Dissociation of Some of the Biological Activities of Porcine and Human Growth Hormones by Cyanogen Bromide Cleavage12

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Abstract
Highly purified porcine (PGH) and human (HGH) growth hormones were reduced,S-aminoethylated and cleaved with cyanogen bromide. The two largest fragments resulting from the cleavage of PGH were separated from the reaction mixture and purified. These were porcine fragment A (about 112 residues) beginning at the fifth residue from the N-terminus and fragment B (55 residues), which follows fragment A in the sequence of the PGH molecule. The largest fragment resulting from the cleavage of HGH, human fragment A (111 residues), was also prepared in purified form. Human fragment A and porcine fragment A represent roughly equivalent portions of their respective hormone molecules. These purified fragments were then tested for biological activity. Porcine fragment A, when injected intraperitoneally into hypophysectomized rats, stimulated the in vivo incorporation of 14C-leucine into the protein of the diaphragm and liver. Also, both porcine fragment A and human fragment A stimulated the in vitro incorporation of leucine into the protein of diaphragms removed from rats that received intraperitoneal injections of the fragments. In this test system, porcine fragment A appeared to be 1/40–1/400 as potent as native PGH on a molar basis. When tested at a high dose, porcine fragment B was inactive on this system. Human fragment A also exhibited some in vitro activity in several test systems. At concentrations of 1–1.5 mg/ml, it increased the rates of uptake of α-aminoisobutyric acid and 3-0-methyl-glucose by isolated diaphragms of hypophysectomized rats. The magnitude of the stimulation produced was much less than that produced by much lower concentrations of native growth hormone, however. This fragment also stimulated 14C-glucose oxidation to 14CO2 by isolated epididymal adipose tissue of hypophysectomized rats. Despite their ability to stimulate protein synthesis in liver and muscle, porcine fragment A and human fragment A failed to promote growth in hypophysectomized rats. Porcine fragment B was also devoid of growthpromoting activity. Further, all three fragments failed to stimulate the in vitro incorporation of 3H-thymidine into the DNA of costal cartilage removed from rats treated with doses of fragment greater than 600 times the effective dose of native hormone. These results clearly demonstrate that following cyanogen bromide cleavage of the porcine and human growth hormone molecules some of the biological activities of the native hormones are retained by the largest of trie fragments, while the ability to stimulate the skeletal system is lost. (Endocrinology90: 1202, 1972)

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