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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↗

PubMed1976

Studies on phenolic studies in human subjects. XX. In vivo conjugation and metabolism of estradiol-17beta in the human kidney.

Labeled estradiol-17beta (E2) was injected into one of the renal arteries of two human subjects. At the same time, an equimolar amount of differently labeled E2 was injected into a peripheral vein. The urinary metabolites were analyzed by DEAE-Sephadex A-25 column chromatography, countercurrent distribution (CCD) and enzyme hydrolyses. Identification was made by statistical analysis of data from CCD, thin layer chromatography (TLC) and co-crystallization upon admixture with authentic compounds. The major urinary metabolites were E2-17glucosiduronate (E2-17G), E2-3G and estriol-16G (E3-16G). The E2-17G was excreted immediately following injection of 14C-E2 into the renal artery of subject no. 1, at a rate which decreased gradually with time; whereas 3H-E2-17G did not appear in the urine until 5 min after injection of 3H-E2 into a peripheral vein. The excretion of 14C-E2-17G was very prominent as opposed to that of 3H-E2-17G; however, the excretion of both 14C- and 3H-E2-17G terminated within 30 min. 14C-E2-3G was excreted immediately following injection, whereas 3H-E2-3G did not appear until 5 min after the injection. Also, the excretion of 14C-E2-3G was more prominent as opposed to that of 3H-E2-3G. The excretion of these compounds was rapid in the initial 15 min after injection and then continued slowly for 1 h. On the other hand, 14C- and 3H-E2-16G appeared at 30 min after injection and the 3H/14C ratio was almost the same as that of the injected compounds. When subject no.2 was injected with the labeles reversed, the results were very similar to those described above. The results indicate that E2 is conjugated directly in the human kidney to form the 17G and 3G and excreted into the urine, whereas the conversion of E2 to E3 occurs systematically rather than in the kidney. In contrast of E3, the kidney appears to play a minor role (no more than 10% of the total E2 is involved) in the conjugation and/or metabolism of E2 in the human.

Estradiol↗

PubMed1985

Use of aqueous two-phase partition to detect cell surface changes during growth of Dictyostelium discoideum.

Changes in the cell surface properties of amoebae of Dictyostelium discoideum during growth in different culture conditions have been studied by aqueous two-phase partitioning on a thin-layer countercurrent distribution apparatus. Changes in cell surface properties were not dependent on the source of nutrients but only on cell density. There was a progressive increase in cell surface hydrophobicity with cell density in both axenic cultures and cultures grown with a bacterial substrate. It is proposed that it is these cell-density-related surface changes that account for the ability of amoebae grown in different conditions to sort out during subsequent development in a manner related to cell fate.

Bacteria↗

PubMed1985

Analysis of urinary aldosterone metabolites in the rabbit by gas chromatography-mass spectrometry.

After a large amount of aldosterone was injected into a male rabbit, urine was collected for 48 h. Separation of urinary aldosterone metabolites into monoglucosiduronate fraction and monosulphate fraction was carried out by a combination of countercurrent distribution and DEAE-Sephadex A-25 column chromatography. Each fraction was hydrolyzed with enzyme and free steroids released were separated by Sephadex LH-20 column chromatography. The free steroid was then identified by gas chromatography-mass spectrometry. In monoglucosiduronate fraction, 3 alpha, 5 beta-tetrahydroaldosterone and 3 beta, 5 alpha-tetrahydroaldosterone were found. On the other hand, 3 alpha, 5 beta-tetrahydroaldosterone was the only aglycone detected in monosulphate fraction. These findings comfirmed results in the preceding paper, where the free steroid was characterized on the basis of the mobility of the steroid and its derivatives on paper chromatography.

Aldosterone↗

PubMed1985

The metabolism of aldosterone and 3 alpha, 5 beta-tetrahydroaldosterone in the rabbit.

Analysis of urinary metabolites of [1, 2-3H]-aldosterone and [1, 2-3H]-3 alpha, 5 beta-tetrahydroaldosterone was performed in male rabbits. The preliminary separation of urinary metabolites was carried out by submitting these metabolites to countercurrent distribution. Further separation of each fraction thus obtained was achieved by means of DEAE-Sephadex A-25 column chromatography. The separated peak was then hydrolyzed with the enzyme and the free steroid released was identified on the basis of the mobilities of the steroid and its derivatives on paper chromatography. After the injection of [1, 2-3H]-aldosterone, a major urinary metabolite was characterized as monosulphate of 3 alpha, 5 beta-tetrahydroaldosterone. In addition, a small amount of the monoglucosiduronate fraction was found in the urine. 3 alpha, 5 beta-tetrahydroaldosterone and 3 beta, 5 alpha-tetrahydroaldosterone were detected as aglycones in this fraction. After the injection of [1, 2-3H]-3 alpha, 5 beta-tetrahydroaldosterone, a similar pattern of urinary radiometabolites was observed. The close similarity between the profile of urinary metabolites of [1, 2-3H]-aldosterone and that of [1, 2-3H]-3 alpha, 5 beta-tetrahydroaldosterone suggests that the conversion of aldosterone to 3 alpha, 5 beta-tetrahydroaldosterone is needed before the conjugation processes take place.

Aldosterone↗

PubMed1987

Analysis of urinary aldosterone metabolites in the guinea-pig.

Analysis of urinary metabolites of [1, 2-3H]-aldosterone was performed in the male guinea-pig. Separation of urinary metabolites was carried out by countercurrent distribution followed by DEAE-Sephadex A-25 column chromatography. A major component was obtained which was both hydrolyzable with sulphatase from Helix pomatia and solvolyzable. Paper chromatography of freed steroids revealed the presence of at least two components and the major aglycone cochromatographed with 3 beta, 5 alpha-tetrahydroaldosterone. In order to get more information about the structure of urinary metabolites, a total of 68 mg of aldosterone was injected into three male guinea-pigs and separation of urinary metabolites was performed in a similar manner. A major component obtained showed the color reaction positive for sulphate (modified rhodizonic acid test) and negative for glucosiduronate (naphthoresorcinol test). Gas chromatographic-mass spectrometric analysis of aglycones released from this conjugate revealed the presence of 3 beta, 5 alpha-tetrahydroaldosterone and an another aglycone, tentatively identified as 21-deoxy-tetrahydroaldosterone. Taken together, it was concluded that 3 beta, 5 alpha-tetrahydroaldosterone-monosulphate and 21-deoxy-tetrahydroaldosterone-monosulphate comprised most of urinary conjugated metabolites of aldosterone in the male guinea-pig.

Aldosterone↗

PubMed1977

The topography of porcine lactate dehydrogenase isoenzyme H4. The identification of lysines on the surface.

Porcine lactate dehydrogenase isoenzyme H4 was treated with methyl 6-(2,4-dinitrophenylamino)hexanimidate hydrochloride and the lysines modified hereby were identified. For this purpose 20 chymotryptic-tryptic N epsilon-[6-(2,4-dinitrophenylamino)hexanimidoyl]lysine containing peptides were isolated by means of gel chromatography, countercurrent distribution, thin-layer chromatography and ion-exchange chromatography. Their amino acid composition, the amino end groups and their electrophoretic mobilities were determined. With these data, the known primary structure of the procine lactate dehydrogenase isoenzyme H4 and the 6-A resolution structure analysis performed by Rossmann et al.[1] we identified the following lysines on the surface of the quarternary structure of the enzyme: no. 4, 6, 60, 77, 82, 121, 157, 179, 226, 230, 241, 306, 308, 316, 327 and 330. No modified lysine peptides were found in the intersubunit binding sites.

Amino Acid Sequence↗

PubMed1975

Isolation and characterization of a new pancreatic polypeptide hormone.

A method is described for isolation, from chicken pancreas, of an avian pancreatic polypeptide which may be a new hormone. This method involves acid-alcohol extraction, gel filtration, DEAE-cellulose chromatography, and droplet countercurrent distribution. The peptide contains 36 amino acids, has a molecular weight of 4240 and the isoelectric point if pH 6 to 7. The average amount of avian pancreatic polypeptide extractable from chicken pancreas was 4 mg/100 g of pancreas. The amino acid sequence of the peptide is Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-Arg-Phe-Tyr-Asp-Asn-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr-NH2.

Amino Acid Sequence↗

PubMed1975

The carboxylic acid groups of bovine luteinizing hormone. The effects of their modification on receptor site binding and subunit-subunit interaction.

The modification of the carboxyl groups of the subunits of bovine luteinizing hormone to neutral derivatives by carbodiimide-mediated coupling with glycine methyl ester has been studied. The modified alpha subunit, which has 8 residues of glycine methyl ester incorporated, will no longer recombine with native beta (hormone-specific) subunit, but the modified beta subunit, with 6 to 7 glycine methyl esters incorporated, will recombine with native alpha to yield a partially active hormone. Derivatization of the intact hormone results in dissociation to subunits together with formation of a major side product which is covalently cross-linked. Significant cross-linked product was not obtained during modification of individual subunits, thus indicating an orientation between an activated carboxyl group(s) and a nucleophile(s) in the intact hormone which favors coupling. Separation of subunits from the derivatized, noncross-linked fraction by countercurrent distribution reveals a heterogeneous preparation of the modified alpha subunit which also will not recombine with either a native or modified beta subunit. The beta subunit from the modified intact hormone was indistinguishable from the modified isolated beta subunit in amino acid composition and in ability to recombine with native alpha subunit. The results are consonant with data from this and other laboratories in which various modifications of the alpha chain, the subunit common to the glycoproteins, more seriously affect recombination than similar modifications of the beta subunits. The number of carboxyl groups modified in each subunit is compatible with but not in total agreement with assignments of amides reported from sequence studies.

Amino Acid Sequence↗

PubMed1990

Isolation and structural elucidation of biotransformation products from acarbose.

Following oral administration the a-glucosidase inhibitor acarbose (O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl) -2-cyclohexen-1-yl]amino]-a-D-glucopyranosyl-(1----4)-O-a-D-glu copyranosyl-(1----4)-D-glucopyranose, Bay g 5421) is degraded by digestive enzymes and/or intestinal microorganism. The effect of anaerobic intestinal bacteria can be studied in an in vitro model which involves the incubation of acarbose with human or animal intestinal flora. Acarbose and nine biotransformation products can be isolated from the incubation mixture. These products were identified by nuclear magnetic resonance and mass spectrometry as so-called component 2 (loss of the terminal glucose), component 1 (loss of both glucose rings), hexose homologues of acarbose and component 2, methyl homologues of acarbose, butyric acid ester of component 2, basic disaccharide (loss of the cyclitol ring of component 2), delta-aminovaleric acid and gamma-aminobutyric acid. Following oral administration of [14C]-acarbose to healthy volunteers, 35% of the radioactivity was excreted in the form of at least 13 metabolites in the urine. Three of the metabolites were isolated by Craig countercurrent distribution and ion-pair HPLC and characterized by virtue of their nuclear magnetic resonance and mass spectra as derivatives of 4-methylpyrogallol. Two were shown to be monomethylether-monosulphates while the third was a monosulphate-monoglucuronide. The synthesis of ten reference substances and the comparison of HPLC and UV data clearly indicated that the majority of the non-isolated metabolites were also 4-methylpyrogallol derivatives. The peculiarities of the nuclear magnetic resonance and mass spectra of this type of compound are discussed.

Acarbose↗

PubMed1985

Enrichment of a fraction toxic to guinea-pigs from Pachystigma pygmaeum (Schltr.) Robyns.

Pachystigma pygmaeum is one of several species of rubiaceous plants which cause delayed heart failure among ruminants after their ingestion at relatively high doses. Using guinea-pigs for toxicity determinations, we were able to separate and enrich a toxic fraction from a fermentation extract of the plant material by countercurrent distribution. It contained virtually no potassium salts, passed through a 500 dalton selective membrane, exhibited lability under acid conditions and was toxic at 1 g/kg per os, with a delayed response of 3-4 days.

Animals↗

PubMed1979

Partial synthesis of harringtonine.

The partial synthesis of harringtonine from cephaltotaxine has been described. A key intermediate, 5, 5-dimethyl-2-hydroxytetrahydrofuran-2-carboxylic acid (V), prepared from 4-methyl-1, 4-valerolactone, was dehydrated smoothly to give 5, 5-dihydrofuran-2-carboxylic acid (VI), Through its sodium salt, VI was converted into the corresponding acyl chloride VIII, which reacted with cephalotaxine in the presence of pyridine to give ester IX. After being treated with hydrichloric-acetic acid, ester IX unerwent Reformatsky reaction to give a mixture (XIV) of harringtonine and its diastereoisomer (epiharringtonine) as the final product which was purified either by countercurrent distribution or column chromatography on neutral alumina. The amounts of the two epimers in the mixture shown by TLC were roughly equal.

Alkaloids↗

PubMed1978

[Hemoglobins, XXI: sequence analysis of porcine hemoglobin (author's transl)].

The hemoglobin of a bavarian domestic pig (Suidae) was isolated. The chains were separated by countercurrent distribution, then cleaved with trypsin. The isolated peptides were sequenced with hydrophilic phenylisothiocyanate I and IV, or with a dimethylaminopropyne program in the sequenator. The sequences of the chains are given. Some methodical aspects of automatic sequencing are discussed and the sequences of human porcine hemoglobin are compared. The role of adult pig haemoglobin as foetal hemoglobin is discussed.

Animals↗

PubMed1976