Effects of bone in vitro of bovine parathyroid hormone and synthetic fragments representing residues 1-34, 2-34 and 3-34.

The biological activities of bovine parathyroid hormone (BPTH) and fragments comprising portions of its amino-terminal sequence have been compared in three different assay systems using embryonic rat bone in vitro. Whereas the 3-34 fragment was without significant activity the 1-34 fragment caused all the actions characteristic of BPTH 1-84, extending to bone previous evidence that the amino-terminal residues are sufficient for expression of the biological effects of intact parathyroid hormone. However, the relative potencies of the fragment and the intact hormone were different in the various systems. BPTH 1-34 showed relatively low osteolytic activity and induced anabolic effects in both osteoblasts and cartilage cells of cultivated embryonic mouse radii which were not evoked by the intact hormone. Further work is required to determine the mechanisms responsible for these interesting alterations in relative potency of fragment and native hormone.     

Preparation and properties of biologically active radioiodinated parathyroid hormone

A constant-current microelectrolytic radioiodination method was used to label bovine parathyroid hormone (BPTH) with ¹²⁵I to an overall iodination ratio of 1:1 iodide atoms per PTH molecule. Such iodinated preparations were shown to be fully active in several bioassay systems: in vitro adenylate cyclase activation in rat renal and skeletal membranes, in vitro calcium release from rat calvaria, and the in vivo hypercalcemic response in chickens. Analysis by Sephadex G-15 chromatography after enzymatic digestion showed the radioiodine to be incorporated predominantly as monoiodotyrosine. Bioassay of iodinated preparations from which uniodinated hormone had been removed by isoelectric focusing showed essentially full hormonal activity. Such methods can be used to consistently produce radioiodinated biologically active preparations of BPTH 1–84 with high specific activity (2000 Ci/mmol).     

An atrial natriuretic factor-like activity in rat posterior hypophysis

An atrial natriuretic factor-(ANF) like immunoreactivity (IR-ANF), is present in the posterior hypophysis of the rat. In order to obtain more direct information on the presence and biological activity of this new posterior hypophysis peptide, we applied a procedure similar to that described for rat atria, to extract an ANF-like material from the posterior hypophysis of the rat. An analysis of the tissue extracts by reverse-phase high performance liquid chromatography (RP-HPLC) suggested that, in this organ, the ANF-like peptides may be present in multiple forms: a low molecular weight peptide which had a RP-HPLC pattern similar to that of the synthetic rat 28 amino acid C-terminal (Ser 99-Tyr 126) ANF, and an unidentified higher molecular weight peptide. The partially purified low molecular weight peptide was found to have a potency similar to that of synthetic rat ANF in the inhibition of adrenocorticotropin-stimulated aldosterone secretion in dispersed zona glomerulosa cells, suggesting that the ANF-like peptide was biologically active. Immunohistochemical visualization of the ANF-like peptides revealed the distribution of the peptide within the posterior hypophysis. There was no immunohistochemical staining for ANF in the intermediate lobe. These results suggest the existence of biologically active ANF-like peptides within the posterior hypophysis of the rat. It is possible that these peptides may modulate locally the posterior hypophysis hormone secretion.     

人修饰型降钙素和鼠酰胺化酶基因在昆虫细胞中的偶联表达

人降钙素 (hCT)是 32氨基酸的多肽激素 ,C-端为α脯氨酰胺结构 ,具有调节体内钙、磷代谢等许多重要生理功能。用重组昆虫杆状病毒表达系统 ,偶联表达合成的人修饰型降钙素 (hmCT)基因与GST融合基因和大鼠酰胺化酶 (PAM)基因 ,再用抗hmCT或抗PAM抗体 ,既检测到由昆虫细胞表达的GSThmCT产物也检测到PAM产物。经GSH 琼脂糖凝胶亲和层析 ,分离纯化GSThmCT融合蛋白。这种蛋白修饰酶与底物在真核细胞偶联表达也将适用于其他生物活性肽的体外表达     

Anti-insulin-like (diabetogenic) peptides in pituitary extracts: role of oxytocin

A peptide has been extracted and characterized from whole bovine pituitaries that has anti-insulin-like activities when assayed in rat adipocytes. This peptide has been purified approximately 100,000-fold, is homogeneous by thin-layer chromatography in three separate solvent systems, and shows a single peak by reverse-phase high-performance liquid chromatography. By these chemical criteria, as well as biological activity criteria (14CO2 production from D-[U-14C]glucose and D-[U-14C]glucose incorporation into glycogen in rat adipocytes], the peptide is indistinguishable from oxytocin. It reacts with anti-oxytocin antibody, and has an amino acid composition indistinguishable from purified oxytocin. The relationship between this material and other previously described anti-insulin or diabetogenic peptides is discussed, but it was not possible to conclude that this peptide, which has been purified to homogeneity and constant specific activity, is related to these previously described factors.     

The role of the tryptophyl residue in the hypotensive peptide xenopsin

The role of the Trp⁶ residue in the biological activity of the hypotensive peptide xenopsin (<Glu-Gly-Lys-Arg-Pro-Trp-Ile-Leu-OH) was investigated. This residue was satisfactorily reduced to 2,3-dihydro-Trp on treatment with excess pyridine-borane in trifluoroacetic acid without any detectable change in other parts of the molecule. The analogous peptide, (Lys², Gly³) xenopsin, was also reduced in a similar manner. Both reduction products were purified by gel filtration and characterized by UV absorption, amino acid composition, and structural analysis.The reduced peptides were assayed on the fundus strip of isolated rat stomach and were found to possess less than 1 percent of the activity of the original peptides. Although each of the reduced analogs had an indoline substituted for an indole in the tryptophyl residue, their biological activity was virtually lost. This suggests that the tryptophyl residue of xenopsin is crucial for its biological activity.     

Phospholamban: dissociation of the 22,000 molecular weight protein of cardiac sarcoplasmic reticulum into 11,000 and 5,500 molecular weight forms

Structural characterization of a 40 amino acid peptide with high intrinsic growth hormone releasing activity isolated from a human pancreatic tumor which had caused acromegaly was accomplished by gas phase sequence analyses of the intact peptide and its carboxy terminal cyanogen bromide digestion fragment. High pressure liquid chromatography of the native peptide and synthetic replicates showed that the molecule possessed a free acid rather than an amidated carboxy terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly- Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-OH using 1.8 nmoles of material. The structural identity of this material with a previously characterized fragment of a larger growth hormone releasing peptide isolated from a different human tumor is discussed.     

Bioactive atrial natriuretic factor-like peptides in rat anterior pituitary

The presence of biologically active atrial natriuretic factor (ANF)-like peptides was demonstrated in rat anterior pituitary. ANF-like immunoreactivity was detected in rat anterior pituitary by specific radioimmunoassay and was extracted from rat anterior pituitary homogenates by heat-activated Vycor glass beads; extracts were purified by reverse-phase high performance liquid chromatography. Two peaks containing ANF immunoreactive material were obtained. The first peak was eluted from the C18 mu Bondapak column at a position similar to the 28-amino acid carboxy terminal peptide (Ser99-Tyr126)-ANF of prohormone. The second peak had the same pattern of elution as the 126-amino acid prohormone, (Asn1-Tyr126)-ANF. The biological activity of the smaller molecular weight peptide (28 amino acid) was assessed by its inhibitory effect on 10(-8) M ACTH-stimulated aldosterone secretion in rat zona glomerulosa cell suspension. This ANF-like material also displaced I125-labelled ANF from rat glomerular receptors with a potency similar to synthetic (Arg101-Tyr126)-ANF. Immunocytochemical localization revealed a distribution of ANF-stained cells similar in pattern and location to that of gonadotrophs. These results suggest the existence of biologically active ANF-like peptides and ANF prohormone within the anterior pituitary. However, their role remains to be elucidated.     

The importance of the C-terminal amide structure of rat pancreastatin to inhibit pancreatic exocrine secretion

A C-terminal fragment of rat pancreatatin, a 26 residue peptide amide and a fragment without a C-terminal amide were synthesized by Fmoc-based solid phase methods and their biological activities were compared. The rat C-terminal fragment inhibited pancreatic exocrine secretions produced by the intravenous injection of 2-deoxy-D-glucose (a central vagal nerve stimulation), whereas the fragment without a C-terminal amide showed no effect on pancreas. These results indicate that the C-terminal amide of this peptide is necessary to reveal its biological activity.     

Characterization of N alpha-acetyl methionyl human growth hormone formed during expression in Saccharomyces cerevisiae with liquid chromatography and mass spectrometry

We found a new variant of human growth hormone (hGH) from the recombinant hGH expression process in Saccharomyces cerevisiae. The variant was identified as N(alpha)-acetyl methionyl hGH which may be formed by N(alpha)-acetylation of met-hGH during the intracellular expression of hGH in S. cerevisiae. The variant was isolated from manufacturing process of LG Life Sciences' hGH product. The variant was subjected to trypsin digestion and RP-HPLC analysis, resulting in a delayed retention time and an increased mass (173 Da) of T1 tryptic peptide. The amino acid composition and amino acid sequence of the peptide showed the same result with T1 peptide of met-hGH except the N-terminal modification on methionine in the variant peptide. With collision induced dissociation (CID) experiments of the variant T1 tryptic peptide, we found the sequence and the a(1) fragment of N-terminal residue matched with those of acetyl-methionyl hGH. Within our production process, we produce the methionyl hGH first and then use the aminopeptidase to cut the N-terminal methionine. So the acetylation may inhibit the aminopeptidase to remove methionine and produces N(alpha)-acetyl methionyl hGH. And the biological activity of the variant was comparable to one of the unmodified hGH when tested by rat weight gain bioassay.     

Rapid assay for in vitro degradation of luteinizing hormone releasing hormone

A radiochemical method for measuring luteinizing hormone releasing hormone (LHRH) degrading enzymatic activity in vitro was developed using LHRH labeled at the N-terminal 5-pyrrolidone-2-carboxylic acid (<Glu) residue. The intact labeled peptide is separated from the labeled fragments formed by cleavage by a cation-exchange batchwise procedure. The assay reflects the degradation of LHRH specifically in terms of inactivation of hormonal activity, is more rapid than a radioimmunoassay, is independent of LHRH concentration, and is not influenced by high protein concentrations. It can be used for studying the degradation of LHRH by subcellular fractions and enzymes. With this assay a highly active enzymatic degradation system was detected in the rat ovary, a recently discovered target organ for LHRH.     

Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of des-arginine 166 erythropoietin

Recombinant human erythropoietin (rhEPO) has been purified to apparent homogeneity from a Chinese hamster ovary cell line expressing a cDNA clone of the human gene. NH2-terminal sequencing of the recombinant hormone indicates that the 27-residue leader peptide is correctly and consistently cleaved during secretion of the recombinant protein into conditioned medium, yielding the mature NH2 terminus (Ala-Pro-Pro-Arg...). Analysis of the COOH terminus of rhEPO by peptide mapping and fast atom bombardment mass spectrometry (FABMS) demonstrates that the arginyl residue predicted to be at the COOH terminus (based on confirmation of both genomic and cDNA sequences) is completely missing from the purified protein. The truncated form of the recombinant hormone, designated des-Arg166 rhEPO, displays an in vivo specific activity of greater than 200,000 units/mg protein. Structural characterization of natural human urinary EPO (uEPO) by peptide mapping and FABMS reveals that the urinary hormone is also missing the COOH-terminal Arg166 amino acid residue, a modification that remained undetected until now. There is no evidence of further proteolytic processing at the COOH terminus beyond specific removal of the Arg166 amino acid residue in either rhEPO or uEPO. On the basis of the FABMS data, we propose that the physiologically active form of the hormone circulating in plasma and interacting with target cells in vivo is des-Arg166 EPO.     

A lymphocyte chemotactic peptide released from immunoglobulin G by neutrophil neutral thiol protease.

A thermostable and dialyzable peptide, released from rabbit IgG by rabbit neutrophil neutral thiol protease, exhibited a distinct chemotactic activity for rat lymphocytes; it was assumed to be derived from the Fc fragment (but not from the Fab fragment) by the enzyme. This substance seemed to be effective for adherent cells (B cells) from rat spleen, but not for nonadherent cells (T cells). The chemotactic peptide was purified by gel filtration on Sephadex G-50 and G-15 and then by high-voltage paper electrophoresis. As previously described, the IgG residue after release of dialyzable peptide(s) exhibited chemotactic activity for neutrophils but not for macrophages.     

Aurintricarboxilic acid as a tool for investigating the template-bound and unbound forms of RNA polymerase I in permeabilized cells

Several polypeptide hormones of apparently diverse structure and function have a number of similarities which suggest that there may be common features in their mechanism of action. These hormones are all composed of a single linear sequence of about 30 amino acids; their hydrophobic amino acids are regularly spaced at every third or fourth amino acid residue, allowing them to form amphipathic structures which can interact with phospholipids; a fragment at or near their N-terminus is required for biological activity. These hormones include glucagon, beta-endorphin, parathyroid hormone and calcitonin. A model is proposed in which all regions of the hormone bind to the receptor with comparable affinity except for a small segment which, when intact, triggers a conformational change in the receptor resulting in a further stabilization of the hormone-receptor complex. The activity of partial sequences and chemically modified forms of beta-endorphin, parathyroid hormone and glucagon are discussed in relation to this model.     

Purification and characterization of the active fragment from Bacillus thuringiensis delta-toxin

Limited tryptic hydrolysis of a partially purified delta-toxin (Mr = 100,000) from Bacillus thuringiensis, has produced a polypeptide fragment of Mr = 60,000 containing the full biological activity. The fragment was the only polypeptide observed in the polyacrylamide-gel electrophoresis of the delta-toxin after treatment with trypsin and could be purified by DEAE-cellulose chromatography. Amino acid and partial sequence analyses indicate that the 60,000 Mr fragment has been derived from the mid-section of the holotoxin peptide; over 80% of Lys, 65% of Pro and 50% of His residues in the holotoxin have been lost in the active fragment. This section must contain the active site since its specific insecticidal activity is approximately twice that of the holotoxin. The active fragment shows complete cross-reactivity with the antiserum raised against the native toxin, and appeared to possess higher thermal stability than the mother protein. It provides a powerful tool for studies of the structure involved in the insecticidal activity.     

Synthesis of truncated analogues of preptin-(1–16), and investigation of their ability to stimulate osteoblast proliferation

Preptin, a 34-amino acid residue peptide hormone is co-secreted with insulin from the β-pancreatic cells and is active in fuel metabolism. We have previously established that a shorter fragment of preptin, namely preptin-(1–16), stimulates bone growth by proliferation and increasing the survival rate of osteoblasts. This was demonstrated in both in vitro and in vivo models. These findings suggest that preptin-(1–16) could play an important role in the anabolic therapy of osteoporosis. However, due to the large size of the peptide it is not an ideal therapeutic agent. The aim of this study was to identify the shortest preptin analogue that retains or even increases the bone anabolic activity as compared to the parent preptin-(1–16) peptide. Truncations were made in a methodical manner from both the N-terminus and the C-terminus of the peptide, and the effect of these deletions on the resulting biological activity was assessed. In order to improve the enzymatic stability of the shortest yet active analogue identified, ruthenium-catalysed ring closing metathesis was used to generate a macrocyclic peptide using allylglycine residues as handles for ring formation. We have successfully identified a short 8-amino acid preptin (1–8) fragment that retains an anabolic effect on the proliferation of primary rat osteoblasts and enhances bone nodule formation. Preptin (1–8) is a useful lead compound for the development of orally active therapeutics for the treatment of osteoporosis.     

Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue

Ghrelin, a novel peptide purified from stomach, is the endogenous ligand for the growth hormone secretagogue receptor and has potent growth hormone-releasing activity. The Ser3 residue of ghrelin is modified by n-octanoic acid, a modification necessary for hormonal activity. We established two ghrelin-specific radioimmunoassays; one recognizes the octanoyl-modified portion and another the C-terminal portion of ghrelin. Using these radioimmunoassay systems, we found that two major molecular forms exist-ghrelin and des-n-octanoyl ghrelin. While ghrelin activates growth-hormone secretagogue (GHS) receptor-expressing cells, the nonmodified des-n-octanyl form of ghrelin, designated as des-acyl ghrelin, does not. In addition to these findings, our radioimmunoassay systems also revealed high concentrations of ghrelin in the stomach and small intestine.     

21-arginine-A] insulin: a biologically active analog.

An analog of sheep insulin which differs from the parent molecule in that the C-terminal amino acid residue of the A chain, asparagine, is replaced by arginine, has been synthesized and isolated in highly purified form. The [Arg21] A chain of sheep insulin was synthesized by the fragment condensation approach and isolated as the S-sulfonated derivative. Conversion of the latter into the sulfhydryl form and interaction with the S-sulfonated B chain of bovine (sheep) insulin yielded [Arg21-A] sheep insulin, which was purified by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. The [Arg21-A] sheep insulin shows potencies of 10.5--12.5 IU/mg when assayed by the mouse convulsion method and 8.6 IU/mg by the radioimmunoassay method (cf. 23--25 IU/mg for the natural hormone). It has been suggested that in the insulin molecule the A21 asparagine participates in salt bridge- and hydrogen bond-forming interactions which are critical in the biological activity of the hormone. Although the [Arg21-A] analog still retains these interactions, it is only ca. 50% as active as the natural hormone. It is speculated that other factors than the above mentioned interactions come into play, which involve the side chain of the A21 amino acid residue and affect the biological activity of the hormone.     

Silkworm diapause hormone, structure—activity relationships indispensable role of C-terminal amide

To determine the structure—activity relationships of the silkworm diapause hormone, a series of peptide analogs having different chain lengths starting from the parent C-terminus and analogs having identical sequences with free acid C-termini were chemically synthesized by solid-phase Fmoc methodology and were further purified by HPLC. Bioassay showed that the analogs with free acid C-termini were non active. The retained activities of those shorter chains were shown only with amidated C-terminal analogs among which the potency depended on the length of the chain. The active peptides required two minimal elements; namely the sequence near and the amidation of the C-terminus. There was no difference in enzymatic digestion of the C-terminally amidated or free acid analogs in pupal haemolymph. Hence the absence of DH activity of the free acid analogs was not because of being selectively hydrolyzed faster than the C-terminally amidated peptides. This suggested that existence of a certain higher order structure could be involved in expressing hormonal activity, or that the negative charge of the free acid terminus may be deleterious to a proper ligand receptor interaction. Since most of the hydrophobic amino acids were located near the C-terminal portion, both the hydrophobicity of the portion near and the amidation of the C-terminus were indispensable structures for diapause hormone activity.     

Glycine-extended anglerfish peptide YG (aPY) a neuropeptide Y (NPY) homologue may be a precursor of a biologically active peptide

The 37 residue peptide YG (aPY), isolated from anglerfish endocrine pancreas, bears distinct sequence homology to the pancreatic polypeptide family of hormones. However, instead of a carboxyl-terminal tyrosine-amide, aPY has a free carboxyl-terminus ending with glycine. Towards studying the structure-activity relationship of this hormone, we have synthesized aPY by solid phase methodology using Boc-amino acid derivatives and phenylacetamidomethyl resin. The crude peptide was purified to homogeneity in 20% yield by reversed phase chromatography. The purified peptide had the expected amino acid composition and sequence, and was found to be identical with the natural aPY by analytical HPLC and peptide mapping of proteolytic digests. Neither the snythetic nor the natural aPY exhibited the characteristic vasoconstrictor activity of the related pancreatic polypeptide family of hormones. However, [Des37-Gly]-aPY, isolated from the anglerfish pancreas, caused vasoconstriction in rats. Based on these results and by analogy to the glycine-extended gastrin peptides, it may be suggested that aPY is a precursor of a biologically active peptide, namely [Des37-Gly]-aPY-amide.