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At least 163 records · Page 9Linked to original sources

PubMed1988

Effect of rapidity of phase separation on the efficiency of cell fractionation by partitioning in aqueous two-phase systems.

Partitioning in two-polymer aqueous phase systems is an established method for the separation, purification and characterization of biomaterials. Because of the relatively slow settling rates of these phases, a consequence of the slight difference in density between them, effort has been directed to speeding up phase separation by various means (e.g., the development of a thin-layer countercurrent distribution apparatus). This has resulted in the more rapid processing of materials. Unlike soluble materials, biological particulates (e.g., cells) generally partition between one of the bulk phases and the interface. The mechanism of cell partitioning involves cell-specific adsorption to droplets of one phase suspended in the other, subsequent to phase mixing, and the delivery of adsorbed cells to the bulk interface as the droplets settle. In this communication we show, using erythrocytes as a model, that speeding up phase separation is counterproductive when partitioning cells and results in reduced efficiency of their separation or subfractionation. The most likely reason for this result is that increasing the speed of phase settling removes the droplets of one phase suspended in the other more rapidly than cells can attach to them, thereby interfering with the mechanism whereby cells partition.

Animals↗

PubMed1975

Improved high-performance liquid chromatographic method for polypeptide antibiotics and its application to study the effects of treatments to reduce microbial levels in bacitracin powder.

Improvements were made in the high-performance liquid chromatographic (HPLC) method to obtain baseline separation of chromatographic peaks of structurally similar polypeptide components in bacitracin. The improved method uses a 30-cm-long stainless-stell column packed with muBondapak C18. The theoretical plates of the column are approximately 140,000 per meter for the bacitracin A peak. The resolution function between bacitracins B1 and B2 and that between bacitracins A and B2 have been improved 418 and 225%, respectively. The components of bacitracin, bacitracins A, B, C, D, E, F, and G, were fractionated by the countercurrent distribution technique. These components, together with Compound X, a compound separated on a carboxymethylcellulose column, and bacitracin F, obtained by degrading bacitracin A sample at neutral pH, were used to identify peaks in the HPLC chromatogram. Effects of processing methods used to reduce microbial contamination levels in bacitracin powders were evaluated. Heat treatment caused a significant loss of antimicrobial activity (35% reduction), bacitracins A, B1, and B2 were reduced by 37, 22, and 21%, respectively. A significant increase (2.8 times) of bacitracin F, an oxidative degradation compound, was show. Irradiation by 60Co at 1.8 Mrad caused no loss of potency nor change in any of the bacitracin components. Ethylene oxide treatment, on the other hand, caused considerable (46%) reduction of potency. Substantial reduction of areas under the peak of bacitracins A, B1, and B2 (50, 24 and 37%, respectively) were noted. The chromatograms showed numerous unresolved peaks around bacitracins A, B1 and B2,; however, no significant increase in the bacitracin F peak, nor appearance of non-UV absorbing peaks were observed. Peptide antibiotics of the polymyxin group, circulin, colistin, and polymyxin, were also analyzed using the muBondapak C18 column with a linear-gradient elution. A UV monitor was used for polymyxin. A moving-wire flame ionization detector was used to monitor circulin and colistin. A sample of polymyxin, circulin, and colistin may be analyzed in less than 20 min of chromatographic time.

Anti-Bacterial Agents↗

PubMed1976

Isolation and structure of somatostatin from porcine hypothalami.

The isolation and structure of somatostatin (GH-RIH) from pig hypothalami are described. This hormone was purified by preparative gel filtration, solvent extraction, countercurrent distribution in two solvent systems, ion-exchange and partition chromatography, and analytical gel filtration. The somatostatin activity was followed by in vitro bioassays and a radioimmunoassay. The isolated product was homogeneous chromatographically and had biological and immunological properties similar to synthetic somatostatin corresponding to the ovine hormone. The primary structure of porcine somatostatin was shown to be H-Ala-Gly-cyclo-(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH. Other immunologically and biologically active form(s) of somatostatin were also detected.

Amino Acid Sequence↗

PubMed1977

Incorporation of [2-3H] ethanolamine into rat muscle phosphoglycerides.

The contribution of phosphatidylethanolamine methylation to phosphatidylcholine biosynthesis in rat muscle was investigated by studying the incorporation of [2-3H] ethanolamine. The specific radioactivities of individual molecular species of muscle phosphoglycerides were measured by a combination of argentation thin-layer chromatography and countercurrent distribution. The specific radioactivity of phosphatidylethanolamine was approximately one thousand times that of phosphatidylcholine. Amongst individual phosphatidylethanolamines, hexaenoic species possessed the highest specific radioactivities and tetraenoic the lowest. Because of the very low incorporation into phosphatidylcholine, the specific radioactivities of combined rather than of individual fractions were measured. The results indicate that the contribution of phosphatidylethanolamine methylation to the overall biosynthesis of phosphatidylcholine in muscle is of minor importance.

Animals↗

PubMed1968

Isolation of crustecdysone (20R-hydroxyecdysone) from a crayfish (Jasus lalandei H. Milne-Edwards).

1. A small amount (2mg.) of crustecdysone, a moulting hormone of crustaceans, was isolated from 1 ton of crayfish waste. 2. The purification procedure used was developed with the aid of crustacean and insect bioassays. 3. CM-Sephadex was found to be superior to Sephadex and very effective for the chromatographic separation of crustecdysone from other non-ionic compounds. The higher efficiency of CM-Sephadex is attributed to the greater number of carboxyl groups available for hydrogen-bonding. 4. Reversed-phase chromatography, with butan-1-ol-cyclohexane mixtures as the stationary phase and water as the flowing phase, proved superior to countercurrent distribution with these solvents for the fractionation of purified extracts. 5. A second moulting hormone, deoxycrustecdysone, and the red-concentrating hormone were obtained in a partially purified form.

Animals↗

PubMed1969

The isolation, identification and synthesis of two metabolites of guanethidine formed in pig and rabbit liver homogenates.

1. Two metabolites of radioactively labelled guanethidine were isolated from rabbit and pig liver homogenates by ion-exchange chromatography on a sulphonic acid resin. 2. One of the metabolites was eluted from the column with ammonia and identified as 2-(6-carboxyhexylamino)ethylguanidine on the basis of the elemental analysis, i.r. spectrum and pH titration curve of the pure compound, and the observed partial loss of tritium for ring-labelled guanethidine during the formation of this metabolite. 3. This identification was confirmed by synthesis. 4. 2-(6-Carboxyhexylamino)ethylguanidine underwent ring-closure in hot alkaline solution to 1-(6-carboxyhexyl)-2-iminoimidazolidine. 5. The other metabolite of guanethidine was eluted from the ion-exchange column with 6m-hydrochloric acid along with the unchanged drug. It was purified by countercurrent distribution and shown to be identical with synthetic guanethidine N-oxide. 6. The two metabolites and the product of ring-closure had less than one-tenth of the antihypertensive activity of guanethidine in the renal-hypertensive rat and are unlikely to contribute to the pharmacological properties of the drug.

Animals↗

PubMed1974

A comparative study of cytokinesins I and II and zeatin riboside: a reply to Carlos Miller.

Two cell-division-promoting factors, which have partition coefficients of 1.9 and 2.75 determined by 500-tube countercurrent distribution in a butanolwater system, have been repeatedly isolated in this laboratory from crown gall tumor tissues of Vinca rosea L. These substances have been given the trivial names cytokinesin I and cytokinesin II, respectively. Chemical and mass spectrometric analyses suggest that both cytokinesins are substituted hypoxanthines and are thus very different compounds from the 6-substituted adenyl cytokinins. Carlos Miller, using a very different and far more drastic isolation procedure, obtained one main cell-division-promoting factor from these same tumor tissues, which he identified as ribosyl-trans-zeatin. On the basis of this finding, and without an attempt to repeat our studies, questions have been raised by Miller concerning the existence of the cytokinesins as biologically active substances. We have, therefore, compared some pertinent physical, chemical, and biological properties of the cytokinesins with those of zeatin riboside, have demonstrated that these three substances can be cleanly separated from one another by a number of different methods and that each behaves as a pure substance in the several systems, and, finally, we have shown that the cytokinesins are not contaminated with ribosyl-trans-zeatin and thus do not owe their biological activity to such a contaminant.

Amino Alcohols↗

PubMed1978

Quantitative determination of cross-linkage of bacteriophage DNA and protein by ionizing radiation.

Coliphage T7 was dissolved in tryptone broth and exposted to 60C gamma-radiation. Cross-linkage of DNA and protein of the virion was assayed using phenol-water countercurrent distribution. The results are interpreted in terms of a statistical model of cross-linkage and double-strand breaks. It was found that protein--DNA cross-links accumulate linearly with dose at a rate of o.74 X 10(-11) cross-links per rad per nucleotide pair, which is of the order of 5 per cent of the formation rate of double-strand breaks.

Cobalt Radioisotopes↗

PubMed1974

Preparation and characterization of an immuno-electron microscope tracer consisting of a heme-octapeptide coupled to fab.

A heme-octapeptide (mol wt 1,550) has been obtained from cytochrome c by successive pepsin and trypsin hydrolysis and purified by gel filtration and countercurrent distribution. It possesses peroxidatic activity characterized by an apparent K(m) of 0.2 M, an apparent v(max) of 4 mmol/min per mg of peptide, and a pH optimum of 7.0. Using a novel two-step conjugation procedure, the heme-octapeptide was coupled to rabbit Fab antibody fragments by first derivatizing it with the N-hydroxysuccinimide ester of p-formylbenzoic acid and subsequently allowing it to form a Schiff base with the amino groups of Fab. Stable covalent linkages were then obtained by reduction of the Schiff bases with sodium borohydride. The conjugate consists of approximately 2 heme-octapeptides attached to each Fab molecule. The molecular weight is 45,000 daltons when coupled to sheep Fab and 50,000 daltons with a Stokes radius of 32 A, when conjugated to rabbit Fab. Its peroxidatic activity is characterized by an apparent K(m) of 0.4 M, an apparent v(max) of 0.4 mmol/min and per mg of attached heme-octapeptide and a pH optimum of 7.0. The conjugate has been used for the localization at the electron microscope level of secretory immunoglobulins in the mammary gland of lactating rabbits.

Animals↗

PubMed1979

Further characterization of the ovine lutropin alpha and beta subunits prepared by the salt precipitation method.

The subunits of ovine lutropin prepared by acid dissociation and salt precipitation were characterized by end group analysis, tryptic peptide mapping, SDS gel electrophoresis and biological activity. No evidence of internal peptide cleavage was found in the alpha subunit. The subunits possessed low activity. The alpha and beta subunits recombined effectively to generate a complex that had full receptor binding activity and in vitro biological activity. The recombinants of subunits prepared by countercurrent distribution showed only 50% activity in both assays. The salt precipitation method alpha subunit could be completely reduced and reoxidized in the absence of denaturants. The reoxidized alpha subunit combines with the native beta subunit generating full activity. However, this recombined hormone tends to lose activity with time, suggesting that the reoxidation may not fully restore the native structur of the reduced alpha subunit. The native lutropin alpha subunit effectively combined with follitropin beta subunit generating complete follitropin activity.

Amino Acid Sequence↗

PubMed1980

Synthesis of somatostatin and [D-Trp8]-somatostatin.

Experimental details for practical syntheses of somatostatin and D-Trp8-somatostatin are described. The peptides were assembled from three fragments which permit further syntheses of analogs with modifications at positions 1, 2 or 8. N alpha-Bpoc protecting groups were used for the two major fragments and these were selectively removed in the presence of the tert.-butyl derived amino acid side chain functionalities. The two cysteine residues were protected by acetamidomethyl groups. All the peptide intermediates were fully characterized and a 10-g synthesis of the protected tetradecapeptide is reported. Major fragments were coupled by the azide method in good yield. Dihydrosomatostatin and D-Trp8-dihydrosomatostatin were isolated, purified, characterized and cyclized. Polymeric side-product was successfully recycled (by reduction with dithiothreitol and reoxidation) to give an overall yield for the oxidation of 52%. Somatostatin and D-Trp8-somatostatin were purified by gel filtration or countercurrent distribution and the final products were fully characterized and determined to be > 97% pure by reversed phase high performance liquid chromatography.

Amino Acid Sequence↗

PubMed1980

Solid-phase peptide synthesis of somatostatin using mild base cleavage of N alpha-9-fluorenylmethyloxycarbonylamino acids.

Experimental details for the "Fmoc solid phase peptide synthesis" of somatostatin are described. The 9-fluorenylmethyloxycarbonyl group was rapidly and quantitatively cleaved by 55% piperidine in dimethylformamide and monitored (u.v.) manually. For a kinetic study, a centrifugal reactor with a photometric control system and reference cell was used at each stage. The symmetrical anhydride coupling reaction was rapid and either acetic anhydride or fluorescamine termination was incorporated to minimize formation of deletion peptides. Anchor-bond cleavage was effected with trifluoroacetic acid which simultaneously removed all the acid labile tert.-butyl side chain protecting groups. N alpha-9-fluorenylmethyloxycarbonyl peptides may be obtained by omitting the piperidine deprotection step after the last cycle of synthesis. From several syntheses, analytically pure di-S-protected somatostatin 14-peptide was obtained in 55-60% overall yield. The S-protecting groups were removed and the product was purified by gel filtration to give homogeneous dihydrosomatostatin (91%) yield. Oxidation of dihydrosomatostatin with potassium ferricyanide and purification by countercurrent distribution provided analytically pure homogeneous somatostatin.

Amino Acid Sequence↗

PubMed1975

The primary structure of tRNAIIArg from brewers' yeast. 1. Complete digestions with pancreatic and T1 ribonucleases.

tRNAIIArg purified from bulk brewers' yeast tRNA by countercurrent distribution followed by two column-chromatographic steps was completely digested with pancreatic and T1 ribonucleases. Isolations of the products have been carried out either by column chromatography or by high-voltage electrophoresis. Analyses of the isolated nucleotides and olignoucleotides were in good agreement and indicate that this tRNA is composed of 76 nucltotide residues including 13 minor nucleotides. Overlaps resulting from the end-products of the two complementary digests led to a sequence of 25 residues. The primary structure of tRNAIIArg has been determined after partial digestion with T1 ribonuclease as described in the following paper.

Arginine↗

PubMed1973

Bromomonamycins, unnatural analogues of the monamycin cyclodepsipeptide antibiotics: production, isolation, and biological activity.

Growth of Streptomyces jamaicensis in a low-chloride medium supplemented with NaBr resulted in the biosynthesis of brominated analogues of the natural chlorinated monamycins. Purification of these new compounds was undertaken by Craig countercurrent distribution studies. Diffusion and dilution assays indicated that antibiotic activity of a monamycin was inversely proportional to its unsolvated molecular weight. The bromo analogues were the least active of the series.

Anti-Bacterial Agents↗

PubMed1984

Na+/H+ antiporter in membrane populations resolved from a renal brush border vesicle preparation.

A conventional brush border membrane preparation, obtained by divalent cation precipitation of homogenates of rabbit renal cortex, was analyzed by countercurrent distribution in an aqueous dextran:polyethylene glycol two-phase system. The resulting fractions were assayed for the presence of the Na+/H+ antiporter and for a variety of biochemical marker enzymes. This analysis revealed four physically distinct membrane populations (A-D). Population A consisted of two subpopulations, A' and A", which were enriched an average of 49-fold in maltase; they were also highly enriched in alkaline phosphatase, leucine aminopeptidase, and Na+/H+ antiporter. On the basis of their marker contents, populations A' and A" appear to represent highly purified, functional brush border membrane vesicles. Population B was enriched twofold in NADPH-cytochrome c reductase and population C was enriched 12-fold in galactosyltransferase. Populations B and C accounted for 25% of the protein in the starting material and appear to reflect contamination of the brush border membrane preparation by subpopulations of endoplasmic reticulum and Golgi fragments. Population D was enriched in Na+/H+ antiporter, alkaline phosphatase, leucine aminopeptidase, Na-K-ATPase, and acid phosphatase but not maltase, NADPH-cytochrome c reductase, galactosyltransferase, or succinate dehydrogenase. Its identity is unclear, and it might consist of a multiplicity of populations from different origins.

Animals↗

PubMed1977

Isolation of gamma-amino butyric acid from pig hypothalami and demonstration of its prolactin release-inhibiting (PIF) activity in vivo and in vitro.

A non-retarded fraction with prolactin-release inhibiting factor (PIF) activity obtained by chromatography of a concentrate of porcine hypothalami on carboxymethyl-cellulose was chromatographically distinct from catecholamines. This fraction was purified further by six steps involving chromatography on Sephadex G-25, countercurrent distribution, free-flow electrophoresis, and chromatography on triethylaminoethyl cellulose. The PIF-active substance was isolated and identified as gamma-amino-butyric acid (GABA) by: 1) amino acid analyses using sodium as well as lithium-based buffers for resolution of biological fluids, 2) thin-layer chromatography of underivatized material as well as phenylthiocarbamyl derivatives, and 3) mass spectroscopy. Natural and synthetic GABA inhibited prolactin, but not LH release in vitro from isolated rat pituitary halves at doses as low as 0.1 microgram/ml. The inhibition was proportional to the dose; natural and synthetic GABA possessed identical PIF activity. Synthetic GABA also decreased prolactin release in monolayer cultures of rat pituitary cells and inhibited TRH-stimulated prolactin release. The inhibition of prolactin release in vitro by GABA could not be blocked by perphenazine, which inhibits PIF activity of catecholamines. GABA also suppressed prolactin release in vivo, although large doses were needed. Either rapid iv injection or infusion of GABA in doses of 1 to 100 mg in rats significantly decreased serum prolactin levels, which were previously elevated by pretreatment with monoiodotyrosine perphenazine, chlorpromazine, haloperidol, or sulpiride. beta-hydroxy GABA significantly depressed prolactin release, but beta-(p-chlorophenyl)-GABA (Lioresal, CIBA) and 4 other analogs of GABA were not effective in vivo and/or in vitro. The results indicate that GABA can inhibit prolactin release by a direct action on the pituitary gland, but whether this effect is physiologically meaningful still remains to be determined.

Animals↗

PubMed1976

Isolation and characterization of follicle-stimulating hormone and luteinizing hormone and its subunits from snapping turtle (Chelydra serpentina) pituitaries.

Highly purified luteinizing hormone and follicle-stimulating hormone have been isolated from extracts of snapping turtle (Chelydra serpentina) pituitaries. Both hormones are potent in non-mammalian gonadotropin bioassays (1.8 X NIH-LH-S1 and 30 X NIH-FSH-S1). The materials have been characterized by polyacrylamide gel electrophoresis, amino terminal group analysis, amino acid and carbohydrate content, and, in the case of turtle luteinizing hormone, ultracentrifugation. The luteinizing hormone was shown to dissociate and subunits were prepared by the countercurrent distribution technique and characterized. Biological activity of the hormone could be regenerated by recombination of the subunits. In addition, it was shown that the snapping turtle luteinizing hormone subunits could be combined with subunits from ovine luteinizing hormone with generation of significant biological activity. Comparisons in properties of the turtle gonadotropins have been made with ovine gonadotropins, showing, in many cases, similarities in properties, suggesting structural features which have been conserved during evolution.

Animals↗

PubMed1992

Heterogeneity in porcine pituitary luteinizing hormone: amino acid and carbohydrate analysis.

Pituitary LH from porcine pituitary glands was purified by a buffered ethanol extraction procedure, ion exchange on DEAE- and carboxymethyl-cellulose, and molecular exclusion on Sephacryl S-200. Purity was assessed by amino acid composition, N-terminal sequence, and polyacrylamide gel electrophoresis. Subunits were isolated by countercurrent distribution and reverse phase HPLC. Four major forms of the alpha-subunit were detected: 1-96 (50%), 3-96 (23%), 4-96 (16%), and 7-96 (11%). [The original sequence report described only the 7-96 form, but we have detected the other forms in our studies of porcine FSH and in this and other species of LH.] Comparable N-terminal heterogeneity was not observed for the beta-subunit. Additional heterogeneity was observed for both subunits, attributable to heterogeneity in the N-linked oligosaccharide moieties. The isolated subunits were submitted to detailed compositional carbohydrate analysis, using pulsed amperometric detection of the HPLC-resolved sugar monomers after trifluoroacetic acid hydrolysis. Sialic acid and sulfate esters were estimated on separate hydrolyzates. The compositional data suggest that the two alpha-subunit N-linked moieties are hybrid complex biantennary structures with sulfated N-acetylgalactosamine (40-50%). Sixty to 70% of the alpha-subunit oligosaccharides are fucosylated. The beta-subunit of porcine LH has a single glycosylation site, which contains a mixture of biantennary oligosaccharide chains (80-90%) ending in N-acetylgalactosamine, half of which are sulfated. The balance (10-20%) are hybrid chains ending in sialylated galactose. The majority of the oligosaccharide on the beta-subunit is fucosylated.

Amino Acid Sequence↗