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Biomedical subjects

H P Bennett

Publications and source records attributed to H P Bennett.

At least 73 records · Page 4Linked to original sources
PubMed1986

Isolation, characterization, and corticotropic activity of rabbit adrenocorticotropin.

Rabbit (r) ACTH was extracted from 600 pituitaries, and 2 forms of immunoreactive ACTH were identified with the least polar form accounting for approximately 90% of the total. Peptide mapping and sequence analysis indicated that three tryptic peptides had retention times identical to those obtained from human (h) ACTH. The least polar tryptic fragment from rACTH had a shorter retention time than the corresponding one from hACTH. Sequence analysis indicated that rACTH differed from hACTH at three different loci, namely, an Asn in place of Asp in position 29; a Val in place of Leu in position 37, and a Val in place of Phe in position 39. Biological activity of the ACTH was compared with synthetic hACTH in 2 bioassays with adrenals from 10-day-old pups, the first using dispersed rabbit adrenal cells and the second using monolayer adrenal cells in culture. The biological potencies of the two ACTH preparations were identical with respect to corticosterone (B) release in the short term bioassay, with an ED50 value of 1.67 X 10(-10) M. The ED50 value for cortisol (F) release for rACTH and hACTH were 1.1 X 10(-10) M and 1.67 X 10(-10) M, respectively, which were not statistically different. The biological potency of rACTH in the monolayer adrenal cell system for both F and B was significantly greater than the hACTH, and the ED50 values were 4.4 X 10(-10) M and 8.9 X 10(-10) M, respectively. There was a progressive decrease in the B/F ratios with increasing concentrations of ACTH in both the bioassay systems suggesting that ACTH stimulated the 17 alpha-hydroxylase activity even when the exposure of cells to ACTH was as short as 2 h.

Adrenocorticotropic Hormone↗
PubMed1985

Use of reversed-phase and ion-exchange batch extraction in the purification of bovine pituitary peptides.

Bovine posterior pituitaries were extracted with an acidic medium designed to maximize solubilization of peptides while precipitating high-molecular-weight protein. The supernatant was then extracted with C18 reversed-phase cartridges to generate a peptide-enriched fraction. Cartridge eluates were subjected to ion-exchange extraction, using a batch procedure which fractionated the peptides into basic, acidic, and neutral pools. Amino-terminal fragments of bovine pro-opiomelanocortin were found to be resolved into separate pools by this method. The 1 to 49 fragment was eluted in the acidic pool while the 1 to 77 fragment was eluted in the basic pool. The 1 to 77 fragment was purified by reversed-phase high-performance liquid chromatography. Amino acid analysis of the fragments, generated from trypsin and V8 protease digestion of the 1 to 77 fragment, permitted assignment of cystine bridges between residues 2 and 24 and between residues 8 and 20. Results from amino sugar analysis were consistent with the presence of an O-linked oligosaccharide at threonine45 and an N-linked oligosaccharide at asparagine65.

Amino Acid Sequence↗
PubMed1984

Isolation and characterization of the 1 to 49 amino-terminal sequence of pro-opiomelanocortin from bovine posterior pituitaries.

An amino-terminal fragment of pro-opiomelanocortin (POMC) has been isolated from bovine intermediate pituitaries by reversed-phase high-performance liquid chromatography. Peptide mapping and amino acid analysis indicated that the primary sequence corresponds exactly to that predicted by cDNA techniques for the first 49 residues of POMC including the presence of four cysteine residues. This peptide is almost certainly generated together with the gamma-melanotropins during the biosynthetic processing of the 16K amino-terminal fragment of bovine POMC. Careful analysis of the fragments resulting from V8 protease and trypsin digestion has permitted assignment of cystine bridges between residues 2 and 24 and between residues 8 and 20.

Amino Acid Sequence↗
PubMed1984

Phosphorylation of parathyroid hormone by human and bovine parathyroid glands.

Human and bovine parathyroid gland slices were incubated in vitro for varying time periods with inorganic 32P and [35S]methionine or [3H]serine. Tissue was then extracted with aqueous medium, and parathyroid hormone (PTH) purified. Incorporated 32P was found to coelute with immunoreactive PTH in multiple chromatographic systems, and a peak of phosphorylated material could be resolved from the nonphosphorylated hormone by reversed phase high pressure liquid chromatography. The amino acid composition of both the phosphorylated and the nonphosphorylated entities conformed to that of the major glandular species of PTH, and phosphorylated hormone accounted for 10-20% of the total. A time course revealed slow incorporation of 32P into hormone, and after a 4-h preincubation with inorganic 32P, co-elution of 32P with both PTH and its precursor was observed. Phosphoserine was identified in purified PTH labeled with [3H]serine. Additionally dilute acid hydrolysis of PTH, labeled with [35S]methionine and containing 32P, generated an 35S-labeled fragment with which 32P co-chromatographed. The results are consistent with in vitro phosphorylation of PTH on serine residues within the NH2-terminal region of the hormone by both human and bovine glands and suggest that phosphorylation of the prohormone occurs as well.

Amino Acids↗
PubMed1983

Isolation of pituitary peptides by reversed-phase high-performance liquid chromatography. Expansion of the resolving power of reversed-phase columns by manipulating pH and the nature of the ion-pairing reagent.

The reversed-phase liquid chromatographic behaviour of pituitary peptides at various pH values was compared. The retention times of peptides at pH 1, 3 and 4.5 were essentially as expected for acidic solvent systems. However, at pH 5.5 or 7, radically different elution characteristics were observed. This is probably brought about by the increase in polarity that accompanies ionization of the peptide carboxyl groups at higher pH values. Basic hydrophobic ion-pairing reagents, such as tetrabutylammonium ion, can be used to maximize differences in polarity. We have previously shown that the use of acidic hydrophobic ion-pairing reagents at low pH (e.g. trifluoroacetic and heptafluorobutyric acids) enhances the basic polarity of peptides. The combination of both approaches facilitated the reversed-phase purification of posterior pituitary glycopeptide and alpha-N-acetyl-beta-endorphin1-27 from an extract of 200 rat neurointermediate pituitaries. The separation of these two peptides at low pH, which is particularly difficult, was achieved by the sequential use of solvent systems containing trifluoroacetic acid (0.1%), heptafluorobutyric acid (0.13%) and finally triethylamine acetate (0.01 M, pH 5.5).

Animals↗
PubMed1983

Human phosphoserine 31 corticotropin1-39. Isolation and characterization.

Two distinct forms of corticotropin1-39 (ACTH) were isolated and purified from an extract of three adult human pituitaries by reversed-phase chromatographic techniques. Structural studies indicated that the more polar form of ACTH was phosphorylated at serine residue 31. Approximately 30% of the ACTH was found in the phosphorylated form. A similar proportion of phosphorylated ACTH was observed in extracts of three pituitaries from human fetuses of 15, 17, and 20 weeks gestation. Phosphorylated and nonphosphorylated human ACTH1-39 were found to be steroidogenically equipotent using both an isolated rat adrenal cell bioassay and a cultured human fetal adrenal cell bioassay.

Adrenocorticotropic Hormone↗
PubMed1983

alpha-N-acetyl-beta-endorphin1-26 from the neurointermediary lobe of the rat pituitary: isolation, purification, and characterization by high-performance liquid chromatography.

The neurointermediary lobes from 190 rat pituitaries were homogenized in an acidic medium which inhibits peptidase activity and maximizes the solubilization of undamaged peptides. Octadecylsilyl-silica (ODS-silica) was used to extract the supernatant of the tissue homogenate. The ODS-silica eluate, now largely protein and salt free, was subjected to reversed-phase high-performance liquid chromatography (HPLC) employing 0.1% trifluoroacetic as counter ion. The column eluates were monitored for beta-endorphin immunoreactivity. Five immunoreactive components were observed. The most hydrophobic of these was repurified on the same HPLC column using 0.13% heptafluorobutyric acid as counter ion. Characterization of the purified peptide by gel permeation HPLC, amino acid analysis, and tryptic fragmentation indicated that it corresponded in structure to alpha-N-acetyl-beta-endorphin1-26. Amino acid analysis of the native peptide and its trypsin and carboxypeptidase fragments indicated that an alanyl residue occupies position 26. This finding is in contrast to the sequence predicted for the beta-lipotropin/corticotropin precursor by recombinant DNA techniques which suggests that the 26th residue of the beta-endorphin molecule should be valine.

Amino Acid Sequence↗
PubMed1982

Characterization of the rabbit renal receptor for native parathyroid hormone employing a radioligand purified by reversed-phase liquid chromatography.

Iodinated native bovine parathyroid hormone (bPTH(1-84)) was separated from uniodinated hormone by reversed-phase liquid chromatography techniques after lactoperoxidase labeling. Analysis of iodinated residues after enzymatic digestion indicated that the major labeled product was largely monoiodinated on the sole tyrosine residue. This material retained full bioactivity in an in vitro renal adenylate cyclase assay. Binding of 125I-bPTH(1-84) to rabbit renal membranes at 4 degrees C was proportional to membrane protein concentration and was saturable and dissociable. Radioligand binding was inhibited by concentrations of unlabeled bPTH(1-84) required to stimulate adenylate cyclase in the same membrane preparation but was not inhibited by non-PTH peptides other than adrenocorticotropin at high concentrations (greater than 10 microM). Synthetic NH2-terminal analogues of bPTH(1-84) all elicited approximately equivalent inhibition of radioligand binding which was, however, less potent than unlabeled bPTH(1-84), suggesting a role for the carboxyl region of the molecule in the interaction of bPTH(1-84) with its receptor. Activity of the NH2-terminal agonists was similar to bPTH(1-84) in stimulating adenylate cyclase. Although substitution in sequence position one, of serine in human PTH(1-34) for alanine in bPTH(1-34), reduced activity in the adenylate cyclase assay, inhibition of 125I-bPTH(1-84) binding by both peptides and by an analogue of bPTH(3-34) was equivalent, consistent with a minimal contribution of the first 2 residues for receptor binding of the NH2-terminal region of PTH. The results illustrate the utility of the radiolabeled preparation of native bPTH we have developed and emphasize the importance of probing the PTH receptor with an intact hormone to maximize information concerning the mechanism of PTH action.

Animals↗
PubMed1982

Characterization of eight forms of corticotropin-like intermediary lobe peptide from the rat intermediary pituitary.

The isolation and characterization of eight forms of corticotropin-like intermediary lobe peptide (CLIP, adrenocorticotropin18-39) from the intermediary lobe of the rat pituitary has been accomplished by using reversed phase high performance liquid chromatography. The eight forms are the result of all combinations of the presence or absence of three post-translational modifications. These are glycosylation, phosphorylation, and removal of the carboxyl-terminal amino acid. The sites of phosphorylation and glycosylation are at serine 31 and asparagine 29, respectively. The eight forms (in order of elution from the reversed high performance liquid chromatography column) are glycosylated, phosphorylated CLIP18-38; glycosylated, nonphosphorylated CLIP18-38; nonglycosylated, phosphorylated CLIP18-38; nonglycosylated, nonphosphorylated CLIP18-38; glycosylated, phosphorylated CLIP18-39; glycosylated, nonphosphorylated CLIP18-39; nonglycosylated, phosphorylated CLIP18-39; and nonglycosylated, nonphosphorylated CLIP18-39.

Adrenocorticotropic Hormone↗
PubMed1982

Fate of human corticotrophin immediately after intravenous administration to the rat.

The distribution and degradation of tritium-labelled human 1-39 corticotrophin have been studied after intravenous administration to the rat. Within 5 min of injection of single major metabolite, 3-39 corticotrophin, appears in the circulation. This metabolite, however, has only 3.6% of the steroidogenic potency of 1-39 corticotrophin and the evidence suggests that it is formed in muscle and skin. By 5 min, extensive degradation had occurred in all the major tissues in the body (muscle, skin, liver, kidney, gut).

Adrenocorticotropic Hormone↗
PubMed1981

Isolation and characterization of corticotropin- and melanotropin-related peptides from the neurointermediary lobe of the rat pituitary by reversed-phase liquid chromatography.

A novel procedure utilizing reversed-phase high-performance liquid chromatography for the extraction and purification of peptides from biological tissues has been applied to the isolation of corticotropin-like intermediary lobe peptide (CLIP) and alpha-melanocyte-stimulating hormone (alpha-MSH) from the neurointermediary lobe of the rat pituitary. The isolation and characterization of two major forms of CLIP and two major forms of alpha-MSH are described. The isolated peptides have been identified by using enzymatic digestions and peptide mapping. The main form of ClIP is a peptide which has been modified by phosphorylation of the serine residue at position 31. This is the first peptide of endocrine origin reported to be modified in such a manner. A non-phosphorylated form of CLIP was also present at lower concentrations. The main form of alpha-MSH was found to be N,-O-diacetyl-alpha-MSH, with the more familiar mono-N-acetyl-alpha-MSH present to a much smaller extent. Thus, in the rat neurointermediary lobe, the two main corticotropin-related peptides present are mostly in modified forms which are the result of posttranslational modifications. It is only by the use of methodology such as that described in this paper that small alterations in peptide structure may be identified.

Adrenocorticotropic Hormone↗
PubMed1981

Isolation and analysis of human parathyrin in parathyroid tissue and plasma. Use of reversed-phase liquid chromatography.

Reversed-phase liquid chromatography techniques have been used to extract and purify human parathyrin from parathyroid adenomas and to analyse the circulating forms of human parathyrin in plasma. Both the supernatant from tissue homogenates, and plasma were extracted with octadecylsilyl-silica (ODS-silica) in a batch procedure. Extracts were subjected to reversed-phase high-pressure liquid chromatography (h.p.l.c.) employing solvent systems composed of aqueous acetonitrile containing trifluoroacetic acid or heptafluorobutyric acid as hydrophobic ion-pairing reagents. The volatile solvents facilitated the radioimmunoassay, bioassay in vitro and amino acid analysis of column fractions and permitted monitoring for u.v. absorbance at 210nm. Isolated glandular parathyrin was found to be homogeneous by sodium dodecyl sulphate/urea/polyacrylamide-gel electrophoresis, to have an amino acid composition conforming to that of human parathyrin-(1--84)-tetraoctacontapeptide and to be bioactive in both renal adenylate cyclase and cytochemical bioassays. ODS-silica extraction permitted examination of large plasms samples by reversed-phase h.p.l.c., facilitating the resolution of the various circulating molecular forms of parathyrin according to their hydrophobic character. Because of its rapidity, excellent recovery and high resolving power, the methodology utilized is uniquely suited to the purification and analysis of parathyrin in tissues and body fluids.

Adenoma↗
PubMed1981

Biosynthesis of phosphorylated forms of corticotropin-related peptides.

Phosphorylated forms of corticotropin[ACTH (1-39)], corticotropin-like intermediary lobe peptide[CLIP, ACTH (18-39)], and the common precursor for ACTH and beta-lipotropin (beta-LPH) have been identified in extracts of rat pituitaries, 32P-Labeled inorganic phosphate was successfully incorporated into ACTH (1-39), CLIP, and the ACTH/beta-LPH precursor in rat neurointermediary lobe explants and into ACTH (1-39) in isolated rat anterior pituitary cells. After peptidase digestion of the labeled CLIP and ACTH, the radioactive phosphate was recoverable as O-phosphoserine. The serine residue at position 31 was the only amino acid found to be phosphorylated in CLIP and ACTH (1-39). The unphosphorylated forms of both peptides were also synthesized. The demonstration of he incorporation of [32P]phosphate into CLIP, ACTH (1-39), and the ACTH/beta-LPH precursor is consistent with the hypothesis that, within the rat intermediary lobe, phosphorylated CLIP is derived from a phosphorylated form of the common precursor, with phosphorylated ACTH (1-39) acting as a biosynthetic intermediate.

Adrenocorticotropic Hormone↗