A Hybrid Noncovalent Complex of Fragments of Porcine and Human Growth Hormones with Diabetogenic Activity*

Abstract

Although porcine GH (pGH) and human GH (hGH) are structurally related, their structures differ to the extent that, unlike hGH, pGH is not active in primates and does not exhibit lactogenic activity. Therefore, it was of interest to determine whether hybrid noncovalent complexes could be formed by complementation of large fragments of pGH with fragments of hGH and to study the immunological and biological properties of such hybrids. For this purpose, pGH was digested with bovine thrombin conjugated to Sepharose. After reduction and S-carbamidomethylation of the digested hormone, two large fragments consisting of residues 1-133 (p1-133T) and 134-191 (p134-191T) were isolated by gel filtration. The human GH fragments used in this work were S-carbamidomethylated peptides 1-134 (h1-134T) and 135-191 (h135-191T) isolated from thrombin-digested hGH. Noncovalent complementation of the peptide fragments was attempted by dissolving equimolar amounts of the materials in 0.5% (wt/vol) ammonium bicarbonate solution containing 6 M guanidine-HCl and then gradually removing the guanidine-HCl by dialysis. Using this method, a hybrid noncovalent complex of peptides p1-133T and h135-191T was produced in 50% yield by weight of starting material. Attempts to produce the reciprocal complex between h1-134T and p134-191T, as well as attempts to recombine the contiguous pGH fragments, p1-133T, and p134-191T, were unsuccessful. The hybrid complex (p1-133T + h135-191T) had 40% the cross-reactivity of native pGH in a rat GH RIA, but it was inert in an hGH RIA. It had very low growth-promoting activity in the 9-day weight gain test in hypophysectomized rats and no detectable in vitro ability to stimulate [14C]phenylalanine incorporation into the protein of the diaphragm of the hypophysectomized rat. When tested for insulin-like activity (stimulation of glucose oxidation in vitro in adipose tissue of hypophysectomized rats), the hybrid complex had less than 1% the activity of native pGH. In contrast, the hybrid complex had approximately 10-20% the diabetogenic activity of native pGH in the ob/ob mouse. The fact that the hybrid complex has substantial diabetogenic activity, although lacking significant anabolic or insulin-like activities, suggests that the structural requirements differ for the expression of these various activities of GH.