ABSTRACT
An improved RP-HPLC method was developed for the determination of the configuration and stereochemical purity of cysteine residues in peptides. The method consists of oxidation of cysteine and cystine residues to cysteic acid, followed by hydrolysis and pre-column chiral derivatization with Val-Marfey's reagent.
Subject(s)
Chromatography, High Pressure Liquid/methods , Cysteine/analysis , Cysteine/chemistry , Oxytocin/chemistry , Peptide Fragments/chemistry , Cysteic Acid/analysis , Cysteic Acid/chemistry , Fluorescence , Hydrolysis , Isomerism , Molecular Conformation , Oxidants/metabolism , Oxytocin/analogs & derivatives , Oxytocin/chemical synthesis , Peptide Fragments/chemical synthesis , o-Phthalaldehyde/chemistryABSTRACT
Small cell lung cancer (SCLC) cell lines produce and secrete various peptide hormones, e.g. bombesin (BN)/gastrin releasing peptide (GRP) like peptides that are proposed to function as their autocrine growth factors. To inhibit the proliferative effect of these hormones we have synthesized short chain BN[7-14]-analogues replacing the C-terminal peptide bond by a methylene-amino (-CH2NH-) unit and introducing D-Phe or D-Ser into position 12. As several substance P (SP) analogues were found to inhibit the growth of SCLC cells, some short chain SP-analogues have been synthesized. (Pseudo)octapeptides were synthesized in solution, by fragment condensation using the DCC/HOPfp method. Fragments and SP-analogues were synthesized stepwise using pentafluorophenyl esters. The resistance to hydrolysis of the reduced peptide bond made permitted exact quantification of the Leupsi(CH2NH)Leu pseudopeptide in hydrolysates. The binding ability of both types of peptides to BN-receptors on Swiss 3T3 mouse fibroblast cells and their antiproliferative effect on NCI-H69 human SCLC cell line have been tested and compared with a short chain SP-antagonist pHOPA-D-Trp-Phe-D-Trp-Leu-Leu-NH2 (R) previously described as a potent inhibitor of SCLC proliferation. While BN-analogues showed weak activity in inhibition of proliferation of SCLC cells, SP-analogues 6: D-MePhe-D-Trp-Phe-D-Trp-Leu(psi)(CH2NH)-Leu-NH2 and 7: D-MePhe-DTrp-Phe-D-Trp-Leu-MPA, in spite of greatly diminished affinity towards the BN-receptor, inhibited SCLC proliferation more effectively than R (6: IC50 = 2 microM, 7: IC50 = 5 microM and R: IC50 = 10 microM). Moreover, 6 inhibited the respiratory activity of SK-MES 1 epithelial type of lung carcinoma cells in proliferating but not in the quiescent state, suggesting that the antiproliferative effect of these compounds is not due to simple cytotoxicity. These short chain analogues of SP might be promising candidates as therapeutic agents in the treatment of SCLC.
Subject(s)
Amides , Carcinoma, Small Cell/pathology , Lung Neoplasms/pathology , Peptides/chemical synthesis , 3T3 Cells , Amino Acid Sequence , Animals , Bombesin/analogs & derivatives , Bombesin/antagonists & inhibitors , Bombesin/chemistry , Bombesin/pharmacology , Cell Division/drug effects , Humans , Hydrolysis , Mice , Molecular Sequence Data , Substance P/analogs & derivatives , Substance P/chemistry , Substance P/pharmacology , Tumor Cells, CulturedABSTRACT
Chromogranin A (CGA) is a ubiquitous 48-kDa secretory protein present in adrenal medulla, anterior pituitary, central and peripheral nervous system, endocrine gut, thyroid, parathyroid, and endocrine pancreas. Recently, we have demonstrated that the protein could be a precursor of bioactive peptides capable of modulating catecholamine secretion from cultured adrenal medullary chromaffin cells. Here we cleaved CGA purified from bovine chromaffin granules with endoproteinase Lys-C, and we isolated and partially sequenced the peptide inhibiting catecholamine secretion from cultured chromaffin cells. A corresponding synthetic peptide composed of the first 20 N-terminal amino acids produced a dose-dependent inhibition in the 10(-9) to 10(-6) M range (with an ID50 of 5 nM) of the catecholamine secretion evoked by carbamoylcholine or by potassium at a depolarizing concentration. This peptide affected secretagogue-induced calcium fluxes but did not alter sodium fluxes. It was found to increase desensitization of cell responses and to modify the kinetics of catecholamine release. Our results indicate that the peptide is extracellularly generated from CGA by a calcium-dependent proteolytic mechanism. We suggest that this peptide, named chromostatin, may be an endocrine modulator of catecholamine-associated responses.
Subject(s)
Adrenal Medulla/metabolism , Chromogranins/pharmacology , Norepinephrine/metabolism , Peptide Fragments/pharmacology , Adrenal Medulla/drug effects , Amino Acid Sequence , Animals , Biological Transport, Active/drug effects , Calcium/metabolism , Carbachol/pharmacology , Cattle , Cells, Cultured , Chromatography, High Pressure Liquid , Chromogranin A , Exocytosis/drug effects , Molecular Sequence Data , Peptide Fragments/isolation & purification , Potassium/pharmacology , Sodium/metabolismABSTRACT
Sodium-liquid ammonia reduction has been used for over 50 years for removal of benzyl-type protecting groups in peptide chemistry. Up until now a definitely blue end-point has generally been accepted for detection of the completion of reaction. Systematic investigation with model compounds has revealed that this is not only unnecessary for the complete removal of the protecting groups but also that the application of sodium in excess results in many undesired transformations which can simply be suppressed or even eliminated by optimizing the sodium consumption. Cleavage of tert.-butyloxycarbonyl group and N-C alpha bond, reduction of carboxamide groups to carbinol derivatives, transpeptidation and formation of a hydantoin derivative have been observed in model experiments by using sodium in excess.
