Self-cleavage of p2Sp1 RNA with Mg2+ and non-ionic detergent (Brij 58)

The precursor of an RNA molecule from T4-infected E. coli cells (p2Sp1 RNA) has the capacity to cleave itself at specific positions [(UpA (139-140) and CpA (170-171)], within a putative loop and stem structure. This sequence-specific cleavage requires at least a monovalent cation and non-ionic detergents. We studied the self-cleavage reaction of an RNA fragment (GUUUCGUACAAAC) (R1) with the sequence corresponding to the p2Sp1 RNA in the presence of Mg2+ and non-ionic detergents. It requires Mg2+ and is aided by a non-ionic detergent, Brij 58. The cleavage reaction is time, temperature, and pH-dependent. The cleavage occurs at the phosphodiester bond between UpA and CpA on the RNA fragment (GUUUCGUACAAAC) (R1). Furthermore, the maximum of cleavage of R1 occurs at a very low Mg2+ concentration (< or = 5 mM).     

Effects of polyoxyethylene detergent (Brij 58) on platelet aggregation,release and clotting activity

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet-rich but had no effect on primary aggregation. Thrombin-induced aggregation of washed human platelets suspended in Tyrode's buffer was inhibited after incubation of cells with 4 · 10^?6 M detergent. Efflux of [¹⁴C]serotonin, ⁴⁵Ca²⁺ and labile aorta contracting substance (thromboxane A₂) and development of prothrombin-converting activity (platelet factor 3) were abolished concomitantly. Aggregation of washed platelets either by sodium arachidonate or by collagen was also inhibited by the same concentration of Brij 58 which inhibited thrombin aggregation. This concentration did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 4 · 10^?5 M, lysed the cells liberating lactate dehydrogenase, serotonin and Ca²⁺. When albumin was included as a platelet stabilizer in the suspending medium the concentration of detergent required for the inhibitory effects was increased ten-fold. This could be attributed to competitive binding of the detergent to albumin, demonstrated with [¹⁴C]acetylated Brij 58. A variety of other polyoxyethylene detergents, at concentrations from 8 · 10^?4 to 5 · 10^?3 M, also inhibited platelet aggregation induced by thrombin. It is concluded that low concentrations of Brij 58 stabilize the platelets against the action of aggregation agents, while higher concentrations produce membrane destabilization and cell lysis.     

Effects of a polyoxyethylene detergent (Brij 58) on platelet aggregation, release and clotting activity

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet-rich plasma but had no effect on primary aggregation. Thrombin-induced aggregation of washed human platelets suspended in Tyrode's buffer was inhibited after incubation of cells with 4.10(-6) M detergent. Efflux of [14C]serotonin, 45Ca2+ and labile aorta contracting substance (thromboxane A2) and development of prothrombin-converting activity (platelet factor 3) were abolished concomitantly. Aggregation of washed platelets either by sodium arachidonate or by collagen was also inhibited by the same concentration of Brij 58 which inhibited thrombin aggregation. This concentration did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 4.10(-5) M, lysed the cells liberating lactate dehydrogenase, serotonin and Ca2+. When albumin was included as a platelet stabilizer in the suspending medium the concentration of detergent required for the inhibitory effects was increased ten-fold. This could be attributed to competitive binding of the detergent to albumin, demonstrated with [14C]acetylated Brij 58. A variety of other polyoxyethylene detergents, at concentrations from 8.10(-4) to 5.10(-3) M, also inhibited platelet aggregation induced by thrombin. It is concluded that low concentrations of Brij 58 stabilize the platelets against the action of aggregating agents, while higher concentrations produce membrane destabilization and cell lysis.     

Nitroxide spin labeled analogs of the non-ionic detergent triton X-100

Two nitroxide spin labeled analogs of the non-ionic detergent, Triton X-100, have been synthesized. Electron spin resonance spectra of these compounds in solution, in egg lecithin multilayers, and in aqueous dispersion of dimyristoylphosphatidylcholine vesicles are described.     

Evidence for cooperativity of protein dissolution in Brij 58 permeabilized L929 cells

Mouse L929 cells were exposed to the nonionic detergent Brij 58. As has been shown in some other cell types, protein leaked from Brij 58 exposed cells only after a lag phase. In the current study we have extended the observations of the kinetics of protein efflux using cultured L cells subjected to treatment with buffers containing Brij 58. The results show that while the cells become permeable essentially at first exposure to the detergent, proteins do not escape immediately. This lag in efflux is at least partly dependent on the concentration of detergent such that a greater lag is seen in cells exposed to the lowest concentrations of Brij. Data are presented that are most readily interpreted as protein leakage having occurred fairly rapidly from individual cells and that show that the time course of protein efflux results, to a large extent, from different sensitivities of individual cells to the detergent. The permeabilized suspension cells consist of only two types, whereas the conversion of cells from one type to the other occurs through the loss of protein to the permeabilization medium. Only two bands are seen in continuous density gradients and there is a conversion of the more dense type to the less dense with longer exposure to detergent. Moreover, the less dense cells contained about half of the protein per cell as the bottom banding cells, and the proteins of the more dense cells appear to be the sum of those released into the permeabilization medium plus those found in the less dense cells.     

Phosphorus NMR analysis of human white matter in mixed non-ionic detergent micelles

In order to study the lipid composition of human white matter, we have developed a 31P NMR spectroscopy method, which allows the determination and quantitation of the main phospholipids found in biological membranes. The technique is based upon the use of a non-ionic detergent (Triton X-100) which induces, in aqueous media, the formation of mixed micelles that are magnetically isotropic. The linewidths and chemical shifts depend on both the molar ratio detergent/phospholipid and the pH of the suspension. After determination of the optimum values for these two parameters, 31P NMR spectra were recorded, in which all phospholipid resonances were resolved. After determining precise chemical shifts for each phospholipid, concentrations were measured by comparing the peak areas with that of an internal standard. Analysis of the complex phospholipid composition of human white matter using this method gave values very close to that found in the literature for such tissue. Moreover this nondestructive method proved to be very sensitive since less than 1 mg of a mixture of phospholipids was needed.     

The major sialoglycoprotein of the human erythrocyte membrane. Release with a non-ionic detergent and purification

The major sialoglycoprotein of the human erythrocyte membrane has been selectively released by the non-ionic detergent Tween 20 and further purified in detergent-free buffers by hydroxyapatite chromatography and, finally, by hydrophobic interaction chromatography on pentyl-Sepharose. The purified glycoprotein shows one main zone, PAS-1, and up to three minor zones after staining both for protein and carbohydrate in polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The relative staining intensities are concentration dependent. When the purified glycoprotein has been heated to 100 °C in dodecyl sulfate, more stain appears in the most rapid zone, PAS-2, and less in the slower zones, indicating a disaggregation of oligomeric forms of this glycoprotein, including a dinier, PAS-1.     

Solubilisation of human erythrocyte band 4.1 protein in the non-ionic detergent Tween 20

Extraction of spectrin-depleted erythrocyte membranes with the non-ionic detergent Tween 20, in a 0.1 M glycine-NaOH buffer (pH 9.8) leads to the solubilization of band 4.1 and the sialoglycoproteins. The comigration of band 4.1 with the sialoglycoproteins in gel filtration and detergent-free electrophoresis indicated that these proteins may be associated as complexes of high molecular weight. Although treatment of intact membranes with Tween 20 under the same conditions does not lead to direct solubilization of proteins, severe disruption of the membranes was observed under phase contrast microscopy. Suspension of the treated membranes in 5 mM phosphate buffer (pH 8.0) leads to the solubilization of band 4.1, spectrin, actin and the sialoglycoproteins. High molecular weight complexes of band 4.1 and the sialoglycoproteins were isolated from these extracts, suggesting a possible interaction between band 4.1 and sialoglycoproteins which may be important for linking the cytoskeleton to the membrane.     

The major sialoglycoprotein of the human erythrocyte membrane. Release with a non-ionic detergent and purification.

The major sialoglycoprotein of the human erythrocyte membrane has been selectively released by the non-ionic detergent Tween 20 and further purified in detergent-free buffers by hydroxyapatite chromatography and, finally, by hydrophobic interaction chromatography on pentyl-Sepharose. The purified glycoprotein shows one main zone, PAS-1, and up to three minor zones after staining both for protein and carbohydrate in polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The relative staining intensities are concentration dependent. When the purified glycoprotein has been heated to 100 degrees C in dodecyl sulfate, more stain appears in the most rapid zone, PAS-2, and less in the slower zones, indicating a disaggregation of oligomeric forms of this glycoprotein, including a dimer, PAS-1.     

Effect of Brij 58 on the hydrolysis of methyl butyrate by lipase from Pseudomonas fluorescens

Purified Pseudomonas fluorescens lipase [EC 3.1.1.3] exhibited slight activity on water-soluble esters such as methyl butyrate, and this activity was increased on addition of Brij 58 (20 oxyethylene hexadecyl ether) to the solution. This stimulating effect of Brij 58 on hydrolysis of various esters (dimethyl succinate, butyl n-acetate, and tributyrin) in aqueous solution was unspecific. Hydrolysis of methyl butyrate depended on the molecular ratio of Brij 58 to lipase, being maximal (about 8 times the basal level at 37 degrees C with 80 mM substrate in 0.1 M NaCl solution) with 30 mol of Brij 58 per mol of lipase. Comparative studies showed that all polyoxyethylene (POE) alkyl ethers tested, stimulated the methyl butyrate hydrolyzing activity and that the Adekatol SO series (dihydric normal alcohol ethoxylate) also stimulated the appreciably active, whereas Triton X-100, sodium cholate, sodium deoxycholate, sodium dodecylsulfate, POE, and fatty acids had no effect. Comparison of the effects of Brij 58 on the methyl butyrate hydrolyzing activities of various lipolytic enzymes indicated that its effect was specific for this lipase. Brij 58 had no detectable effect with emulsified esters, such as supersaturated methyl butyrate and triolein.     

Crossed immunoelectrophoresis of bovine milk fat globule membrane protein solubilized with non-ionic detergent.

Detergent solubilized bovine milk fat globule membrane material studied by crossed immunoelectrophoresis combined with histochemical techniques revealed four major protein complexes. All four were found to bind to concanavalin A and three were identified as sialoglycoproteins. Xanthine oxidase activity was associated with the non-sialoglycoprotein precipitate. Immunoabsorption with intact milk fat globules showed an internal location of the xanthine oxidase, whereas the three other main proteins plus Mg2+-ATPase and 5'-nucleotidase were disposed on the outer membrane surface. The major proteins from milk fat globule membrane and membrane material isolated from skim milk showed immunochemical identity.     

Characteristics of n-octyl beta-D-thioglucopyranoside, a new non-ionic detergent useful for membrane biochemistry.

n-Octyl beta-D-thioglucopyranoside, a new non-ionic detergent, was synthesized. Properties, and applicability to membrane proteins, of this detergent were investigated. The detergent was easily removed by dialysis. The solubilizing power of this detergent for Escherichia coli membrane proteins was similar to that of n-octyl beta-D-glucopyranoside, which has been widely used in membrane biochemistry. No inactivation of proteins was observed after the solubilization. n-Octyl beta-D-thioglucopyranoside was superior to n-octyl beta-D-glucopyranoside in that it was much more stable and could be synthesized at much lower cost.     

Non-lytic, non-ionic detergent extractions of plasma membrane constituents from normal and transformed fibroblasts

A technique has been developed for the selective extraction of plasma membrane protein constituents from normal and transformed cells employing non-ionic detergents. The extraction procedure does not damage cells as judged by cell viability, ⁵¹Cr release, and trypan blue staining. Lactoperoxidase-catalyzed iodina- tion followed by detergent extraction permits demonstration of a 100 000 dalton protein which is found on the surface of normal but not transformed hamster and mouse fibroblasts.