A Method for Solubilization of a Human Growth Hormone Analogue from Plerocercoids of Spirometra mansonoides

Abstract

The curious phenomenon of body growth stimulation of rodents infected with plerocercoids of the tapeworm, Spirometra mansonoides, was first reported by Mueller (1963, Ann. N.Y. Acad. Sci. 113: 217-233). Plerocercoids produce a substance which increases somatomedin and body growth (Garland et al., 1971, Endocrinology 88: 924-927) while suppressing endogenous growth hormone (GH) (Garland and Daughaday, 1972, Proc. Soc. expl Biol. Med. 139: 497-499). The unique nature of plerocercoid growth factor (PGF) stimulated interest in its isolation and characterization. We and others (Phares and Ruegamer, 1973, Proc. Soc. expl Biol. Med. 142: 374-377; Chang et al., 1983, Proc. Soc. expl Biol. Med. 143: 457-479) showed that biologically active PGF could be collected by incubating plerocercoids in vitro. Efforts to purify PGF were hampered by the lack of a sensitive assay. This problem was solved after the demonstration that PGF displaces human GH (hGH) from receptors on rabbit liver microsomal membranes (Tsushima et al., 1974, Biochem. biophys. Res. Commun. 59: 1062-1068). These investigators incubated plerocercoids in media fortified with 3% fetal calf serum, changed the media once each week for 6 mo, pooled the collected media, and found the equivalent of 183 ng/ml of hGH. Incubation media without serum contained less activity (24-90 ng/ml). In the present study, PGF activity was determined in a radioreceptor assay (RRA) (Tsushima and Friesen, 1973, J. clin. Endo. Metab. 37: 334-337). Activity of PGF is expressed as ng equivalents (ngE) of a hGH standard (NIADDK-hGH-I-1). Human GH was iodinated by a modification of the chloramine-T method of Hunter and Greenwood (1962, Nature [London] 194: 495-496). After 45 sec the reaction mixture was passed over a 0.9 cm x 30 cm Sephadex G-50 column previously equilibrated in 0.05 M barbital buffer, pH 8.6. This chromatographic procedure stops the reaction and separates the [125I]hGH from unreacted iodide. A specific activity of 50-80 ,tCi/,ug is routinely obtained. Another fundamental difficulty with purification was the lack of an efficient method to obtain material with sufficient amounts of active PGF to initiate purification procedures. Attempts to concentrate large volumes of media containing small amounts of PGF by lyophilization and flash evaporation resulted in the loss of 75-100% of the activity. This report presents a method to extract large amounts of active PGF from plerocercoids. Plerocercoids were removed from laboratory mice, the anterior 0.5 cm cut off and injected into other mice. Tsushima et al. (loc. cit.) found no binding activity in media from cultures containing only the tails of plerocercoids. However, we found no difference in the amount of active PGF obtained between intact plerocercoids and tails. The reinjected anterior portions will grow new tails and can be used repeatedly to produce additional plerocercoid mass. Plerocercoids were incubated in a variety of media without serum ranging from simple (bicarbonate-buffered saline) to complex (minimum essential medium with Hanks' balanced salts fortified with amino acids, fatty acids, vitamins and antibiotics) at several temperatures (30-45 C) and pH's (7.0-8.5). The more complex media allowed the longest incubation times (up to 6 days) but did not allow collection of the greatest amount (ngE/g of tails) of active PGF. The incubation conditions and media which produced the greatest amount of active PGF were found following incubation in 0.1 M NaHCO3 pH 7.6, 0.12 M NaCl at 43 C for 24 hr or in Krebs-Ringer bicarbonate, pH 7.4 at 43 C for 48 hr which yielded 1,755 and 1,450 ngE/g of tails respectively. Incubations were terminated when the tails began to disintegrate. The fact that the greatest amount of total activity was obtained from incubations where the tails disintegrated relatively rapidly prompted us to attempt to collect active PGF by homogenization of tails. Attempts to obtain active PGF by homogenization in a wide variety of buffers followed by differential centrifugation