Distribution and metabolism of chlorphentermine-C14 in rats and mice.
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Adipose Tissue↗
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Effect of some poly(ethylene glycol)-bound and dextran-bound affinity ligands on the partition of synaptic membranes in aqueous two-phase systems.
Ligands with an apparent affinity for various structural elements on the surface of synaptic membrane fragments have been bound to the polymers poly(ethylene glycol) and dextran. The ligand-polymer derivatives have been included in aqueous two-phase systems composed of water, poly(ethylene glycol) and dextran. The uneven distribution of the polymers resulted in the concentration of the polymer-bound ligand in one of the two phases. The effect of the ligand-polymer on the partition of membranes was studied by using synaptic membranes from calf brain, obtained by standard centrifugation methods. By using ligand-containing two-phase systems for nine-step counter-current distribution of membranes, it was shown that the distribution behaviour of various parts of the membrane preparation could be affected. The distribution was followed by determination of opiate binding, acetylcholinesterase, and total membrane (using protein and light-scattering measurements).
Acetylcholinesterase↗
Liquid-liquid extraction of membranes from calf brain using conventional and centrifugal counter-current distribution techniques.
Neural membranes isolated from calf brain have been partitioned in aqueous two-phase systems containing dextran and polyethyleneglycol. When the partition was repeated several times, using counter-current distribution technique, the distribution of the membranes between the upper phase and the interface changed in a non-ideal manner and in favour of the interface. By using a centrifugal counter-current distribution device the time for the experiment could be reduced by a factor of 7-8 and the distribution was similar to what could be expected for ideally behaving membranes. The time-dependent change of the membranes is discussed in terms of aggregation and lateral membrane perturbations. Despite this effect a certain fractionation has been achieved as deduced from analysis of cholesterol content, opiate receptor activity and acetylcholinesterase activity along the counter-current distribution row of fractions. Compared to the starting material these activities were enriched some two-fold in certain fractions.
Animals↗
Lateral heterogeneity of rat liver plasma membranes analysed by counter-current distribution.
The lateral heterogeneity of rat liver plasma membranes was examined by fragmentation and fractionation by counter-current distribution in an aqueous two-phase polymer system. The distribution pattern was analysed by plotting the relative specific activities of marker components against each other. By this analysis asialo-orosomucoid receptors were found in a domain separated from domains containing 5'-nucleotidase and leucine aminopeptidase by another domain devoid of these markers. 5'Nucleotidase and leucine aminopeptidase resided in adjacent but separate domains. The experimental data were compared with corresponding plots of markers in model membranes. The model membranes yielded plots of different shapes depending on marker distribution and fragment size. This method of analysis should be useful for examining the lateral heterogeneity also of other membranes.
5'-Nucleotidase↗
Partition and counter-current distribution of membrane particles in aqueous dextran-poly(ethylene glycol) two-phase systems with special reference to synaptosomes.
Aqueous two-phase systems composed of water, dextran and poly(ethylene glycol) can be used for the separation of biological particles. The adjustment of the partition of such particles between the two phases and the interface between them has been studied by using a preparation of synaptosomes (from calf brain cortex) also containing free mitochondria. The partition has been affected by variation of polymer concentrations and addition of salts, e.g. phosphates and chloride. The time for separation of the phases showed a bimodal behaviour with an initially rapid formation of bulk phases followed by a slow phase separation. The relative amount of mixed phases at the time of the transition was proportional to the amount of particles included. Counter-current distribution with moderate time for the phase separation was carried out in such way that the interface material travelled with approximately half the speed of the moving upper phase. In this way the distribution of the particles between the upper phase and the interface as well as between the interface and the lower phase could be studied in the same experiment. The heterogeneity of the synaptosome preparation was clearly demonstrated by counter-current distribution at low polymer concentrations while no separation was obtained when the system contained larger amounts of polymers. Possible reasons for this behaviour are discussed.
Animals↗
Fractionation of wheat proteins by counter-current distribution using aqueous two-phase systems containing propionic acid.
Wheat proteins, soluble in diluted acid (glutenins), have been fractionated by counter-current distribution (CCD) using an aqueous two-phase system. The phase system is based on poly(ethylene glycol) and dextran but contains also 1% propionic acid and 6 mM magnesium sulfate. Approximately half of the bulk proteins partitioned to the upper phase while starch and other particles were recovered only into the lower phase. Whole wheat flour could be applied as sample for the CCD and 57 transfers were carried out. Starch and insoluble proteins remained stationary, while proteins followed the mobile phase to various degrees giving rise to a distribution pattern. The CCD pattern of the proteins showed distinct differences when various kinds of wheat flour were analysed. The patterns indicate that at least six subpopulations of proteins can be obtained by using two-phase extraction.
Acids↗
Rat bone marrow erythroid cell fractionation by counter current distribution in non-charge-sensitive two-phase systems.
Counter-current distribution in non charge-sensitive aqueous poly(ethylene glycol)-dextran two phase systems allows the fractionation of rat bone marrow cells into two broad cell subpopulations with different distribution coefficients in a relatively short time. Morphological identification and enzymatic studies suggest that erythroid cells are mainly present in the subpopulation with the higher distribution coefficient. The distribution coefficient and, therefore, surface hydrophobicity of these cells, apparently increase in parallel with an increase in their degree of differentiation and maturation.
Animals↗
Heterogeneity of smooth endoplasmic reticulum from rat liver studied by two-phase partitioning.
Smooth microsomal membranes, prepared from rat liver by sucrose-density-gradient centrifugation, were subfractionated by counter-current distribution in an aqueous two-phase system consisting of poly(ethylene glycol) and Dextran T500. A comparison of the distribution curves of marker enzymes, together with theoretically calculated curves, indicated the presence of at least five membrane subfractions, differing in the ratios of the marker enzymes. Glucose-6-phosphatase and arylesterase distributed in one manner, and NADPH-cytochrome c reductase and NADH-ferricyanide reductase in another. Evidence for further heterogeneities in the distribution of marker enzymes in smooth microsomes was obtained by analysing the membrane domain structure using a recently described method [Albertsson (1988) Q. Rev. Biophys. 21, 61-98]. Phenobarbital treatment did not influence the behaviour of the marker enzymes.
Animals↗
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↗
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↗
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↗
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↗
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↗
[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↗
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↗
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↗
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↗
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↗