brij-58 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

We have investigated the possibility of cell-fee synthesis of membrane proteins in the absence of a membrane and in the presence of detergent. We used the bacterial mechanosensitive channel MscL, a homopentamer, as a model protein. A wide range of nonionic or zwitterionic detergents, Triton X-100, Tween 20, Brij 58p, n-dodecyl beta-D-maltoside, and CHAPS, were compatible with cell-free synthesis, while n-octyl beta-D-glucoside and deoxycholate had an inhibitory effect. In vitro synthesis in the presence of Triton X-100 yielded milligram amounts of MscL per milliliter of lysate. Cross-linking experiments showed that the protein was able to oligomerize in detergents. When the purified protein was reconstituted in liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similar to that of the recombinant protein produced in Escherichia coli. Cell-free synthesis of membrane proteins should prove a valuable tool for the production of membrane proteins whose overexpression in heterologous systems is difficult.

Topics: Cell Membrane Permeability; Cell-Free System; Cetomacrogol; Cholic Acids; Deoxycholic Acid; Detergents; Escherichia coli Proteins; Genetic Vectors; Glucosides; Ion Channels; Lipid Bilayers; Mechanotransduction, Cellular; Octoxynol; Patch-Clamp Techniques; Polymers; Polysorbates; Solubility

Unlike most other mammalian species, domestic cats glucuronidate phenolic compounds poorly and are therefore highly susceptible to the toxic side effects of many drugs, including paracetamol. In this study, we evaluated the role of enzyme constraint, a characteristic that limits the activity of all uridine 5'-diphosphoglucuronosyltransferase (UGT) enzymes, in the aetiology of this species-dependent defect of drug metabolism. Detergent activation experiments were performed using hepatic microsomes from cats (4), dogs (4), man (4), and 6 other mammalian species (1 liver each). In addition, we used microsomes from Gunn rats which are sensitive to paracetamol toxicity because of a genetic defect affecting all family 1 UGTs. Increase in paracetamol-UGT activity at optimum concentrations of detergent was used as an index of enzyme constraint. Native activity (measured in the absence of detergent) was less than one-sixth in cats compared with other species. Optimum detergent treatment tended to enhance rather than abolish this difference, however, indicating relatively lower levels of constraint of paracetamol-UGT in cats compared with other species. Similarly, detergent treatment failed to reduce the native activity difference between homozygous mutant and normal Gunn rats. Initially CHAPS (3-(3-cholamidopropyl)-dimethylammonio-1-propanesulphonic acid) was used as the detergent activator; in 3 of 4 microsomal preparations from man, however, inhibition rather than activation was observed at all detergent concentrations used. Studies were repeated using the non-ionic detergent, Brij 58 (polyoxyethylene 20-cetyl ether), which resulted in similar although more profound activation and no inhibition. We conclude that deficient paracetamol glucuronidation in cats does not result from increased paracetamol-UGT constraint in this species compared with other mammalian species. Other causes, such as differences in enzyme protein concentration or substrate affinity might be responsible.

Topics: Acetaminophen; Analgesics, Non-Narcotic; Animals; Cats; Cattle; Cetomacrogol; Cholic Acids; Detergents; Dogs; Female; Glucuronosyltransferase; Horses; Humans; Macaca; Male; Mice; Microsomes, Liver; Mutation; Rabbits; Rats; Rats, Gunn; Species Specificity; Surface-Active Agents; Swine