Two-dimensional gel electrophoresis of neurosecretory polypeptides in crustacean eyestalk

Summary A knowledge of the precise location of neurosecretory cell bodies is a prerequisite for studying the synthesis and subsequent processing of neurosecretory polypeptides stored in axon terminals comprising the sinus gland of the crustacean eyestalk. Structural data establish that the X organ in the medulla terminalis ganglion (mtXo) of the crayfish eyestalk represents 90–95% of the cell bodies actively synthesizing neurosecretory vesicles stored in the neurohemal sinus gland (Fig. 4). These cell bodies transport rather than accumulate neurosecretory vesicles as judged by light and electron microscopy suggesting that neurohormone precursors, but not subsequently stored products, might be found there. Two-dimensional electrophoresis of sinus gland and mtXo homogenates support this hypothesis. In crayfish, lobster and blue crab, stained two-dimensional gels display a number of sinus gland-specific polypeptides whose high concentrations and low molecular weights are consistent with stored neurosecretory material (Table 1). These neuropeptides are not detected in mtXo homogenates or in non-neurosecretory neural tissue with Coomassie Blue staining. By decreasing the porosity of the second dimension, the two-dimensional gel technique has proven useful in determining the molecular weights of a variety of neurosecretory polypeptides stored in the sinus gland. The crayfish and lobster store several polypeptides of ca. 7,000 Dalton. The blue crab stores two 7,000, two 13,000 and three 20,000 Dalton sinus gland polypeptides detected in stained gels.Following a 4 h incubation in³H-labelled amino acids, predominantly labelled 19,000–21,000 Dalton polypeptides are detected in crayfish mtXo homogenates by 2-D gel autoradiography (Fig. 12). Concomitantly, three labelled polypeptides (4,000–10,000 Dalton) appear in the sinus gland (Fig. 13), suggesting that they are cleaved from 19,000–21,000 Dalton molecules. This study is the first to examine neurosecretory precursors and their putative cleavage products in the Crustacea.Abbreviations mtXo medulla terminalis X organ - NEPHGE non-equilibrium pH gradient electrophoresis - PAF paraldehyde fuchsin - SDS sodium dodecylsulfate