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Fragments of Human Growth Hormone Produced by Digestion with Thrombin: Chemistry and Biological Properties^*

Departments of Biochemistry and Physiology, Emory University School of Medicine Atlanta, Georgia 30322

Address requests for reprints to: Dr. Jack L. Kostyo, Department of Physiology, The University of Michigan Medical School, Ann Arbor, Michigan 48109.

Abstract

Human GH (hGH) was digested with bovine thrombin conjugated to Sepharose, resulting in the cleavage of only one peptide bond between amino acid residues 134 and 135 in the large disulfide loop of the molecule. hGH was quite sensitive to the proteolytic action of thrombin, with digestion going essentially to completion. Thrombin-digested hGH (TDhGH) was equipotent with hGH in promoting growth in hypophysectomized rats, in stimulating the in vitro oxidation of glucose by isolated adipose tissue of hypophysectomized rats, in inducing N-acetyllactosamine synthase activity in mammary tissue explants from pregnant mice, and in competing with [^l25I]hGH for binding to hGH antibodies. TD-hGH was reduced and alkylated to produce S-carbamidomethylated (RCAM-TDhGH), S-aminoethylated (RAE-TD-hGH), and S-carboxymethylated (RCM-TD-hGH) derivatives. These materials showed substantial alterations in their profiles of biological activity when compared to intact hGH. RCAM-TD-hGH and RAE-TD-hGH had 50% of the growth-promoting activity of hGH but only 20% of the insulin-like activity. In contrast, their lactogenic activity and immunoreactivity were equivalent to those of intact hGH. RCM-TD-hGH had greatly reduced growth-promoting and insulin- like activities but retained a high degree of lactogenic activity and immunoreactivity.

When TD-hGH was reduced and S-carbamidorriethylated in 6 M urea or 6 M guanidine-HCl and then chromatographed in the presence of these same agents, the effective separation of peptides 1–134 and 135–191 was achieved in high yield. Guanidine- HCl proved to be the dissociating agent of choice, since exposure of peptide 1–134 to 6 M urea promoted aggregation of the peptide. Peptide 1–134, isolated by the above means, had a low order of growth-promoting and insulin-like activities and greatly reduced immunoreactivity, whereas peptide 135–191 was essentially inert. However, peptide 1–134 was found to retain approximately 25–50% of the diabetogenic activity (ability to reduce glucose tolerance) of intact hGH in the ob/ob mouse.

Footnotes

^* This work was supported by Grants HD-04485, HD-01231, and HD-08230 from the NIH and a grant from the Kroc Foundation.

Received December 6, 1979.

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