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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↗
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↗
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↗
PubMed1987

Separation by counter-current distribution of young-chick erythrocyte populations and numerical resolution of distribution curves.

The presence of two red cell populations in young chicks has been demonstrated after increasing the settling time used during counter-current distribution with charged 5% Dextran--4% poly(ethylene glycol) two-phase systems. A Fortran program using statistical methods was applied to show the resolution in two peaks (with two or three subpopulations assumed) of the counter-current distribution curves.

Animals↗
PubMed1986

Fractionation of rat liver plasma-membrane regions by two-phase partitioning.

Rat liver plasma membranes, enriched in blood-sinusoidal or bile-canalicular regions by differential and sucrose-gradient centrifugation, were further purified by partitioning in an aqueous polymer two-phase system. This method separates membranes according to differences in surface properties rather than size and density. A several-fold increase in the ratio of leucine aminopeptidase (a bile-canalicular marker) and 5'-nucleotidase to asialo-orosomucoid binding (a blood-sinusoidal marker) was obtained in one fraction, whereas another fraction gave a 2-3-fold increase in ratio of blood-sinusoidal to bile-canalicular markers. Furthermore, the markers for both regions of the plasma membrane, as well as markers for Golgi membranes and lysosomes, showed a heterogeneous behaviour on counter-current distribution.

Animals↗
PubMed1978

[Method of partitioning polyene macrolide antibiotics].

Levorin and mycoheptin, polyenic antibiotics were separated into 3 and 7 heptaenic components respectively by the method of disc electrophoresis in 7.5 per cent polyacryl amide gel prepared with tris-HCl buffer, pH 8.9. It was shown that nystatin had different composition as dependent on the organism producing it. The results of electrophoresis confirmed the data of the counter-current distribution.

Antifungal Agents↗
PubMed1992

Surface heterogeneity of rat sperm during maturation.

Rat sperm isolated from the caput and caudal epididymis and the vas deferens were subjected to multiple partition in aqueous two-phase systems. The technique was used to reveal heterogeneity of a sperm population with respect to particular surface properties. Sperm from all three regions gave broad distributions indicative of heterogeneous cell populations. Greatest heterogeneity was observed for cauda sperm with caput and vas sperm producing similar distributions. Following multiple partition sperm from different regions of the distribution profiles were immunostained with three antibodies known to recognise maturation antigens. The results show that some antigens are acquired during epididymal transit whilst others are present throughout. The partition (surface heterogeneity) seen cannot therefore be explained solely by the distribution of the antigens recognized by 2D6, 6B2 and 3D5.

Animals↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1992

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↗