Triton Phase Research Articles

Species dependent aqueous biphasic extraction of some heavy metals

A clean liquid–liquid extraction system was designed for extraction of three different heavy metals (Hg2+, Pb2+ and Bi3+) using an Aqueous Biphasic System consisting of a nonionic surfactant Triton X-100 in combination with two different salts viz., magnesium sulphate and sodium citrate. The extraction was monitored in two ways, by direct partitioning as well as by forming a complex with diphenylthiocarbazone (dithizone) for each metal ion in the Triton phase and measuring the colour intensity using a spectrophotometer. The change in extraction pattern at different pH of the salt rich phase was monitored. The possible change in speciation of these metal ions with pH was checked with the software programme ‘CHEAQS’ (Chemical Equilibria in Aquatic Systems). These heavy metals were also found to have higher extraction in the Triton phase when a prior complexation with dithizone was performed. The method may find extensive applications for the removal and decontaminations of the above mentioned toxic heavy metals from a sample.

The 40- and 90-kDa membrane proteins (ORF6 gene product) of Mycoplasma pneumoniae are responsible for the tip structure formation and P1 (adhesin) association with the Triton shell

After Triton X-100 treatment of Mycoplasma pneumoniae cells, a portion of the adhesin P1 (transmembrane protein) proved to remain tightly associated with the Triton insoluble material (Triton shell) as shown previously by several authors. However, the spontaneous loss of two cytadherence-associated membrane proteins of 90 and 40 kDa (gene product of the open reading frame 6 of the P1 operon) in a hemadsorption-negative mutant, designated M5, resulted in a 100% release of the P1 protein into the Triton phase and in the lack of the characteristic tip-like attachment organelle of M. pneumoniae indicating an essential role of the open reading frame 6 gene product in tip structure formation.

The 40- and 90-kDa membrane proteins (ORF6 gene product) of Mycoplasma pneumoniae are responsible for the tip structure formation and P1 (adhesin) association with the Triton shell.

After Triton X-100 treatment of Mycoplasma pneumoniae cells, a portion of the adhesin P1 (transmembrane protein) proved to remain tightly associated with the Triton insoluble material (Triton shell) as shown previously by several authors. However, the spontaneous loss of two cytadherence-associated membrane proteins of 90 and 40 kDa (gene product of the open reading frame 6 of the P1 operon) in a hemadsorption-negative mutant, designated M5, resulted in a 100% release of the P1 protein into the Triton phase and in the lack of the characteristic tip-like attachment organelle of M. pneumoniae indicating an essential role of the open reading frame 6 gene product in tip structure formation.

Purification and partial biochemical characterization of a Mycoplasma fermentans-derived substance that activates macrophages to release nitric oxide, tumor necrosis factor, and interleukin-6

Mycoplasmal products may exert a number of diverse in vitro effects on cells of the immune system. A macrophage-activating substance from Mycoplasma fermentans was described in this laboratory and named mycoplasma-derived high-molecular-weight material (MDHM). Using synthesis of nitric oxide by peritoneal cells from endotoxin low-responder mice as an assay system, MDHM was purified as follows. After freeze-thawing of M. fermentans, MDHM activity was sedimented with the membrane fraction. Membranes were delipidated with chloroform-methanol, and MDHM activity was extracted with octyl glucoside. Coextracted proteins were degraded by proteinase K. MDHM was further purified by reversed-phase high-pressure liquid chromatography and eluted in one major and one minor peak of activity. Neither carbohydrates nor amino acids were found as constituents. MDHM had the following properties: it partitioned into the phenol phase upon phenol-water extraction and into the Triton phase after extraction with Triton X-114. MDHM was not inactivated by either phospholipase A2 or triglyceride lipases. However, mild periodate treatment led to a > 95% loss of activity. Also, alkaline hydrolysis at 25 degrees C completely abolished MDHM activity with a half-life of 2 min. MDHM activity was spread out over a wide molecular weight range upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes, whereas after proteinase treatment MDHM activity migrated close to the front. These features of MDHM, taken together, speak in favor of an amphiphilic molecule with a lipid moiety carrying fatty acids in ester linkage and a polyol moiety of unknown character. MDHM was active in the nanogram-per-milliliter range, activating macrophages to release nitric oxide, interleukin-6, and tumor necrosis factor.

A FULLY AUTOMATED DUAL‐WAVELENGTH PHOTOMETER FOR PHYTOCHROME MEASUREMENTS AND ITS APPLICATION TO PHYTOCHROME FROM CHLOROPHYLLCONTAINING EXTRACE

Abstract The photometer described here measures phytochrome photoreversibility of as many as 20 samples fully automatically. Monochromatic light of two laser diodes (670 and 780 nm) is used as a source for the measuring light beam. The whole measuring procedure is controlled by a computer linked to a lock‐in amplifier. This arrangement permits a rather simple construction and allows automatic zeroing and sample change.In addition the application of a Triton phase partitioning method to allow phytochrome photoreversibility mea surements of extracts of green tissue is described. A single phase partitioning with a chlorophylYphytochrome mixture reduced chlorophyll concentration to less than 10%. Repeated phase partitioning of crude extracts from green moss protonemata reduced chlorophyll content to less than 2% while leaving the phytochrome concentration almost unchanged. In some instances this method has advantages over polyethyleneimine precipitation, which is commonly used to obtain chlorophyll‐free extracts.

Additional GPI-anchored glycoproteins on human platelets that are absent or deficient in paroxysmal nocturnal haemoglobinuria

In order to detect novel glycophosphatidylinositol (GPI)-anchored platelet proteins, human platelets were incubated with PI-specific phospholipase C (PI-PLC) and the supernatant was analysed by PAGE and silver-staining for additional protein bands. PI-PLC treatment resulted in the appearance of at least two additional novel GPI-linked glycoproteins (GP), GP500 and GP175, in the supernatant. Their presence on the platelet plasma membrane surface was demonstrated by periodate/[ 3H]borohydride surface-labelling. Activation of platelets did not enhance the amount of GP500 and GP175 that could be cleaved by PI-PLC. In Triton X-1 14 phase partitioning of platelet membranes the membrane form of GP175, mfGP175, was in the Triton phase while mfGP500 was found in the water phase. Neither GP500 nor GP175 were present in the supernatant of surface-labelled platelets treated with PI-PLC from 4 patients, diagnosed as having paroxysmal nocturnal haemoglobinuria (PNH), but the supernatant from platelets from healthy volunteers treated the same way contained both.

A STUDY OF MEMBRANE‐ASSOCIATED PHYTOCHROME: HYDROPHOBICITY TEST AND NATIVE SIZE DETERMINATION

Abstract—We confirmed that after extraction in the absence of added Mg2+, a small fraction of phytochrome was associated with pelletable, hydrophobic membranes. When microsomal material of several plant species was subjected to Triton X‐114 phase partitioning, a part of phytochrome migrated into the hydrophobic Triton phase in contrast to soluble phytochrome. The amount of bound phytochrome partitioning into the Triton phase varied from 6% for oats to 30% for zucchini and 50% for mustard and maize (0.5–10% of total phytochrome). Membrane‐associated phytochrome could be solubilized by the zwitterionic detergent CHAPS and with the nonionic detergent dodecylmaltoside. Subjected to gel filtration on Superose‐6/FPLC, oat phytochrome of the CHAPS solubilized sample was eluted in three different molecular weight ranges. There was a main fraction with the molecular weight of purified phytochrome, another fraction (approximately 20%) with a higher molecular weight, and a third small fraction appearing immediately after the void volume. Gel filtration after solubilization by dodecylmaltoside resulted in two distinct fractions: the one eluted at the position of the phytochrome dimer, and the other (approximately 15%) with an apparent molecular weight of 800 kDa. Phytochrome was detected, separated and quantified by SDS‐PAGE, and western blotting with the monoclonal antibody Z‐3B1. We assume that the distinct, phytochrome positive, high molecular weight fractions contain phytochrome associated with hydrophobic protein.

Triton's surface properties: A preliminary analysis from ground‐based, Voyager photopolarimeter subsystem, and laboratory measurements

The surface properties of Triton have been determined from a threefold approach involving analysis of ground‐based and Voyager photopolarimeter subsystem observations of its phase curve, theoretical modeling, and laboratory measurements of the phase functions of a wide variety of materials. The physical parameters derived include the single‐scattering albedo, the single‐particle phase function, the compaction state of the optically active portion of the regolith, and the degree of macroscopic roughness. We find that Triton has a high single‐scattering albedo (0.96 ± 0.01 at 0.75 μm) and an unusually compacted surface possibly similar to that of Europa. Preliminary results suggest that Triton's single‐particle phase function and the macroscopically rough character of its surface are Similar to those of most other icy satellites. Laboratory measurements of the phase curve of a sample of a high‐albedo, compact volatile‐contaminant analogue are in reasonable agreement with Triton's phase curve and provide evidence that the model parameters derived for Triton's surface are physically realistic.

Human rhinovirus mutants resistant to low pH

Mutants of human rhinovirus serotype 14 (HRV14) with increased resistance to treatment at low pH were obtained by repeated cycles of exposure to pH 4.5 and propagation in HeLa cells. Whereas wild-type virus lost more than 5 logs of infectivity upon incubation at pH 4.3, the three isolates examined were essentially unaffected. Conformational change of the viral capsid upon exposure to low pH was assessed as an increase of hydrophobicity by partition between an aqueous phase and a Triton X-114 phase; the mutants required exposure to a much lower pH to accumulate in the Triton phase than wild-type HRV14. The sequence of the capsid region was determined for three isolates; two isolates were found to have the changes Thr17 to lle in VP2 and Asn100 to lle in VP1. The third isolate also had the change Thr17 to lle in VP2; however, in VP1, Asp101 was replaced by Glu. Separate introduction of the mutations into full length cDNA clones of the wild-type sequence of HRV14 showed that only the changes in VP1 were necessary for the increased stability at pH 4.5. The implications of the mutations for the three-dimensional structure of the viral capsid are discussed.

Glucosidase II, a protein of the endoplasmic reticulum with high mannose oligosaccharide chains and a rapid turnover.

Glucosidase II is regarded as a resident protein of the endoplasmatic reticulum. The enzyme removes alpha-1-3-linked glucose from high mannose oligosaccharides N-linked to asparagine residues of glycoproteins. Monospecific antibodies raised against the pig kidney enzyme are used to study the metabolism of the enzyme in a rat hepatoma cell line. These antiglucosidase II antibodies specifically immune precipitate glucosidase II as a 100,000-Da species from [35S]methionine-labeled cells. In addition, protein blotting and immune staining of cell extracts from both rat liver and human and rat hepatoma cell lines show identity in apparent Mr (100,000). Glucosidase II synthesized in the presence of tunicamycin is approximately 94,000 Da, indicating the presence of one or more N-linked oligosaccharide chains. Cell-free protein synthesis of rat hepatoma total RNA demonstrates that glucosidase II is synthesized as a slightly higher molecular weight species as compared to the polypeptide synthesized in whole cells in the presence of tunicamycin, indicating that the enzyme has a cleavable signal sequence. Using a pulse-chase protocol, the apparent molecular weight does not change upon longer chase periods. In addition, the 100,000-Da protein remains sensitive to endo-beta-N-acetylglucosaminidase H regardless of prolonged chase periods. The cells incorporate [3H]mannose into the enzyme; after release with endo-beta-N-acetylglucosaminidase H, most of the radioactivity comigrates with Glc1-Man9-GlcNAc on a gel filtration column. Phase separation in Triton X-114 shows a partition between the aqueous and the Triton phase, the major portion being separated in the aqueous phase. In rat hepatoma cells glucosidase II has a half-life of 50 min. This value is not altered if the cells are grown in the presence of monensin nor of methyl-deoxynoijirimycin. However, tunicamycin and low concentrations or primaquine (raising the pH of acidic compartments) causes a 100% increase in half-life of glucosidase II. We conclude that glucosidase II is a hydrophilic, probably not a transmembrane membrane, protein with a short half-life. It is the first example of an oligosaccharide-processing enzyme not being an integral membrane protein.