Amino acid sequence of a prothoracicotropic hormone of the silkworm Bombyx mori

Abstract

We have determined the complete amino acid sequence of 4K-PTTH-H, one of three forms of the Mr 4400 prothoracicotropic hormone of the silkworm Bombyx mori, active to brainless pupae of Samia cynthia ricini. Like vertebrate insulin, it consists of two nonidentical peptide chains (A and B chains). The A chain consists of 20 amino acid residues. The B chain is a mixture of four microheterogeneous peptides, two of which consist of 28 residues, and the other two, of 26 residues. 4K-PTTH-II has considerable sequence homology (40%) with human insulin, and it resembles porcine relaxin both in the carboxyl-terminal cysteine residue of the A chain and in the amino-terminal pyroglutamic acid residue of the B chain. The identical distribution ofthe six cysteine residues also indicates that 4K-PTTH-ll belongs to the insulin family. Carboxypeptidase A Digestion. A suspension of carboxypeptidase A (treated with diisopropyl fluorophosphate, Sigma) in water was centrifuged at 1000 x g for 5 min and the supernatant was removed to eliminate free amino acids. The precipitated carboxypeptidase A was dissolved in 0.1 M ammonium acetate (pH 9.0) at a concentration of 1 ,g/,u and 1 ,ul of this solution was added to 19,ul of the same buffer containing 4K-PTTH-II (0.7 nmol), DA (1.1 nmol), DB1 (0.6 nmol), or DB2 (1.0 nmol). The digestions were performed at 37°C. Aliquots (5 ,ul each) were taken at 0.5, 1, 2, and 4 hr after the initiation of incubation, and the released amino acids were quantified by RP-HPLC (Senshu Pak SEQ-4; Senshu Kagaku, Tokyo) after derivatization to phenylthiocarbamoyl amino acids (7).Pyroglutamate Aminopeptidase Digestion. Tryptic peptides T2 (1.8 nmol) and T3 (5.0 nmol), and DB2 (0.5 nmol) were dissolved in 10 ,ul of 0.1 M phosphate buffer (pH 7.5) with 30 mM dithiothreitol. To these solutions, 4 ,ug of pyroglutamate aminopeptidase (Boehringer Mannheim) in 10 Al of the same buffer was added. After incubation at 35°C for 1 hr, the mixture was subjected to RP-HPLC on a TSK ODS-120T column developed with a gradient of 5-22.5% acetonitrile in 0.1% trifluoroacetic acid.Chymotrypsin Digestion. DB1 (3.0 nmol) and DB2 (7.0 nmol) were dissolved in 20 ,ul of 0.1 M Tris'HCl buffer (pH 7.8). To these solutions, 0.5 jig of a-chymotrypsin (Sigma) in 1 ,ul of 1 mM HCO was added. After incubation at 25°C for 3 hr, the resulting peptides were separated by RP-HPLC on a Develosil 5-ODS column with a linear gradient of 5-40% acetonitrile in 10 mM ammonium acetate.Amino Acid Analyses. Peptides (30-200 pmol) were hydrolyzed in constant-boiling HCO (Pierce) at 110°C for 20 hr.Abbreviations: PTTH, prothoracicotropic hormone; RP-HPLC, reversed-phase high-performance liquid chromatography. 5840The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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cDNA structure of an insulin-related peptide in the Pacific oyster and seasonal changes in the gene expression

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