Journal of Neuroscience, Vol 7, 2773-2784, Copyright © 1987 by Society for Neuroscience
Identification and purification of two precursors of the insect neuropeptide adipokinetic hormone
S Hekimi and M O'Shea
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.