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Detergents are amphiphilic molecules widely used to solubilize biological membranes and/or extract their components. Nevertheless, because of the complex composition of biomembranes, their solubilization by detergents has not been systematically studied. In this review, we address the solubilization of erythrocytes, which provide a relatively simple, robust and easy to handle biomembrane, and of biomimetic models, to stress the role of the lipid composition on the solubilization process. First, results of a systematic study on the solubilization of human erythrocyte membranes by different series of non-ionic (Triton, CxEy, Brij, Renex, Tween), anionic (bile salts) and zwitterionic (ASB, CHAPS) detergents are shown. Such quantitative approach allowed us to propose Re sat—the effective detergent/lipid molar ratio in the membrane for the onset of hemolysis as a new parameter to classify the solubilization efficiency of detergents. Second, detergent-resistant membranes (DRMs) obtained as a result of the partial solubilization of erythrocytes by TX-100, C12E8 and Brij detergents are examined. DRMs were characterized by their cholesterol, sphingolipid and specific proteins content, as well as lipid packing. Finally, lipid bilayers of tuned lipid composition forming liposomes were used to investigate the solubilization process of membranes of different compositions/phases induced by Triton X-100. Optical microscopy of giant unilamellar vesicles revealed that pure phospholipid membranes are fully solubilized, whereas the presence of cholesterol renders the mixture partially or even fully insoluble, depending on the composition. Additionally, Triton X-100 induced phase separation in raft-like mixtures, and selective solubilization of the fluid phase only.

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.

In this study, we have reported the effect of nonionic, anionic, cationic, and zwitterionic detergents on the enzymatic activity and structural stability of Rhizopus niveus lipase. Secondary structural changes were monitored by Far-UV CD which shows that surfactant induces helicity in the Rhizopus niveus lipase protein which was maximum in case of CTAB followed by SDS, CHAPS, and Brij-35. Similarly, tertiary structural changes were monitored by tryptophan fluorescence. We also carried out enzyme kinetics assays which showed that activity was enhanced by 1.5- and 1.1-fold in the presence of CHAPS and Brij-35, respectively. Furthermore, there was a decline in activity by 20 and 30 % in case of SDS and CTAB, respectively. These studies may be helpful in understanding detergent-lipase interaction in greater detail as lipases are used in many industrial processes.

Mature transforming growth factor beta1 (TGF-β1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-β1/C77S (further termed TGF-β1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding enteropeptidase recognition site. High-level expression (~1.5 g l(-1)) of Trx/TGF-β1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-β1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-β1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-β1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-β1m from bacteria with a yield of about 2.8 mg from 100 ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-β1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.

CHAPS is a zwitterionic surfactant derivative of bile salts, widely used in membrane protein isolation. While some studies regarding CHAPS self-aggregation suggest continuous increase in micelle size at increasing CHAPS concentration, other works point to the existence of two definite micelle types. In this work, stearic acid spin labels (5, 12, or 16-SASL) were added to CHAPS solutions to obtain information about the micellar structure using electron paramagnetic resonance spectroscopy. The spectra of 12-SASL were processed using Principal Factor Analysis, and at all concentrations they could be reproduced as linear combinations of only three fundamental spectra, the first one corresponding to free 12-SASL in aqueous solution. This fact suggests only two hydrophobic environments that host 12-SASL, assigned to primary and secondary CHAPS micelles. The relative populations of the label in each environment were obtained as a function of CHAPS concentration. Our results suggest barrel-shaped primary micelles with a minimum mean radius of 1.46 nm, and a critical micelle concentration cmcI = 4 mM. Secondary micelles are formed by aggregation of primary ones, with cmcII = 10 mM. They have several elongated hydrophobic pockets, with similar dimensions for all aggregate sizes. Our results contribute to the understanding of the mechanism of interaction of chain amphiphiles with CHAPS micelles. Fil: Rodi, Pablo Marcelo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Bocco Gianello, María D.. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Gennaro, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Física del Litoral; Argentina

The reconstitution of large amounts of integral proteins into lipid vesicles is largely prompted by the complexity of most biological membranes and protein stability. We optimized a particular system which maximized the incorporation efficiency of large soluble amounts of KcsA potassium channel in Escherichia coli membranes. The effects of two detergents, octylglucoside and 3-[(cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), on KcsA reconstitution were compared. Reconstitution efficiency was found to be incredibly high for CHAPS-treated proteoliposomes followed by dialysis at room temperature. This approach may allow more accurate investigation of integral membrane proteins in their natural membrane environment via biophysical or biochemical techniques.

Protein extraction is a critical step in any proteomics study. Since most fungi possess a robust cell wall, efficient isolation of total proteins has become challenge to fungal proteomics. To circumvent this bottleneck of fungal proteomics, we standardized a protocol named as Mg/CHAPS extraction by comparing with an established method of protein extraction (Tris/EDTA extraction), using 2-DE and MALDI-TOF MS. Total mycelial proteins were isolated using both protocols from Magnaporthe grisea (causal agent of rice blast disease). Six hundred forty two proteins were resolved on two 2-DE gels corresponding to mycelial proteomes isolated by Mg/CHAPS and Tris/EDTA. Mycelial proteome extracted by Mg/CHAPS showed higher number protein spots than to Tris/EDTA. Quantitative analysis of mycelial proteome, histogram and MS analyses of a protein spot suggested that Mg/CHAPS extraction is more effective than the widely used protocol i.e. Tris/EDTA.

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.

3-[(3-cholamidopropyl)-dimethylammoniol-1-propane sulfonate (CHAPS) can be used as an effective chiral selector for the separation of dansyl-amino acids by capillary electrophoresis (CE). While CHAPS can serve as an chiral selector, better enantiomeric separation can be performed by using CHAPS not as the sole chiral selector but as one of a [CHAPS-SDS-cyclodextrin] three-component system. In this CHAPS-SDS-CD system, enantiomeric separations of the amino acids can be readily accomplished by judiciously adjusting the pH of the solution, concentrations of CHAPS and SDS, and the concentration and type of CD. All amino acids can be baseline resolved in less than 15 minutes with resolution as high as 2.01 at pH 6.5 with 50 mM of CHAPS and 75 mM of SDS. The resolution is also dependent on the size of the CD. Substantial increase in the resolution can be readily achieved by replacing β-CD with γ-CD. For example, theR s for Leu was increased by four-folds (from 1.65 to 6.29) while the elution time still remains as short as 20 min when β-CD was replaced by γ-CD.

A method for measuring intermolecular NOEs in protein complexes based on asymmetric sample deuteration is described. 13C/1H-I,L,V-methyl, U-2H labeled protein is produced using the biosynthetic precursors [γ-13C]-α-ketobutyrate and [γ,γ′-13C2]-α-ketoisovalerate. The labeled protein is mixed with its unlabeled binding partner and a 3D 13C-HMQC-NOESY is recorded, yielding unambiguous intermolecular aromatic/methyl NOEs. A simple synthesis of the biosynthetic precursors via reaction of diethyl oxalate with alkyl Grignard compounds is reported. The method is demonstrated for a 35 kDa heterodimeric protein complex dissolved in a CHAPS micelle. This approach will facilitate the solution structure determination of protein/protein, protein/ligand or protein/nucleic acid complexes.

Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-dependent protein kinase (PKA) occurs in intact cells, the purified enzyme in vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of different detergents and ligands on the stoichiometry of the phosphorylation and activity of Na,K-ATPase from duck salt glands (alpha1beta1-isoenzyme) were comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent ratio in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol Pi/mol of alpha-subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the detergent inhibits the Na,K-ATPase. A correlation was found between the inclusion of P(i) into the alpha-subunit and the loss of activity of the Na,K-ATPase.

When 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) was removed from the mixed CHAPS/EggPC micelles, large vesicles were prepared by dialysis or by slow step-by-step dilution, but small vesicles were prepared by fast one-step dilution. When sodium cholate was removed from the sodium cholate/EggPC micelles, small vesicles formed either by dialysis or by dilution; however, in the presence of 5 mM Ca2+ large vesicles were produced by dialysis, while small vesicles were prepared by dilution. The size growth was related to a detergent-induced fusion of the vesicles containing a large amount of detergent. Using spectrophotometry, quasielastic light scattering and freeze–fracture electron microscopy the fusion events were investigated both through the process of vesicle solubilization by adding detergent and through the process of vesicle formation by diluting a mixed micelle. The results suggest that a rapid CHAPS-induced fusion of the vesicles led to the large resultant vesicles and that no fusion of vesicles containing sodium cholate is responsible for the formation of small vesicles. Furthermore, the ultimate vesicle size related to rapid or slow detergent removal is dependent on the kinetic aspects of the fusion.

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 mRNA cap-binding protein eIF4E is the limiting factor in the eIF4F translation initiation complex, which mediates the binding of the 40S ribosome to the mRNA. 15N relaxation studies have been used to characterize the backbone dynamics of deuterated eIF4E in a CHAPS micelle for the apoprotein, the m7GDP-bound form, and the dinucleotide (m7GpppA)-bound form, as well as for CHAPS-free eIF4E. Large differences in overall correlation time between the CHAPS-free form (11.8 ns) and samples containing different concentrations of CHAPS (15.9–19.4 ns) indicate that eIF4E is embedded in a large micelle in the presence of CHAPS, with a total molecular weight in the range of 40–60 kDa. CHAPS seems to restrict the mobility of the a2–b3 and a4–b5 loops which are thought to be embedded in the micelle. No significant changes in overall mobility were seen between the m7GDP-bound form, the m7GpppA-bound form, and the apoprotein. Amide hydrogen exchange data indicate the presence of slowly exchanging amides in two surface-exposed helices (a2 and a4), as well as the a4–b5 loop, indicating protection by the CHAPS micelle. The micelle covers the convex side of the protein away from the cap-binding site.

Previous studies showed that bile salts had apromoting effect on colon cancer development and thiseffect was inhibited by ursodeoxycholate (UDC). Werecently found that both human colorectal adenomas and carcinomas were associated with a specificdecrease in alkaline sphingomyelinase activity. In thiswork, we compared the effects of ursodeoxycholate andother bile salts on the levels of rat intestinal alkaline sphingomyelinase both in theintestinal loops and after oral administration. Bilesalts at different concentrations were injected intointestinal loops and the dissociation of alkalinesphingomyelinase from the mucosa was assayed. We found that bilesalts, including taurocholate, taurodeoxycholate,glycocholate, glycochenodeoxycholate, and3-(3-cholamidopropyl dimethylammonio)-1-propanesulonate(CHAPS), dose dependently dissociated alkalinesphingomyelinase from the intestinal mucosa. UDC alonedid not dissociate the enzyme but significantlyinhibited the dissociation caused by other bile saltsand CHAPS. Feeding rats with 0.3% (w/w) taurocholate forfour days decreased peak activity of intestinal alkalinesphingomyelinase by 39% and total activity in theintestine by 20% and increased the output of the enzyme in the feces. In contrast, feeding 0.3%(w/w) UDC for four days increased the peak activity ofalkaline sphingomyelinase in the small intestine by 87%and the activity in the colon by 187% . The total activity of alkaline sphingomyelinase wasincreased by 80% and the output of the enzyme in thefeceswas only slightly increased by UDC administration.The changes in alkaline phosphatase after feeding taurocholate and UDC were much smaller. Ourresults indicate that UDC and other bile salts havedifferent effects on the levels of alkalinesphingomyelinase, which may be implicated in theirdifferent influences on cancer development reportedpreviously.

Absorption spectra of the D1-D2-cytochrom b559 complex at 4°C were investigated at pressures up to 300 MPa. Pressure effects were mostly reversible and independent of the detergent used (CHAPS or dodecyl-β-D-maltoside). Red-shifts were observed under pressure for the chlorophyll Qy- and the β-carotene S0 → S2 bands. The relatively small Qy-shift of approximately 0.15 cm-1/MPa is an indication for the absence of strongly coupled chlorophyll dimers within the reaction center and supports earlier reports from low-temperature measurements (Chang HC, Jankowiak R, Reddy NRS and Small GJ (1995) Chem Phys 197: 307–321). The carotene red-shift (seen in CHAPS) is much larger (0.5 – 0.6 cm-1/MPa) and within the range observed for excitonically coupled chlorophylls. However, since carotenes are more sensitive to changes of refractive index, we do not consider this evidence for excitonically coupled carotenes. Varying the pH and the detergent induced only small effects. Pigment exchange using high pressure instead of elevated temperature was not possible under the conditions tested.

Phospholipid/cholesterol vesicles were solu-bilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Above 30 mol% cholesterol (Ch) in the lipid vesicles several remarkable changes of the solubilization process were observed. (i) Two modes of solubilization: The effective detergent to lipid ratio Rc(M) for the formation of mixed micelles decreased from Rc(M) = 43 ± 3 at low lipid concentrations, [L]≤ 0.15 mm, to Rc(M) = 2.4 ± 0.3 above [L] = 0.5 mm (40 mol% Ch, T = 20 °C). (ii) At subsolubilizing CHAPS concentrations, filamentous and helical microstructures were formed, similar to those which were observed in native and model bile. (iii) The number of observed fibers was about two orders of magnitude higher in the presence of the negatively charged lipids phosphatidylglycerol (PG) and phosphatidic acid (PA) compared to the zwitterionic phosphatidylcholine (PC). Fiber formation began after 16–18 h using PG and PA compared to 3–4 days in the presence of PC. Screening of the charged lipids by NaCl effectively reduced the formation of fibers. Assuming binding of Na+ to the charged lipid aggregates, an intrinsic binding constant Kint = 0.6 M–1 was determined by applying the Gouy-Chapman theory. After the addition of CHAPS to PG/Ch vesicles, a fast initial solubilization of the vesicles ( 15 h). The microstructures are visualized by darkfield and electron microscopy. The method of vesicle solubilization is compared to the dilution of concentrated micellar solutions, which is usually applied to model bile systems.

Cyclic 2,3-diphosphoglycerate (cDPG) hydrolase activity was demonstrated in cofactor-free extract of Methanobacterium thermoautotrophicum (strain ΔH), but not in crude extract. Only after ultrafiltration or dialysis of crude extract cDPG hydrolase activity could be shown. cCPG hydrolysis was optimal at pH 6.0 and 60°C. Hydrolysis of cDPG occurred under nitrogen or hydrogen atmosphere and was completely inhibited by oxygen. Phosphate and potassium chloride were also strong inhibitors: 50% inhibition occurred at 0.6–0.7 mM phosphate or 0.2 M KCl. The enzyme was localized in the membrane fraction and could be solubilized for approximately 60% by treatment with 25 mM of the detergent CHAPS. The Km and the Vmax for cDPG were determined at 60°C and were 59 mM and 216 mU/mg, respectively. Furthermore, cDPG hydrolase was dependent on the presence of Co2+. The role of cDPG and cDPG hydrolase is discussed.

We have characterized a phosphatidic acid phosphatase (PAP, EC 3.1.3.4) that is associated with cell membranes from rat brain using [32P]phosphatidic acid as substrate in a simple assay. The enzyme could be activated by Triton X-100, cholic acid and Chaps and inhibited by Lubrol PX and sodium dodecyl sulfate. The optimal pH was between 6.0 and 7.0 Mg2+ was not essential for enzyme activity. The enzyme activity was decreased by about 50% by Ca2+ at concentrations of 0.1 to 1 mmol/l. Zn2+ inhibited the enzyme by 50% at concentrations of about 10 mumol/l in the absence of, and 100 nmol/l in the presence (3 mmol/l) of, Triton X-100. NaF decreased the activity by about 50% at concentrations between 0.3 and 1 mmol/l when Triton X-100 was added, but did not inhibit the enzyme if the detergent was not present. N-Ethylmaleimide (NEM) did not affect the enzyme. In the absence of Triton X-100, propranolol and metoprolol enhanced the PAP activity. In the presence of 3 mmol/l Triton X-100, the enzyme was inhibited by about 50% by propranolol at a concentration of 10 mmol/l, whereas metoprolol caused only a slight inhibition of PAP. The Km for phosphatidic acid was 150 mumol/l and was changed to 20 mumol/l by 3 mmol/l Triton X-100 without the Vmax being changed. Enzyme activity could be solubilized by 1-5% (w/v) Triton X-100. Gel filtration chromatography showed a M(r) of 320,000. This membrane-associated PAP from neuronal tissue probably belongs among the NEM-insensitive forms of PAP enzymes which have been proposed to play a role in transmembrane signal transduction via phospholipase D.

Bombesin/gastrin releasing peptide (BN/GRP) receptors were solubilized and purified from human glioblastoma (U-118) and lung carcinoid cell lines (NCI-H720). The U-118 cells, when extracted with CHAPS/cholesterol hemisuccinate (CHS), bound (125I-Tyr4)BN with high affinity (Kd = 2 nM) to a single class of sites (Bmax = 150 fmol/mg protein). Specific (125I-Tyr4)BN binding was inhibited with high affinity by BN, GRP, GRP14-27, and receptor antagonists such as (D-Phe6)BN6-13methylester(ME) and (D-Phe6)BN6-13 propylamide(PA) (IC50 = 2, 22, 3, 1 and 2 nM, respectively) but not GRP1-16 or BN1-12. The solubilized and cellular receptor bound peptides with similar affinity. The solubilized receptor was purified using (Lys0, Gly1-4, D-Ala5)BN and (Lys3, Gly4,5, D-Tyr6)BN3-13 PA affinity resins. When eluted from the affinity resins by NaCl, the receptor bound (125I-D-Tyr6)BN6-13ME with high affinity. The NCI-H720 BN/GRP receptor was purified 86,000-fold after extraction with CHAPS/CHS and purification using both affinity resins. SDS-PAGE analysis indicated that major 65 and 115 kDa proteins were purified. These data indicate that BN/GRP receptors can be solubilized from human cells and purified using affinity chromatography techniques with retention of ligand binding activity.

Ca2+ release from skeletal sarcoplasmic reticulum (SR) could be regulated by at least three mechanisms: 1) Ca2+, 2) calmodulin, and 3) Ca2+/calmodulin-dependent phosphorylation. Bell-shaped Ca2+-dependence, of Ca2+ release from both actively- and passively-loaded SR vesicles suggest that opening and closing of the Ca2+ release channel could be regulated by [Ca2+ o] . The time- and concentration-dependent inhibition of Ca 2+ release from skeletal SR by calmodulin was also studied using passively-Ca2+ loaded SR vesicles. Up to 50% of Ca 2+ release was inhibited by calmodulin (0.01–0.5 µM); this inhibition required 5–15 min preincubation time. The hypothesis that Ca2+/calmodulin-dependent phosphorylation of a 60 kDa protein regulates Ca2+ release from skeletal SR was tested by stopped-flow fluorometry using passively-Ca2+-loaded SR vesicles. Approximately 80% of the initial rates of Ca2+-induced Ca2+ release was inhibited by the phosphorylation within 2 min of incubation of the SR with Mg·ATP and calmodulin. We identified two types of 60 kDa phosphoproteins in the rabbit skeletal SR, which was distinguished by solubility of the protein in CHAPS. The CHAPS-soluble 60 kDa phosphoprotein was purified by column chromatography on DEAE-Sephacel, heparin-agarose, and hydroxylapatite. Analyses of the purified protein indicate that the CHAPS-soluble 60 kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding isoforms of PGM were cloned and sequenced using synthetic oligonucleotides. Two types of PGM isoforms (Type I and Type 11) were identified. The translated amino acid sequences show that Type II isoform is SR-form. Our results are significant in terms of understanding evidence of an association of glycolytic and glycogenolytic enzymes with SR and a role in the regulation of SR functions. (Mol Cell Biochem 114: 105-108, 1992)

Characteristics of silica gel dynamically modified with a bile acid derivative have been investigated in liquid chromatography. 3-[(3-cholamidopropyl) dimethylamonio]-1-propane-sulfonate (CHAPS) is selected as modifier. CHAPS possesses a quaternary ammonium group which can be attached to the silica gel surface by cation exchange. The stationary phase modified with CHAPS has been applied to the separation of 1,1′-binaphthyl-2,2′-diyl-hydrogen phosphate enantiomers.

At the concentration needed for NMR, the calcium-saturated form of calcineurin B dissolved in water shows resonance line widths that indicate aggregation of this protein. Although the line width or aggregation state can be influenced to some degree by temperature, pH, and salt concentrations, in the absence of detergent no conditions could be found where the protein behaved as a monomeric unit. In the presence of a 10- to 20-fold molar excess of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), resonance line widths were considerably narrower and were compatible with a protein of approximately 25 kDa. The presence of the NMR signals of the non-deuterated CHAPS does not interfere with modern isotope-directed NMR studies as the signals from protons not attached to 15N or 13C are removed by isotope filtering and purge pulses.

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.

The potential usefulness of a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHPAS), in the stabilization of acidic and basic fibroblast growth factors (FGFs) was examined. Among several detergents, CHAPS was found to be not only non-cytotoxic but also most useful in handling the diluted preparations of FGFs. The advantages are as follows: 1) at lower concentrations than 0.01% CHAPS did not affect growth factor activity of calf serum (CS) and the growth rate of BALB/c 3T3 cells. The primary culture of rat prostate epithelium and colony formation of NRK-49F cells were hardly influenced by CHAPS lower than 0.003%; 2) the loss of FGFs that usually occurs due to their adherence to the surface of storage containers was effectively prevented by inclusion of 0.1% CHAPS; 3) the recovery of FGFs after storage or dialysis was significantly enhanced by inclusion of 0.1% CHAPS; 4) CHAPS at lower concentrations than 0.1% does not interfere with amino acid analysis, except that Thr may be misled only when the ratio of protein/CHAPS is low; 5) amino acid sequence analysis was hardly disturbed by CHAPS up to 0.5%. These results indicate that CHAPS is useful as a stabilizing agent for various kinds of polypeptides capable of showing biological activity at a low concentration.

Addition of the animal ether phospholipid platelet-activating factor, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, (PAF) stimulates medium acidification in cultured soybean (Glycine max L.) cells. The pH of the medium after 8-10 hours is on the average one pH unit lower than in controls. With fusicoccin an average pH difference of 1.7 units is reached. Phospholipids, glycerol, 1-oleyl-2-acetyl-sn-glycerol, 1-0-hexadecyl-sn-glycerol, and triolein at the same concentrations as PAF had no stimulatory effect on medium acidification. The detergents CHAPS and deoxycholate lead to alkalinization of the medium whereas lysophosphatidylcholine (LPC), a detergent with structural similarity to PAF, shows no effect.

[3H]Spiperone [( 3H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [3H]tyramine [( 3H]PTA), thus indicating that [3H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [3H]PTA binding and [3H]DA uptake processes were markedly decreased, with no effect on [3H]mazindol [( 3H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo and in vitro inhibitor of [3H]PTA binding, did not affect the [3H]MAZ binding process. This further supported the suggestion that while [3H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [3H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.

The Stokes radii and the apparent molecular weights of the CHAPS or Triton X-100 solubilized benzodiazepine receptors are calculated by gel exclusion chromatography. The results suggest that the molecular receptor complex solubilized by CHAPS is much larger than the complex solubilized by Triton X-100.

Mammary carcinoma cell membranes were solubilized by a zwitterionic detergent, 3-(3-cholamidopropyl)-dimethyl-ammonio)-propanesulfonate (CHAPS). The solubilized membrane components were resolved by high pressure liquid chromatography (HPLC) using a styrogel protein column. One of the fractions was found to react specifically with anti-F9-antiserum in a solid-phase immunosorbent assay. Following recycling this fraction was purified to homogeneity and had an N-terminal amino acid residue of lysine. Its molecular weight was estimated to be 15,000 daltons. No comparable fraction could be detected in the solubilized membrane components of nonmalignant mammary tissue.