damaged sub-basement membrane tissue, showed decreased confluence and an atypical phenotype. The choice of detergents used for tissue decellularization can have a marked effect upon the integrity of the BMC of the resultant bioscaffold. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A feature of transmissible spongiform encephalopathies is the accumulation of infectious prion proteins (PrP(Sc)), which are formed by the conversion of physiological prion proteins (PrP(C)). As PrP(C), which is modified posttranslationally with various types of glycoproteins, serves as the substrates for PrP(Sc) conversion, various PrP(C) subtypes may play a role in the formation of PrP(Sc) and species-specific transmission; the cattle disease BSE is transmissible naturally to humans, but the sheep disease scrapie is not. To reveal new mechanisms modulating prion conversion, we analyzed the PrP(C) profiles by determining the differential PrP(C) protein solubilities in the anionic and nonionic detergents N-lauroylsarcosine, N-octyl-β-D-glucopyranoside, CHAPS and deoxycholic acid. We compared the resulting solubility profiles of human PrP(C) with the solubility profiles of PrP(C) from sheep and cattle. The PrP(C) subtypes were differentially soluble. However, non-glycosylated PrP(C) from cattle and human was found explicitly in the insoluble fraction, while non-glycosylated ovine PrP(C) was detected in the soluble fraction. These findings indicate the existence of low-solubility PrP(C) phenotypes in cattle and humans.

Increasing evidence has linked membrane cholesterol to amyloid precursor protein (APP) processing. β-Sitosterol (BS) is one of the most common forms of plant sterols, with the structure very similar to that of cholesterol. Using HT22 mouse hippocampal cells, this study investigated whether the substitution of membrane cholesterol with BS influences APP metabolism. It was found that cholesterol/BS substitution promoted nonamyloidogenic processing of APP without affecting membrane fluidity. Additional experiments suggest that the effect of membrane BS on APP metabolism is associated with the migration of APP from lipid rafts toward non-raft regions. Given that dietary BS can enter the brain and accumulates in the plasma membrane of brain cells, these results suggest a potential use of BS in the prevention of Alzheimer's disease.

Novel paradigms for CYP24A1 inhibitor development are needed to circumvent existing efficacy and toxicity issues related to human therapeutics in this class. We hypothesize that improved structural knowledge of CYP24A1 in complex with natural substrates, inhibitors and/or its redox partner protein, adrenodoxin (Adx) is required to facilitate the next generation of CYP24A1 inhibitor design. To this end, we have developed truncated expression constructs for both rat CYP24A1 (Δ51) and bovine Adx (Δ108), which allow us to purify a stable and reversible state of the CYP24A1:Adx complex, for use in ongoing X-ray crystallographic studies. Spectral characterization of the reversible complex revealed that Adx binding enhanced the stability of the enzyme-substrate complex, despite lowering the ligand binding affinity of the free enzyme, for 1,25(OH)2D2, over 9-fold. Truncation of CYP24A1's flexible N-terminus (Δ51) improved the enzyme's ability to recruit substrate, without altering Adx's ability to stabilize the ligand-bound form. We also found that several common crystallization detergents, including CHAPS, inhibit ligand binding to the CYP24A1:Adx complex at concentrations well below their reported critical micelle concentration (CMC) values. Ultimately, this research provides a useful platform and framework for the study of conformationally complex, membrane-protein complexes, in the ligand-bound state.

International audience; AIM: Evidence that brain-derived neurotrophic factor (BDNF), a neurotrophin largely involved in cognition, is expressed by cerebral endothelial cells led us to explore in rats the contribution of the cerebral microvasculature to BDNF found in brain tissue and the link between cerebrovascular nitric oxide (NO) and BDNF production.METHODS: Brain BDNF protein levels were measured before and after in situ removal of the cerebral endothelium that was achieved by brain perfusion with a 0.2% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulphonate) solution. BDNF protein and mRNA levels as well as levels of endothelial NO synthase phosphorylated at serine 1177 (P-eNOSser1177 ) were measured in cerebral microvessel-enriched fractions. These fractions were also exposed to glycerol trinitrate. Hypertension (spontaneously hypertensive rats) and physical exercise training were used as experimental approaches to modulate cerebrovascular endothelial NO production.RESULTS: CHAPS perfusion resulted in a marked decrease in brain BDNF levels. Hypertension decreased and exercise increased P-eNOSser1177 and BDNF protein levels. However, BDNF mRNA levels that were increased by exercise did not change after hypertension. Finally, in vitro exposure of cerebral microvessel-enriched fractions to glycerol trinitrate enhanced BDNF production.CONCLUSION: These data reveal that BDNF levels measured in brain homogenates correspond for a large part to BDNF present in cerebral endothelial cells and that cerebrovascular BDNF production is dependent on cerebrovascular endothelial eNOS activity. They provide a paradigm shift in the cellular source of brain BDNF and suggest a new approach to improve our understanding of the link between endothelial function and cognition.

The alpha-v beta-6 (αvβ6) integrin has been identified as playing a key role in the activation of transforming growth factor-β (TGFβ) that is hypothesised to be pivotal in the development of cancer and fibrotic diseases. Therefore, the αvβ6 integrin is an attractive therapeutic target for these debilitating diseases and a drug discovery programme to identify small molecule αvβ6 selective arginyl-glycinyl-aspartic acid (RGD)-mimetics was initiated within GlaxoSmithKline. The primary aim of this study was to pharmacologically characterise the binding to αvβ6 of a novel clinical candidate, compound 1, using a radiolabelled form. Radioligand binding studies were completed with [(3)H]compound 1 against the human and mouse soluble protein forms of αvβ6 to determine accurate affinity estimates and binding kinetics. The selectivity of compound 1 for the RGD integrin family was also determined using saturation binding studies (αvβ1, αvβ3, αvβ5, αvβ8, α5β1 and α8β1 integrins) and fibrinogen-induced platelet aggregation (αIIbβ3 integrin). In addition, the relationship between divalent metal cation type and concentration and αvβ6 RGD site binding was also investigated. Compound 1 has been demonstrated to bind with extremely high affinity and selectivity for the αvβ6 integrin and has the potential as a clinical tool and therapeutic for investigating the role of αvβ6 in a range of disease states both pre-clinically and clinically. In addition, this is the first study that has successfully applied radioligand binding to the RGD integrin field to accurately determine the affinity and selectivity profile of a small molecule RGD-mimetic.

The serotonin1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive and behavioral functions. Solubilization of the hippocampal serotonin1A receptor by CHAPS is accompanied by loss of cholesterol that results in a reduction in specific agonist binding activity. Replenishment of cholesterol to solubilized membranes restores membrane cholesterol content and significantly recovers specific agonist binding. In order to test the stringency of cholesterol requirement, we solubilized native hippocampal membranes followed by replenishment with desmosterol. Desmosterol is the immediate biosynthetic precursor of cholesterol in the Bloch pathway differing only in a double bond at the 24th position. Our results show that replenishment with desmosterol restores ligand binding of serotonin1A receptors. This is consistent with earlier results showing that desmosterol can replace cholesterol in a large number of cases. However, these results appear to be contradictory to our earlier findings, performed by sterol manipulation utilizing methyl-β-cyclodextrin, in which we observed that replacing cholesterol with desmosterol is unable to restore specific ligand binding of the hippocampal serotonin1A receptor. We discuss the possible molecular mechanism, in terms of nonannular lipid binding sites around the receptor, giving rise to these differences.

Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed inSpodoptera frugiperda(Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.

Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a membrane bound enzyme utilizing long-chain fatty acyl-coenzyme A and cholesterol to form cholesteryl esters and coenzyme A. Previously, we had expressed tagged human ACAT1 (hACAT1) in CHO cells and purified it to homogeneity; however, only a sparse amount of purified protein could be obtained. Here we report that the hACAT1 expression level in H293 cells is 18-fold higher than that in CHO cells. We have developed a milder purification procedure to purify the enzyme to homogeneity. The abundance of the purified protein enabled us to conduct difference intrinsic fluorescence spectroscopy to study the binding between the enzyme and its substrates in CHAPS/phospholipid mixed micelles. The results show that oleoyl CoA binds to ACAT1 with Kd=1.9 μM, and elicits significant structural changes of the protein as manifested by the significantly positive changes in its fluorescence spectrum; stearoyl CoA elicits a similar spectrum change with much lower in magnitude. Previously, kinetic studies had shown that cholesterol is an efficient substrate and an allosteric activator of ACAT1, while its diastereomer epicholesterol is neither a substrate nor an activator. Here we show that both cholesterol and epicholesterol induce positive changes in the ACAT1 fluorescence spectrum; however, the magnitude of spectrum changes induced by cholesterol is much larger than epicholesterol. These results show that stereospecificity, governed by the 3beta-OH moiety in steroid ring A, plays an important role in the binding of cholesterol to ACAT1.

Rat synaptosomal plasma membranes were extracted with a detergent (CHAPS, a zwitterionic derivative of cholic acid). mu and delta opioid receptor binding and adenylate cyclase activities were tested in the intact membranes and in the supernatants from detergent treated membranes. The 6000 X g/8 min. supernatant contained mu receptor binding equal to 33% of the mu receptor binding measured in the untreated membranes. When the detergent treated membranes were sedimented at (50,000 X g/10 min.), 23% of the mu receptor binding was recovered in the supernatant. After a 100,000 X g/30 min. centrifugation the supernatant contained 10% of the mu receptor binding when compared to untreated membranes. Of the delta receptor binding found in intact membranes, 10% or less was recovered in the 3 supernatants described above. Furthermore, the mu and delta receptor binding were distributed differently among particles in the supernatants. This indicates differences in the chemical properties of the mu and delta opioid receptors. Adenylate cyclase assays showed that the G/F site of this enzyme complex was inactivated in the supernatants from detergent treated membranes parallel to the delta receptor binding decrease. However, the catalytic part of adenylate cyclase was present in the supernatants and seemed resistant to the detergent.

Mutations in the NPC1 gene cause Niemann-Pick type C disease, which appears to result from a defect in intracellular cholesterol trafficking. NPC1 is a member of the resistance-nodulation-cell division (RND) permease superfamily and contains a sterol-sensing domain, yet its cellular function and the identity of its substrates remain unknown. FLAG-tagged human NPC1 was purified from NPC1-expressing Chinese hamster ovary cells by solubilization in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), followed by affinity chromatography. Purified NPC1 in detergent solution appeared to be oligomeric as determined by gel filtration fast protein liquid chromatography and was photolabeled by an azido-cholesterol derivative. Fluorescent cholesterol analogs, including dehydroergosterol, cholestatrienol, and 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol), displayed enhanced fluorescence upon binding to NPC1 and also resulted in saturable, concentration-dependent quenching of NPC1 intrinsic Trp fluorescence. The apparent binding affinity for these three sterols was in the 0.5-6 microm range. Binding of NBD-cholesterol to NPC1 at low detergent concentration (2 mm CHAPS) was of high apparent affinity (0.5-0.6 microm) and occurred rapidly (1 min). However, binding of a BODIPY-labeled cholesterol derivative was very slow, requiring approximately 3 h to reach equilibrium. The apparent NBD-cholesterol binding affinity was greatly reduced at higher detergent concentration. The stoichiometry of NBD-cholesterol binding to NPC1 was approximately 1. Various sterols, including native cholesterol and 25-hydroxycholesterol, inhibited NBD-cholesterol binding, suggesting that they compete for binding to the protein. Dynamic quenching studies showed that bound NBD-cholesterol was almost completely shielded from the aqueous medium, suggesting that it is buried in a deep hydrophobic pocket in NPC1. The use of fluorescent cholesterol analogs provides novel information on the molecular properties of the sterol-binding site in the full-length NPC1 protein.

AIMS/BACKGROUND: Biliary phospholipid secretion involves predominant segregation of canalicular phosphatidylcholine into bile. We tested the hypothesis that micellar concentrations of the major physiologic bile salt taurocholate can preferentially solubilize phosphatidylcholine from the canalicular rat liver plasma membrane. METHODS: Subcellular fractions from rat liver and kidney were isolated with standardized procedures, incubated in vitro with taurocholate or 3-[(3-cholamidopropyl)dimethylammonio]-propane-1-sulphonate (CHAPS) and released phospholipids determined after centrifugation. RESULTS: After incubation of canalicular (cLPM) and basolateral (blLPM) rat liver plasma membrane vesicles with 6 and 8 mM taurocholate, the proportion of phosphatidylcholine released was about two-fold higher as compared with its relative contribution to the overall lipid composition of the membranes. Quantitatively, this taurocholate-induced preferential phosphatidylcholine release was about four-fold higher in cLPM (117 nmol) as compared with blLPM (28 nmol). Comparison of membranes from different organs showed that increased sphingomyelin content reduced taurocholate-induced phosphatidylcholine release. Furthermore, phosphatidylcholine release from cLPM did not fit an inverse exponential relationship between membrane sphingomyelin content and phosphatidylcholine release from different starting material, indicating that cLPM is especially prone to taurocholate-induced phosphatidylcholine release. In contrast, in rat liver microsomes and kidney brush border membranes, taurocholate released phospholipids in proportion of their membrane contents, indicating an unspecific membrane solubilizing effect only. Similarly, CHAPS had an unselective lipid solubilizing effects in cLPM and blLPM. CONCLUSION: These results support the concept that the very last step of canalicular phospholipid secretion is mediated in vivo by bile salt-induced vesiculation of phosphatidylcholine-enriched microdomains from the outer leaflet of cLPM.

The G-electrode-loading method (GELM) is a technique enabling a large number of proteins from rat liver to enter an immobilized pH gradient (IPG) gel strip for isoelectric focusing (IEF). In this method, three slips containing the sample solution are placed on the cathodic edge of an IPG gel strip and a slip containing Chaps solution, a filtration membrane, and an electrode slip are placed on top. Finally, a G-electrode is placed on these slips. The Chaps solution (an amphoteric compound) is supplied gently to the sample solution during IEF and helps the proteins in the sample solution to enter the IPG gel strips with a high solubilization capacity. This method was compared with traditional slip-loading and in-gel rehydration, and it showed the best results for protein separation, including high-molecular-mass proteins.

Detergents are indispensable solubilizing agents in the purification and analysis of membrane proteins. For mass spectrometric identification of proteins, it is essential that detergents are removed prior to analysis, necessitating an in-gel digestion step. Here, we report a procedure that allows use of detergents and in-solution digestion of proteins. Crude membrane preparations from mouse brain were solubilized with Triton X-100, CHAPS, or SDS, and the detergents were depleted from the membrane proteins using a desalting column equilibrated with 8 M urea. Following digestion with endoproteinase Lys-C, the resulting peptides were analyzed by LC-MS/MS on Linear ion trap-Orbitrap instrument. Applying stringent identification criteria, in single-LC-MS-runs, 1059 +/- 108 proteins, including 797 +/- 43 membrane proteins, were mapped from mouse brain. The identified proteins represented a broad spectrum of neurotransmitter receptors and other ion channels. The general applicability of the method is demonstrated by profiling of membrane proteins from four other mouse organs. Single-run analyses of eye, liver, spleen, and skeletal muscle allowed identification of 522 +/- 9, 610 +/- 7, 777 +/- 8, and 307 +/- 7 membrane proteins. Our results demonstrate that membrane proteins can be analyzed as efficiently as soluble proteins.

Polyspecific organic cation and anion transporters of the SLC22 protein family are critically involved in absorption and excretion of drugs. To elucidate transport mechanisms, functional and biophysical characterization of purified transporters is required and tertiary structures must be determined. Here, we synthesized rat organic cation transporters OCT1 and OCT2 and rat organic anion transporter OAT1 in a cell free system in the absence of detergent. We solubilized the precipitates with 2% 1-myristoyl-2-hydroxy- sn-glycero-3-[phospho- rac-(1-glycerol)] (LMPG), purified the transporters in the presence of 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or octyl glucoside, and reconstituted them into proteoliposomes. From 1 mL reaction vessels 0.13-0.36 mg of transporter proteins was purified. Thus, from five to ten 1 mL reaction vessels sufficient protein for crystallization was obtained. In the presence of 1% LMPG and 0.5% CHAPS, OCT1 and OAT1 formed homo-oligomers but no hetero-oligomers. After reconstitution of OCT1, OCT2, and OAT1 into proteoliposomes, similar Michaelis-Menten K m values were measured for uptake of 1-methyl-4-phenylpyridinium and p-aminohippurate (PAH (-)) by the organic cation and anion transporters, respectively, as after expression of the transporters in cells. Using the reconstituted system, evidence was obtained that OAT1 operates as obligatory and electroneutral PAH (-)/dicarboxylate antiporter and contains a low-affinity chloride binding site that stimulates turnover. PAH (-) uptake was observed only with alpha-ketoglutarate (KG (2-)) on the trans side, and trans-KG (2-) increased the PAH (-) concentration in voltage-clamped proteoliposomes transiently above equilibrium. The V max of PAH (-)/KG (2-) antiport was increased by Cl (-) in a manner independent of gradients, and PAH (-)/KG (2-) antiport was independent of membrane potential in the absence or presence of Cl (-).

The serotonin1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive, behavioral, and developmental functions. Solubilization of the hippocampal serotonin1A receptor by 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) is accompanied by loss of membrane cholesterol which results in a reduction in specific agonist binding activity. Replenishment of cholesterol to solubilized membranes restores the cholesterol content of the membrane and significantly enhances specific agonist binding activity. In order to test the stringency of the requirement of cholesterol in this process, we solubilized native hippocampal membranes followed by replenishment with 7-dehydrocholesterol (7-DHC). 7-DHC is an immediate biosynthetic precursor of cholesterol differing only in a double bond at the 7th position in its sterol ring. Our results show, for the first time, that replenishment of solubilized hippocampal membranes with 7-DHC does not restore ligand binding activity of the serotonin1A receptor, in spite of recovery of the overall membrane order. This observation shows that the requirement for restoration of ligand binding activity is more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane sterols with this important neuronal receptor under pathogenic conditions such as the Smith-Lemli-Opitz syndrome.

We previously provided preliminary evidence for the presence of a putative membrane ecdysone receptor (mEcR) anchored in the plasma membranes of anterior silk glands (ASGs) in Bombyx mori. This receptor may act in concert with the conventional EcR in 20E-dependent programmed cell death of these glands. We report here, for the first time, the solubilization of mEcR from ASG membranes using the zwitterionic detergent CHAPS in the presence of NaCl. Our results show by ligand binding assay that mEcR solubilized this way is functionally active and retains 75% of its native binding activity. We also defined experimental conditions that yielded protein/detergent complexes with partial binding activity, which makes it possible to purify the membrane-bound ecdysone binding protein. © 2007 Zoological Society of Japan.

A mixture of thiourea, urea and CHAPS (TUC) is an excellent solvent compatible with isoelectrofocusing (IEF) separation of water-insoluble protein extracts, and their subsequent two-dimensional gel electrophoresis is an important step in proteomic studies. The main aim of this work was to quantify extremely low amounts of water-insoluble proteins contained, for instance, in samples collected in bio-aerosol samplers. High CHAPS concentrations solubilize many proteins. However, enzyme-linked immunosorbent assay (ELISA), which is the most popular immunodetection method of quantifying antigens, is unfortunately not compatible with these high CHAPS concentrations and with the low protein concentrations of TUC extracts. The most common mixture used to solubilize these proteins contains 2 mol l − 1 thiourea, 7 mol l − 1 urea and 5% w/v CHAPS. This paper shows that these components inhibit the adsorption and/or recognition of proteins on microtitration plates, preventing antigen quantification under classic ELISA conditions. We have tried several solvents (ethanol, isopropanol, acetonitrile and trichloroacetic acid) to make the TUC-soluble proteins stick to the ELISA plates, and ethanol was shown to be the most appropriate. In this study, we have defined a new ELISA protocol allowing rapid and sensitive detection of low concentrations (60–500 ng ml − 1 ) of water-insoluble proteins extracted with high concentrations of TUC.

Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol (CH)-depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with detergent-resistant membranes (DRMs) into two groups. Group A proteins (occludin, claudin-2 and -3) were detected in large, intermediate and small aggregates in both detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH altered the distribution of group A and B proteins among the three size categories in a detergent-specific manner. In lysates produced with octyl glucoside, a detergent that selectively extracts proteins from DRMs, group A proteins were undetectable in large aggregates and CH depletion did not alter the distribution of either group A or B proteins in intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in intimate enough contact with CH to be cross-linked to [(3)H]-photo-cholesterol. However, antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein. Unlike claudins, occludin's presence in TJs and DRMs did not require palmitoylation. Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergent-related differences in the densities of TJ-bearing DRMs. There was little or no change in those densities after CH depletion. Removing CH from the plasma membrane increased tyrosine and threonine phosphorylation of occludin, and transepithelial electrical resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER fell below control values. We conclude that the association of integral TJ proteins with DRMS, pelleted at low speeds, is partially CH-dependent. However, the buoyant density of TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH.

Two isoforms of ryanodine receptors are expressed in skeletal muscles, RyR1 and RyR3. We investigated the relative level of expression of RyRs in developing murine skeletal muscles using [3H]ryanodine binding and immunoprecipitation experiments. In the diaphragm RyR3 accounted for 11% of total RyRs in 5-day-old mice and for 3% of total RyRs in 60-day-old mice. In hindlimb muscles, RyR3 accounted for 3% and 1% of total RyRs in 5-day-old and adult mice, respectively. The activity of RyR1 channels in native microsomal vesicles from murine muscles was found to be as low as 35% of that measured after CHAPS exposure, while no inhibition was observed for RyR3. CHAPS sensitivity of recombinant RyR1 and RyR3 expressed in HEK293 cells was also investigated. The activity of recombinant RyR1 but not RyR3 channels was found to be inhibited in native conditions, suggesting that this property may not be dependent on a muscle environment.

Gangliosides and glycosylphosphatidylinositol (GPI)-anchored proteins contain lipid tails that tether them to the outer side of the cell membrane. This mode of association with the cell membrane enables them to take part in the organization of lipid rafts, but it also permits gangliosides and GPI-anchored proteins to be actively released from one cell and inserted into the membrane of an another cell. Recently we have reported that under conditions of lipid raft isolation Triton X-100 causes significant redistribution of both gangliosides and GPI-anchored proteins. Aiming to find a less disruptive detergent we have evaluated effects of CHAPS, Saponin, deoxycholic acid, Trappsol, Tween 20, Triton X-100, Brij 96V, Brij 98, and SDS on the distribution of GM1 and GD1a gangliosides and Thy-1 (GPI-anchored protein) in brain tissue sections at room temperature and at 4º ; C. At room temperature all detergents (in 1% concentration and incubation for 2 h) extracted significant amounts of both gangliosides and Thy-1 from the membranes. At 4º ; C the extraction was weaker, but Triton X-100, CHAPS and deoxycholic acid caused significant redistribution of GD1a and Thy-1 from grey matter into the white matter. Both redistribution and extraction were significantly augmented if sections were incubated with detergents in the presence of primary antibodies. Other detergents like Trappsol and Tween 20 apparently cause less redistribution but they are also less efficient in revelation of cryptic epitopes. Neither one of the nine tested detergents is the ideal choice. However, Brij 96V appears to be able to sufficiently reveal myelin epitopes, while causing the least amount of artifacts.

Membrane proteins play a large variety of func- tions in life and represent 30% of all genomes sequenced. Due to their hydrophobic nature, they are tightly bound to their biological membrane, and detergents are always required to extract and isolate them before identification by mass spectrometry (MS). The latter, however remains difficult. Peptide mass fingerprinting methods using tech- niques such as MALDI-TOF MS, for example, have be- come an important analytical tool in the identification of proteins. However, PMF of membrane proteins is a real challenge for at least three reasons. First, membrane proteins are naturally present at low levels; second, most of the detergents strongly inhibit proteases and have deleterious effects on MALDI spectra; and third, despite the presence of detergent, membrane proteins are un- stable and often aggregate. We took the mitochondrial uncoupling protein 1 (UCP1) as a model and showed that differential acetonitrile extraction of tryptic peptides com- bined with the use of polystirene Bio-Beads triggered high resolution of the MALDI-TOF identification of mitochon- drial membrane proteins solubilized either with Triton- X100 or CHAPS detergents.

Lipid phosphate phosphatases (LPPs), integral membrane proteins with six transmembrane domains, dephosphorylate a variety of extracellular lipid phosphates. Although LPP3 is already known to bind to Triton X-100-insoluble rafts, we here report that LPP1 is also associated with lipid rafts distinct from those harboring LPP3. We found that LPP1 was Triton X-100-soluble, but CHAPS-insoluble in LNCaP cells endogenously expressing LPP1 and several LPP1 cDNA-transfected cells including NIH3T3 fibroblasts. In addition to the non-ionic detergent insolubility, LPP1 further possessed several properties formulated for raft-localizing proteins as follows: first, the CHAPS-insolubility was resistant to the actin-disrupting drug cytochalasin D; second, the CHAPS-insoluble LPP1 floated in an Optiprep density gradient; third, the CHAPS insolubility of LPP1 was lost by cholesterol depletion; and finally, the subcellular distribution pattern of LPP1 exclusively overlapped with that of a raft marker, cholera toxin B subunit. Interestingly, confocal microscopic analysis showed that LPP1 was distributed to membrane compartments distinct from those of LPP3. Analysis using various LPP1/LPP3 chimeras revealed that their first extracellular regions determine the different Triton X-100 solubilities. These results indicate that LPP1 and LPP3 are distributed in distinct lipid rafts that may provide unique microenvironments defining their non-redundant physiological functions.

Objective To characterize the major membrane protein of goat spermatozoa. Design Basic research. Setting Samples collected from local slaughterhouse and study conducted in an academic research environment. Patient(s) Goat epididymal tissue. Intervention(s) Goat epididymal tissues were collected immediately after slaughter and the spermatozoa were isolated within 2 hours. Sperm immobilization test was performed with motile spermatozoa at 32°C within 3–4 hours of collection and isolation of the cells. Main Outcome Measure(s) The heparin-binding sperm membrane protein (HBSM) of goat is insensitive to trypsin and its antisera immobilize spermatozoa in presence of complement. Result(s) Forty-two percent of membrane protein could be extracted with 0.25% (wt/vol) 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) from the isolated sperm membrane. By heparin-affinity chromatography, 46% of the extracted protein was recovered. Positive hybridization with radiolabeled heparin on western transfer confirmed the heparin-binding property of the protein (HBSM). Heparin binding to HBSM is an ionic bondage and can be disrupted by 1 M NaCl, as revealed by 86% recovery of the radiolabeled heparin in trichloroacetic acid–precipitated supernatant of [ 125 I] heparin-HBSM conjugate. Heparin-binding sperm membrane protein is localized at the anterior region of the spermatozoal head. No detectable proteolytic fragment of HBSM was detected after limited digestion by trypsin. Heparin-binding sperm membrane protein antisera (1:10,000 titer) developed from rabbit did not recognize the denatured protein. The antisera inhibited spermatozoal motility in a complement-dependent manner. Conclusion(s) We suggest that the heparin-binding motif of the spermatozoal membrane protein might be required in modulation of the spermatozoal motility.

Export of macromolecules from the endoplasmic reticulum (ER) lumen into the cytosol is a major aspect of the quality control systems operating within the early secretory system. Glycopeptides are exported from the ER by an ATP- and GTP-dependent pathway, which shares many similarities to the protein export system. Significantly, for glycopeptides, there is no requirement for cytosolic factors, biochemically distinguishing the glycopeptide and protein paths and probably reflecting the lower conformational complexity of the former substrate. Genetic studies in yeast, and biochemical data from higher eukaryotes, indicate that glycopeptides utilise the Sec61 translocon. Here, we report a new system allowing access to lumenal ER components, facilitating assessment of their importance in glycopeptide retrotranslocation and potentially other processes. Saponin, in combination with CHAPS, but not saponin alone, facilitated removal of >95% of lumenal protein disulphide isomerase (PDI) and BiP. Upon resealing, these microsomes retained glycopeptide export competence. These data suggest that the majority of lumenal components of the ER are most likely nonessential for glycopeptide export. In addition, export competence was highly sensitive to the addition of external protease, indicating a role for protein factors with cytoplasmically exposed determinants.

Summary A major antigen of the brush border membrane of Torpedo marmorata kidney was identified and purified by immunoprecipitation. The sequence of its 18 N terminal amino acids was determined and found to be very similar to that of mammalian aminopeptidase N (EC 3.4.11.2). Indeed aminopeptidase N activity was efficiently immunoprecipitated by monoclonal antibody 180K1. The purified antigen gives a broad band at 180 kDa after SDS-gel electrophoresis, which, after treatment by endoglycosidase F, is converted to a thinner band at 140 kDa. This antigen is therefore heavily glycosylated. Depending on solubilization conditions, both the antigen and peptidase activity were recovered either as a broad peak with a sedimentation coefficient of 18S (2% CHAPS) or as a single peak of 7.8S (1% CHAPS plus 0.2 % C12E9), showing that Torpedo aminopeptidase N behaves as an oligomer stabilized by hydrophobic interactions, easily converted into a 160 kDa monomer. The antigen is highly concentrated in the apical membrane of proximal tubule epithelial cells (600 gold particles/μm2 of brush border membrane) whereas no labeling could be detected in other cell types or in other membranes of the same cells (basolateral membranes, vacuoles or vesicles). Monoclonal antibodies prepared here will be useful tools for further functional and structural studies of Torpedo kidney aminopeptidase N.

1. The serotonin1A (5-HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. 2. We report here, for the first time, the solubilization of 5-HT1A receptors stably expressed in Chinese Hamster Ovary (CHO) cells using the zwitterionic detergent CHAPS in presence of NaCl followed by polyethylene glycol (PEG) precipitation. We show by ligand-binding assay that the 5-HT1A receptor solubilized this way is functionally active. We have optimized the efficiency of solubilization with respect to total protein and NaCl concentration. 3. Our results show that careful control of salt and protein concentration is crucial in optimal solubilization of membrane receptors heterologously expressed in cells in culture. The effective solubilization of important neurotransmitter receptors such as 5-HT1A receptors which are present in very low amounts in the native tissue may represent an important step in characterizing membrane receptors expressed in mammalian cells in culture.

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic effects of steroid hormones. We have previously described that the low-affinity glucocorticoid binding protein (LAGS), present in the endoplasmic reticulum of the male rat liver, has pharmacological and biochemical properties different from those of nuclear receptors. The LAGS is under multihormonal regulation and binds glucocorticoids, progestins, and synthetic steroids but is unable to bind either estradiol, testosterone, or triamcinolone acetonide. In this study, we have solubilized the LAGS and investigated their pharmacological and hydrodynamic properties and their peptide composition. We found that LAGS is an integral protein bound to the endoplasmic reticulum. CHAPS provided its optimal solubilization without changes in its pharmacological properties. Hydrodynamic properties of LAGS showed that it has a molecular mass of at least 135 kDa. SDS-PAGE of covalently-labeled LAGS showed that [3H]dexamethasone binds two peptides of 53 and 37 kDa, respectively. Thus, the LAGS appears as an oligomeric protein under multihormonal regulation. The availability of solubilized LAGS and the fact that it can be induced in vivo represent major steps toward purification and understanding the functional significance of this unique steroid-binding protein.

The present study reports a comparison of recently described solubilizing methods, to set up a simple protocol for obtaining two-dimensional (2-D) gel electrophoresis maps of brain tissue. Different protocols were used for preparing rat brain homogenates and the resulting maps were compared by image analysis. Three different detergents, two delipidation methods, and introduction of a fractionation step based on different protein solubility in surfactants, were evaluated. When using efficient zwitterionic detergents (3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, CHAPS; amidosulfobetaine 14, ASB-14), the patterns obtained by direct loading of total extracts were qualitatively overlapping with patterns obtained from fractionated samples. In contrast, a weaker nonionic agent (Nonidet P-40, NP-40) produced a different protein pattern in the collected fractions. Delipidation did not improve the results for all the different extraction methods. Immunoblots performed with antibodies recognizing cytosolic and membrane-spanning proteins, which were detected as nondegraded spots, showed that membrane proteins with intermediate molecular mass could be recovered. We suggest, as a simple and efficient method for preparing rat brain maps, the homogenization in a solution containing an efficient zwitterionic surfactant, which allows to solubilize cytosolic and membrane proteins in a single step. Alternatively, a fractionation can be carried out on samples homogenized by a weak solubilizing agent, a more labor-intensive effort resulting in a larger number of proteins on two maps.

The serotonin1A (5-HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioural and cognitive functions. This paper reports an efficient strategy to solubilize 5-HT1A receptors from bovine hippocampal membranes using the zwitterionic detergent CHAPS which is mild and non-denaturing. Since high concentration of CHAPS has earlier been shown to induce dissociation and depletion of G-protein sub-units, a low (pre-micellar) concentration of CHAPS was used for solubilizing 5-HT1A receptors in the presence of NaCl followed by PEG precipitation. This results in solubilization of 5-HT1A receptors with a high degree of efficiency and gives rise to high affinity, functionally active G-protein-sensitive solubilized receptors. Optimal solubilization of the receptor from the native source with high ligand binding affinity and intact signal transduction components may constitute the first step in the molecular characterization of the 5-HT1A receptor in particular, and G-protein-coupled receptors in general.

Toxicology is increasingly focused on molecular events comprising adverse outcome pathways. Atrazine activates the hypothalamic-pituitary adrenal axis, but relationships to gonadal alterations are unknown. We characterized hormone profiles and adrenal (intact and castrate) and testis (intact) proteomes in rats after 3 days of exposure. The adrenal accounted for most of the serum progesterone and all of the corticosterone increases in intact and castrated males. Serum luteinizing hormone, androstenedione, and testosterone in intact males shared a non-monotonic response suggesting transition from an acute stimulatory to a latent inhibitory response to exposure. Eight adrenal proteins were significantly altered with dose. There were unique proteomic changes between the adrenals of intact and castrated males. Six testis proteins in intact males had non-monotonic responses that significantly correlated with serum testosterone. Different dose-response curves for steroids and proteins in the adrenal and testis reveal novel adverse outcome pathways in intact and castrated male rats.

The zwitterionic detergent CHAPS was used to solubilize the human mu-opioid receptor (hMOR) from SH-SY5Y neuroblastoma cells and recombinant hMOR-CHO (CHO-T7-hMOR) and hMOR-SH-SY5Y (SH-SY5Y-T7-hMOR) cell membranes. Agonist stimulation and G-protein activation by the mu-selective opioid agonist DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) were recovered after removing of CHAPS after polyethylene glycol (PEG) precipitation. Binding assays show that hMOR solubilized and reconstituted this way was functional and able to interact with both agonist peptides and with G-protein. The effective solubilization and reconstitution of hMOR from mammalian cells, without truncation and extensive modification, represent an essential step toward the purification of a receptor bearing important post-translational modifications.

Association of cyclophilin A with renal brush border membranes: Redistribution by cyclosporine A. Background Administration of the immunosuppressive agent cyclosporine A (CsA) is associated with nephrotoxicity. The main target for CsA, cyclophilin A (CypA), was found in high levels in epithelial cells of renal proximal tubules. In the present study, CypA was immunodetected and characterized following CsA treatment in subcellular fractions of renal cortex. Method The renal content and distribution of CypA was evaluated in untreated rats and in rats treated with a subcutaneous injection of CsA (10 mg · kg -1 · day -1 ) for 10 days. Results In untreated rats, membrane-bound CypA represents 0.25% of total brush border membrane (BBM) proteins, similar to the proportion found in the soluble fraction. High ionic strength treatment was unable to extract CypA from BBMs, whereas alkaline treatment (Na 2 CO 2 , pH 11) and detergent 3 – [(3 – cholamidopropyl) – dimethyl – ammonio] – 1 – propanesulfate (CHAPS) released it from BBMs. These results indicate that CypA is associated with renal BBMs, and that hydrophobic interactions are involved in this association. The CypA distribution was strongly modified in both BBMs and the soluble fraction after CsA treatment, but its affinity for CsA estimated by photoaffinity labeling was unaffected. The CypA expression level decreased by 45% in BBMs, while it increased by 33% in the soluble fraction, compared with control rats. CypA remained associated with the membranes following in vitro incubation of renal BBMs with CsA. However, incubation of CypA with one of its substrates released CypA from renal BBMs. Conclusions These experiments suggest that renal BBMs contain a significant amount of CypA and chronic exposure to CsA, and acute exposure to one of CypA substrates may modify its subcellular distribution.

Acyl-CoA:cholesterol acyltransferase (ACAT) is a key enzyme in cellular cholesterol homeostasis and in atherosclerosis. ACAT-1 may function as an allosteric enzyme. We took a multifaceted approach to investigate the subunit composition of ACAT-1. When ACAT-1 with two different tags were co-expressed in the same Chinese hamster ovary cells, antibody specific to one tag caused co-immunoprecipitation of both types of ACAT-1 proteins. Radioimmunoprecipitations of cells expressing the untagged ACAT-1 or the 6-histidine-tagged ACAT-1 yielded a single radiolabeled band of predicted size on SDS-polyacrylamide gel electrophoresis. These results show that ACAT-1 exists as homo-oligomers in intact Chinese hamster ovary cells. We solubilized HisACAT-1 with the detergent deoxycholate or CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid), performed gel filtration chromatography and sucrose density gradient centrifugations in H(2)O and D(2)O, and determined the Stokes radii and sedimentation coefficients of the HisACAT1-detergent complexes. The estimated molecular mass of HisACAT-1 is 263 kDa, which is 4 times that of the HisACAT-1 monomer (69 kDa). Finally, cross-linking experiments in intact cells and in vitro show that the increase in cross-linker concentrations causes an increase in size of the HisACAT-1-positive signals, forming material(s) 4 times the size of the monomer, supporting the conclusion that ACAT-1 is a homotetrameric enzyme.

Diabetes-associated hyperglycemia causes glycation of proteins at reactive amino groups, which can adversely affect protein function. Although the effects of glycation on soluble proteins are well characterized, there is no information regarding membrane-associated proteins, mainly because of the lack of reproducible methods to determine protein glycation in vivo. The current study was conducted to establish such a method and to compare the glycation levels of membrane-associated proteins derived from normal and diabetic tissue. We present a detailed sample preparation protocol based on the borohydride-periodate assay, modified to allow manipulation of animal tissue. Assay noise associated with extraction protocols and nonproteinaceous buffer components was eliminated by the using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) as a membrane detergent, applying desalting columns, and including a protein precipitation step. The glycation level of membrane proteins from diabetic rats is elevated to 4.89 nmol/mg protein (standard deviation [SD] 0.48) compared with normoglycemic control tissue (2.23 nmol/mg protein, SD 0.64). This result is consistent with and correlated to the total glycated hemoglobin levels in diabetic and normoglycemic rats. Using100 microg protein, the described methods allow further study of protein glycation effects on the function of individual transporter proteins and the role of these modifications in diabetes.

The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver.

The metabolism and synthesis of an important mycobacterial lipid component, phosphatidylinositol (PI), and its metabolites, was studied in Mycobacterium smegmatis and M . smegmatis subcellular fractions. Little is known about the synthesis of PI in prokaryotic cells. Only a cell wall fraction (P60) in M . smegmatis was shown to possess PI synthase activity. Product was identified as PI by migration on TLC, treatment with phospholipase C and ion exchange chromatography. PI was the only major product (92.3%) when both cells and P60 fraction were labeled with [ 3 H]inositol. Also, a neutral lipid inositol-containing product (4.1% of the total label) was identified in the P60 preparations. Strangely, PI synthase substrates, CDP-dipalmitoyl-DAG and CDP-NBD-DAG, added to the assay did not stimulate [ 3 H]PI and NBD-PI yield by M . smegmatis . At the same time, addition of both substrates to rat liver and Saccharomyces cerevisiae PI synthase assays resulted in an increase in the product yield. Upon addition of CHAPS to the mycobacterial PI synthase assay, both substrates were utilized in a dose-dependent manner for the synthesis of NBD-PI and [ 3 H]PI. These results demonstrate a strict substrate specificity of mycobacterial PI synthase toward endogenous substrates. K m of the enzyme toward inositol was shown to be 25 μM; Mg 2+ stimulated the enzyme to a greater degree than Mn 2+ . Structural analogs of myo -inositol, epi -inositol and scyllo -inositol and Zn 2+ were shown to be more potent inhibitors of mycobacterial PI synthase than of mammalian analogs. Lack of sequence homology with mammalian PI synthases, different kinetic characteristics, existence of selective inhibitors and an important physiological role in mycobacteria, suggest that PI synthase may be a good potential target for anti-tuberculosis therapy.

RPE65 is a major membrane associated protein found in the vertebrate retinal pigment epithelium (RPE). Various studies have shown this protein to be essential for visual function, possibly at the level of the processing of retinoids. The pigment epithelium is the anatomical site in which the visual chromophore 11-cis retinal is generated. The two critical RPE enzymes in the isomerization pathway are lecithin retinol acyl transferase (LRAT) and isomerohydrolase, which processes all-trans-retinyl esters into 11-cis-retinol. Both enzymes are membrane bound. It is shown here that RPE65 can be largely extracted (90–95%) from RPE membranes by 1 M KCl by itself, or with added detergent CHAPS. The almost quantitative extraction of RPE65 from RPE membranes has little or no effect on in vitro LRAT and isomerohydrolase activities in quantitative enzymatic assays using RPE membranes, suggesting that RPE65 may not have an important role to play in the enzymatic processing of all-trans-retinol into 11-cis-retinol in vitro.

Inhibition of binding of the labelled antagonist (-)[3H]CGP 12177 by the full agonist (-)isoproterenol results in shallow competition curves, characteristic of the presence of both high- and low-affinity states of beta-adrenoceptors (betaAR). When in excess, the GTP analog 5'-guanylylimidodiphosphate (GppNHp) is expected to convert all receptors in the high-affinity state to the low-affinity state. However, in the rat cortex and cerebellum synaptosomes, a proportion of the betaAR in the high-affinity state was GppNHp-insensitive. This apparent GppNHp-insensitivity decreased with decreasing temperature of incubation. Moreover, it was totally abolished by the gentle treatment of membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). We propose that a protein factor interacts with the betaAR/Gs protein complex and that it induces the GppNHp-insensitivity. This factor would be released by CHAPS in a functional form because it may regenerate the GppNHp-insensitivity after concentration and reconstitution with CHAPS-treated membranes. It is likely that the factor acts as a stabiliser of betaAR in the high-affinity state.

Hepatic retinyl ester hydrolase (REH) activity was isolated from porcine and human liver and characterized, and some of its properties were compared with those of other retinyl-ester-splitting enzymes. Sequence analysis revealed that the REH proteins are structurally similar to non-specific carboxylesterases and distinct from bile salt-activated lipases and cholesterol esterases. Pig REH, a 64-kDa protein, hydrolyzed retinyl palmitate at a rate of 595 nmol x h(-1) x mg(-1) protein in the presence of 100 mM Chaps with an apparent Km value for retinyl palmitate of 27.5 microM. The pH optimum was 7.0-9.2. Its human counterpart has a molecular mass of 65 kDa and a pH optimum near 6.5. In the presence of Chaps, pig REH activity was stimulated up to 1.7-fold by various non-ionic detergents. The ranking order of retinyl palmitate cleavage initiated by the stimuli was n-dodecylglucoside > octanoyl-N-methylglucamide > n-octyglucoside > n-dodecylmaltoside > Triton X-114 > Triton X-100. Porcine REH was effectively inhibited by alpha-tocopherol and bis-(4-nitrophenyl) phosphate [(Np)2P]. The structural, immunological and catalytic features, pH dependence, and the effect of (Np)2P on enzyme activity of pig REH are similar to those reported for the non-specific carboxylesterase ES-4. However, ES-4 differed from REH in molecular mass and the requirement of Chaps or Chaps-like detergents as cofactor. Judging from these results, pig REH may be a non-specific carboxylesterase isoform.

Prolactin receptor from rat liver (PRL-R, 42 kDa) was cross-linked to a radiolabeled azidophenacyl derivative of human growth hormone ([ 125 I]AP-hGH) to yield a 63 kDa adduct. In addition, a protein of Mr 50–52 K was detected as a 73 kDa complex. Microsomes incubated with either (a) increasing amounts of [ 125 I]AP-hGH, or (b) a fixed amount of photoprobe and increasing concentrations of unlabeled hGH, showed that the 73/63 kDa band intensity ratio remains constant (0.71–0.77). Once transferred onto nitrocellulose membranes, only the 42 kDa protein is able to bind [ 125 I]AP-hGH or [ 125 I]hGH. Two anti-PRL-R monoclonal antibodies fail to cross-react with proteins of Mr 50–52 K. In membranes solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), a significantly lower amount of the 73 kDa complex is detected. Thus, the 50–52 kDa protein appears to be structurally unrelated to, but is presumably associated with the PRL-R. The 73 kDa complex is also detected under low membrane fluidity conditions (1 °C), indicating that PRL-R associates to this 50–52 kDa protein prior to hormone binding. Perfusion of rat liver with [ 125 I]AP-hGH shows that this associated protein accompanies the receptor along its intracellular pathway.

We purified extensively 25-hydroxyvitamin D3 1α-hydroxylase (calcidiol, NADPH: oxygen oxidoreductase (1-hydroxylating), EC 1.14.13.13) from kidney mitochondria of rachitic rats and disclosed its peculiar properties as a P450. The final preparation was identified as a 55 kDa protein having an intense absorption at 417 nm characteristic of P450. The specific activity was 4.8 nmol/min/mg of protein indicating a 350-fold purification. Specific content of P450 was 1.1 nmol/mg of protein and turnover number was 4.4 min−1.