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PubMed1993

Fractionation of erythroblasts with affinity-mediated modifications of their electrical properties using counter-current distribution.

We have previously reported the possibility of modifying the electrical properties of cells by means of their interaction with a specific ligand carrying a polyelectrolyte (Anal Biochem 200: 280-285). This selective modification of receptor-containing cells changed their partition in a charge-sensitive aqueous two-phase system. We here present the fractionation of electrically modified erythroblasts by the use of an automatic multiple-partition procedure, counter-current distribution. The cells were fractionated according to the degree of differentiation of erythroblasts as evaluated from the hemoglobin content as well as the relative activities of the two enzymes, 3-phosphoglycerate kinase and bisphospho-glycerate mutase.

Animals↗
PubMed1993

Recognition and separation of isoenzymes by metal chelates. Immobilized metal ion affinity partitioning of lactate dehydrogenase isoenzymes.

Poly(ethylene glycol) (PEG)-bound chelated metal ions partition preferentially into the top, PEG-rich, phase of a PEG-salt or PEG-dextran aqueous two-phase system. Extraction by this soluble affinity ligand of proteins is due to a selective interaction of the chelated metal ion with accessible histidine residues on the protein surface. Using Cu-iminodiacetate-PEG (Cu-IDA-PEG) the surface of lactate dehydrogenase (LDH) isoenzymes from different species was probed for the presence of metal chelate binding sites. It was demonstrated that the homotetramers (LDH-1)(H4) from rabbit, bovine and pig displayed weak binding to chelated copper whereas the M4-type isoenzymes (LDH-5) bound strongly to this ligand. The binding of the different heterotetramers increases as the number of M-type subunits increases. In contrast, the human isoenzymes are bound to chelated copper in a reversed sequence. The comparison of the affinity partitioning effect of Cu-IDA-PEG in PEG-salt and PEG-dextran systems revealed that the discriminatory effect of copper is promoted by high salt concentrations. Resolution of isoenzymes by multiple extraction using counter-current distribution provides valuable data on the partitioning of enzymes relative to that of the bulk proteins. The efficacy of metal chelate affinity partitioning for the purification of LDH from tissue samples by batchwise extraction was also demonstrated.

Animals↗
PubMed1985

Studies on the ATP-sensitivity of yeast phosphofructokinase by means of affinity partitioning using polymer bound Cibacron blue F3G-A.

Yeast phosphofructokinase was partitioned in an aqueous two-phase system composed of polyethylene glycol and dextran in which a small amount of the first polymer was replaced by Cibacron Blue F3G-A substituted polyethylene glycol. It was found that the partition coefficient, K, of the enzyme determined immediately after adding the enzyme to the system and within a series of time intervals (min) shifts from a lower to a higher value. This effect was amplified when the enzyme was preincubated with fructose 6-phosphate in millimolar concentration range. Other effectors, like ATP, ADP, AMP, fructose 1,6-bisphosphate and protons were without significant influence on the partition. A similar behaviour in lowering the partition coefficient was found with an enzyme form desensitized to ATP-inhibition. The existence of enzyme forms of different affinity to Cibacron Blue depending on the fructose 6-phosphate could be manifested by counter-current distribution. Because of the competition in the binding of Cibacron Blue and ATP to the nucleotide binding sites the conclusion was drawn that the partition of phosphofructokinase reflects the alteration of the affinity of the enzyme to ATP.

Adenosine Triphosphate↗
PubMed1969

Lipid metabolites of carbon tetrachloride.

5 min after intravenous injection into rats of (14)C- or (36)Cl-carbon tetrachloride, liver lipids were found labeled. Most of the radioactivity was found in the phospholipid fraction. The metabolites were shown to comprise a heterogeneous group of branched long-chain chlorinated fatty acids, probably containing the trichloromethyl side chain. Surviving liver slices also formed these metabolites. In a simple chemical system which generates trichloromethyl free radicals, carbon tetrachloride added to methyl oleate to form esters which behaved like the metabolites during counter-current distribution and urea adduction. The evidence strongly suggests the formation of these metabolites by free radical attack on unsaturated lipids. The relation of these observations to current theories of carbon tetrachloride intoxication is discussed.

Animals↗
PubMed1990

Fractionation in two-phase systems of red cells during rat development: changes in pyruvate kinase and bisphosphoglycerate mutase activities in relation to red cell switching.

An inverse relationship between 2,3-bisphosphoglycerate levels and the ratio calculated from pyruvate kinase and bisphosphoglycerate mutase activities has been observed in red populations of rats during animal development. Counter-current distribution in aqueous two-phase systems of these cells populations shows a displacement of distribution profiles towards the high-numbered cavities of the rotor as animal ages. Heterogeneity of cells after distribution is only observed during the switching process from fetal to adult red cells taking place along the postnatal stage of development. Values for the pyruvate kinase/bisphosphoglycerate mutase ratio in these fractions suggest the separation of fetal (liver) from adult (bone marrow) red cells.

Animals↗
PubMed1976

[Isolation and physicochemical characteristics of antibiotics from the LIA-0773 and LIA-0780 s trains].

Individual components of the antibiotics from strains LIA-0773 and LIA-0708 were isolated with the method of counter-current distribution. Physico-chemical and biological properties of these substances were studied. It was shown that the substances differed from each other and chondamycin, and antifungal antibiotic by the values of their chromatographic mobility, physico-chemical and biological properties.

Actinomycetales↗
PubMed1987

Laterobasal membranes from intestinal epithelial cells: isolation free of intracellular membrane contaminants.

A simplified method for isolating highly purified laterobasal membranes (LBM) from enterocytes is based on treatment of membranes with 8 mM CaCl2 concentration in order to aggregate intracellular membrane contaminants. The resultant LBM showed an average 15-fold enrichment and constituted 8% of the original K-stimulated phosphatase in the initial crude homogenate. It showed typical LBM migration on counter-current distribution (CCD) and was essentially free of contamination with endoplasmic reticulum and Golgi membranes. This method is highly efficient and yields sufficient purified LBM to allow comprehensive analysis of enterocyte membrane events.

Animals↗
PubMed1979

Protein-protein interactions studied by counter-current distribution. I. Theoretical computations.

Many biological macromolecules are known to interact either with themselves, with other macromolecules or with small compounds. A simple equilibrium method for detecting and quantifying these interactions is to study the mutual influence of the molecules on their respective counter-current distribution in liquid-liquid biphasic systems. The theoretical counter-current distribution patterns for the components in an interacting system, A + B in equilibrium AB, have been calculated for two models in order to establish the boundary conditions and to optimize the experimental procedure. The patterns have been calculated for a range of association constants, partition coefficients and initial concentrations of the two reactants.

Countercurrent Distribution↗
PubMed1975

A study of DNA from chloroplasts separated by counter-current distribution.

Three types of chloroplasts (Peak I, Peak II, and Peak III chloroplasts) isolated by counter-current distribution in aqueous polymer two-phase systems have been studied with respect to DNA content. (The characterization was performed by studying the buoyant density, the melting profile and the amount of DNA of each type). The DNA was found to differ between the three types of chloroplasts.

Centrifugation, Density Gradient↗
PubMed1992

Management of sedimentation in centrifugal counter-current distribution of sperm cells in an aqueous 2-phase system.

It is generally assumed that centrifugal counter-current distribution (CCCD) in aqueous two-phase systems cannot be employed for analyzing or fractioning cell populations, due to large particles of sediment in the system caused by enhanced gravity. The present work was undertaken to find out whether addition of Percoll to a two-phase system would be a useful method to avoid this cell sedimentation. The results obtained show that bull spermatozoa partition as a unique peak in a CCCD using a Dextran T500-poly(ethylene glycol) 6000 system, and that sedimentation takes place significantly in the upper phase during the process. Addition of increasing concentrations of Percoll made this unique peak wider and two different populations of bull spermatozoa were finally obtained when Percoll concentration rose to 13.6%. This management of cell sedimentation in CCCD could be of great interest for analyzing cell heterogeneity, since the shortening of the time required for counter-current distribution should prevent the loss of cell viability during the separation process. Finally, the results obtained suggest that an increase of viscosity rather than of density is the phase feature which has greater influence on managing cell sedimentation in CCCD.

Animals↗
PubMed1990

Settling-time dependence of rat bone marrow cell partition and counter-current distribution in charge-sensitive aqueous two-phase systems. Relationship with the cell partitioning mechanism.

Differences in the settling-time dependence of single and multiple cell partitions have been found between heterogeneous (bone marrow cells) and homogeneous (erythrocytes) populations when using charge-sensitive dextran-poly(ethylene glycol) aqueous two-phase systems. The cell populations were partitioned using both single test-tube experiments and multiple thin-layer counter-current distribution. Lengthening the settling time, to favour phase separation, and decreasing the upper phase volume are more effective in fractionation by the counter-current distribution of heterogeneous cell populations than increasing the interfacial tension, although all three were employed to speed phase settling. On the basis of these results, the original cell partitioning mechanism proposed for non-charge-sensitive systems has been extended to charge-sensitive systems.

Animals↗
PubMed1989

Heterogeneity of a crude synaptosomal preparation, studied by affinity partitioning using hexaethonium-poly(ethylene glycol).

The heterogeneity of a synaptosomal preparation was studied by the use of affinity partitioning in combination with centrifugal counter-current distribution. Hexaethonium-poly(ethyleneglycol) was used as the extracting agent. The fractions were analyzed for: light scattering, protein, choline acetyltransferase, L-glutamate decarboxylase, glutamine synthetase, 2',3'-cyclicnucleotide-3'-phosphohydrolase, acetylcholinesterase and succinate dehydrogenase. The material was fractionated into three main fractions which differed in their content of marker-enzymes.

Animals↗
PubMed1994

Biochemical characterization of human erythrocytes fractionated by counter-current distribution in aqueous polymer two-phase systems.

The fractionation of normal human erythrocytes by counter-current distribution (CCD) in charge-sensitive dextran-polyethylene glycol two-phase systems was confirmed and extended to red blood cells from heterozygous beta-thalassaemic patients. The differences between the distribution profiles of normal (homogeneous) and abnormal (heterogeneous) red blood cells reflect their different surface-charge properties. As suggested by the decline of membrane sialic acid released after neuraminidase treatment and the specific activities of two age-dependent enzymes (membrane acetylcholinesterase and intracellular pyruvate kinase) in the distribution profiles (from the left- to the right-hand side fractions), the fractionation seems to be according to red blood cell age. A constancy of the 2,3-bisphosphoglycerate level was observed in ageing red blood cells.

2,3-Diphosphoglycerate↗
PubMed1978

Portal circulations and their relation to counter-current systems.

We have reviewed the distribution of portal circulations throughout the animal body; they are commoner than is generally supposed. Most portal circulations consist of two serial capillary beds connected by one or more larger vessels. We have called these 'convergent' portal circulations: examples are hepatic portal, placental, hypophysial, renal, ovarian and testicular circulations, as are parts of the lymphatic circulation. A second type of portal circulation, which is less common, consists of two serial capillary beds that are not connected by larger vessels. These we have called 'continuous' portal circulations: adrenal and pancreatic circulations are examples of this type. When a countercurrent concentrating mechanism exists in the body it is always part of the primary or secondary bed of a convergent portal circulation, though some convergent portal circulations are not associated with countercurrent mechanisms.

Adrenal Glands↗
PubMed1981

Separation by counter-current distribution of rat and chicken erythrocytes of different age, and its application to the assay of enzyme activities.

The separation of cells with different ages from erythrocyte populations of adult rats and young or adult chickens have been achieved by counter-current distribution (CCD). A thin-layer CCD apparatus has been employed. Erythrocytes from blood samples taken at different times after 59Fe i.p. injection were separated by CCD. By compilation in a "composite curve" of the hemoglobin and radioactivity CCD profiles obtained for each erythrocyte population, the distribution of cells according to age can be inferred. Young erythrocytes of rats are located at the right part of the CCD curves, while older cells are distributed towards the left. An opposite distribution has been found for erythrocytes from adult or young chickens. As a first attempt for the application of the CCD procedure to the assay of enzyme activities, it was found a decrease in phytase activity as the age of chicken erythrocytes increases and an increase in phosphoglycerate kinase and phosphofructokinase as the age of rat erythrocytes increases.

6-Phytase↗
PubMed1978

Biochemical studies on rat liver Golgi apparatus. III. Subfractionation of fragmented Golgi apparatus by counter-current distribution.

Vesicular fragments of Golgi apparatus, smooth- and rough-surfaced microsomes from rat liver are differently partitioned in aqueous polymer two-phase systems consisting of dextran, polyethylene glycol, and sodium phosphate buffer. At a given polymer concentration, the amount of material partitioned in the top phase increases in the following order: rough microsomes less than smooth microsomes less than Golgi fragments. Counter-current distribution of Golgi fragments in the system consisting of 6.8% (w/w) dextran T500 and 6.8% polyethylene glycol 4,000 results in the separation of the fragments into three fractions; i.e. Fractions I, II, and III. NADH- and NADPH-cytochrome c reductase activities are detected almost exclusively in Fraction I, whereas the activities of galactosyltransferase, acid phosphatase, 5'-nucleotidase, and thiamine pyrophosphatase are maximal in Fraction III and minimal in Fraction I. The distribution of these enzymes suggests that Fraction I is similar to, though not identical with, microsomes, Fraction III resembles plasma membrane and lysosomes, and Fraction II is between the two. It is concluded that NADH- and NADPH-cytochrome c reductases are localized in a restricted region of the Golgi structure and that intra-Golgi differentiation seems to proceed in a discontinuous manner.

Acid Phosphatase↗
PubMed1988

Partitioning behaviour in aqueous two-phase systems and fractionation by counter-current distribution of chick-embryo erythrocytes with numerical resolution of distribution curves.

The partition of chick-embryo and young-chick erythrocytes in dextran-poly-(ethylene glycol) two-phase systems depends on the interfacial tension and electrical potential differences between the phases. Counter-current distribution with charged 5% dextran-poly(ethylene glycol) systems has proved to be an adequate method for the separation of primitive and definitive erythrocytes present in chick embryos when a phase settling time of 20 min is used. The computer-aided numerical resolution of experimental curves has shown the existence of subpopulations which could not have been detected by using conventional methods.

Animals↗
PubMed1991

Mutagenic contaminants in synthetic peptides obtained by an azide coupling.

Hormone-like peptides are, almost by definition, not mutagenic. It was, therefore, unusual to find that some batches of peptides synthesized by azide coupling were mutagenic in the Ames test. One of these peptides, eledoisin, showed mutagenic activity particularly in Salmonella typhimurium TA 1535 without metabolic activation. This activity was independent of the peptide purity determined by HPLC and a dose response relationship was observed at concentrations over the solubility limit of the peptide in the assay medium. We therefore suggested that the mutagenic effect might be due to the presence of chemically undetectable, water-soluble impurities, which could be removed by counter-current distribution. If, however, the same final coupling was carried out by the mixed anhydride procedure, no mutagenic activity was observed. Consequently, we considered that the mutagenicity detected was due to traces of hydrazoic acid salts arising during azide formation in the coupling step. In fact only the product of the coupling reaction between the pivotal intermediates was mutagenic.

Amino Acid Sequence↗