Biosynthesis and axonal transport of proteins and identified peptide hormones in the X-organ sinus gland neurosecretory system

Abstract

In vitro biosynthesis of neurosecretory peptides specific to the X-organ sinus gland (XOSG) of the land crabCardisoma carnifex was studied. The neurosecretory somata comprising the X-organ were selectively exposed to saline containing3H-leucine, glucose and antibiotics for 5 h (pulse) followed by 19–24 h of superfusion with nutrient media (chase). Analysis of extracts of somata and terminal regions by high voltage paper electrophoresis demonstrated the uptake and incorporation of3H-leucine into protein by X-organ somata and its axonal transport to terminal regions. Tritiated-leucine incorporation was reduced 90% by 50 μg/ml puromycin, indicating ribosome-mediated synthesis. Axonal transport was blocked by ligation of the axons; this did not affect the rate of3H-leucine incorporation by the somata. Radiolabelled peptide hormones were identified by comigration of radiolabel with RPLC and/or SDS-PAGE determined regions of bioactivity and by comparison of RPLC elution patterns with those of sinus gland peptides purified and analyzed for amino acid composition by Newcomb (1983a). Erythrophore concentrating hormone (ECH) and at least two 6,000 Dalton peptides having crustacean hyperglycemic hormone activity (CHH) were identified among peptides biosynthetically radiolabelled. Comparison of XOSG systems receiving 5 h of incubation with those receiving a pulse-chase (5 h∶19 h) regime demonstrates the existence of polypeptide precursors to the XOSG peptide hormones.