Insulin-like growth factor I (IGF-I) and insulin stem from a common precursor, are structural homologues, act through similar receptors and elicit insulin-like and growth-promoting effects in vitro and in vivo. Serum IGF-I levels are controlled by growth hormone, insulin and nutrition. Insulin-deficient growth-arrested diabetic animals have reduced serum IGF-I levels which are restored towards normal by insulin but not by growth-hormone treatment. Here we show that normal growth of diabetic rate is restored by infusion of recombinant human (rh)IGF-I without normalization of the blood sugar level and that insulin acts via an increase of IGF-I synthesis on growth of diabetic rats. We describe a new mechanism of endocrine control of growth in which IGF-I is the major stimulator at the cellular level. Growth hormone and insulin act mainly by modulating the hepatic synthesis of IGF-I.

Natural human insulin-like growth factor (IGF) I is a relatively large single chain peptide (mol. wt 7649) with a known sequence of 70 amino acids. C6----C48, C47----C52 and C18----C61 assignments have been previously proposed for the three disulphide bonds linking six cysteine residues (C6, C18, C47, C48, C52 and C61), on the basis of analogy (and homology) with proinsulin. In this work, IGF I synthesized by recombinant DNA technology (r-IGF I, with identical biological activity and chromatographic behaviour) was subjected to a three-step mass spectrometric analysis in combination with degradation methods for structural verification. Firstly, the correct molecular weight of the intact peptide was determined by high-mass fast atom bombardment (FAB) analysis. Secondly, twofold enzymatic degradation (chymotrypsin followed by V8 protease, 'FAB mapping' of the cleavage products) was employed in order that fragments with 'isolated' S-S bonds would be produced which allow an unambiguous assignment. This immediately established the C18----C61 linkage as it was contained in a singly bridged two-chain peptide. The two other S-S bonds, which cross-link C6 and the 'tight' C47 to C52 segment, remained 'unresolved' within a more complex, doubly bridged triple-chain peptide. Thirdly, further degradation of this structural block, in which cleavage of the C47-C48 bond was required to discern these bonds, was carried out by using FAB tandem mass spectrometry and (for additional corroboration) manual Edman degradation. Both procedures confirmed the original C6----C48/C47----C52 prediction.(ABSTRACT TRUNCATED AT 250 WORDS)

Recently, gastrin releasing peptide (GRP), Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-TrpAla-Val-Gly-His-Leu-Met-NH2, a mammalian bombesin, was isolated from porcine gastric mucosa and sequenced by McDonald et aL9 This polypeptide was manually synthesized by solid-phase methodology, using a benzhydrylamine-styrene-1% divinylbenzene copolymer. Deprotection and cleavage from the resin were accomplished by HF.The crude peptide was purified by gel filtration and reverse-phase, high-performance liquid chromatography (RP-HPLC). Homogeneity of the synthetic peptide was demonstrated by RP-HPLC, sequence analysis, peptide mapping, and amino acid analysis. The peptide was further characterized by thin-layer chromatography, paper electrophoresis, optical rotation, ultraviolet spectroscopy, and 300-MHz Fourier transform proton nuclear magnetic resonance spectroscopy. The circular dichroism spectra of GRP indicated that the polypeptide chain was largely random with no evidence for a-helical structure. The primary structure was confirmed by amino acid analysis of the tryptic peptide fragments, sequence analysis of GRP and its Met(0) derivative using a modified 890 C spinning-cup sequencer, and C-terminal end group determination. GRP released gastrin when administered systemically and decreased gastric acid secretion when given intracisternally in rats. GRP also mimicked CNS-mediated actions of bombesin to influence thermoregulation or glucoregulation, most likely because of the common C-terminal homology of these peptides. This assumption was supported by the observation that the synthetic acetylated octapeptide [Ac-HisZ0]-GRP (20-27) showed pharmacological effects similar to those exhibited by GRP and amphibian bombesin.Bombesin, a tetradecapeptide first isolated and characterized from the skin of the frog Bombina bombina2 has been shown in mammals to influence various neural and visceral functions. Acting through the central nervous system, bombesin is a potent stimulus to disrupt therm~regulation,~ to stimulate adrenal epinephrine ~e c r e t i o n ,~ which results in lowering of plasma insulin and elevation of plasma glucagon and glucose? and to suppress gastric acid secretiom6 Bombesin when injected peripherally also stimulates gastrin release, gall bladder contraction, antidiuresis, and intestinal motor activity.' It is now recognized on the basis of immunologic data that bombesin has counterparts in mammalian brain and gastrointestinal tissues.* McDonald et a1.9 have recently isolated and sequenced a porcine intestinal, 27 amino acid containing peptide, termed gastrin releasing peptide (GRP), which stimulates the release of gastrin (1) The conventions and nomenclature used are those of the IUPAC-IUB Commission on Biochemical Nomenclature ("Collected Tentative Rules and Recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature", 2nd ed.; American Rivier, J.; Brown, M. hoc. Natl. Acad. Sci.(7) Enpamer, V.; Melchiorri, P.; Erspamer, G. F.; Negri, L. "Gastrointestinal Hormon...

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A simple process of in vitro folding has been developed for the preparation of hirudin dimer. A variant of recombinant hirudin with Asp33 replaced by Cys was expressed in yeast and isolated by HPLC. Crude Cys"-hirudin contains heterogeneous products that are made of one species of primary sequence. They were together reduced/denatured, and allowed to re-fold in the sodium bicarbonate buffer @H 8.3) alone. Active, homogeneous Cys33-hirudin monomer folded spontaneously with a first order rate constant of0.05 f 0.01 mm-', followed by the oxidation of two Cys3' to produce the pure dimer. The folding yield was 90%. On an equal weight basis, both Cys33-hirudin monomer and the dimer exhibit thrombin inhibitory activity comparable to that of wild-type hirudin. Due to the presence of an extra cysteine, the folding of active hirudin monomer (formation of three native disulfides) was accelerated by at least 1Zfold.Hirudin dimer; Protein folding; Disulfide-linked hirudin dimerization