Use of Receptor Affinity Chromatography in Purification of the Growth Hormone-Like Factor Produced by Plerocercoids of the Tapeworm Spirometra Mansonoides

Abstract

The plerocercoid stage of the tapeworm Spirometra mansonoides produces a functional analog of human growth hormone (hGH). Among the similarities between plerocercoid growth factor (PGF) and hGH is competition for the same receptors on rabbit liver membranes. To take advantage of this characteristic in a purification scheme for PGF, rabbit liver microsomes were solubilized in Triton X-100 and the hGH receptors were purified over an hGH affinity column. The purified receptors from six rabbit livers were coupled to Affi-Gel-10 to create a receptor affinity column which was used to purify PGF. Chromatography of crude PGF over the receptor column resulted in a 1044 fold increase in specific activity. SDS-PAGE in the presence of 2-mercaptoethanol showed that the affinity-purified PGF contained three protein bands with apparent Mrs of 27.5 K, 22 K, and 16.7 K. Injections of the partially-purified PGF into hypophysectomized rats produced a dose-dependent growth response and 400 ng eq of PGF each day for 10 days stimulated a growth response not significantly different from that produced by 250 micrograms of bovine GH each day. Receptor affinity chromatography was an effective method to purify small amounts of PGF in a single step with negligible loss of biological activity.

The Growth Factor from Plerocercoid Larvae of the Tapeworm, Spirometra mansonoides, Stimulates Growth But Is Not Diabetogenic

“…This seems unlikely as it is clear from our current results and earlier reports that regardless of whether PGF is administered via plerocercoid infection ( 1 1, 12,22) or by injection of partially purified material (14, 25,26), both growth-stimulating and insulin-like activities are expressed. This seems unlikely as it is clear from our current results and earlier reports that regardless of whether PGF is administered via plerocercoid infection ( 1 1, 12,22) or by injection of partially purified material (14, 25,26), both growth-stimulating and insulin-like activities are expressed.…”

“…Furthermore, in support of the effective dosage used on mice, previous studies show that doses of PGF as low as 50 ng eq/day stimulated significant increases in body growth of hypophysectomized rats (25,26) and induced acute and chronic insulin-like effects in adipose tissue and skeletal muscle of normal and hypophysectomized rats (14). The maximum PGF dose used (200 ng eq/day) was eight times the absolute amount required to stimulate a dramatic and unequivocal body growth response in normal mice, but was not diabetogenic.…”

“…All stages of the life cycle of S. mansonoides are maintained in our laboratory based on methods described by Mueller (24). Previous studies involving in vitro metabolic assays ( 13,14) and body weight gain assays (25,26) indicate that the effective dose of PGF is not dependent on its purity (specific activity), but rather on the total activity used. Previous studies involving in vitro metabolic assays ( 13,14) and body weight gain assays (25,26) indicate that the effective dose of PGF is not dependent on its purity (specific activity), but rather on the total activity used.…”

“…For the current studies, PGF solubilized from purified plerocercoid membranes (25) was used without further purification. The quantity of PGF was expressed as the binding activity in nanogram equivalents (ng eq) of the hGH standard (25). Quantitation of PGF was based on a radioreceptor assay using liver membranes from late pregnant rabbits, '*'I-labeled hGH and hGH (NIH, hGH-1-1 2.2 IU/mg) as the standard ( 15).…”

In Vitro Insulin-Like Actions of the Growth Factor from the Tapeworm, Spirometra mansonoides

“…Plerocercoids were removed from mice, homogenized, and a membrane fraction was separated and solubilized in the nonionic detergent Triton X-100 (Sigma Chemical Co., St. Louis, MO) as described previously (2). Plerocercoids were removed from mice, homogenized, and a membrane fraction was separated and solubilized in the nonionic detergent Triton X-100 (Sigma Chemical Co., St. Louis, MO) as described previously (2).…”