Identification and purification of two precursors of the insect neuropeptide adipokinetic hormone

Abstract

Our objective was to establish a system for the investigation of neuropeptide biosynthesis in an insect. To achieve this we developed an in vitro organ culture system for the corpora cardiaca (CC) of the locust (Schistocerca gregaria). The CC are the neurosecretory structures containing the adipokinetic hormones AKH I and AKH II. Tritiated amino acids were added to the glands, and we studied the development of label in newly made proteins using size-exclusion and reverse-phase liquid chromatography. We performed pulse-chase and pulse- translation block experiments using 3H-tryptophan as tracer. We also raised an antiserum to a synthetic AKH analog and used this in combination with liquid chromatography to identify 2 precursor polypeptides, P1 and P2. Size-exclusion chromatography indicated the presence of a major component of the CC of about 8 kDa that incorporates 3H-tryptophan before AKH I and II. Moreover, in both pulse- chase and pulse-translation block experiments we showed that label is transferred from this 8 kDa component into AKH I. We call this component proAKH; it is recognized by anti-AKH serum in a radioimmunoassay (RIA). Further fractionation of tritium-labeled proAKH by reverse-phase chromatography yielded 2 polypeptides, P1 and P2. Both are AKH-immunoreactive and contain 3H-tryptophan after in vitro pulse labeling, and both are proposed precursors of AKH I. The in vitro system we have developed may be a model system for the study of the processes of neuropeptide biosynthesis and its regulation in an intact neurosecretory tissue of an insect.