polidocanol and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

The effect of 26 different membrane-perturbing agents on the activity and phase distribution of inositol phosphorylceramide synthase (IPC synthase) activity in crude Candida albicans membranes was investigated. The nonionic detergents Triton X-100, Nonidet P-40, Brij, Tween, and octylglucoside all inactivated the enzyme. However, at moderate concentrations, the activity of the Triton X-100- and octylglucoside-solubilized material could be partially restored by inclusion of 5 mM phosphatidylinositol (PI) in the solubilization buffer. The apparent molecular mass of IPC synthase activity solubilized in 2% Triton X-100 was between 1.5 x 10(6) and 20 x 10(6) Da, while under identical conditions, octylglucoside-solubilized activity remained associated with large presumably membrane-like structures. Increased detergent concentrations produced more drastic losses of enzymatic activity. The zwitterionic detergents Empigen BB, N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB), Zwittergent 3-10, and amidosulfobetaine (ASB)-16 all appeared capable of solubilizing IPC synthase. However, these agents also inactivated the enzyme essentially irreversibly. Solubilization with lysophospholipids again resulted in drastic losses of enzymatic activity that were not restored by the inclusion of PI. Lysophosphatidylinositol also appeared to compete, to some extent, with the donor substrate phosphatidylinositol. The sterol-containing agent digitonin completely inactivated IPC synthase. By contrast, sterol-based detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and taurodeoxycholate (tDOC) had little or no effect on the enzyme activity. The IPC synthase activity in C. albicans membranes remained largely intact and sedimentable at CHAPS concentrations (4%) where >90% of the phospholipids and 60% of the total proteins were extracted from the membranes. At 2.5% CHAPS, a concentration where approximately 50% of the protein and 80% of the phospholipids are solubilized, there was no detectable loss of enzyme activity, and it was found that the detergent-treated membranes had significantly improved properties compared to crude, untreated membranes as the source of IPC synthase activity. In contrast to assays utilizing intact membranes or Triton X-100 extracts, assays using CHAPS- or tDOC-washed membranes were found to be reproducible, completely dependent on added acceptor sub

Topics: 4-Chloro-7-nitrobenzofurazan; Biochemistry; Butyrates; Candida albicans; Centrifugation, Density Gradient; Ceramides; Cetomacrogol; Cholic Acids; Chromatography; Detergents; Dose-Response Relationship, Drug; Glucosides; Hexosyltransferases; Kinetics; Lipids; Lysophospholipids; Octoxynol; Phospholipids; Polidocanol; Polyethylene Glycols; Polysorbates; Quaternary Ammonium Compounds; Taurodeoxycholic Acid; Time Factors

The multidrug resistance protein (ABCC1 or MRP1) causes resistance to multiple drugs through reduced drug accumulation. We have previously demonstrated direct interaction between MRP1 and unmodified drugs using photoreactive drug analogues. In this study, we describe the use of [125I]iodoaryl azido-rhodamine123 (IAARh123)-a photoactive drug analogue of rhodamine 123, to study the effects of mild detergents and denaturing agents on MRP1-drug binding in membrane vesicles prepared from HeLa cells transfected with the MRP1 cDNA. Our results show that the zwitterionic detergent CHAPS and a nonionic detergent Brij35 inhibited the photolabeling of MRP1 with IAARh123. Sodium deoxycholate (SDC) and octyl-beta-glucoside (OG), structurally similar to CHAPS and Brij35 and disrupting the lipid bilayer, showed a modest increase of MRP1 photolabeling with IAARh123. Proteolytic digestion of IAARh123 photolabeled MRP1 labeled in the presence or absence of various detergents (CHAPS, SDC, or OG) revealed identical photolabeled peptides. Consistent with the drug-binding results, non-toxic concentrations of CHAPS and Brij35 reversed vincristine and etoposide (VP16) toxicity in MRP1 expressing cells. Taken together, the results of this study show that MRP1-drug interaction can occur outside the lipid bilayer environment. However, this interaction is inhibited with certain mild detergents.

Topics: Antineoplastic Agents; Binding Sites; Cell Survival; Cholic Acids; Detergents; Drug Interactions; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Etoposide; HeLa Cells; Humans; Lipid Bilayers; Multidrug Resistance-Associated Proteins; Photoaffinity Labels; Polidocanol; Polyethylene Glycols; Protein Structure, Tertiary; Rhodamine 123; Vincristine

Adenylyl cyclase (AC) was extracted from human myometrium with either non-ionic (Lubrol-PX or Triton X-100) or zwitterionic (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS) detergents. The soluble enzyme was stimulated by forskolin, a hydrophobic activator, in the presence of Mg2+ indicating that the catalytic subunit had not been damaged after solubilization. The enzyme was also activated by 5'-guanylyl imidodiphosphate (Gpp(NH)p) showing that the catalytic unit was not separated from stimulatory guanine nucleotide binding protein (Gs) during the extraction. Both activators showed different effects on the stimulatory efficacy and potency of AC activity solobulized with detergents. Gel filtration of Lubrol-PX and CHAPS extracts over a Sepharose CL-2B column partially resolved AC and its complexes. The chromatographic profile for Lubrol-solubilized AC presented a main peak of about 200 kDa whereas CHAPS-solubilized AC showed a dominant peak of about 1100 kDa. The heterodisperse peaks obtained revealed that the catalytic AC subunit was not separated from Gs proteins after gel filtration, and that AC could be associated with other cellular proteins. When Lubrol extract was submitted to anionic-exchange chromatography, the enzyme was purified about 7.5 fold (enzymatic activity of 48.1 pmol/min/mg of protein). The catalytic subunit was co-eluted with both AC-activating proteins Galphas large (52.2 kDa) and Galphas small (48.7 kDa). This is the first demonstration of the stable physical association of AC with both alphas subunits of G proteins in human myometrium.

Topics: Adenylyl Cyclases; Cholic Acids; Chromatography, Agarose; Detergents; Female; GTP-Binding Protein alpha Subunits, Gs; Humans; Myometrium; Octoxynol; Polidocanol; Polyethylene Glycols; Solubility

The differential effects of representative, commonly available ionic (SDS), nonionic (Brij 35, Tween 20, and Triton X-100), and zwitterionic (Chaps) detergents on the catalytic activity and properties of human cathepsins B, L, S, and K were examined. The presence of detergents in the assay buffer affected the activity of cathepsins to variable extents; Chaps enhanced the activity of all the enzymes while SDS was most detrimental. Tween 20 lowered cathepsin S activity, while it slightly enhanced that of all other cathepsins studied. The presence of detergents in the activation buffer was clearly beneficial to both cathepsins L and K, possibly by favoring the release of the enzyme from the walls of the incubation vessel. Overall, the results indicate that Chaps is the optimal detergent for use with this family of enzymes.

Topics: Cathepsin B; Cathepsin K; Cathepsin L; Cathepsins; Cholic Acids; Cysteine Endopeptidases; Detergents; Dose-Response Relationship, Drug; Endopeptidases; Humans; Kinetics; Octoxynol; Polidocanol; Polyethylene Glycols; Polysorbates; Recombinant Proteins; Sodium Dodecyl Sulfate; Spectrometry, Fluorescence

Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to rat brain membranes (RBM) is enhanced nine-fold by EDTA/water dialysis and 1.3- to 4.2-fold by 50 nM ketosteroid R 5135, or 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or related piperazine-N-alkanesulfonate buffers, or extensive washing with NaCl/Na phosphate or Na phosphate/citrate solution. About one-fifth of the [35S]TBPS binding capacity appears in the soluble fraction whereas the rest remains in particulate form on treatment of the EDTA/water-dialyzed RBM with 20 mM CHAPS. Similar KD values (64-86 nM) are obtained for the original EDTA/water-dialyzed membranes and the CHAPS-treated and/or -solubilized preparations. The Bmax of the EDTA-treated RBM is reduced five-fold on solubilization with CHAPS. The potency for displacement of [35S]TBPS changes in the presence of CHAPS or on CHAPS solubilization: gamma-aminobutyric acid (GABA) and muscimol inhibit specific [35S]TBPS binding more strongly in the absence than in the presence of CHAPS: TBPS, picrotoxinin, and photoheptachlor epoxide are almost equally active with RBM, RBM + CHAPS, and RBM solubilized with CHAPS. Levels of (1R, alpha S)-cis-cypermethrin and dimethylbutylbarbiturate which are inhibitory with RBM are moderately stimulatory after TBPS receptor solubilization. Thus CHAPS defines three regions of the GABA receptor-ionophore complex, i.e., the GABA and benzodiazepine receptors, the TBPS/picrotoxinin/polychlorocycloalkane receptor(s), and the sites at which the alpha-cyano pyrethroid and the barbiturate interact with TBPS binding.

Topics: Animals; Binding Sites; Brain; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Bridged-Ring Compounds; Cholic Acids; Edetic Acid; Kinetics; Male; Membranes; Polidocanol; Polyethylene Glycols; Rats; Receptors, GABA-A; Solubility