Insulin-Like Peptides of the Cerebropleural Ganglion of the Mollusc Anodonta cygnea: Isolation, Purification, and Radioligand Analysis

Cerebropleural ganglia from 4000 individuals of the mollusc Anodonta cygnea were submitted to procedures developed for isolation of vertebrate pancreatic insulins: homogenization and extraction, stage-like isoelectrical sedimentation, and ion-exchange chromatography. As a result of purification of the obtained preparation, using high-effective liquid chromatography, there were identified 7 protein peaks differing by time of retention on the reverse-phase sorbent in acetonitryl gradient and designated as insulin-related peptides (IRP), IRP1-IRP7. The material was characterized by the peptide ability to inhibit specific binding of ¹²⁵I-insulin and of insulin-related factor-1 (¹²⁵I-IGF-1) by plasma membranes of the rat liver and brain. The IC₅₀ value of peptide concentration (nM) able to replace 50% of the labeled hormone bound with the receptor amounted in the insulin radioreceptor system for IRP1 to 330, for IRP3 to 130, for IRP4 to 17, for IRP5 to130, for IRP6 to 420 nM. Peptide IRP7 at a maximal concentration (10⁴ ng/ml) replaced less than 50% of labeled hormone, whereas in IRP2 no inhibitory ability was detected under these experimental conditions. The IC₅₀ value in the case of ¹²⁵I-IGF-1 amounted for IRP1, IRP4, and IRP5 to17, for IRP2 to 50, for IRP3 to 83, for IRP6 to 133 nM. IRP7 at a concentration of 10⁴ ng/ml replaced less than 50% of labeled hormone. The same high relative affinity of the peptide IRP4 (12% of activity of standard insulin and IGF-1) to both receptor types is revealed. The results of analysis in two types of hormonal test systems indicate the ability of the insulin-related peptides of the anodonta cerebropleural ganglion to interact with the vertebrate receptor of insulin and IGF-1. This gives grounds to suggest the presence of the metabolic and growth-stimulating properties in these peptides. For the first time, the IGF-1 activity is revealed in insulin-like molecules in invertebrates. Taking into account the chromatographically revealed differences of physicochemical characteristics of individual IRP as well as predominance of their IGF-1-binding properties, there is suggested another organization of the IRP receptor-binding domains in IPR of this mollusc species, as compared with mammalian insulins.