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PubMed1965

Isolation and identification of a pepsitensin.

1. From ox plasma incubated with pepsin a highly purified pepsitensin has been isolated by fractional precipitation, solvent extraction, column chromatography, countercurrent distribution and paper chromatography. 2. Comparison of the properties of this substance with those of synthetic valyl-5 angiotensin I showed: (a) the same specific pressor activity; (b) the same amino acid composition; (c) identical paper-electrophoretic mobilities at various pH values. 3. N- and C-Terminal studies carried out on the intact polypeptide and on the products of chymotrypsin digestion established the following sequence for the amino acids present in the molecule: Asp-Arg-Val-Tyr-Val-His-Pro-Phe-His-Leu. This structure is identical with that of valyl-5 angiotensin I.

Journal Article↗

PubMed1967

An octadecatrienoic acid from Lamium purpureum L. seed oil containing 5,6-allenic and trans-16-olefinic unsaturation.

1. Lamium purpureum L. (Labiatae) seed oil contains 16% of a new acid characterized as (-)-octadeca-5,6-trans-16-trienoic acid (proposed trivial name ;lamenallenic acid') (Ia). The acid was isolated as its methyl ester by countercurrent distribution by using a combination of recycle-single withdrawal techniques. Methyl lamenallenate (Ib) is strongly laevorotatory. 2. The structure was established by infrared spectroscopy, nuclear-magnetic-resonance spectroscopy, quantitative hydrogenation and oxidative cleavage data of the original acid and of hydrazine partial reduction products. 3. Other unsaturated esters identified by their cleavage products were oleate, linoleate and linolenate. 4. A very small amount (less than 1%) of methyl laballenate [(-)-methyl octadeca-5,6-dienoate] was also isolated and identified.

Journal Article↗

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↗

PubMed1984

Changes in the DNA content of amoebae of Dictyostelium discoideum during growth and development.

A fluorimetric assay has been used to determine the DNA content of amoebae of Dictyostelium discoideum during growth and development. Amoebae grown in axenic culture tended to be multinucleate and had a greater DNA content than amoebae grown with a bacterial substrate, which were mononucleate. During the first 10 h of development there was little change in the DNA content of amoebae grown with a bacterial substrate, but the average DNA content per cell in amoebae grown axenically decreased as the amoebae became virtually mononucleate. Amoebae at 10 h development that had been harvested during exponential axenic growth were divided into two populations by countercurrent distribution in a polymer two-phase system. DNA content indicated that one population was largely in the G2-phase of the cell cycle, whereas the other population was largely in the G1-phase. Similar results were obtained at 10 h development with amoebae harvested during the stationary phase of axenic growth, although these amoebae start development all in the G2-phase of the cell cycle. Spores had a low DNA content, indicating that they were in G1-phase. It is proposed that all amoebae in G2-phase after early development differentiate, after mitosis, into spores and that stalk cells are formed from amoebae that remain in G1-phase after 10 h development.

Cell Nucleus↗

PubMed1997

Surface changes of ram spermatozoa by adsorption of homologous and heterologous seminal plasma proteins revealed by partition in an aqueous two-phase system.

Ram spermatozoa freed from seminal plasma by a dextran 'swim-up' procedure were incubated with Tween 20 and fractionated into motile (PEG-rich) and stationary (dextran-rich) fractions by centrifugal countercurrent distribution (CCCD) in an aqueous dextran-Ficoll-polyethylene glycol (PEG) two-phase system. Increasing concentrations of Tween 20 resulted in greater amounts of extracted protein and lower cell viability. Addition of bull seminal plasma increased the proportion of live cells, whereas ram seminal plasma increased the proportion of stationary cells. Proteins isolated from each type of seminal plasma restored the CCCD profile of treated spermatozoa to the right, this effect being reduced when proteins were thermally denatured. Bovine serum albumin induced a slight displacement to the left. No restoration of profile was achieved when ram spermatozoa were exposed to proteins from bull seminal plasma in the presence of protein-free ram seminal plasma. Adsorption of seminal plasma proteins by spermatozoa previously stripped of surface proteins by exposure to detergent reversed the detergent effect on motility. The findings are consistent with the concept that ram seminal plasma contains a factor that interferes with protein adsorption on the cell surface and prevents the protective effect of seminal plasma proteins on maintenance of cell viability.

Adsorption↗

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↗

PubMed1954

The purification of hypertensin I.

The purification of hypertensin I has been described. The final product which is four times as powerful a pressor agent as l-arterenol, is obtained with an over-all recovery of 40 per cent. The product consists of a single component in countercurrent distribution, having a nitrogen content of 15.97 per cent and a specific activity of 7050 Goldblatt units per mg. of N or 1125 units per mg. of solid. Acid hydrolysis and paper chromatography indicate in a preliminary fashion that there are about nine amino acids present in the intact polypeptide.

Amino Acids↗

PubMed1955

Amino acid composition and electrophoretic properties of hypertensin I.

A preparation of hypertensin I was purified by countercurrent distribution and was shown to migrate as a single component in starch blocks at pH 9.3 and 4.2. It had an isoelectric point of 7.7. Quantitative analysis by ion exchange column chromatography showed eight amino acids in approximately unimolar proportion: aspartic, proline, valine, isoleucine, leucine, tyrosine, phenylalanine, and arginine. There were in addition two moles of histidine.

Amino Acids↗

PubMed1956

The amino acid composition of hypertensin II and its biochemical relationship to hypertensin I.

Preparations of hypertensin II, obtained from the treatment of hypertensin I by the action of the hypertensin converting enzyme of plasma and purified by countercurrent distribution, were quantitatively analyzed for their amino acid content. Chromatography on ion exchange columns showed the presence of equimolar amounts of aspartic acid, proline, valine, isoleucine, tyrosine, phenylalanine, histidine, and arginine. Hypertensin I was found to contain one mole of leucine and one mole of histidine in addition to the amino acids of hypertensin II. These two amino acids were isolated from the conversion products of hypertensin I and identified as the peptide histidylleucine. Carboxypeptidase digestion of hypertensin I showed the carboxyl terminal sequence of amino acids to be residue-phenylalanyl-histidylleucine. Similar studies of hypertensin II demonstrated residue-phenylalanine. It was concluded that the conversion of hypertensin I by the plasma hypertensin converting enzyme involved hydrolysis of the phenylalanyl-histidine bond to form hypertensin II and histidylleucine. The further removal by carboxypeptidase of phenylalanine from hypertensin II destroyed all of the vasoconstrictor activity.

Amino Acids↗

PubMed1963

The purification and partial characterization of several forms of hog renin substrate.

Hog renin substrate has been separated into three major (A, B, and C) and two minor forms (D and E) by DEAE cellulose chromatography. Two of the major forms (B and C) have been further fractionated into two additional types (1 and 2) by countercurrent distribution. The purification of substrates A, C(1), and C(2) has been completed. Analysis shows that all three are glycoproteins with molecular weights of about 57,000, and have similar amino acid compositions. Differences exist in the sialic acid, glucosamine, and neutral hexose content, which may account for different physical properties. All the forms of the substrates are attacked by renin at similar rates, and appear to yield the same angiotensin I.

Amino Acids↗

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↗

PubMed2002

Isolation of a new member of the ecdysteroid glycoside family: 2-deoxy-20-hydroxyecdysone 22-O-beta-D-glucopyranoside.

A new ecdysteroid glycoside, 2-deoxy-20-hydroxyecdysone 22-O-beta-D-glucopyranoside, is isolated from the herb Silene italica ssp. nemoralis (Waldst. and Kit.) Nyman. The compound is purified with multistep chromatography, such as classical column chromatography on alumina and droplet countercurrent distribution. Also, it is expanded using twice low-pressure reversed-phase liquid column chromatography. Chromatography in four steps results in the purified 2-deoxy-20-hydroxyecdysone 22-O-beta-D-glucopyranoside. Two other ecdysteroids have also been separated, including the formerly identified integristerone A and 24(28)-dehydromakisterone A.

Caryophyllaceae↗

PubMed2004

The constant proportion of grana and stroma lamellae in plant chloroplasts.

The relative proportion of stroma lamellae and grana end membranes was determined from electron micrographs of 58 chloroplasts from 21 different plant species. The percentage of grana end membranes varied between 1 and 21% of the total thylakoid membrane indicating a large variation in the size of grana stacks. By contrast the stroma lamellae account for 20.3 +/- 2.5 (sd)% of the total thylakoid membrane. A plot of percentage stroma lamellae against percentage of grana end membranes fits a straight line with a slope of zero showing that the proportion of stroma lamellae is independent of the size of the grana stacks. That stroma lamellae account for about 20% of the thylakoid membrane is in agreement with fragmentation and separation analysis (Gadjieva et al. Biochim. Biophys. Acta 144: 92-100, 1999). Chloroplasts from spinach, grown under high or low light, were fragmented by sonication and separated by countercurrent distribution into two vesicle populations originating from grana and stroma lamellae plus end membranes, respectively. The separation diagrams were very similar lending independent support for the notion that the proportion of stroma lamellae is constant. The results are discussed in relation to the composition and function of the chloroplast in plants grown under different environmental conditions, and in relation to a recent quantitative model for the thylakoid (Albertsson, Trends Plant Sci. 6: 349-354, 2001).

Journal Article↗

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↗