Purification and characteristics of the chloride transport stimulating factor from locust corpora cardiaca: a new peptide

Abstract

Corpora cardiaca (CC), cAMP, and hemolymph all increase short-circuit current (Isc) and electropotential difference (PD) across locust rectum by stimulating electrogenic transport of Cl− from the lumen. Using ΔIsc as a bioassay, we have purified the water-soluble stimulant (CTSH) from CC using gel filtration chromatography, DEAE-Sephadex anion exchange, cellulose acetate electrophoresis, and thin-layer chromatography. A single peak of CTSH activity was observed after all these procedures, although small amounts of CTSH activity occasionally remained in the high molecular weight (MW) protein precipitate. CTSH was purified more than 100-fold on Bio-Gel P-30 columns. It has a MW of 8 000 – 12 000, is destroyed by trypsin digestion, and has a net negative charge over the pH range (5–10) at which it is most stable. Various properties (i.e., stability at 20 °C, localization in CC, MW, Rf values) and reciprocal bioassay s indicate that CTSH is different from diuretic, antidiuretic, and adipokinetic hormones from locust CC. No difference in the properties of CTSH from glandular (GL) and storage lobes (SL) of CC were noted, although 80% of activity was in the SL. The concentration of purified CTSH required to cause maximal stimulation of rectal Isc is less than 7 nM.