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PubMed1988

Heterogeneity in the human erythrocyte band 3 anion-transporter revealed by Triton X-114 phase partitioning.

Triton X-114 phase partitioning used in conjunction with countercurrent distribution was utilized to examine the phasing properties of the human erythrocyte Band 3 anion-transport protein. Phase partitioning and countercurrent distribution of Band 3 protein followed by electrophoresis and immunoblotting revealed that Band 3 protein possesses biphasic properties with approx. 65% of the Band 3 97,000-Mr species being localized in the detergent phase and 35% isolated in the aqueous phase. The bidirectional phasing of the anion-transporter does not appear to be a result of glycosylation or phosphorylation, since treatment of alkali-washed ghosts with glycosidases or phosphatase respectively did not significantly alter the phasing profiles. Chymotrypsin treatment of erythrocytes followed by the purification of the 60,000-Mr fragment, and exposure of this fragment to phase separation and countercurrent distribution also revealed biphasic partitioning with 70% of the species being isolated in the aqueous phase and 30% in the detergent phase. These data demonstrate that the human erythrocyte Band 3 anion-transport protein is heterogenous by Triton X-114 phase partitioning and that this heterogeneity is preserved in the 60,000-Mr chymotryptic fragment of Band 3 protein.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1979

Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems.

Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging. The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6 degrees C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maxium occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.

Animals↗

PubMed1976

Structural studies on RNA from Bombyx mori L. I. Nucleoside composition of enriched tRNA species from the posterior silkgland purified by coutercurrent distribution.

A large scale fractionation of tRNA from the posterior silkgland of the silkworm Bombyx mori L. by countercurrent distribution is described. One single 1,500 transfer distribution carried out with Phosphate buffer-Fromamide-Isopropanol (PFI) solvent system yields highly enriched isoaccepting species with increasing mobility order: tRNA1Gly, tRNA1-2Ala, tRNATyr, tRNA2Gly, tRNA1Ser and tRNA2Ser with 75%, 70%, 90%, 60%, 60%, and 90% purities respectively. Nucleosides fingerprint analysis of each iso-tRNA species confirms the anticodon structures previously suggested for tRNA2Ala (IGC), tRNA2bGly (U-CC) (U-CC) and tRNA2bSer (IGA). Twenty two minor nucleosides, three of them with unknown structure, have been detected. They are: m5C in tRNA1Gly, m1I in all tRNAAla species, polar A and U called X in tRNATyr, polar U derivative in tRNAGly2, mt6A in tRNASer1 and i6A tRNA2Ser. Both tRNASer species have m3C and ac7C. We do not detect Q, Y and thiol derivatives. The elution characteristics of silkgland tRNA species may be expressed in a semilogarithmic diagram where log K (K is the partition coefficient) is related to the base ratio A/Y) and the coding properties. The distribution pattern of silkgland tRNAs has been compared with that of Yeast and Rat liver tRNAs fractionated by countercurrent distribution with the PFI and PMB (Potassium phosphate buffer, 2-methoxy ethanol, 2-butoxy ethanol) solvent systems.

Animals↗

PubMed1976

Partition in two-polymer aqueous phases reflects differences between membrane surface properties of erythrocytes, ghosts and membrane vesicles.

Partition of cells (defined as the percentage of added cells in the top phase) in dextran-polyethylene glycol aqueous two-phase systems is an extremely sensitive indicator of cell surface properties. By appropriate choice of polymer concentrations and salt composition and concentration one can, to some extent, select the membrane properties (e.g. charge (Walter, H. (1975) in Methods in Cell Biology (Prescott, D.M., ed.), Vol. 9, pp. 25-50, Academic Press, New York), lipid composition (Walter, H, Krob, E.J. and Brooks, D.E. (1976) Biochemistry, 15, 2959-2964)) that determine the partition behavior of cells (or membranes). In the present experiments we have obtained by partition information on separability, alteration and ensuing heterogeneity during preparation of human erythrocytes, ghosts and rightside-out and inside-out vesicles from human erythrocyte membranes. A phase system in which partition is greatly (although not exclusively) dtermined by membrane charge was used. It was found that: 1. The partition coefficients (and hence the membrane surfaces) of ghosts and of rightside-out vesicles differ from those of the erythrocytes from which they are derived. 2. Rightside-out and inside-out vesicles have different countercurrent distribution patterns with the rightside-out vesicles having the higher partition coefficient (probably reflecting higher membrane charge of the latter). 3. Rightside-out vesicles are highly heterogeneous membrane populations as evidenced by broader than theoretical countercurrent distribution curve which, in some experiments, even split into two populations outright. This heterogeneity is particularly interesting since the enzyme markers used to establish vesicle sidedness (Steck, T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed), Vol. 2, pp. 245-281, Plenum Press, New York) indicate "pure" rightside-out preparations. 4. Inside-out vesicles could, under no conditions tested, be rendered totally free of rightside-out vesicles according to the enzyme markers used to indicate vesicle sidedness (Steck, T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed), Vol. 2, pp. 245-281, Plenum Press, New York). A discussion of factors involved in the partition of the vesicles and ghosts together with analogies to the partition behavior of stored or in vitro modified human red blood cells is presented.

Acetylcholinesterase↗

PubMed1986

Membrane surface properties of lymphocytes of normal (DBA/2) and autoimmune (NZB/NZW)F1 mice: effects of L-canavanine and a proposed mechanism for diet-induced autoimmune disease.

Partitioning cells in a dextran polyethylene glycol aqueous two-phase system (countercurrent distribution, CCD) is a sensitive method for learning about cell surface membrane properties and for subfractionating cell populations. In this study, we subjected lymphocytes from normal DBA/2 mice and autoimmune F1 New Zealand black/New Zealand white [NZB/NZW)F1) mice to countercurrent distribution and found that T cells partition to the right and B cells partition to the left of the CCD curve. We found no difference between the CCD patterns of normal and autoimmune mice. When the murine lymphocytes were exposed to a cationic dietary amino acid (L-canavanine) in vitro, L-canavanine selectively affected the CCD pattern of autoimmune B cells, reflecting an alteration in surface membrane properties. We separated these lymphocytes with altered surface membrane properties by CCD. Impaired B-cell immune responses associated with L-canavanine were isolated to this lymphocyte fraction. This study provides the first evidence that alterations in the charged surface membrane properties are associated with abnormal (auto) immune response.

Animals↗

PubMed1980

Correlation between phagocytic and membrane surface properties reflected by partitioning of human peripheral blood monocytes in two-polymer aqueous phases.

Human peripheral blood monocyte-enriched fractions (identified by staining for peroxidase and by sizing) were obtained by velocity sedimentation at unit gravity of peripheral blood mononuclear cells. They were then fractionated by countercurrent distribution (a multiple-extraction procedure) in a charged Dextran/poly(ethylene glycol) aqueous phase system. The monocytes remained viable after the separation (order of 90%). Cells obtained from different cavities along the extraction train were tested for their ability to phagocytize latex particles. With increasing partition coefficient (presumably higher charge-associated membrane properties) the ratio of monocytes that phagocytized to monocytes that did not phagocytize increased appreciably. When, however, monocytes were permitted to phagocytize particles prior to countercurrent distribution, an increase in partition coefficient was associated with an appreciable decrease in the above-specified ratio. Control experiments indicate that the observed change in partitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitiartitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitioning) and their ability to ingest particles. Furthermore, an alteration in the surface charge-associated properties of monocytes as a consequence of phagocytosis is indicated by the cells' reduced partition coefficient.

Cell Membrane↗

PubMed1980

Human proinsulin, VII: synthesis of two protected peptides corresponding to the sequences 1--45 and 46--96 of the prohormone.

The synthesis of two protected large peptides 1--45, and 46--86 covering the entire amino acid sequence of human proinsulin is described. Peptide 1--45, was synthesized from two intermediate fragments 1--23 and 24--45 and purified by countercurrent distribution in dimethylformamide system (K = 0.5). Peptide 46--86 was synthesized from two intermediate fragments 46--70 and 71--86 and purified by countercurrent distribution in two solvent systems, the dimethylformamide system (K = 0.06), and the toluene system (K = 0.16).

Amino Acid Sequence↗

PubMed1972

Separation of prodigiosenes and identification as prodigiosin syntrophic pigment from mutant pairs of Serratia marcescens.

Countercurrent distribution is capable of resolving mixtures of closely related prodigiosene pigments. Syntrophic pigment produced by several pairs of Serratia marcescens color mutants was identified as prodigiosin (2-methyl-3-amyl-6-methoxyprodigiosene) by countercurrent distribution, soda lime pyrolysis, and other techniques. The metabolic block of mutant strain H-462, derived from parent strain HY, was located between the blocks of mutant strains OF and WF, both derived from parent strain Nima.

Bacteriological Techniques↗

PubMed1997

Calmodulin-stimulated Ca(2+)-ATPases in the vacuolar and plasma membranes in cauliflower.

The subcellular locations of Ca(2+)-ATPases in the membranes of cauliflower (Brassica oleracea L.) inflorescences were investigated. After continuous sucrose gradient centrifugation a 111-kD calmodulin (CaM)-stimulated and caM-binding Ca(2+)-ATPase (BCA1; P. Askerlund [1996] Plant Physiol 110: 913-922; S. Malmström, P. Askerlund, M.G. Plamgren [1997] FEBS Lett 400: 324-328) comigrated with vacuolar membrane markers, whereas a 116-kD caM-binding Ca(2+)-ATPase co-migrated with a marker for the plasma membrane. The 116 kD Ca(2+)-ATPase was enriched in plasma membranes obtained by aqueous two-phase partitioning, which is in agreement with a plasma membrane location of this Ca(2+)-ATPase. Countercurrent distribution of a low-density intracellular membrane fraction in an aqueous two-phase system resulted in the separation of the endoplasmic reticulum and vacuolar membranes. The 111-kD Ca(2+)-ATPase co-migrated with a vacuolar membrane marker after countercurrent distribution but not with markers for the endoplasmic reticulum. A vacuolar membrane location of the 111-kD Ca(2+)-AtPase was further supported by experiments with isolated vacuoles from cauliflower: (a) Immunoblotting with an antibody against the 111-kD Ca(2+)-ATPase showed that it was associated with the vacuoles, and (b) ATP-dependent Ca2+ uptake by the intact vacuoles was found to be CaM stimulated and partly protonophore insensitive.

Calcium-Transporting ATPases↗

PubMed1982

A method for separating commercial colistin complex into new components: colistins pro-A, pro-B and pro-C.

Commercial colistin was separated into three major components as well as lipid components by reversed phase adsorption chromatography on Diaion HP-20 AG, a macroreticular styrene-divinylbenzene copolymer, without any inorganic salts or detergents, in aqueous-organic solvent as mobile phase. These expected components were colistins A, B and C; there were, however, appreciable differences between these components and colistins A, B and C, isolated by countercurrent distribution. The newly isolated components showed slightly higher potency than colistins A, B and C; their molecular weights, as determined by gel permeation chromatography of 2,4-dinitrophenyl derivatives on TSK-GEL G2000H (mobile phase: dimethylformamide), were also slightly higher. Accordingly, they were tentatively named colistins pro-A, pro-B and pro-C. During purification by countercurrent distribution (solvent system: sec-BuOH - n-BuOH - 0.1 N HCl, 30:6:40), colistin pro-A was converted to colistin A. Similarly, colistin pro-B was converted to colistin B, and colistin pro-C to colistin C. Therefore, we concluded that colistins A, B and C are artifacts.

Amino Acids↗

PubMed1974

Separation of erythromycin-resistant and -susceptible subpopulations of Escherichia coli 15 by partition in two-polymer aqueous phases.

Partition of cells in two-polymer aqueous phases depends on subtle differences in the cells' surface properties (primarily surface charge). A culture of Escherichia coli 15 arg(-) was subjected to countercurrent distribution in a dextranpolyethylene glycol aqueous phase system and found to consist of two well-differentiated subpopulations. Clones derived from these two subpopulations (designated clones 5 and 6) exhibited characteristic partitions and were stable on subculture. Clone 5 cells were found to be susceptible to erythromycin and clone 6 cells were resistant. When a culture of clone 5 was exposed to erythromycin, resistant mutants were selected with the same partition as clone 6. Countercurrent distribution in two-polymer aqueous phase systems is thus shown to be a sensitive method for detecting some heterogeneities of bacterial populations and resolving such mixtures. Possible clinical implications of changes in bacterial surface properties associated with acquired drug resistance are discussed.

Chemical Phenomena↗

PubMed1984

Studies on the metabolic fate of [14C]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the mouse.

Studies on the Metabolic Fate of [14C]2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in the Mouse. KOSHAKJI, R. P., HARBISON, R. D., and BUSH, M. T. (1984). Toxicol. Appl. Pharmacol. 73, 69-77. After a single po dose (135 micrograms/kg; 62 microCi/kg) of 14C-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in male ICR/Ha Swiss mice, 67 to 76% of the administered dose was eliminated via the feces and 1 to 2% in the urine during the first 24 hr following treatment. It seems likely that most of this material was simply not absorbed. Much of the remaining chemical was then excreted slowly in the urine (2%) and feces (7%) during the next 10 days, partly as the unchanged compound and partly as metabolites. One of the metabolites (Fraction II) appears to be a single polar, acidic metabolite characterized in urine (0.4 +/- 0.1%) and feces (2.2 +/- 0.2%), and is also likely excreted as a glucuronide conjugate. The rest of the radioactivity was in the form of unchanged TCDD in the animal body (17 +/- 2%). Steady rates of decline in the concentrations of the 14C as well as of the unchanged TCDD were reached in the feces and urine after the fifth day following the administration of the chemical. Based on this steady rate, the half-life of the radioactivity in the body was approximately 20 days. Urine, feces, and whole body were analyzed by solvent extraction, 14C counting, thin-layer chromatography, and countercurrent distribution.

Animals↗

PubMed1979

Metabolism and distribution of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in pregnant mice.

Pregnant mice were treated with a single oral dose of [carboxy-14C]2,4,5-T (100 mg/kg; 1.22 mu Ci/mg) on day 12 of gestation and sacrificed after 0.25, 0.5, 2 and 24 hours. Maternal blood, embryos, placentas and yolk sacs were analyzed by solvent extraction, TLC, and countercurrent distribution. Expressed as percentage of the administered dose/g tissue, the unchanged 2,4,5-T found in maternal blood, placentas, yolk sacs, and embryos was 3, 0.5, 0.5, and 0.2%, respectively, after 0.25 hours, and 4, 2, 2, and 0.5%, respectively, after 24 hours. No major metabolites of 2,4,5-T were detected. Urine and feces were also collected and analyzed. Radioactivity was largely eliminated in the urine, 69-78% of the administered dose in 7 days. Feces contained 5-9% of the dose. In the urine unchanged 2,4,5-T accounted for 35-44% of the dose, and 22-23% as very polar material. Unchanged 2,4,5-T in the feces was 3-5% and 1-2% as polar material. 2,4,5-T administered to pregnant mice is largely distributed and eliminated as 2,4,5-T and very polar material.

2,4,5-Trichlorophenoxyacetic Acid↗

PubMed1988

Counter-current distribution of sonicated inside-out thylakoid vesicles.

Inside-out thylakoid vesicles were isolated from spinach chloroplasts, and fragmented by sonication. Different fragments were separated by counter-current distribution and analyzed for chlorophyll and P700. The inside-out vesicles had a chlorophyll a/b ratio of 2.2-2.4 (original chloroplasts 2.8-3.0). After further fragmentation of the inside-out vesicles by sonication and separation by countercurrent distribution three populations of vesicles were obtained having chlorophyll a/b ratios of 1.7, 1.9 and 2.5 respectively. The P-700 was depleted in fractions with lower chlorophyll a/b ratio and was nearly absent in the fraction having a chlorophyll a/b ratio of 1.7 (chlorophyll/P700 greater than 4500 mol/mol). That PSII membrane vesicles, with such a low chlorophyll a/b ratio and lacking PSI, can be prepared by a non-detergent method provides strong support for the notion that PSI and PSII are segregated along the thylakoid membrane. A plot of P700 per chlorophyll against chlorophyll b/(a + b) fits a straight line connecting the pure PSI membrane (chlorophyll a/b = 6; P700/chlorophyll = 5.6 mmol/mol) with the pure PSII membrane (chlorophyll a/b = 1.7; P700 = 0). These two membranes can be considered as separate phases of a two-dimensional phase system. Models for the thylakoid membrane are discussed.

Chloroplasts↗

PubMed1978

Isolation, characterization and distribution of adenosine 3':5'-cyclic monophosphate from Pinus radiata.

Cyclic AMP was extracted in 0.1 M-HCl from tissues of Pinus radiata and purified by gel filtration on Sephadex G-10, and chromatography on Dowex AG1 (X2) and polyethyleneimine-cellulose in two separate solvent systems. Presumptive cyclic AMP from 10kg batches of pine needles was characterized by countercurrent distribution in the presence of cyclic [8-3H]AMP. Statistical analysis of the curves for radioactivity and mass (determined by the Gilman competitive-binding assay) showed that the fit of the curves was highly significant for seven degrees of freedom. The distribution of cyclic AMP within P. radiata and various other plant tissues was determined by the Gilman procedure. The results suggest that there is no relationship between variations in cyclic AMP concentrations and the known function of the tissue in which it was measured.

Chromatography↗

PubMed1984

Surface differences between erythrocytes from arbitrarily chosen (presumably hematologically normal) individuals detected by cell partitioning.

Differences in membrane surface properties (both charge-associated and lipid-related) of erythrocytes from any two arbitrarily selected individuals can be detected by use of a purely physical method: cell partitioning in dextran-poly(ethylene glycol) aqueous phase systems. The procedure consists of isotopically labeling (with [51Cr]-chromate) aliquots of red blood cell populations to be compared. Such labeled cells are mixed with an excess of unlabeled erythrocytes from the other individual and the mixtures subjected to countercurrent distribution in either a charge-sensitive or a non charge-sensitive aqueous phase system. As control we also prepare mixtures of labeled cells with unlabeled cells from the same individual to ascertain that the label per se has no influence on the cells' partitioning behavior. The distribution curves are analyzed for total cells (in terms of hemoglobin absorbance) and labeled cells (in terms of counts/min). Changes in the relative specific activities through the distribution curves are routinely obtained when cells from different individuals are used and are indicative of subtle differences in surface properties of such erythrocyte populations.

Cell Separation↗

PubMed1986

Synthesis and application of a poly(ethylene glycol)-antibody affinity ligand for cell separations in aqueous polymer two-phase systems.

The possibility of producing biospecific affinity ligands for separating cells in two polymer aqueous phase systems on the basis of cell surface antigens was investigated. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated monomethyl poly(ethylene glycol) (PEG) fractions (molecular weights approximately 200, 1900, and 5000) at various molar ratios of PEG to protein lysine groups. The partition coefficient of the protein in a Dextran/PEG two-phase system increased with increasing degree of modification and increasing PEG molecular weight. There was a concomitant loss in ability to agglutinate human erythrocytes. The ability of the modified IgG to bind to a DEAE-cellulose column was almost eliminated by reaction with the PEG 5000, and was decreased to a lesser extent by PEG 1900. This PEG 1900-modified IgG substantially increased the partition of fresh or fixed human erythrocytes into the PEG-rich phase of a suitable phase system, while having no effect on rabbit cell partition. The partition increase could be inhibited by unmodified anti-human red cell IgG but not by nonspecific unmodified human IgG, demonstrating that the ligand effects were specific for the cell type against which the antibody was raised. A mixture of rabbit and human erythrocytes, which ordinarily have very similar partitions in the phase systems used, could be separated on a countercurrent distribution apparatus using the modified IgG. These results demonstrate the feasibility of producing immunologically specific affinity partition ligands for cell separation.

Affinity Labels↗

PubMed1986

Heterogeneity in the surface properties of B16 melanoma cells from sublines with differing metastatic potential detected via two-polymer aqueous-phase partition.

When mixed in aqueous solution at low concentrations, the neutral polymers dextran and poly(ethylene glycol) (PEG) rapidly form a two-phase system, consisting of a dextran-enriched lower phase and a PEG-enriched upper phase. Two B16 mouse melanoma cell lines, B16-F1 (low lung colonizing capability) and B16-F10 (high lung colonizing capability) were found to partition differentially into the upper phase in a variety of two-phase systems. Upper-phase partition depends primarily on either hydrophilic (i.e., surface charge density) or hydrophobic (i.e., affinity for the hydrocarbon chain of a PEG-fatty acid ester) cell surface properties, depending on the system used. In single-step partition studies, cells of the B16-F10 subline displayed a greater preference than B16-F1 cells for the upper phase in the hydrophilic system and less preference in systems sensitive to hydrophobic properties. Countercurrent distribution (CCD) experiments, performed with [125I]deoxyuridine DNA-labelled cells, were consistent with single-step partition results. These CCD results demonstrated that B16-F10 cells exhibited greater DNA synthesis than B16-F1 cells and that considerable heterogeneity, in both hydrophobic and hydrophilic surface properties, was present in subpopulations of cells of both sublines. The data also showed considerable enrichment of 125I-specific cell activity in certain sections of the distributions, indicating that differences in cellular DNA synthesis are reflected in the surface properties to which partition is sensitive.

Animals↗