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H P Bennett

Publications and source records attributed to H P Bennett.

At least 91 records · Page 5Linked to original sources
PubMed1980

Separation of collagen cyanogen bromide-derived peptides by reversed-phase high-performance liquid chromatography.

A new technique for separation of the CNBr cleavage products of collagen is described. It involves the use of a 30 nm pore reversed-phase high-performance liquid chromatography column eluted with a linear gradient of acetonitrile/water containing 0.01 M-heptafluorobutyric acid. The separation of type I, type II and type III CNBr peptides is described. Resolution is particularly good for the low-molecular-weight peptides. The method is fast, quantitative and sensitive, and the complete volatility of the eluent facilitates the recovery of the separated peptides.

Acetonitriles↗
PubMed1979

Distribution and degradation of two tritium-labelled corticotrophin analogues in the rat.

The distribution and degradation of corticotrophin-(1--24)-tetracosapeptide specifically labelled with tritium at Tyr2, Phe7 or Tyr23 and [D-Ser1, Lys17, Lys18]-corticotrophin-(1--18)-octadecapeptide amide labelled at Tyr2 were studied at various times after intravenous injection into rats. By characterizing the radioactivity in plasma and various tissues, an overall picture of the metabolic handling of the two peptides emerged. The peptides left the circulation rapidly, entering mainly muscle and skin where they were extensively degraded. The D-Ser1-containing analogue was less rapidly degraded and intact peptide persisted in muscle and skin for up to 1 h. This peptide probably returned to the circulation giving rise to the sustained plasma levels observed after injection of the D-Ser1-substituted octadecapeptide but not after injection of the tetracosapeptide. Although initially the kidneys did not clear such large amounts of peptide as did muscle and skin they played an important role by continuously and, on the basis of existing evidence, irreversibly clearing the peptides and peptide fragments from the circulation and degrading them.

Adrenocorticotropic Hormone↗
PubMed1977

Use of octadecasilyl-silica for the extraction and purification of peptides in biological samples. Application to the identification of circulating metabolites of corticotropin-(1-24)-tetracosapeptide and somatostatin in vivo.

Peptides can be adsorbed on octadecasilyl-silica from large volumes of aqueous solution and eluted with aqueous solvent mixtures containing methanol or acetonitrile. These properties may be used for the extraction and purification of peptide fragments in plasma samples collected from rats. After intravenous injection of Synacthen [corticotropin-(1-24)-tetracosapeptide], it was shown that within 2 min the main circulating products were intact peptide and its sulphoxide. In addition, a number of fragments indicative of cleavage at the N- and C-termini were present. Most of the products formed from Synacthen have low biological activity. Somatostatin was rapidly cleaved in vivo and in vitro to a single product, which probably retains biological activity. The absence of other circulating products suggests that somatostatin is only inactivated once it leaves the circulation.

Adrenocorticotropic Hormone↗
PubMed1977

Renal uptake and metabolism of adrenocorticotrophin analogues in the rat: an autoradiographic study.

Renal resorption of tritiated adrenocorticotrophin analogues was studied in the rat using light microscopic and quantitative electron microscopic autoradiography. The synthetic corticotrophins used were Synacthen (corticotrophin-(1-24)-tetracosapeptide) and C41795-Ba ([D-Ser1,Lys17,Lys18]-corticotrophin-(1-18)-octadecapeptide amide), the tetracosapeptide being tritiated in either the tyrosine residue of position 2 or 23 or the phenylalanine of position 7 and the octadecapeptide in the tyrosine of position 2. Inspection of autoradiographs showed that peptides injected intravenously were resorbed into proximal tubules by endocytosis to produce vesicles whose radiolabel later appeared in lysosomes, a route previously elucidated for other peptides and proteins. The use of two techniques for analysis of electron microscopic autoradiographs, however, suggested that apical tubules also acquire label and are in some way involved in the transfer of resorbed labelled material from endocytotic vesicles to lysosomes. In addition, the autoradiographic analyses revealed that the duration of lysosomal labelling depends upon the position of tritium in the chain. Thus, when the CO2H-terminus of Synacthen was labelled, silver grains were more transiently associated with lysosomes than was the case when the NH2-terminal or core regions were tritiated, indicating a greater resistance of these portions of the peptide to attack by intracellular peptidase. The label from the chemically protected C 41795-Ba was also less readily expelled from the lysosomes of the proximal tubules.

Adrenocorticotropic Hormone↗
PubMed1976

On the metabolism of two adrenocorticotrophin analogues.

The metabolism of Synacthen (corticotrophin-(1--24)-tetracosapeptide) and C-41795-Ba ([D-Ser1,Lys17,Lys18]-corticotrophin-(1--18)-octadecapeptide amide) have been compared in the rat following intravenous injection. Using Synacthen and C-41795-Ba labelled with tritium it was possible to follow the tissue distribution of the two peptides. The kidney was shown to concentrate intact peptide and fragments of both peptides. Autoradiography of perfused kidney sections demonstrated the uptake of radioactivity into the lysosomes of the kidney proximal tubule cells. Comparison of the distribution of radioactive products formed from the tetracosapeptide labelled in different positions indicates that, prior to renal uptake, metabolism is taking place at other sites in the body. It is suggested that rapid cleavage occurs extracellularly at or near the N- and C-termini. Then large fragments remaining in the circulation, together with any intact material, are filtered through the glomerulus and are taken up by endocytosis into the proximal tubule cells of the kidney. The synthetic N- and C-terminal protection of the octadecapeptide appears to inhibit the extracellular attack and so the concentration of intact peptide in the kidneys is initially higher than the tetracosapeptide. Although concentrations of radioactivity in other tissues are low in comparison with the kidneys, the quantities are quite high when the weight of the tissues are considered. Chromatographic analysis of this radioactivity reveals that the octadecapeptide gives rise to much higher tissue levels of intact peptide and we believe that this acts as a depot and gives rise to the sustained blood concentrations and prolonged biological effects observed with this peptide.

Adrenocorticotropic Hormone↗
PubMed1974

Fate of corticotrophins in an isolated adrenal-cell bioassay and decrease of peptide breakdown by cell purification.

1. The fate of corticotrophins in a trypsin-dispersed rat adrenal-cell assay system was investigated with a view to establishing whether differences in the rate of inactivation might contribute to potency differences observed between analogues. 2. Corticotrophin-(1-24)-tetracosapeptide and to a lesser extent synthetic 1-39 corticotrophins were found to be inactivated during incubation with cell suspension. 3. Peptide fragments were isolated by using [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide as a marker. The fragments indicate a peptidase with a predominantly tryptic specificity. 4. The peptidase is present in the extracellular fluid and is released from cells when they are damaged. 5. Cells were fractionated on an albumin gradient. Cells from the zona fasciculata and the zona intermedia or reticularis were present in fractions which produced fluorogenic steroids in response to corticotrophin. 6. Purification of the cells by centrifugation through albumin decreased degradation by peptidases, so that if the assay is carried out with a dilute suspension of purified cells peptide breakdown should not affect the observed potencies of adrenocorticotrophin analogues. 7. No binding of [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide to cells could be detected at low concentrations of the peptide. This indicated that less than 120 receptors/cell are occupied during stimulation by a dose that would elicit approx. 80% of the maximal response.

Adrenal Glands↗
PubMed1974

The isolation and amino acid sequence of an adrenocorticotrophin from the pars distalis and a corticotrophin-like intermediate-lobe peptide from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias.

An adrenocorticotrophic hormone (ACTH) was isolated from extracts of the pars distalis of the pituitary of the dogfish Squalus acanthias by gel filtration and ion-exchange chromatography. It had 15% of the potency of human ACTH in promoting cortico-steroidogenesis in isolated rat adrenal cells. Sequence analysis revealed it to be a nonatria-contapeptide with the following primary structure: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Met-Gly-Arg-Lys-Arg-Arg-Pro-Ile-Lys-Val-Tyr-Pro-Asn-Ser-Phe-Glu-Asp-Glu-Ser-Val-Glu-Asn-Met-Gly-Pro-Glu-Leu. The N-terminal tridecapeptide sequence was identical with the proposed structure of dogfish alpha-melanocyte-stimulating hormone (alpha-MSH). On comparison with human ACTH eleven amino acid differences were seen, nine of which are in the 20-39 region of the molecule which is not essential for the steroidogenic activity of ACTH. A peptide identical with the 18-39 portion of this new ACTH was similarly isolated from the neurointermediate lobe of the pituitary where considerable amounts of dogfish alpha-MSH were found. This supported our view that ACTH as well as having a distinct biological role of its own is also the precursor of alpha-MSH.

Adrenal Cortex Hormones↗
PubMed1974

Structural studies of alpha-melanocyte-stimulating hormone and a novel beta-melanocyte-stimulating hormone from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias.

A melanocyte-stimulating hormone (MSH) has been isolated from extracts of the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias by gel-filtration and ion-exchange chromatography. It had approximately 1% of the potency of mammalian alpha-MSH on bioassays in vitro on frog skin and dogfish skin. Sequence analysis revealed it to be a hexadecapeptide with the following primary structure: Asp-Gly-Asp-Asp-Tyr-Lys-Phe-Gly-His-Phe-Arg-Trp-Ser-Val-Pro-Leu. It appears to be related to the beta-MSH species of mammalian species but has only the sequence -His-Phe-Arg-Trp- in common with the heptapeptide core -Met-Glu-His-Phe-Arg-Trp-Gly- which is characteristic not only of the MSH peptides but also of the adrenocorticotrophins and lipotrophins studied so far. An alpha-MSH was also isolated, 50% of which was amidated at the C-terminus group. Sequence data from this study taken in conjunction with those from a previous study (Lowry & Chadwick, 1970b) revealed it to be a tridecapeptide which is identical with the N-terminal sequence of dogfish adrenocorticotrophin.

Adrenocorticotropic Hormone↗
PubMed1973

Pituitary peptide.

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Adrenocorticotropic Hormone↗
PubMed1973

Confirmation of the 1-20 amino acid sequence of human adrenocorticotrophin.

The tryptic fragments of natural human adrenocorticotrophin, were separated by countercurrent distribution and a correction in positions 25, 26, 27 and 30 was made by Riniker et al. (1972) in a study of the fragment containing residues 22-39. We have purified the remaining tryptic fragments, namely residues 1-8, 9-15, 16-21 and 17-21, by using ion-exchange chromatography on CM-cellulose and have carried out sequence determination by using the subtractive Edman degradation procedure and digestion with aminopeptidase M and carboxypeptidase B. These results have confirmed the proposed sequence for human adrenocorticotrophin in regions 6-7, 10-14 and 17-20, which had previously been arrived at only by analogy with the invariant sequence found in the three other mammalian adrenocorticotrophin species that had been investigated.

Adrenocorticotropic Hormone↗