Studies on the Apparent Human Chorionic Gonadotropin-Like Factor in the CrabOvalipes ocellatus*

Abstract

The presence of hCG-like factors in extracts of the various organs of aquatic organisms was determined by RIA and radioreceptor assay (RRA). Extracts of the stomach and hepatopancreas of the lady crab (Ovalipes ocellatus) demonstrated parallel dose-response curves to hCG (CR-119) in a homologous hCG RIA system and with RRA using bovine corpus luteum membranes. However, there was a significant deviation of the dose-response curves with crab extract and with hCG (CR-119) in homologous hCGβ RIA system. On the other hand, the dose-response curves with crab extract and hCG (CR-119) in the RIA system utilizing antisera against hCGβ COOH-terminal peptide were practically parallel. CG isolated from human placental extract was adsorbed on a Concanavalin A-Sepharose column, while the hCG-like factor isolated from crab stomach extract was not adsorbed, suggesting that the crab factor contains little or no mannose or glucose moieties. On gel filtration through a Sephadex G-100 column, the apparent hCG-like factor present in the crab stomach extract was retarded compared to [125I]iodo-hCG (used as a marker), indicating that the apparent molecular weight of the crab CG-like factor was less than that of hCG. Upon acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the apparent hCG-like material, which was further purified by ion exchange chromatography on a DEAE-Sephadex A-50 column, showed a single major band with a migratory rate corresponding to trypsin, indicating that the molecular weight of the hCG-like factor is identical to that of trypsin. The hCG-like factor in the crab stomach extract showed no biological activity when assayed by the mouse uterine weight method. After incubation of the crab stomach extract with inhibitors of trypsin-like protease, i.e. N-α-tosyl-L-lysine chloromethyl ketone, antipain, leupeptin, ovomucoid, and soybean trypsin inhibitor, the hCG-like activity of the crab material, as measured with RIA and RRA, was reduced drastically. These findings demonstrate that a trypsinlike protease(s) can mimic hCG in the in vitro assay systems and give rise to false positive results.