Presence Of Nonionic Detergent Research Articles

Determination of inorganic phosphate in the presence of nonionic detergents: A modified isobutanol-benzene extraction method

Inorganic phosphate can be determined either radiometrically or spectrophotometrically after extraction of its complex with molybdate into an organic phase. Triton X-100 interferes with this extraction. Determination of the inorganic phosphate can be carried out in the presence of up to 0.8% ( w v ) Triton X-100 by modification of the method: After addition of the silicotungstate, the sample is centrifuged, the yellow oily phase removed, and a sufficient amount of silicotungstate added again. Lubrol WX interfered even more drastically than Triton X-100, but the modified method was effective in the presence of 0.04% ( w v ) Lubrol WX. The method was useful in biochemical assays such as those of ATPase and cyclic nucleotide-dependent phosphodiesterase.

Purification and Properties of Boar Acrosin

An isolation procedure in the presence of non-ionic detergents has been developed for the large-scale preparation of boar acrosin. Five steps including hydrophobic interaction chromatography on phenyl-Sepharose resulted in a 161-fold purification of the enzyme with an accumulation yield of 41%. The resultant acrosin preparation had a molecular weight of 38,000, an isoelectric point of 10.5 and a specific activity of 37 U/mg. Apparent homogeneity was judged by sodium dodecyl sulfate electrophoresis and isoelectric focusing. A polarity index of 41.2% was calculated from the amino acid composition. Acrosin was stable at pH 5.5 in the presence of 0.1% Triton X-100. In the absence of detergent acrosin was strongly adsorbed on plastic surfaces.

An improved method for the preparation of undegraded polysomes and active messenger RNA from immature chicken erythrocytes

A simple method for the large-scale isolation of undegraded polysomes and active mRNA from immature hen erythrocytes is described. The method is based on the removal of white cells from heparinized blood by adsorption on sulfoethyl- or sulfopropyl-Sephadex. Red cells thus purified can be stored frozen and lysed in the presence of nonionic detergents. The yield of polysomes is about twice that obtained by the usual mild lysis procedures designed to prevent lysis of lysosomes from contaminating white cells.

Procaine, a Local Anesthetic Interacting with the Cell Membrane, Inhibits the Processing of Precursor Forms of Periplasmic Proteins in Escherichia coli

Treatment of Escherichia coli cells with procaine (0.55%, w/v) results in the accumulation of precursor in addition to mature forms of two periplasmic proteins, alkaline phosphatase and glutamine-binding protein. The precursor form of alkaline phosphatase has a higher molecular weight than the mature form by about 2600. An experimental technique is described to isolate and purify precursor forms of any presumably exported protein. After the membrane solubilization step in the presence of nonionic detergent, a peptidase is stimulated, resulting in partial cleavage of the precursors. The products of this cleavage have been identified as the mature protein and presumably the signal peptide in the case of alkaline phosphatase. The amino acid composition of this peptide, which is comprised of 25 residues, has been determined. Procaine (0.55%, w/v) causes an increase in molecular packing of lipid molecules in the membrane which might result in an alteration of membrane fluidity sufficient for selective inhibition of processing of precursors of exported proteins.

Membrane biogenesis: cotranslational integration of the bacteriophage f1 coat protein into an Escherichia coli membrane fraction.

The coat protein (CP) of bacteriophage f1 is integrated into an Escherichia coli plasma membrane fraction consisting of inverted vesicles when it is synthesized in a cell-free, coupled transcription--translation system supplemented with the inverted vesicles. By using proteolytic enzymes as probes, we found by subsequent peptide mapping and determination of the sequence of the proteolytic products that CP was inserted into the inverted vesicles in an orientation indistinguishable from that in inverted vesicles prepared from infected E. coli: only a COOH-terminal portion of approximately 10 residues was accessible to proteolysis, whereas the remainder of CP (CP') was entirely protected. Protection of CP' was dependent on the integrity of the vesicle membrane, because it was abolished when proteolysis was done in the presence of nonionic detergents. Insertion was observed when the inverted vesicles were present during translation in the cell-free system, not when they were added after translation. Thus, the asymmetric insertion of this type of integral membrane protein is strictly coupled to translation. These findings are discussed with respect to prokaryotic membrane biogenesis and are related to bacteriophage f1 assembly and infection.

Lymphocyte Associated β2‐Microglobulin Studied by Crossed Radioimmunoelectrophoresis

A sensitive radioimmunoelectrophoretic assay was employed in the study of beta 2-microglobulin (beta2m) from human blood lymphocytes, allowing the detection of 2.5 pmol of beta2m corresponding to 3 x 10(6) lymphocytes. The intrinsic (amphiphilic) membrane proteins were solubilized and examined in crossed immunoelectrophoresis against rabbit antisera in the presence of non-ionic detergent. The beta2m associated precipitate was traced by post-electrophoretic incubation with 125I-labelled antibodies to beta2m and autoradiography. A polyspecific rabbit antiserum to human lymphoid cells retained its capacity to precipitate lymphocyte beta2m after absorption with isolated beta2m (on an immunosorbent column), showing that lymphocyte beta2m is complexed to other molecules. No free beta2m was found on lymphocytes when examined against the absorbed antiserum to human lymphoid cells in an intermediate gel and anti-beta2m in a reference gel.

Sialyltransferases in rat brain: intracellular localization and some membrane properties.

Abstract— Total rat cerebral homogenate, with nuclei removed, yielded sialyltransferase activity peaks that were distinct from the protein distribution profile in a continuous sucrose density gradient. Marker enzyme studies and electron microscopic examinations on the gradient fractions suggested that most of the sialyltransferase activities were not associated with the synaptosomes.The sialyltransferases appeared to be localized in the smooth microsomal membranes and the Golgi complex derivatives. The sialyltransferase activities were stimulated by non‐ionic detergent mixture, Triton CF‐54/Tween 80 (2/1, w/w), the effect being much more pronounced with exogenous substrates. The stimulatory effect was dependent on detergent concentration. With 1 mg detergent mixture per mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; exogenous GM1a, 700; exogenous DS‐fetuin, 230. The action of the nonionic detergents appears to be on a hydrophobic segment of the enzyme molecule, bearing the active site, which is buried in the membrane lipid bilayer. This was substantiated by the partial trypsin resistance of the sialyltransferase activities and the abolition of that resistance when trypsiniza‐tion was performed in the presence of nonionic detergents. Furthermore, the sialyltransferase activities were markedly inhibited by organic solvents; and these inhibitory effects were inversely proportional to the solvent dielectric constants.

Structural data concerning the major rat brain myelin proteolipid P7 apoprotein

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Characteristics and hormonal control of some glycoprotein glycosyl-transferase reactions in male reproductive organs

UDP-galactose: glycoprotein galactosyltransferase and GDP-focuse: glycoprotein fucosyltransferase activities were found to be exceptionally high in homogenates of rat ventral prostate and certain other male secondary sexual organs. Nearly all of the intracellular forms of these glycosyltransferases in prostate were associated with readily sedimentable membranous structures and were maximally active only in the presence of non-ionic detergents. Rat and human prostatic secretions, and especially human seminal plasma, also are strikingly rich, in comparison with blood serum, in glycoprotein galactosyl-transferases that are present in these fluids in soluble forms that are not activated by detergents. Very little galactosyltransferase is associated with spermatozoa. The properties of prostatic fluid and seminal plasma galactosyl-transferases are very similar to those of the enzyme present in mammary gland and milk, although functional modifier proteins resembling α-lactalbumin appear to be absent from male genital glands and their fluids. The glycoprotein galactosyltransferase and fucosyltransferase activities of rat prostate decline fairly slowly after orchiectomy and are gradually enhanced in castrates by androgen treatment in a manner which suggests that their levels vary coordinately with elaboration of endoplasmic reticulum and Golgi membranes in this tissue. Prolactin does not affect prostatic galactosyltransferase in hypophysectomized animals or its enhancement by androgens.

Subunit structure of the glycoprotein complex of avian tumor virus

Envelope glycoprotein of avian tumor virus is linked by disulfide bonds in a structure that we have designated VGP to stand for viral glycoprotein. VGP appears to contain one molecule of gp85 and one of gp37. Under nonreducing conditions, VGP is the only glycoprotein component that is stable in the presence of ionic detergent, although in the presence of nonionic detergent two or more VGPs are associated in discrete complexes. The disulfide bonds linking viral glycoprotein are formed before release of virus from infected cells.

Assays for cellular antigens in the presence of detergents

AbstractWhen red blood cells or lymphocytes were fixed with glutaraldehyde, binding assays for determinants recognized by antibodies could be carried out in the presence of nonionic detergents. Specific inhibition of the binding assays by detergent‐solubilized membranes and cell extracts was possible, thus allowing rapid routine assays of detergent extracts during the purification of membrane components. The assay system was established for rat histocompatibility antigens and for cell‐associated immunoglobulin.

Protein synthesis in the presence of non-ionic detergents in a cell-free system from rabbit reticulocytes.

Protein synthesis in the presence of non-ionic detergents in a cell-free system from rabbit reticulocytes.

An efficient procedure for the isolation of polyribosomes from tissue culture.

In this paper an efficient procedure is described for the isolation of polysomes from tissue culture cells. Optimal yields, especially of the heavier classes of polysomes can only be obtained at a rather high KC1 concentration in the presence of nonionic detergent. Evidence is provided that the lower polysomal yield after extraction at low salt concentration is not due to breakdown of the polysomes by RNAase.Furthermore, the yield of polysomes is pH dependent only at low salt concentration, while at high salt concentration no influence of the pH is observed. Refreshing of growth medium several hours before harvesting the cells, increases the ratio of polysomes to 80 S monomers.

Interactions in the system: Clay‐detergent‐cellulose

AbstractThe McBain equation describing detergency was used to study the process cellulose·clay+detergent= cellulose·detergent+clay·detergent, by taking thoroughly purified cotton, montmorillonite swelling clay, and n−C12H25O (CH2CH2O)−14H as well as other nonionic and anionic detergents. The three binary equilibria which add up to this ternary system were also studied.X‐ray diffraction showed that monionic detergents sorbed by the clay were intercalated between adjacent, 9.5 Å thick lattice layers of clay with their chains parallel to these layers, in sheets one or two molecules thick. The process was accompanied by dehydration and proceeded down to low equilibrium detergent concentrations. The deflocculation of sodium montmorillonite suspensions by polyoxyethylated compounds was shown by an increase in turbidity and decreases in viscosity and sedimentation volume. Cellulose sorbed C12H25O (CH2CH2O)14H reversibly. Its uptake was considerably less than that of clay on a weight basis but was comparable on an area basis.Kaolinite and calcium montmorillonite picked up by cellulose from aqueous suspensions were gradually removed by washing with water, following first‐order kinetics.Of the sodium montmorillonite picked up, 0.13±0.01% could not be removed by washing with water. This level of tenaciously retained clay was independent of clay concentration, rate of stirring, and temperature. The ash of fabric treated with sodium montmorillonite was a shrunken replica of the fabric, preserving microscopic details faithfully. This and comparison of the specific surface areas of cotton and sodium montmorillonite indicate that cotton was completely coated with clay to an average depth of three to four lattice layers. The reason for this thickness is that the primary particles in sodium montmorillonite dispersions are packets of three to four lattice layers.Anionic and cationic detergents removed little or none of the 0.13% sodium montmorillonite. Polyoxyethylated and polyoxypropylated compounds removed part but always left behind at least 0.04%. This is the amount of clay required to cover the cellulose surface completely with a sheet of single, nonoverlapping lattice layers. However, when brought into contact in the presence of nonionic detergents, cellulose retained no sodium montmorillonite at all.

Multiplicity of mitochondrial monoamine oxidases

There have been only two brief reports which presented direct evidence of the existence of more than one monoamine oxidase in mitochondria although this has been suggested by many workers. Gorkin (1963) partially separated p-nitrophenylethylamine oxidase activity from m-nitro-p-hydroxybenzylamine oxidase activity by ion exchange chromatography of mitochondrial extracts in the presence of nonionic detergent (OP-10). Youdim and Sandler (1967) report the appearance of two or three bands of enzyme activity upon polyacrylamide gel electrophoresis of partially purified monoamine oxidase from rat liver or human placental mitochondria. This paper describes the separation of solubilized liver mitochondrial monoamine oxidases from three species by gel filtration in the presence of detergent. Based on substrate specificity there appear to be at least three different monoamine oxidases present in rat and rabbit liver and at least two in beef liver.