Purification, characterization and radioimmunoassay of thyrocalcitonin from rat thyroid glands.

Abstract

A highly purified preparation of rat thyrocalcitonin (TCT) has been obtained from lyophilized thyroid glands by gel chromatography following acid-acetone extraction. Biological activity of Sephadex G-50 eluates appeared in two peaks. The TCT in the major peak was concentrated, and applied to a Bio-Gel P-6 column, and a major protein peak was eluted which coincided with TCT activity. Potency, estimated by bioassay in rats, increase approximately 3500-fold from 0.075 MRC U/mg lyophilized glands to 250-400 MRC U/mg in the final product. The overall yield of TCT activity was about 36%. The purified product was characterized by chemical procedures and evaluated for its antigenic properties and use for radioimmunoassay. The purified rat TCT was used both labeled with 125I and as unlabeled standard. The following results were obtained: 1) Guinea pig antisera to either human or rat TCT were capable of binding 125I-rat TCT or 125I-human TCT; 2) Using either 125I-human or 125I-rat TCT and antisera to either TCT, pg amounts of rat and human TCT reacted in the assay while ng to mug amounts of salmon calcitonin or porcine TCT failed to react; 3) Using 125I-rat TCT and antisera to human or rat TCT, synthetic C-terminal (10-32 or 22-32) fragments of human TCT reacted well, while N-terminal (1-18) or desamide (1-32) derivatives reacted poorly or not at all; 4) Rat TCT was easily detected in normal thyroid venous plasma (5-10 ng/ml) and thyroid gland extracts (similar to 1 mug/gland) but not in peripheral blood; 5) Bioassay and radioimmunoassay of rat thyroid extracts (N equals 18) showed good agreement (r equals 0.86, p less than 0.001). The results support the idea that rat TCT is closely related to human TCT, indicate that major antigenic determinants reside in the C-terminal portion of the molecule, and show that antisera to either human or rat TCT can be used to measure rat TCT.