Real-time monitoring of Metridia luciferase release from cells upon interaction with model toxic substances by a fully automatic flow setup - A proof of concept.

This manuscript reports on a fully automatic sequential injection system incorporating a 3D printed module for real-time monitoring of the release of Metridia luciferase from a modified liver epithelial cell line. To this end, a simple and effective approach for the automation of flash-type chemiluminescence assays was developed. The 3D printed module comprised an apical and a basal compartment that enabled monitoring membrane processes on both sides of the cell monolayer aimed at elucidating the direction of luciferase release. A natural release was observed after transfection with the luciferase plasmid by online measurement of the elicited light from the reaction of the synthesized luciferase with the coelenterazine substrate. Model substances for acute toxicity from the group of cholic acids - chenodeoxycholic and deoxycholic acids - were applied at the 1.0 and 0.5 mmol L-1 levels. The tested cholic acids caused changes in cell membrane permeability that was accompanied by an increased luciferase release. The obtained kinetic profiles were evaluated based on the delay between the addition of the toxic substance and the increase of the chemiluminescence signal. All experiments were carried out in a fully automatic system in ca. 5 min per sample in 30 min intervals and no manual interventions were needed for a sampling period of at least 6 h.

Biological Activities and Molecular Docking of Brassinosteroids 24-Norcholane Type Analogs.

The quest and design of new brassinosteroids analogs is a matter of current interest. Herein, the effect of short alkyl side chains and the configuration at C22 on the growth-promoting activity of a series of new brassinosteroid 24-norcholan-type analogs have been evaluated by the rice leaf inclination test using brassinolide as positive control. The highest activities were found for triol 3 with a C22(S) configuration and monobenzoylated derivatives. A docking study of these compounds into the active site of the Brassinosteroid Insensitive 1(BRI1)-ligand-BRI1-Associated Receptor Kinase 1 (BAK1) complex was performed using AutoDock Vina, and protein-ligand contacts were analyzed using LigPlot+. The results suggest that the hydrophobic interactions of ligands with the receptor BRI1LRR and hydrogen bonding with BAK1 in the complex are important for ligand recognition. For monobenzoylated derivatives, the absence of the hydrophobic end in the alkyl chain seems to be compensated by the benzoyl group. Thus, it would be interesting to determine if this result depends on the nature of the substituent group. Finally, mixtures of S/R triols 3/4 exhibit activities that are comparable or even better than those found for brassinolide. Thus, these compounds are potential candidates for application in agriculture to improve the growth and yield of plants against various types of biotic and abiotic stress.

Synthesis and activity evaluation of a series of cholanamides as modulators of the liver X receptors.

The Liver X receptors (LXRs) are members of the nuclear receptor family, that play fundamental roles in cholesterol transport, lipid metabolism and modulation of inflammatory responses. In recent years, the synthetic steroid N,N-dimethyl-3ß-hydroxycholenamide (DMHCA) arised as a promising LXR ligand. This compound was able to dissociate certain beneficial LXRs effects from those undesirable ones involved in triglyceride metabolism. Here, we synthetized a series of DMHCA analogues with different modifications in the steroidal nucleus involving the A/B ring fusion, that generate changes in the overall conformation of the steroid. The LXRα and LXRß activity of these analogues was evaluated by using a luciferase reporter assay in BHK21 cells. Compounds were tested in both the agonist and antagonist modes. Results indicated that the agonist/antagonist profile is dependent on the steroid configuration at the A/B ring junction. Notably, in contrast to DMHCA, the amide derived from lithocholic acid (2) with an A/B cis configuration and its 6,19-epoxy analogue 4 behaved as LXRα selective agonists, while the 2,19-epoxy analogues with an A/B trans configuration were antagonists of both isoforms. The binding mode of the analogues to both LXR isoforms was assessed by using 50 ns molecular dynamics (MD) simulations. Results revealed conformational differences between LXRα- and LXRß-ligand complexes, mainly in the hydrogen bonding network that involves the C-3 hydroxyl. Overall, these results indicate that the synthetized DMHCA analogues could be interesting candidates for a therapeutic modulation of the LXRs.

New cholic acid derivatives: Biocatalytic synthesis and molecular docking study.

A series of cholic acid derivatives was synthesized by enzyme catalysis. Eleven acetyl and ester derivatives of cholic acid, eight of them new compounds, were obtained through regioselective lipase-catalyzed reactions in very good to excellent yield. The influence of various reaction parameters in the enzymatic esterification, acetylation and alcoholysis reactions, such as enzyme source, alcohol or acylating agent: substrate ratio, enzyme: substrate ratio, solvent and temperature, was studied. Moreover, in order to shed light to cholic acid behavior in the enzymatic reactions, molecular docking of the lipase with cholic acid and some derivatives was carried out.

Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.

In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC). The use of TEVC allows for the recording of macroscopic currents elicited by agonist activation of nAChR-DCs that assemble in the oocyte plasma membrane. Furthermore, we examined the stability of nAChR-DCs, which is obligatory for the nAChR crystallization, using a 30 day FRAP assay in LCP for each detergent. The present results indicate a marked difference in the fractional fluorescence recovery (ΔFFR) within the same detergent family during the 30 day period assayed. Within the cholesterol analog family, sodium cholate and CHAPSO displayed a minimum ΔFFR and a mobile fraction (MF) over 80%. In contrast, CHAPS and BigCHAP showed a marked decay in both the mobile fraction and diffusion coefficient. nAChR-DCs containing phospholipid analog detergents with an alkylphosphocholine (FC) and lysofoscholine (LFC) of 16 carbon chains (FC-16, LFC-16) were more effective in maintaining a mobile fraction of over 80% compared to their counterparts with shorter acyl chain (C12, C14). The significant differences in macroscopic current amplitudes, activation and desensitization rates among the different nAChR-DCs evaluated in the present study allow to dissect which detergent preserves both, agonist activation and ion channel function. Functionality assays using TEVC demonstrated that LFC16, LFC14, and cholate were the most effective detergents in preserving macroscopic ion channel function, however, the nAChR-cholate complex display a significant delay in the ACh-induce channel activation. In summary, these results suggest that the physical properties of the lipid analog detergents (headgroup and acyl chain length) are the most effective in maintaining both the stability and functionality of the nAChR in the detergent solubilized complex.

Differential antigenic protein recovery from Taenia solium cyst tissues using several detergents.

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.

Safety, beneficial and technological properties of Enterococcus faecium isolated from Brazilian cheeses.

This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens . Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results.

Safety, beneficial and technological properties of Enterococcus faecium isolated from Brazilian cheeses

This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens. Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results.

Safety, beneficial and technological properties of Enterococcus faecium isolated from Brazilian cheeses

This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens. Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results.(AU)

Structural properties of CHAPS micelles, studied by molecular dynamics simulations.

Detergents are essential tools to study biological membranes, and they are frequently used to solubilize lipids and integral membrane proteins. Particularly the nondenaturing zwitterionic detergent usually named CHAPS was designed for membrane biochemistry and integrates the characteristics of the sulfobetaine-type detergents and bile salts. Despite the available experimental data little is known about the molecular structure of its micelles. In this work, molecular dynamics simulations were performed to study the aggregation in micelles of several numbers of CHAPS (≤ 18) starting from a homogeneous water dilution. The force field parameters to describe the interactions of the molecule were developed and validated. After 50 ns of simulation almost all the systems result in the formation of stable micelles. The molecular shape (gyration radii, volume, surface) and the molecular structure (RDF, salt bridges, H-bonds, SAS) of the micelles were characterized. It was found that the main interactions that lead to the stability of the micelles are the electrostatic ones among the polar groups of the tails and the OH's from the ring moiety. Unlike micelles of other compounds, CHAPS show a grainlike heterogeneity with hydrophobic micropockets. The results are in complete agreement with the available experimental information from NMR, TEM, and SAXS studies, allowing the modeling of the molecular structure of CHAPS micelles. Finally, we hope that the new force field parameters for this detergent will be a significant contribution to the knowledge of such an interesting molecule.

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane.

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a "facial" detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K=0.06mM(-1)), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.

Isolation of acetylated bile acids from the sponge Siphonochalina fortis and DNA damage evaluation by the comet assay.

From the organic extracts of the sponge Siphonochalina fortis, collected at Bahía Bustamante, Chubut, Argentina, three major compounds were isolated and identified as deoxycholic acid 3, 12-diacetate (1), cholic acid 3, 7, 12-triacetate (2) and cholic acid, 3, 7, 12-triacetate. (3). This is the first report of acetylated bile acids in sponges and the first isolation of compound 3 as a natural product. The potential induction of DNA lesions by the isolated compounds was investigated using the comet assay in lymphocytes of human peripheral blood as in vitro model. The results showed that the administration of the bile acid derivatives would not induce DNA damages, indicating that acetylated bile acids are nontoxic metabolites at the tested concentrations. Since the free bile acids were not detected, it is unlikely that the acetylated compounds may be part of the sponge cells detoxification mechanisms. These results may suggest a possible role of acetylated bile acids as a chemical defense mechanism, product of a symbiotic relationship with microorganisms, which would explain their seasonal and geographical variation, and their influence on the previously observed genotoxicity of the organic extract of S. fortis.

High-throughput screening method for lipases/esterases.

High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, ß-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test.

The use of ASB-14 in combination with CHAPS is the best for solubilization of human brain proteins for two-dimensional gel electrophoresis.

Protein extraction is the most important step to reveal a proteome by Two-Dimensional Gel Electrophoresis. Usually, the urea/thiourea based standard protein extraction buffer (SB) is combined with detergents with the aim of achieving better resolution and solubilization of different classes of proteins. In order to produce better gels and achieve the greatest spot resolution of Human Brain Proteins, comparisons using 2-DE of extracted proteins from Human Brain Frontal Cortex with SB constituents (7M Urea, 2M Thiourea and 100mM DTT) were made, using different detergent compositions in the buffer. SB preparations in combination with CHAPS and ASB-14 as well as with ASB-16 (reported for the first time in 2-DE experiments) have been tested. Our results confirm that the most efficient solubilizing solution for 2-DE analysis of cytosolic and membrane Human Brain Proteins is SB combined with 4% CHAPS and 2% ASB-14.

Effect of surfactants and natural detergents on phosphatidylcholine synthesis in photoreceptor membranes.

The synthesis of phosphatidylcholine (PC) in rod outer segments (ROS) catalysed by lysophosphatidylcholine acyltransferase and phosphatidylethanolamine N-methyltransferase (PE N-MTase) was studied and the effects of natural (FA and lysophospholipids) and synthetic (Triton X-100, deoxycholate and CHAPS) surfactants was evaluated. In all experimental conditions used, incorporation of labelled oleate into lysophosphatidylcholine (lysoPC) was at least 40 times greater than oleate incorporation into any other lysophospholipid. Acylation of lysoPC was slightly affected by Triton X-100 and was totally inhibited in the presence of 10 mM sodium deoxycholate (NaDOC) or CHAPS. Below their critical micelle concentration (cmc) Triton X-100 and NaDOC stimulated acylation of all ROS lysophospholipids analysed. The activity of PE N-MTase was stimulated at detergent concentrations below the cmc and inhibited at concentrations above the cmc for all three detergents tested. The effect of FA with differing degree of unsaturation on PC synthesis was evaluated. Oleic acid (10 microM) inhibited methyl group incorporation into total PC, whereas from 100 microM onward, the methylating activity increased with preferential synthesis of PC. Docosahexaenoic acid, in turn, inhibited PE N-MTase activity at every concentration tested. These results suggest that PC synthesis in ROS membranes is modified by bioregulators and surfactants altering the physico-chemical state of the membrane.

Control by signaling modulators of the sorting of canalicular transporters in rat hepatocyte couplets: role of the cytoskeleton.

Hormonal control of the restoration of hepatocanalicular polarity in short-term cultured hepatocyte couplets was analyzed. One hour following isolation, couplets were unable to accumulate the fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), and showed a nonpolarized distribution of F-actin and mrp2 over the cell body. A progressive, time-dependent restoration of couplet-polarized function and morphology was reached after 4 hours of culture. Both dibutyryl cyclic adenosine monophosphate (DBcAMP) and the Ca(2+)-elevating compound, thapsigargin, accelerated restoration of normal couplet morphology and function. The DBcAMP-mediated stimulus was inhibited by the Ca(2+) chelator, 1, 2-bis-(o-aminophenoxy)-ethene-N,N,N',N'-tetra-acetate tetra-(acetomethyl)ester (BAPTA/AM), but not by the protein kinase A (PKA) inhibitors, KT5720 or H89, suggesting that Ca(2+) elevation rather than PKA activation is involved. N-(6-aminohexyl-5-chloro-1-napththalenesulfonamide (W-7), a calmodulin inhibitor, and the protein kinase C (PKC) activator, phorbol dibutyrate, inhibited both the basal and the DBcAMP-stimulated recovery of functional polarity, whereas staurosporine and Gö 6976, 2 PKC inhibitors, accelerated the basal recovery of polarized function. Disruption of the microtubule cytoskeleton by colchicine induced only minor changes under basal, but not under DBcAMP-stimulated, conditions. The Golgi complex disruptor, brefeldin A, significantly delayed, and the microfilament-disrupting agent, cytochalasin D, fully blocked, both processes. However, DBcAMP stimulated trafficking of vesicles containing CLF to the pericanalicular region under the last condition. Our results indicate that restoration of couplet polarity following isolation occurs via a Ca(2+)-calmodulin-mediated mechanism, which depends on microfilament, but not on microtubule integrity. A second pathway is activated by DBcAMP activation via Ca(2+)-calmodulin formation, whose requirements with respect to cytoskeletal components are opposite. PKC has a negative regulatory role in both pathways.

A membrane protein associated with the prolactin receptor. Studies with a photoactivatable human growth hormone derivative.

Prolactin receptor from rat liver (PRL-R, 42 kDa) was cross-linked to a radiolabeled azidophenacyl derivative of human growth hormone ([125I]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 [125I]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 [125I]AP-hGH or [125I]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 degree C), indicating that PRL-R associates to this 50-52 kDa protein prior to hormone binding. Perfusion of rat liver with [125I]AP-hGH shows that this associated protein accompanies the receptor along its intracellular pathway.

Features of the shuttle pair 11 beta-hydroxyprogesterone-11-ketoprogesterone.

11 beta-hydroxyprogesterone (HOP) and 11-ketoprogesterone (KP) are reversible components of a shuttle pair whose interconversion in rat liver is catalyzed by isoform-1 of 11 beta-hydroxysteroid dehydrogenase. Kidneys also produce this interconversion. The present study was carried out to investigate the shuttle pair and its components in the rat. As in corticosterone/11-dehydrocorticosterone, oxidation is more effective at an alkaline pH, while reduction prevails at a neutral pH. Moreover, both reactions are inhibited by the detergent 3-[(3-cholamido propyl)-dimethylammonio]-1-propane-sulphonate (CHAPS). However, at variance with the 11-ketosteroids cortisone (E) and 11-dehydrocorticosterone (A) thought to be "inactive," KP has slight direct Na(+)-retaining properties, and it, as well as HOP, induces glucocorticoids (11 beta-hydroxycorticoids) to retain sodium. 11-ketoprogesterone exhibits 17 times better affinity for native type 1 mineralocorticoid receptor than HOP and a 3-fold affinity for partially purified (transcortin free) mineralocorticoid receptor. However, KP, in contrast to HOP, binds only weakly to transcortin, not at all to glucocorticoid receptor, and requires reduction at C11 for tyrosine aminotransferase (TAT) induction.

Purification of fibroblast growth factor-2 from human placenta using tri(n-butyl)phosphate and sodium cholate.

Tri(n-butyl)phosphate (TNBP) and sodium cholate (SC) mixtures have been used to inactivate lipid-enveloped viruses like HIV and hepatitis B. We exploited the use of this combination to purify fibroblast growth factor-2 (FGF-2) from human placenta. Human placentas were extracted in the presence of 0.3% TNBP/0.2% SC and the clarified homogenate was adsorbed to S-Sepharose. The active fractions were further loaded onto a heparin-Sepharose column and purified FGF-2 was eluted with 2.0 M NaCl. FGF-2 purified this way was indistinguishable from FGF-2 purified without TNBP/SC in the extraction step in terms of yield, specific activity and biological response. The lipid-enveloped vaccinia virus was used in a parallel experiment to evaluate the inactivation capacity of our protocol. Under the conditions described here, the combined use of TNBP/SC did not eliminate but reduced significantly the number of vaccinia virus PFUs by log 2-3.

Phospholipid solubilization during detergent extraction of rhodopsin from photoreceptor disk membranes.

The solubilization of rhodopsin and phospholipids from disks prepared from bovine retinal rods was studied using five different detergents. The relative amounts of rhodopsin and lipid extracted during membrane solubilization differed dramatically with the nature of the surfactant; the two nonpolar detergents, Emulphogene (polyoxyethylene-10 tridecylether) and octylglucoside, removed more protein than lipid; two bile salt-related detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps) and taurocholate, released relatively more lipid than protein; and digitonin, which shares characteristics with both groups of detergents, extracted more lipid per mole of rhodopsin than the former two but less than the latter two. Solubilization was temperature-dependent with all five detergents, though particularly so with octylglucoside: concentrations adequate for the total micellation of disks at 23 degrees C were ineffectual at 4 degrees C. In total solubilizates of disks, the amount of lipid recovered in rhodopsin-lipid-detergent micelles showed a closer correlation with the critical micellar concentration (CMC) than with the chemical nature of the detergent (octylglucoside > taurocholate > Chaps > digitonin > Emulphogene). The higher the CMC, the larger the amount of lipid associated to the solubilized rhodopsin and the larger the amount of lipid reassociated to rhodopsin upon surfactant dilution. For all five detergents, the lipid progressively extracted from disks during solubilization was relatively richer in phosphatidylcholine (PC) than the lipid in the original membranes. The lipid which tended to be associated with rhodopsin in protein-lipid-detergent mixed micelles was also consistently richer in PC than that present in lipid-detergent micelles. Bleaching of solubilized rhodopsin decreased the amount of lipid in protein-lipid-detergent micelles. Rhodopsin photolytic transitions were faster in nonionic than in bile salt-related detergents.