John B. Mills

Metabolic Effects of Plasmin Digests of Human Growth Hormone in the Rat and Man

As a first step in our study of structure-function relationships among primate and non-primate growth hormones, human growth hormone (hGH) was subjected to the limited digestive activity of human plasmin. The lyophilized whole digest, containing less than 2% of unchanged hormone, had an average of 2.3 new amino-terminal groups per mole. The digest had the same potency as the native hormone (a) in causing weight gain in hypophysectomized rats; (b) in stimulating somatomedin production in hypophysectomized rats; (c) in stimulating upake of [(3)H]leucine into isolated diaphragm of hypophysectomized rats; (d) in accelerating transport of [(14)C]alpha-aminoisobutyric acid into isolated diaphragm of hypophysectomized rats; (e) in stimulating uptake of [3-0-methyl-(14)C]glucose by isolated adipose tissue of hypophysectomized rats; (f) in accelerating conversion of [(14)C]glucose to (14)CO(2) by isolated epididymal adipose tissue of hypophysectomized rats. The digest also caused glucosuria in partially pancreatectomized rats treated with dexamethasone. These metabolic actions of plasmin-digested hGH in the array of animal tests were confirmed by comparable effects elicited in 11 human subjects (nine pituitary-deficient children and adolescents and two nondeficient adults). A single injection of the plasmin digest caused an increase in plasma free fatty acids and a fall in plasma amino acids. Seven daily injections caused positive balances of nitrogen, phosphorous, sodium, and potassium, gain in body weight, and in two of three subjects impairment of glucose tolerance. The potency of the plasmin digest in producing these metabolic effects in man was comparable to that of native hGH.Thus, 2-3 bonds in the hGH molecule can be cleaved by plasmin without impairing the hormones growthpromoting, anabolic, diabetogenic, and adipokinetic actions for rat and man.

Fragments of human growth hormone produced by digestion with bromelain. Chemistry and biological properties.

Abstract In an effort to produce small discrete fragments of human growth hormone (GH), we examine the action of the proteolytic enzyme, bromelain, on this molecule. Purified human GH incubated for 40 min at 22°C with crude bromelain and gel-filtered on Sephadex G-100 resulted in a major digestion product, peak 2. SDS-urea gel electrophoresis in the presence of β-mercaptoethanol suggested that peak 2 was composed of two polypeptide chains. Two polypeptide fractions were isolated by the reduction and S -alkylation of peak 2 in 6 M guanidine-HCl and subsequent chromatography on Sephacryl S-200 in 6 M guanidine-HCl. These two fractions, A and B, had the same mobilities as the two components of peak 2 on SDS-urea gels. Amino-terminal analysis, tryptic peptide mapping, carboxypeptidase digestion, cyanogen bromide cleavage, and amino acid analysis of fraction A indicated that it was peptide 1–135. Amino-terminal analysis and tryptic peptide mapping of fraction B suggested the presence of a mixture of peptides 143–191, 145–191 and 146–191. Thus, peak 2 is heterogeneous and appears to be a mixture consisting of peptide 1–135 + peptide 143–191, peptide 1–135 + peptide 145–191 and peptide 1–135 + peptide 146–191, in each case the N-terminal peptide being joined to the C-terminal peptide by the disulfide bridge between residues 53 and 165. In the weight-gain test in hypophysectomized rats, two preparations of peak 2 appeared to be somewhat less active than the native human GH preparations from which they were derived. Several preparations of peak 2 showed equivalent potency in stimulating [ 14 C]glucose oxidation to 14 CO 2 by isolated epididymal adipose tissue of hypophysectomized rats. Also, most of the peak 2 preparations were somewhat less active than native human GH in displacing 125 I-labeled human GH bound to antibodies to human GH.

Anabolic actions of reduced and S-carbamidomethylated human growth hormone and its plasmin digest in man.

Six children aged 12-15 yr, deficient in endogenous growth hormone, were each treated, after a 7-day control period, for 7 days with 0.0168, 0.052, and 0.168 U/kg body wt3/4 human growth (hGH) (doses A, B, and C, respectively) in separate metabolic balance studies. Doses B and C caused a dose-related retention of N, P, K, Na, and Cl in ratios of 1/0.069/4.5/7.5/5.6. These ratios indicate increments in masses of protoplasm/extracellular fluid (ECF)/bone in ratios of 1/2.0/ less than 0.001. Three of the children were also treated with doses A, B, and C of reduced and carbamidomethylated hGH (RCAM-hGH). Doses B and C caused 1.2-2.8 times as much retention of N, P, and K, and 0.3-0.5 times as much retention of Na and Cl, as did the corresponding doses of hGH. The plasmin digest of RCAM-hGH gave results generally similar to RCAM-hGH. For RCAM-hGH and its plasmin digest, N, P, K, Na, and Cl were retained in ratios of about 1/0.14/5.4/2.2/2.1, indicating increments of protoplasm/ECF/bone of about 1/0.8/0.05. These findings indicate that reduction and carbamidomethylation alter the anabolic actions of hGH in man in both quantitative and qualitative manner. RCAM-hGH is more potent in stimulating enlargement of protoplasm and bone, and less potent in stimulating expansion of ECF, than is the native hormone. The profile of anabolic actions of RCAM-hGH in man does not appear to be further altered by digestion with plasmin.