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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↗
PubMed1965

Sulfur: incorporation into the transfer fraction of soluble ribonucleic acid.

When S(35)-labeled soluble RNA from Escherichia coli K 38 is subjected to gel filtration, four fractions of RNA are obtained by elution. Only one RNA fraction, the transfer RNA, contains sulfur, presumably as thionucleotides. Treatment with ribonuclease suggests that the incorporated sulfur is an integral part of the polynucleotide chain; digestion with alkali yields a mixture of products containing sulfur, the major one being eluted in a position similar to uridine diphosphate upon Dowex-l chromatography. Analysis by countercurrent distribution of S(35)-labeledtransfer RNA from E. coli B reveals that the incorporated sulfur is found in many RNA's that accept amino acids, but the possibility remains that not all acceptor RNA's contain sulfur.

Chromatography↗
PubMed1990

Coupling of poly(ethylene glycol) to albumin under very mild conditions by activation with tresyl chloride: characterization of the conjugate by partitioning in aqueous two-phase systems.

Poly(ethylene glycol) activated with tresyl chloride has been covalently linked to albumin as a result of a 2-h incubation in 0.05 M sodium phosphate buffer, pH 7.5, containing 0.125 M sodium chloride (0.344 OSM). The coupling of poly(ethylene glycol) to albumin was demonstrated by the increase in the partition coefficient of the protein in poly(ethylene glycol)-dextran aqueous two-phase systems. A linear relationship between the log of the partition coefficient of the poly(ethylene glycol)-albumin conjugate and the degree of modification (measured as the amino groups consumed during the coupling step) has been demonstrated. Countercurrent distribution in the two-phase system showed that poly(ethylene glycol)-albumin was heterogeneous with respect to its partitioning behavior, indicating that the albumin was not uniformly modified with poly(ethylene glycol).

Chemical Phenomena↗
PubMedDate not supplied

Isolation and identification of the major metabolite of albuterol in human urine.

The major urinary metabolite of albuterol in man was isolated and purified by ion-exchange chromatography and countercurrent distribution. On the basis of proton magnetic resonance, infrared, and mass-spectral analysis, enzymic hydrolysis, and assay of inorganic sulfate after acid hydrolysis, the metabolite has been identified as the 4'-O-sulfate ester of albuterol.

Albuterol↗
PubMed1979

Synthesis of renin substrate by rat liver.

The present study attempts to determine if the isolated rat liver is capable of synthesizing renin substrate from 14C-labelled amino acids added in the perfusate. The renin substrate is characterized via reaction with renin, forming a substance that is subsequently identified as proangiotensin. Extensive evaluation of the reaction product is carried out by using molecular-sieve chromatography, countercurrent distribution, reactivity with converting enzyme, radioimmunological technique and bioassay. The results demonstrate that isolated rat liver perfused with artificial salt solution is capable of synthesizing a protein that reacts with renin to form a radioactive substance indistinguishable from proangiotensin.

Angiotensinogen↗
PubMed1971

Angiotensin and renin in rat and dog brain.

An enzyme with the characteristics of classical renin was isolated from brain extracts of nephrectomized dogs. This enzyme is thermolabile, nondialyzable, and forms a vasoconstrictive material when incubated with renin plasma substrate at pH 7. A short lasting pressor activity was also found in brain extracts of dogs and rats. This activity was due to a substance identified by chemical and pharmacological tests as angiotensin. Countercurrent distribution of brain extracts of rats showed that 38% of the pressor activity corresponded to angiotensin II and the remainder to angiotensin I. A remarkable correlation was found between angiotensin and norepinphrine concentrations in different portions of the encephalon of the dog.

Angiotensin II↗
PubMed1970

The inhibition of N-acetyl-beta-D-glucosaminase by the (1-4)-and (1-5)-lactones of N-acetylglucosaminic acid.

The inhibition of N-acetyl-beta-d-glucosaminase activity by 2-acetamido-2-deoxy-d-gluconolactone was examined. Separation of the (1-->4)- and (1-->5)-lactones was achieved by using paper chromatography or countercurrent distribution and identification was obtained by examination of the relative stability of the components of separated material. Quantitative measurement of the two lactones was by kinetic titration or by a colorimetric method based on their reaction with hydroxylamine. It was shown that only the (1-->5)-lactone acted as an inhibitor of N-acetyl-beta-d-glucosaminase.

Animals↗
PubMed1987

The partitioning behavior of K-562 cells associated with different stages of culture growth.

Partitioning behavior of cells in dextran-poly(ethylene glycol) aqueous phase systems is a sensitive reflection of the cells' surface properties. A decrease in partition ratio, in charge-sensitive phases, of a variety of cell lines as a function of culture growth has been reported by a number of investigators. The basis for this phenomenon remains unclear. We have now studied the surface properties of K-562 cells (a human cell line originally derived from a patient with chronic myelogenous leukemia in blast crisis) during suspension culture growth by countercurrent distribution. The mean partition ratio of viable cells remained constant during 220 h of culture (i.e., well into stationary phase). The decrease in mean partition ratio of the cell population as a whole during culture, previously observed and reported by others, is attributed to the lower partition ratio of non-viable cells which increase with time of culture.

Cell Division↗
PubMed1978

Fractionation of microsomal membranes on the basis of their surface properties.

Partition in dextran-poly(ethylene glycol) aqueous-phase systems can be used for both membrane subfractionation and gaining information on membrane surface properties [H. Walter (1977) in Methods of Cell Separation (Catsimpoolas, N., ed.), vol. 1, pp. 307-354, Plenum, New York]. Smooth, light rough and heavy rough rat liver microsome (obtained by sucrose-density-gradient centrifugation) were subjected to countercurrent distribution in such a system. Smooth microsomal membranes had the highest, heavy rough microsomal membranes the lowest and light rough microsomal membranes an intermediate partition coefficient. The separation is based primarily on hydrophobic differences in the membrane surfaces of the three preparations and is thus due to microsomal properties not previously utilized in their fractionation. The method permits additional subfractionations of microsomes.

Animals↗
PubMed1975

[The lysis of proteins with cyanogen bromide (author's transl)].

Bovine beta-lactoglobulin-AB was split with cyanogen bromide, and the reaction mixture was analyzed by countercurrent distribution, gel chromatography and finally, chromatography on phosphocellulose. In addition to the previously described splitting products, we obtained three more minor products, with yields of 8, 15 and 25%. The analytical data indicate that these were formed by lysis C-terminal from the tryptophan-19 and the tryptophan-61 of beta-lactoglobulin. The result is discussed.

Amino Acids↗
PubMed1985

Polypeptide compositions of amoebae of the cellular slime mould Dictyostelium discoideum separated by partitioning during development.

Amoebae of the cellular slime mould Dictyostelium discoideum at 8 h or 10 h development were separated into two populations by countercurrent distribution in a dextran-poly (ethylene glycol), two-phase system. Two-dimensional, polyacrylamide-gel electrophoresis was then used to separate the polypeptides from the populations of amoebae. The two populations of amoebae at 8 h development differed 9n polypeptide composition as did the populations separated at 10 h development. This confirms that cell differentiation is initiated in D. discoideum prior to 8 h development.

Countercurrent Distribution↗
PubMed1976

Cross partition and determination of net charge of the isoenzymes of enolase.

Enolase from bakers' yeast was separated into three isoenzymes by countercurrent distribution. The isoenzymes were partitioned in aqueous polymer two-phase systems containing positively charged trimethylamino poly(ethylene glycol) or negatively charged poly(ethylene glycol) sulphonate. The plots of the partition coefficient of each isoenzyme versus pH in the two biphasic systems intersect at pH equal to the isoelectric point. From slopes of the plots, the net charge of the isoenzymes at pH 6.57 was determined to be +2, -3, and -8 respectively.

Animals↗
PubMed1979

Hydroheptin: a water-soluble polyene macrolide. II. Chemical and biological properties.

Hydroheptin, a new polyene macrolide antifungal antibiotic, is co-produced with the antibiotic, chartreusin, by a strain of Streptomyces chartreusis designated as IMRU 3962 isolated in our laboratory. The unique water-solubility of this antibiotic at neutrality, revealing in aqueous solution molecular dispersion and an ultraviolet-visible absorption spectrum characteristic of an all-trans heptaene chromophore, clearly distinguishes it from all previously-described and naturally-occurring heptaene macrolides. The isolation and identification of the amino sugar, mycosamine (3-amino-3,6-dideoxy-D-mannose), in acid hydrolysates of hydroheptin and the absence of an aromatic amine upon retrograde alkaline dealdolization of the molecule certainly characterize the antibiotic as a member of the non-aromatic heptaene macrolide group. Chromatographic and countercurrent distribution studies likewise support its novelty. With little or no demonstrable activity against bacteria, hydroheptin as compared to other non-aromatic heptaene macrolides exhibits excellent but somewhat less activity against a wide variety of yeasts and fungi. Likewise, its parenteral toxicity appears to be less than that of other heptaene macrolides.

Animals↗
PubMed1968

Methods for the separation and identification of mycobactins from various species of mycobacteria.

A single pure component was isolated from mycobactin P by countercurrent distribution; its side chain is n-cis-octadec-2-enoyl; its purity and molecular structure were confirmed by mass spectrometry of its aluminium complex. The separation of ferric and of aluminium complexes of mycobactins by thin-layer chromatography is described. Mycobacterium terrae, M. marinum and M. smegmatis produce mycobactins that differ among themselves and from mycobactins P and T. A nomenclature for the mycobactins and their derivatives is suggested.

Chromatography, Gas↗
PubMed1976

The extraction and purification of a cysteine transfer ribonucleic acid from baker's yeast.

1. A modification of the RPC 1 system of A.D. Kelmers, G.D. Novelli & M.P. Stulberg (1965) (J. Biol. Chem. 240, 3979-3983) is described in which the support medium is a Celite of narrow range particle size treated with dichlorodimethylsilane. 2. By using this system an apparently pure preparation of tRNA Cys was isolated from baker's yeast tRNA. 3. This preparation accepted at least 60% of the theoretical quantity of [3-14C]cysteine in a conventional assay and failed to accept isoleucine, phenylalanine, proline, serine or tyrosine. 4. A theoretical countercurrent-distribution curve calculated by assuming a distribution coefficient K of 2.03 was in excellent agreement with the profiles of E260 and cysteine-acceptor ability after 537 transfers in the 1.85 M-phosphate/formamide/propan-2-ol system of C.M. Connelly & B.P. Doctor (1965) (J. Biol. Chem. 241, 715-719). 5. Chromatography of tRNA Cys on Bio-Gel P100 polyacrylamide beads afforded two components one of which was far less efficient than the other in accepting cysteine. The base compositions of the two were similar.

Amino Acids↗
PubMed1987

Fractionation of K-562 cells on the basis of their surface properties by partitioning in two-polymer aqueous-phase systems.

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 subjected such cells, in the log phase of growth, to countercurrent distribution in a charge-sensitive dextran-polyethylene glycol aqueous-phase system, a method that fractionates cells on the basis of subtle differences in their surface properties, and found that: (1) The cell population is heterogeneous since it is composed of cells with different partition ratios. (2) There is a correlation between increasing cell partition ratios and increasing cell electrophoretic mobilities. (3) Cells under different parts of the distribution curve have dissimilar ratios of cells in different parts of the cell cycle, a phenomenon that may, at least partially, be the basis for the subfractionation of these cells. There is a clear tendency for cells in G0 + G1 + early S to decrease and for those in late S + G2 + M to increase with increasing partition ratios. (4) Sialic acid is a major surface charge component of the cells as evidenced by a dramatic drop in their partition ratios after treatment with neuraminidase.

Cell Cycle↗