Biomedical subjects
Publications and source records attributed to H P Bennett.
The nucleotide sequence of a novel peptide from a rat Leydig cell hypercalcemic tumor H-500 was determined. This cDNA encodes a peptide of 93 amino acids and contains a heparin binding domain similar to histone 2-B. Northern blot analysis showed tissue specific expression of this peptide mRNA.
Seven cationic, cystine-rich peptides of 29 to 32 amino acid residues have been purified from extracts of rat bone marrow (R-1, R-1a, R-1b, R-2, R-3, R-4 and R-5). Structural analysis clearly indicated that all seven peptides belong to the corticostatin/defensin family of leukocyte-derived peptides known to participate in oxygen-independent killing of phagocytosed bacteria. For R-1 to R-5, six cysteine residues were found at characteristic and highly conserved positions. R-1a and R-1b were partially characterized and appear to be structural variants of R-1. Aside from the conserved cysteines, there is a remarkable degree of structural diversity evident within the sequences of those members of the corticostatin/defensin family characterized so far. The structures of the peptides that we have purified can be compared directly with the sequences obtained for rat defensins isolated from extracts of peritoneal neutrophils (Lehrer, Ganz and Selsted, Cell, 64 (1991) 229-230). Some discrepancies are apparent which can be explained in terms of proteolytic cleavage of several of these peptides at both amino- and carboxyl-termini. The corticostatins owe their bioactivity to their ability to compete with corticotropin for occupancy of the corticotropin receptor (Zhu, Hu, Mulay, Esch, Shimasaki and Solomon, Proc. Natl. Acad. Sci. USA, 85 (1988) 592-596). The potency of these peptides can be expressed in terms of their capacity to inhibit the steroidogenic response of isolated rat adrenocrotical cells half-maximally stimulated by corticotropin (i.e., at the ED50 concentration for corticotropin in this assay, namely 33 pM). In this assay, the rat peptides R-1, R-2 and R-3 were shown to be inactive. In contrast, the more cationic peptides R-4 and R-5 were found to inhibit steroidogenesis. R-4 was somewhat less active than rabbit corticostatin (IC50 25 nM) showing an IC50 value of 50 nM. R-5 appeared to be significantly less potent than R-4. The lower yield of R-5 precluded an accurate estimate of the corticostatic potency of this peptide. R-4 differs in structure from R-5 in having an arginine to serine substitution at position 7. It can be concluded that an arginine at this position accounts, at least in part, for the corticostatic activity of R-4.(ABSTRACT TRUNCATED AT 400 WORDS)
A Guard-PAK precolumn system was used for the reversed-phase liquid chromatography purification of a small, synthetic radiolabeled opioid peptide, FK 33-824 (D-Ala2, methyl-phe4, Met (O)ol5 enkephalin) (FK). This procedure involves trace enrichment of iodinated peptide onto the precolumn while iodination reagents are not retained. Radioactive contamination of high-performance liquid chromatography columns and injectors is thus avoided. Precolumn chromatography has sufficient resolving power to separate not only labeled from unlabeled peptide but also mono- from di-iodinated peptide. Purified 125I-labeled FK (estimated specific activity 85.9-153.7 Ci/mmol) showed high specific binding to mouse corpus striatum, neocortex, cingulate cortex, nucleus accumbens septi, diagonal band of Broca, nucleus medialis septi, area preopticus magnocellularis, and the nucleus of the caudate/putamen. Radioligand binding was inhibited by both antagonists (naloxone and naltrexone); and agonists D-Ala2, N-methyl-phe4, gly-ol5-enkephalin [DAGO]; FK; and beta-endorphin at all concentrations tested (1 x 10(-8) to 1 x 10(-4) M). Adrenocorticotropin hormone (ACTH) did not block ligand binding at any concentration tested. Distribution of mu opioid receptors was analyzed by light microscopic autoradiography. Sections incubated with 125I-labeled FK in the presence of agonists and antagonists demonstrated decreasing ligand binding with increasing doses of competitor. ACTH did not block ligand binding at any concentration tested. HPLC analyses of ligand which had been iodinated 1.5 half lives before the date of the experiment demonstrated a single peak similar to that of freshly iodinated ligand. Similar binding kinetics and autoradiographic labeling patterns were observed as compared to those obtained with freshly iodinated peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Three corticostatic peptides (GP-CS1, GP-CS2 and GP-CS3) were purified from extracts of guinea pig bone marrow. Each was identified on the basis of their ability to inhibit the secretion of corticosterone by isolated rat adrenal cells stimulated by ACTH. GP-CS1 and GP-CS2 were found to be 31 residues in length, rich in arginine and to have six cysteines typical of the corticostatin/defensin family of peptides previously purified from phagocytic cells of the immune system. GP-CS1 was found to be identical to GP-CS2 except for having a leucine at position 21 instead of isoleucine. GP-CS3 was also found to be rich in arginine and cysteine but structurally distinct from the other peptides. A combination of endoprotease mapping, ion-spray mass spectrometry and gas-phase sequencing revealed that GP-CS3 was a novel homo-dimer consisting of two 13 amino acid residue subunits cross-linked through eight cysteines in an anti-parellel configuration.
The effect of Ca2+ on the extent and pattern of processing of pro-opiomelanocortin and an N-terminal fragment by a purified pituitary secretory vesicle, soluble aspartic endoprotease, was studied. Ca2+ stimulated the first cleavage of pro-opiomelanocortin by pro-opiomelanocortin-converting enzyme to yield 21-23 kDa adrenocorticotropin and beta-lipotropin, but its effect was minimal. The production of adrenocorticotropin from the 21-23 kDa intermediate was stimulated approximately 2.3-fold in the presence of 10 mM Ca2+, and processing of beta-lipotropin to beta-endorphin was stimulated about 1.3-1.4-fold by 5-10 mM Ca2+. The production of gamma-melanotropin-immunoreactive material from bovine N-pro-opiomelanocortin(1-77) was stimulated approximately 1.3-fold at both 100 microM and 1.5-2.0 mM Ca2+. Further characterization of the gamma-melanotropin-immunoreactive material by HPLC demonstrated that the major products were gamma 3-[Lys]melanotropin and gamma 3-melanotropin at both Ca2+ concentrations. These results indicate that pro-opiomelanocortin-converting enzyme is stimulated by Ca2+.
The amino terminus of bovine pro-opiomelanocortin (N-POMC1-77) is partially processed in the intermediate lobe of the pituitary to N-POMC1-49 and lys-gamma 3-melanotropin. Two pools of N-POMC1-77 were isolated which were differentially glycosylated at threonine45, while N-POMC1-49 isolated from bovine intermediate lobe extracts existed in a non-glycosylated form. This suggested that differential O-linked glycosylation of N-POMC1-77 may regulate cleavage at the Arg49-Lys50 processing site. We tested this hypothesis by incubating N-POMC1-77 glycoforms with purified proopiomelanocortin converting enzyme. Only non-O-glycosylated N-POMC1-77 and O-glycosylated N-POMC1-77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys-gamma 3-melanotropin, identified by high-performance liquid chromatography. These data provide the first functional evidence to support a role for differential O-linked glycosylation in the regulation of the processing of the N-terminus of bovine POMC.
We studied cell-volume changes caused by adding corticostatin (CS) or defensin-like peptides to villus enterocytes isolated in suspension from guinea pig jejunum. Guinea pig CS (10(-9) M) added to villus cells in Na(+)-containing medium reduced volume, but immediate cell swelling was caused by 10(-6) M guinea pig CS. In Na(+)-free N-methyl-D-glucamine-containing medium 10(-9) M guinea pig CS accelerated the initial rate of shrinkage compared with cells in N-methyl-D-glucamine-containing medium alone as well as causing greater cell shrinkage. Guinea pig CS-stimulated cell shrinkage was prevented by a Ca2(+)-channel blocker--5 microM nifedipine, by chelation of extracellular Ca2+ with 100 microM EGTA, or by omega-conotoxin (10(-9) M). The Ca2+ ionophore A23187 (2.5 microM) reduced volume when added to villus cells in N-methyl-D-glucamine-containing medium; this action was prevented by EGTA, or quinine--an inhibitor of K+ conductance, or 9-anthracenecarboxylic acid--a Cl- channel blocker, suggesting that the volume reduction occurred because K+ and Cl- conductances were activated. Guinea pig CS-stimulated volume reduction was also prevented by 100 microM quinine or 9-anthracenecarboxylic acid. We conclude that jejunal villus enterocytes possess a Ca2(+)-activated Cl- conductance and a K+ conductance that need not be stretch-activated. Corticostatic peptides cause volume reduction in villus cells by activating L-type Ca2+ channels; other defensin-like peptides were without effect.
In the last four years corticostatic (anti-ACTH) peptides have been isolated from human, rabbit, guinea pig and rat tissues. These peptides do not act via the cAMP cell signalling system but rather via the inhibition of the binding of ACTH to its receptor most probably through direct competition with the 14-18 sequence of ACTH for receptor binding. ACTH has specific high affinity receptors on adrenal cells but rabbit corticostatin I (CSI) has high capacity, low affinity receptors which are competed for by unlabelled excess CSI but not by excess ACTH. This indicates the presence of specific CSI adrenal cell receptors. The rabbit pituitary, hypothalamus, thalamus, adrenals, lungs and placenta contain sizeable amounts of immunoassayable CSI. Immunochemical localization of CSI indicates that it is present in the large macrophages and in neutrophils in rabbit lung, in macrophages and "supporting" endothelial cells in the spleen and in the adrenals in the cells of the zona reticularis. We have also isolated and identified new peptides which contain 12 cysteines from immune cells of humans, rats and a teleost, the carp. The functions of these peptides are now being determined. This large family of peptides may have many other, yet unidentified functions but at present we can only describe a small number of these.
We have prepared two monoclonal antibodies for the N-terminal glycopeptide of pro-opiomelanocortin 1-77 (N-POMC(1-77)) purified from porcine pituitaries. Antibody 1-244 recognizes an epitope located within the gamma 3-melanotropin (gamma 3-MSH or POMC(51-77)) sequence, whereas antibody 2-197 binds specifically to a determinant in the 1-49 region of N-POMC. These monoclonal antibodies were used to construct a two-site solid-phase radioimmunoassay that can detect as little as 50 pg of N-POMC(1-77). The assay is linear between 0.5 and 5 ng of porcine peptide and recognizes equally well the homologous peptides purified from human and bovine pituitaries. The assay has been used to analyze reversed-phase high pressure liquid chromatography fractions of crude bovine pituitary extracts and detected a peptide with chromatographic properties identical to those of N-POMC(1-77). When used to stain immunoblots of bovine intermediate pituitary extracts, both the 2-197 and 1-244 antibodies could recognize a major peptide comigrating with purified N-POMC(1-77). In addition, antibody 2-197 also detected a peptide with a mobility similar to that of standard N-POMC(1-49). When used in conjunction with a second anti-mouse antibody coupled to colloidal gold particles, antibody 2-197 stained N-POMC immunoreactive material located in granules in thin sections of pituitary.
Three structural variants of the joining peptide (JP) fragment of POMC have been purified from human pituitaries. Ion exchange and reverse phase tissue extraction procedures were combined with reverse phase HPLC to achieve complete purification of each form of JP. Fragments resulting from tryptic hydrolysis of each form were characterized by amino acid analysis and fast atom bombardment mass spectrometry. The predominant form of human JP, accounting for about 50% of the total purified, was found to be conjugated to glutathione through the lone cysteine residue at position 9. The other two variants were identified as human JP with a free cysteine residue and human JP dimer and accounted for 35% and 15%, respectively, of the total purified. Recently, human JP-(1-18) has been suggested as having adrenal androgen-stimulating activity. None of the three JP variants or their respective 1-20 amino-terminal fragments resulting from tryptic hydrolysis showed any ability to promote the secretion of dehydroepiandrosterone sulfate by cultured human fetal adrenal cells. Similarly, no potentiation of the stimulatory effects of ACTH-(1-39) was observed. The three variants of human JP as well as JP purified from rat, porcine, and bovine pituitaries were tested for their ability to stimulate androgenic steroids from dispersed fetal rabbit adrenal cells. None showed any significant biological activity either in stimulating steroid secretion or in potentiating the action of ACTH-(1-39).
The amino-terminal fragment of beta-lipotropin (i.e. beta-lipotropin (1-40)) and joining peptide portions of pro-opiomelanocortin have been purified from extracts of bovine posterior pituitaries. Peptides were purified using a combination of reversed-phase and ion-exchange batch extraction procedures followed by reversed-phase high performance liquid chromatography. beta-Lipotropin (1-40) was found to consist of four major components while joining peptide was found to consist of two major components. Fast atom bombardment-mass spectrometric analysis of the tryptic fragments of both peptides revealed that the observed heterogeneity could be explained in terms of post-translational modifications. beta-Lipotropin (1-40) was found to be sulfated at tyrosine residue 28 to an extent of about 50%. The tyrosine residue in beta-lipotropin (1-40) is situated within an amino acid sequence with a preponderance of glutamate residues. Sulfation of this amino acid residue is entirely compatible with the known primary structure requirements of the sulfotransferase enzyme located in the trans-Golgi fraction. Both beta-lipotropin (1-40) and joining peptide were found to have pyroglutamate at their amino termini to an extent of about 50%. The cDNA sequence for bovine pro-opiomelanocortin predicts the presence of glutamic acid at position 1 of both peptides. Pyroglutamate is normally formed through the cyclization of glutamine. This reaction is thought to be catalyzed by a pyroglutamate forming enzyme located within the secretory granule fraction. Under certain circumstances peptides with glutamate at their amino termini may act as substrates for this enzyme.
The amino acid sequence of chymodenin, a hormone-like peptide from porcine duodenum is reported. The molecule is known to rapidly alter the proportions of digestive enzymes secreted by the rabbit pancreas in vivo and in vitro, by selection of the specific intra-pancreatic source from which the preset mixture of digestive enzymes is secreted. The sequence is identical to that of cytochrome C-oxidase peptide VII (cCoVII) from bovine heart, with the exception of a substitution of threonine for alanine at position 6 and a second substitution of alanine for threonine at position 71. Disulfide bridges link positions 29-64 and 39-53. cCoVII-chymodenin has a pentapeptide (-Ala-Glu-Gly-Thr-Phe-) near the carboxy-terminus which is immediately preceded by an -Arg-Arg- sequence in the porcine and bovine sequences of cCoVII. This peptide is identical to a pentapeptide found close to the amino terminus of the hormones gastric inhibitory peptide (GIP) and glucagon-like peptide I. The identity to cCoVII means chymodenin as isolated is itself unlikely to be a gastrointestinal hormone. However, the partial commonality of sequence with the glucagon-secretin family immediately adjacent to a pro-hormone-like activation site, and the specific actions on the exocrine pancreas, means that the molecule probably mimics the natural actions of an as-yet uncharacterized member of the glucagon family, which exerts a unique action on exocrine pancreatic secretion.
Several peptides are generated from prosomatostatin (proSS) in addition to somatostatin-14 (SS-14) and somatostatin-28 (SS-28). These are SS-28(1-12), proSS(1-76) and, as shown more recently, proSS(1-63) and antrin. Important variations in the proportion of these molecular forms are seen among different tissues and among different species. Processing of the precursor in the human brain yields minimal quantities of SS-28(1-12) and high levels of proSS(1-76), namely in cortical areas and in the bed nucleus of the stria terminalis. This is in contrast with findings in rat brain, where SS-28(1-12) is a predominant molecular form. Antrin, which corresponds to proSS(1-10), reaches its highest concentration in the antral portion of the stomach (117 +/- 13 pmol/g wet weight), where it is found in secretory granules of delta cells. We observed an inverse relationship between levels of antrin and proSS(1-63) after chromatography of various tissue extracts. This suggests a precursor-product relationship between these two peptides.
A simple scheme for the rapid and efficient isolation of rat pro-atrial natriuretic factor (pro-ANF) has been developed. An isolated rat adrenal cell bioassay for ANF was established to optimize heart tissue extraction and chromatography conditions. This assay is based on the ability of ANF to inhibit angiotensin II-stimulated aldosterone secretion. IC50 values for ANF were approximately 320 pM. The protocol that was established consisted of extraction of rat atria in 5 N acetic acid containing protease inhibitors. The extract was lyophilized, resolubilized in 0.1% trifluoroacetic acid containing 1% (w/v) sodium chloride, and subjected to RP-HPLC. Extraction of small batches of atria (i.e., from 10 or 20 rats) resulted generally in a yield of 2 nmol per rat (i.e., approximately 30 micrograms). The identity and purity of the pro-ANF were confirmed by the determination of both the amino acid composition and the amino-terminal sequence. Purified pro-ANF was radioiodinated and the efficiency of the extraction and purification procedure was assessed by adding labeled peptide to the initial tissue extract. The structural integrity and overall recovery of radioactivity were determined by RP-HPLC. The purification scheme provides undamaged pro-ANF of high purity. Purified pro-ANF was compared with synthetic rat ANF in the rat adrenal glomerulosa cell and isolated rat aortic strip bioassays. The peptides were apparently equally active in the adrenal cell system and approximately threefold less potent in relaxing aortic strips. The apparent equipotency in the adrenal cell bioassay may be due to the conversion of pro-ANF to ANF-like peptides during the bioassay incubation.
Gel-permeation high-performance liquid chromatography (GP-HPLC) columns provide rapid high-resolution separations but are frequently limited to analytical tasks because the injection volumes must be small. The reduction of volume required for the loading of solutes can often be impractical and lead to poor recoveries. We have developed a trace-enrichment technique to circumvent this problem. By placing a Waters Guard Pak within the loop of a Valco injector and connecting a pump to the injection port it is possible to concentrate proteins and peptides onto the guard column from relatively large volumes. Enrichment onto a reversed-phase guard column insert is achieved by loading solutes in an aqueous solution or one of low organic solvent concentration. Provided that the GP-HPLC is mean-while equilibrated with a solvent system of sufficiently high organic solvent concentration (i.e., 40% acetonitrile containing 0.1% trifluoroacetic acid) it is possible to elute material that has been loaded in this manner by simply placing the injection loop in line with the column. The solvent strength abruptly increases and the peptide or protein sample is loaded onto the column in a very small volume. We have applied this loading principle to both analytical and semipreparative problems. The amino-terminal fragment of pro-opiomelanocortin (POMC) has been extracted from a single human fetal pituitary (18 weeks gestation) and characterized in terms of its molecular weight. This study indicated that no proteolytic processing of the amino-terminal fragment of POMC takes place at this stage in development. In a larger scale application the amino-terminal fragment of POMC was purified from bovine anterior pituitaries.(ABSTRACT TRUNCATED AT 250 WORDS)
Recombinant human parathyroid hormone (hPTH) was expressed in Escherichia coli harboring a plasmid containing a synthetic human parathyroid hormone gene under the control of the E. coli lac promoter. Three major forms of the hormone were isolated by acid extraction and purified to homogeneity by high performance liquid chromatography. By amino acid analysis and NH2-terminal sequencing, these were identified as hPTH-(1-84), formyl-methionyl-hPTH-(1-84), and hPTH-(8-84). The recombinant hPTH-(1-84) was immunologically indistinguishable from a World Health Organization standard of extracted native hPTH-(1-84). Recombinant hPTH-(1-84) was also bioactive in renal and skeletal adenylate cyclase assays. In the skeletal bioassay performed in UMR 108 osteosarcoma cells its activity was identical to that of an hPTH-(1-84) standard. In this bioassay, formyl-methionyl-hPTH-(1-84) had 10% of the activity of hPTH-(1-84) and hPTH-(8-84) was inactive. The results demonstrate the importance of isolating hPTH-(1-84) from other recombinant forms and metabolites to achieve full hormonal bioactivity and indicate that purified recombinant hPTH-(1-84) can thereby be obtained which should be a useful source of hormone for both basic and clinical studies.
We examined the characteristics of mitogens extracted from human benign prostatic hyperplasia and prostatic adenocarcinoma tissue. Although mitogens for fetal rat skin fibroblasts as well as for rat calvarial osteoblasts and osterosarcoma cells were found, distinct entities that acted selectively in cells of the osteoblast phenotype could be obtained by sequential reverse-phase high performance liquid chromatography. Two peptides with apparent molecular weights of 10,000 and 13,000 D were derived from hyperplastic tissue, whereas a single moiety of 10,000 D was obtained from malignant tissue. These entities increased cell numbers and alkaline phosphatase activity in osteoblastlike cells consistent with effects on both growth and differentiation. Prostatic peptides did not stimulate adenylate cyclase in osteosarcoma cells. Mitogenic activity selective for osteoblastlike cells was identified in postpubertal but not prepubertal normal prostate. The results demonstrate the existence of osteoblastic growth factors in prostatic tissue whose presence may accompany postpubertal development.
A chemical method has been established for the detection of carboxyl-terminally amidated peptides in tissue extracts. Tissue was homogenized in an acidic medium designed to solubilize peptides while precipitating high-molecular-weight protein. The homogenate supernatant was in turn subjected to reversed-phase extraction with C18 Sep-Pak cartridges. The eluates were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). Individual fractions were exhaustively digested with thermolysin, derivatized with phenylisothiocyanate (PITC), and then subjected to ethyl acetate extraction under basic conditions. The phenylthiocarbamyl (PTC)-amino acid amide derivatives were selectively taken up into the organic phase, while the other digestion products remained in the aqueous phase. The organic phase was analyzed by RP-HPLC on a Pico-Tag amino acid analysis column, monitoring eluates at 254 nm. PTC-amino acid amides were identified and quantitated by comparing their elution positions and peak areas, respectively, with those of standards. Their identities were confirmed by amino acid analysis, following hydrolysis with hydriodic acid. The technique was applied to extracts of bovine posterior pituitaries and a human medullary thyroid carcinoma. Vasopressin (-Leu-Gly-amide), oxytocin (-Gly-amide), Lys1 gamma 1-melanotropin (-Phe-amide), and various acetylated and non-acetylated forms of alpha-melanotropin (-Val-amide) were identified in the posterior pituitary extract. Various forms of calcitonin (-Val-Gly-Ala-Pro-amide) were detected in the tumour extract. For vasopressin and calcitonin the thermolytic digest resulted in di- and tetra-peptides, respectively, reflecting thermolytic cleavage at more favoured sites.