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PubMed1987

Subfractionation of human T lymphocyte subpopulations by partitioning: surface heterogeneity of the OKT8/Leu2+ T cell subset.

Human peripheral blood mononuclear cells (PBMC) were subjected to countercurrent distribution in a charge-sensitive dextran-poly (ethylene glycol) aqueous two-phase system. Cells are thereby subfractionated on the basis of their charge-associated surface properties. The helper/inducer T cell subset (OKT4/Leu3+) has a single distribution curve with a low partition ratio (P). In contrast, the suppressor/cytotoxic (OKT8/Leu2+) subset is clearly heterogeneous and gives two peaks, one with a lower and the other with a higher P value. The cells in the latter peak constitute part of a minor subpopulation of cells enriched with natural killer (Leu7+) cells. Double labelling studies indicate that a majority of Leu2+ cells with a high P value also have a marker of natural killer cells (HNK-1), whereas the Leu2+ cells with low partition ratio are depleted with respect to cells bearing both markers.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Effect of surface modification of rat erythrocytes of different ages on their partitioning behavior in charge-sensitive two-polymer aqueous phases.

Partitioning differences between cells in two-polymer aqueous phase systems originate from subtle differences between the surface properties of cells. Because of the exponential relation between the parameters affecting the partition ratio (P) and the P itself, differences in membrane components suspected of effecting the differential partitioning of closely related cell populations cannot be directly established by conventional chemical assay techniques. In order to study the chemical nature of the components responsible for the age-related changes in surface properties of rat red cells we have devised an approach which uses a combination of isotopic labeling of erythrocyte subpopulations of distinct cell age with different enzyme and/or chemical treatments followed by countercurrent distribution in charge-sensitive two-polymer aqueous phase systems. These studies show that: neuraminidase-susceptible sialic acid is not responsible for the cell age-related surface differences detected by partitioning; the component(s) responsible for the cell age-related surface differences can be extracted (from aldehyde-fixed red cells) with ethanol or cleaved with dilute sulfuric acid. Our data are consistent with the hypothesis that ganglioside-linked sialic acid is the chemical moiety responsible for the cell charge-associated surface differences among rat red blood cells of different ages.

Age Factors↗
PubMed1988

Detection of surface differences between closely related cell populations by partitioning. Cultured K-562 cell sublines.

The K-562 cell line is a culture of human leukemia stem cells originally derived from a patient with chronic myelogenous leukemia in blast crisis. We have applied a sensitive method capable of detecting subtle differences in charge-associated and noncharge-related cell surface properties between closely related cell populations to K-562 cells from different sources and having different histories. The method consists of isotopically labeling aliquots of each of two cell populations to be compared with 51Cr-chromate and mixing the labeled cells with an excess of unlabeled cells with which they are to be compared. The mixtures are subjected to countercurrent distribution in either a charge-sensitive or a noncharge-sensitive dextran-poly(ethylene glycol) aqueous two-phase system. The distribution curves are analyzed for total cells (in terms of electronic counts) and labeled cells (in terms of cpm). Alterations in relative specific activities through the distribution curves are indicative of differences in surface properties between such cell populations. Using this method we have found surface differences, both charge-associated and noncharge-related, between any two K-562 cell sublines examined. Interestingly, whereas we observed differences among K-562 sublines, we never witnessed a change in surface properties of the respective sublines. The differences among the sublines examined remained unaltered for more than 40 passages in our hands. It thus appears likely that the event(s) leading to an altered K-562 cell surface, detectable by partitioning, does not occur gradually.

Cell Line↗
PubMed1979

Subfractionation of human peripheral blood lymphocytes on the basis of their surface properties by partitioning in two-polymer aqueous phase systems.

Partitioning of cells in dextran-poly(ethylene glycol) aqueous-aqueous two-phase systems is a sensitive method for separating cells and for obtaining information on their surface properties. Highly purified lymphocytes were obtained by velocity sedimentation of human peripheral blood mononuclear cells and fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged two-polymer aqueous phase system. The lymphocytes remained viable after separation (order of 90%) and the E-rosetting cells responded (after adding back monocytes) to mitogens (PHA, Con A, PWM). Not only was the total lymphocyte population found to be highly heterogeneous (as evidenced by a broad and skewed distribution curve), but we were able to show that cells that rosetted with E, or had complement or Fc receptors were composed of additional subpopulations as well. The bulk of complement-receptor-bearing cells had the lowest partition coefficient (K), E-rosetting cells an intermediate K, and Fc-receptor-containing cells the highest K. The largest lymphocytes were among the subpopulation having the highest K and neither responded to T cell mitogens nor rosetted with E. Our results thus demonstrate that human peripheral blood lymphocytes can be subfractionated by CCD. The fractions are differentially enriched with lymphocyte subpopulations having characteristic surface markers and functional abilities.

Animals↗
PubMed1973

Confirmation of the 1-20 amino acid sequence of human adrenocorticotrophin.

The tryptic fragments of natural human adrenocorticotrophin, were separated by countercurrent distribution and a correction in positions 25, 26, 27 and 30 was made by Riniker et al. (1972) in a study of the fragment containing residues 22-39. We have purified the remaining tryptic fragments, namely residues 1-8, 9-15, 16-21 and 17-21, by using ion-exchange chromatography on CM-cellulose and have carried out sequence determination by using the subtractive Edman degradation procedure and digestion with aminopeptidase M and carboxypeptidase B. These results have confirmed the proposed sequence for human adrenocorticotrophin in regions 6-7, 10-14 and 17-20, which had previously been arrived at only by analogy with the invariant sequence found in the three other mammalian adrenocorticotrophin species that had been investigated.

Adrenocorticotropic Hormone↗
PubMed1986

The application of aqueous two-phase partition to the study of chick limb mesenchymal diversification.

A technique which identifies cells differing in surface character, aqueous two-phase partition using thin-layer countercurrent distribution (TLCCD), has been used to study differentiation and pattern formation in the developing chick limb bud. The TLCCD profiles of cell populations, derived from various regions of morphologically undifferentiated mesenchyme from three different stages of limb development, have been compared. At no stage, or location, has the population been found to be homogeneous. Cells from progress zones and more proximal regions could all be resolved into several populations. The populations from progress zones at three different developmental stages were qualitatively similar but differed in the proportions of cells in each. The most striking differences in cell populations were those obtained from the most proximal region of the limb, closest to the flank, which represents the developmentally most advanced region.

Animals↗
PubMed1971

A rapidly acting metabolite of vitamin D3.

A vitamin D(3) metabolite in intestine more polar than 25-hydroxy-vitamin D(3)(25-OH D(3)) has been detected by countercurrent distribution. The intestinal metabolite is found also after administration of labeled hydroxy D(3), indicating that it arises from vitamin D(3) via the intermediate 25-hydroxy derivative. The more polar metabolite is localized in the nuclear-chromatin fraction and appears in the gut before the physiological response to vitamin D(3).3.5 mug of the intestinal metabolite was isolated from 1250 chickens; the resulting purified material proved to be a potent mediator of calcium absorption. On a weight basis, it was at least 5 times as effective as vitamin D(3), and acted 3 times faster than either hydroxy D(3) or D(3) in stimulating intestinal calcium transport in the rachitic chick. It is proposed that this as yet uncharacterized steroid represents the active form of vitamin D(3) in the intestine.

Animals↗
PubMed1976

Nonuniform biosynthesis of multiple hemoglobins in the adult rat and guinea pig.

Separation of adult rat bone marrow cells by the method of thin layer countercurrent distribution permits the analyses of 59Fe-tagged erythroid cells for the various multiple hemoglobins and the assignment of such hemoglobins to erythroid cells at different stages of their development. Of the six adult red cell hemoglobins, hemoglobin 5 is synthesized most actively in the earliest erythroid cell whereas hemoglobin 4 (the major hemoglobin of the red cell) is synthesized most actively in the latest erythroid cells, e.g. the reticulocyte. Experimental evidence also indicates that maturation of the erythroid cell is accompanied by a decreased rate of synthesis of hemoglobin 5. The earliest erythroid cells of the marrow contain two hemoglobins, 7 and 8, which are absent in the adult red cell. Similar studies with the guinea pig confirm the nonuniform biosynthesis of its two hemoglobins and suggest that the phenomenon may be a general one among mammalian multiple hemoglobins.

Amino Acid Sequence↗
PubMed1979

Role of hemoglobin in proton transfer to the active site of carbonic anhydrase.

The binding of bovine oxyhemoglobin to bovine carbonic anhydrase with a dissociation constant between 10(-5) and 10(-7) M has been determined by countercurrent distribution using aqueous, biphasic polymer systems. This result provides an explanation for the very efficient proton transfer between hemoglobin and carbonic anhydrase, a transfer which enhances the catalytic activity of carbonic anhydrase as measured by 18O exchange between bicarbonate and water at chemical equilibrium (Silverman, D. N., Tu, C. K., and Wynns, G. C. (1978) J. Biol. Chem, 253, 2563-2567). Two rate constants describing 18O exchange activity of carbonic anhydrase at pH 7.5 show saturation behavior when plotted against hemoglobin concentration consistent with a dissociation constant of 2.5 X 10(-6) M between bovine hemoglobin and carbonic anhydrase. Interpretation of these rate constants in terms of a two-step model for 18O exchange indicates that hemoglobin enhances the rate of exchange from carbonic anhydrase of water containing the oxygen abstracted from bicarbonate, but does not affect the catalytic interconversion of CO2 and HCO3- at chemical equilibrium.

Animals↗
PubMed1979

Fractionation of membranes from Acholeplasma laidlawii A on the basis of their surface properties by partition in two-polymer aqueous phase systems.

Acholeplasma laidlawii A consists of pleomorphic cell clusters surrounded by a single membrane. When lysed, a cell gives rise to several membrane fragments which cannot be separated from each other by isopycnic sucrose gradient centrifugation. A heterogeneous lateral organization of the cell membranes was detected by countercurrent distribution of membrane fragments in a two-polymer aqueous phase system. It revealed that the membranes consist of at least two subpopulations with respect to surface properties. Changes in the fatty acid and cholesterol content of the membranes revealed that the resolution of different subpopulations was predominantly due to a critical ratio of monoglucosyldiglyceride to diglucosyldiglyceride. The heterogeneity of the membrane probably depends on lipid-lipid and lipid-protein steric interactions. Charged lipids, an apolar monoglucolipid and the ratio between lipids and proteins also affect membrane partition. The differences in the subpopulations were further reflected by different specific activities of NADH dehydrogenase, NADH oxidase and ATPase. These activities varied independently. Minor quantitative differences in the protein patterns of different subpopulations were apparent. The origin and the preservation of the membrane subpopulations are discussed in terms of lipid-lipid and lipid-protein interactions, their age and energy metabolism.

Acholeplasma laidlawii↗
PubMed1977

[Primary structure of tRNA Thr 1a and b from brewer's yeast].

One of the two major species of brewer's yeast tRNA threonine (tRNA Thr 1) has been purified by countercurrent distribution followed by two chromatographic steps (respectively on a Sepharose 4B and a BD-cellulose column). Complete digestion with pancreatic and T1 RNases and a partial hydrolysis with T1 RNase followed by the isolation and determination of the nucleotide sequences of the resulting fragments permitted the derivation of its primary structure. tRNA Thr 1 is in fact a mixture of two subspecies differing only by a A49-U65 base pair in 50 per cent of the molecules which is replaced by a G49-C65 pair in the other 50 per cent. These two subspecies consist of 76 nucleotide residues including 14 minor nucleotides. They show a characteristic m3C at the 3'terminal end of the anticodon loop, an anticodon I-G-U followed by t6A and C48, uncompletely modified (50 per cent) to m5C within the 5 nucleotides long extra-arm. The minor nucleotides m2G m2 2G are located at positions in which they generally occur in the tRNA structures as does m1A within the T-psi-C loop.

Base Sequence↗
PubMed1976

Properties of chloroplasts isolated by phase partition.

Chloroplasts from spinach can be separated into at least three different populations by countercurrent distribution using polymer two-phase systems. The chloroplast particles of the three populations differ in protein/chlorophyll ratio, ultrastructure and metabolism. One population, peak I, consists of intact chloroplasts surrounded by the chloroplast envelope; the second population, peak II, consists of chloroplasts, which have lost their envelopes and much of their stromal material; the third population, peak III, consists of particles containing intact chloroplasts surrounded by a membrane-bound cytoplasmic layer including mitochondria and peroxisomes. Rapid batch procedures of peak I chloroplasts incorporated 14C almost entirely into glycolate and intermediates of the Calvin cycle and starch synthesis. Only small amounts were found in sucrose and amino acids. On the other hand preparations of peak III chloroplasts have a much broader spectrum of 14C-labelled products. Sucrose, malate and some amino acids contained about 40% of the 14C incorporated. It is concluded from these experiments that sucrose is formed not within the chloroplast but in the cytoplasm from intermediates exported by the chloroplast. The origin of peak III particles and their use for studying the cooperation between the chloroplast and the surrounding cytoplasm including mitochondria and peroxisomes is discussed.

Carbon Dioxide↗
PubMed1969

Studies on phenolic steroids in human subjects. IX. Role of the intestine in the conjugation of estriol.

In order to compare the enteric circulation of estriol-16alpha-glucosiduronate (see preceding paper) with that of estriol (E(3)), labeled estriol was administered to six women by several routes: both injection and infusion (300 min) into the cubital vein, injection into the portal vein system, ingestion and instillation into the jejunum and ileum. Urine, collected from 0-2, 2-4, 4-8, 8-12, and 12-24 hr, was analyzed by countercurrent distribution for its content of radioactive 3- and 16-glucosiduronate (E(3)-3Gl,E(3)-16Gl) and sulfoglucosiduronate (E(3)-3S,16Gl) of estriol. After peripheral injection of E(3), E(3)-16Gl was excreted rapidly and E(3)-3S,16Gl at a slower and more constant rate. E(3)-3Gl was barely detectable after infusion. After injection of E(3) into the portal vein, the excretion of E(3)-3S,16Gl was greater and quicker than after peripheral injection. Even in a subject with a complete bile fistula, the urinary excretion of E(3)-3S,16Gl was essentially unchanged. Ingestion also produced the same result. Only after instillation into the ileum was a large and rapid excretion of E(3)-3Gl obtained, whereas the excretion of E(3)-3S,16Gl, and E(3)-16Gl were depressed. These results together with those of the preceding paper suggest that E(3) does not readily appear in the small intestine except via a hepatoenteric circulation that produces very little E(3)-3Gl. When present in the distal segment of the small intestine, however, absorption, conjugation, and elimination proceed readily.

Biliary Fistula↗
PubMed1975

Abnormal membrane surface properties during maturation of rat reticulocytes elicited by bleeding as measured by partition in two-polymer aqueous phases.

Partition of cells in two-polymer aqueous phases is an extremely sensitive method for the separation of cells and for tracing subtle changes in the cells' membrane surface properties (primarily surface charge) as a function of in vivo processes (e.g. differentiation, maturation, ageing). Combining isotope labelling and cell countercurrent distribution techniques we have examined the membrane surface properties of rat reticulocytes produced in response to erythroid stress by bleeding. It was found that the rapid increase in the partition coefficient of normal reticulocytes subsequent to release into the peripheral blood (which reflects a rapid increase in membrane surface charge) is absent in reticulocytes produced in response to bleeding. In this way the reticulocytes behave like those produced in response to repeated phenylhydrazine injections. 'Stress reticulocytes' never mature to erythrocytes having normal membrane surface properties as judged by partition. The experiments show that our previous results are not due to the phenylhydrazine per se but rather to the production of 'stress reticulocytes'. Further, if remodelling of the phlebotomy-induced reticulocyte cell membrane occurs in the circulation as has been suggested it does not lead to a cell with normal membrane surface properties. Whether the abnormal membrane surface properties of 'stress reticulocytes' and of the erythrocytes to which they give rise affect the cells' life-span is not clear since phenylhydrazine-induced 'stress reticulocytes' have been reported to have at least a component of short-lived cells while the phlebotomy-induced cells are said to have a virtually normal life-span. It is likely that reticulocytes produced in severe anaemias, in general, mature to erythrocytes having abnormal surface properties (i.e. lower membrane surface charge).

Animals↗
PubMed1969

Studies on phenolic steroids in human subjects. 8. Metabolism of estriol-16 alpha-glucosiduronate.

6,7-(3)H-Estriol-16alpha-glucosiduronate-(14)C was administered to eight women (nine studies) by several routes: both injection and infusion (300 min) into the cubital vein, injection into the portal vein system, ingestion and instillation into the duodenum, jejunum, and ileum. Urine, collected from 0-2, 2-4, 4-8, 8-12, and 12-24 hr, was analyzed by countercurrent distribution for its content of radioactive 3- and 16-glucosiduronate (E(3)-3Gl,E(3)-16Gl) and sulfoglucosiduronate (E(3)-3S,16Gl) of estriol as well as for (3)H/(14)C ratio of each conjugate. After peripheral injection 50-60% of the injected E(3)-16Gl was excreted unchanged along with about 5% as E(3)-3S,16Gl with an unchanged (3)H/(14)C ratio, indicating direct sulfation of the injected E(3)-16Gl. During a 300 min infusion, urinary excretion closely resembled that following injection. But 2-4 hr after the end of the infusion excretion of E(3)-3S, 16Gl stopped, excretion of E(3)-3Gl (17%/24 hr) with an elevated (3)H/(14)C ratio started, and excretion of E(3)-16Gl continued (70%/24 hr), but with a rapidly increasing (3)H/(14)C ratio. This indicated sequestration in a sluggishly metabolizing compartment where two processes occurred: (a) extensive hydrolysis of E(3)-16Gl followed by reconjugation at either C3 or C16 with unlabeled uridine diphosphate glucuronic acid (UDPGA), thereby increasing the (3)H/(14)C ratio; and (b) transconjugation from C16 to C3, thereby producing E(3)-3Gl with finite (3)H/(14)C ratios. Instillation into various segments of the small intestine produced results qualitatively similar to those after intravenous infusion, whereas ingestion and intraportal injection resembled peripheral intravenous injection. Therefore, we have postulated the possibility of an enteric circulation (in addition to an enterohepatic circulation) in which the steroid or its conjugates are transported into the small intestine in the succus entericus, modified, and then reabsorbed and excreted in the urine-a process which requires several hours.

Adolescent↗
PubMed1975

Affinity partitioning. A method for purification of proteins using specific polymer-ligands in aqueous polymer two-phase systems.

We describe a method, called affinity partitioning, for the purification of proteins containing specific ligand binding receptor sites. This method adds specificity to the procedures for protein purification with aqueous polymer two-phase systems by introduction of a polymer derivative, coupled to an appropriate ligand. The addition of a polymer-ligand that partitions predominantly into one phase shifts the protein that binds this substance to the same phase. By performing countercurrent distribution in the presence of a polymer-ligand, the protein that binds the polymer-ligand can be separated from a heterogenous mixture. One example of affinity paritioning used dextran as the polymer-ligand. Dextran was chosen since it is a constituent of the most commonly used system for partitioning proteins. In a dextran-poly(ethylene oxide) system, concanavalin A bound dextran and partitioned predominantly into the dextran-rich phase. The addition of the specific competitor, D-mannose, displaced the partition coefficient toward unity, while the application of L-fucose, a noncompetitor, had little effect. Application of affinity partitioning to the purification of another protein required the synthesis of a specific polymer-ligand. To study this we synthesized dinitrophenyl-poly-(ethylene oxide), which binds specifically to S-23 myeloma protein. Addition of dinitrophenyl-poly(ethylene oxide) to the dextran-poly(ethylene oxide) phase system shifted the S-23 myeloma protein into the poly(ethylene oxide)-rich phase. epsilon-N-dinitrophenyl-L-lysine, by competing with binding of dinitrophenyl-poly(ethylene oxide), antagonized the latter's effect on the partition coefficient of S-23 myeloma protein. By adding various amounts of dinitrophenyl-poly-(ethylene oxide), we correlated the partition coefficient with concentration of polymer-ligand. A model of the action of polymer-ligand derivatives on the partition coefficient, derived from thermodynamic considerations, was found to be consistent with the experimental data relating the concentration of polymer-ligand and partition coefficient. Affinity partitioning should prove to be a useful complement to affinity chromatography in the purification of mixtures of proteins. Since cells and subcellular particles may be purified with aqueous polymer two-phase systems, affinity partitioning might be applied to their fractionation by using polymer-ligands specific for unique surface receptors.

Animals↗
PubMed1992

Effect of cell exposure to top or bottom phase prior to cell partitioning in dextran-poly(ethylene glycol) aqueous phase systems: erythrocytes as a model.

Cells exposed to dextran (Dx)-rich bottom phase prior to cell partitioning in Dx-poly(ethylene glycol) (PEG) aqueous two-phase systems have lower partition ratios than cells exposed to PEG-rich top phase. Aspects of this previously observed phenomenon were explored. In the present work charge-sensitive phases made with Dx T500 and PEG 8000 were used exclusively. It was found that: (1) even on countercurrent distribution (CCD) red cells (RBC) loaded in bottom phase have a lower apparent partition ratio, G, than the same cells loaded in top phase; (2) when part of the same cell population is loaded into top phase and part into bottom phase of the same load cavities for CCD, with the cells loaded into top or bottom bearing an isotopic tracer (51Cr), the cells loaded into top phase have a higher G value than the cells loaded into bottom phase; (3) the shift in the CCD curves of human or of rat RBC between cells loaded in top or bottom phase using systems having the same polymer concentration (though different salt compositions) shows no striking difference and is, for the number of experiments run, not statistically significant; (4) when the quantity of cells loaded for CCD is reduced from 10(9) to 10(8), the G value of cells loaded in top phase is reduced slightly while that of cells loaded in bottom phase is diminished more appreciably; (5) increasing polymer concentrations yield larger differences in G values between (rat) RBC loaded in top or bottom phase; (6) when cells exposed to top or bottom phase, respectively, are centrifuged and suspended in bottom or top phase, respectively, their CCD patterns are qualitatively similar to cells exposed to these latter respective phases initially; (7) rat RBC populations containing 59Fe-labeled cells of different but distinct age are fractionated on CCD irrespective of whether loaded in top or bottom phase. An exception are populations containing very young mature labeled cells (e.g., 4-d old) which are resolved when loaded in top phase but not in bottom phase. Thus cell populations exist which can be resolved by CCD when loaded in one of the phases but not when loaded in the other. Glutaraldehyde-fixed rat RBC containing 4-d old labeled cells are fractionated by CCD irrespective of whether loaded in top or bottom phase.

Animals↗
PubMed1992

Aqueous two-phase systems for biomolecule separation.

Over the past thirty years, aqueous polymer two-phase technology has evolved, both experimentally and theoretically, into a separation science with many useful applications in biomolecule purification and bioconversion. This paper summarizes the developments in the applications of aqueous two-phase systems to biotechnology. The main topics to be considered are the phase diagram and its characteristics, fundamentals of biomolecule partition, large-scale and multi-stage aqueous two-phase biomolecule purification, and extractive bioconversions. The first topic involves a discussion of the thermodynamics of aqueous polymer two-phase formation and how it is influenced by such factors as polymer molecular weight and concentration, temperature, and salt type and concentration. Next, the theoretical and experimental aspects of biomolecule partition in aqueous two-phase systems will be discussed in light of the factors which influence biomolecule partition: polymer concentration and molecular weight; temperature; salt type and concentration; the addition of charged, hydrophobic and affinity derivatives. Having reviewed the fundamentals of phase diagram formation and biomolecule partition, the next two topics are applications of aqueous two-phase technology. The first set of applications involve the large-scale extraction of proteins using one to three equilibrium stages and multi-stage purifications using countercurrent distribution, liquid-liquid partition chromatography and continuous countercurrent chromatography. The second application, and very promising area for future aqueous two-phase technology, is the extractive bioconversion which permits the simultaneous production and purification of a biomolecule.

Biotechnology↗