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

Mills, John; Reagan, Charles R.; Rudman, Daniel; Kostyo, Jack L.; Zachariah, Pulimuttil James; Wilhelmi, Alfred E.

doi:10.1172/jci107491

Cited by 35 publications

(14 citation statements)

References 32 publications

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“…Bewley et al, furthermore, did not observe increased activity of RCAM-hGH in the tibia test (6). Moreover, we have not observed increased activity of PD-hGH, RCAM-hGH, or PD-RCAM-hGH in promoting uptake of thymidine into rat costal cartilage, another process related to bone growth (5,9). Thus, we are not able to relate the present observations on augmented P retention by RCAM-hGH and PD-RCAM-hGH in man to the enhanced potency of PDhGH in the rat tibia assay.…”

Section: Discussioncontrasting

confidence: 51%

“…It is tempting to speculate that the increased retention of phosphorus seen in man with RCAMhGH and its plasmin digest is related to the enhanced activity of plasmin digests of native hGH (PD-hGH) when assayed in the rat by the tibia test (17)(18)(19). However, such digests do not cause increased phosphorus retention in man (5). This effect is first seen in man when native hGH is reduced and carbamidomethylated.…”

Section: Discussionmentioning

confidence: 99%

“…The hormone can be digested by plasmin without reducing its potency in stimulating weight gain by the hypophysectomized rat (5), and without lessening anabolic potency in man (5). Furthermore, the disulfide bonds of hGH can be reduced without affecting its capacity to cause the hypophysectomized rat to gain weight, provided the sulfhydryl groups produced by reduction are protected with an uncharged blocking group (6).…”

Section: Introductionmentioning

confidence: 99%

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

Heymsfield¹,

Bethel²,

Hall³

et al. 1977

J. Clin. Invest.

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A B ST RA CT Six children aged 12-15 yr, deficient in endogenous growth honnone, were each treated, after a 7-day control period, for 7 days with 0.0168, 0.052, and 0.168 U/kg body wt314 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/ < 0.00 1.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 RCAMhGH. 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 fuirther altered by digestion with plasmin.

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Section: Discussioncontrasting

confidence: 51%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Heymsfield¹,

Bethel²,

Hall³

et al. 1977

J. Clin. Invest.

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“…The methods used have been described in detail (11). In vitro insulin-like activity was assessed by measuring the ability of the hormone preparations to stimulate D-[U-14C]glucose oxidation to 14CO2 by isolated epididymal adipose tissue of hypophysectomized rats (Charles River Breeding Laboratories) weighing approximately 100 g. The details of the procedure have been described (16). RESULTS Because of the similarities in size and properties of native 22-kDa hGH and 20-kDa hGH, it has been quite difficult in the past to isolate 20-kDa hGH from pituitary extracts in a form free of contamination with small amounts of 22-kDa hGH and other pituitary hormones.…”

mentioning

confidence: 99%

Biosynthetic 20-kilodalton methionyl-human growth hormone has diabetogenic and insulin-like activities.

Kostyo¹,

Cameron²,

Olson³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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The anterior pituitary gland produces a 20-kilodalton (kDa) variant of human growth hormone (hGH) that differs from the predominant 22-kDa form of hGH in that amino acid residues 32-46 are deleted. Previous work has suggested that the 20-kDa variant possesses the full growth-promoting and lactogenic activities of 22-kDa hGH but lacks its intrinsic diabetogenic and insulin-like activities. In the present study, recombinant DNA techniques were used to prepare biosynthetic 20-kDa hGH, and some of the biological properties of the purified hGH variant were examined. The biosynthetic 20-kDa hGH variant was found to share the propensity for aggregation exhibited by its native counterpart. Moreover, like the native variant, biosynthetic 20-kDa hGH possessed full growth-promoting activity in the weight gain test in hypophysectomized rats. However, contrary to previous work suggesting that native 20-kDa hGH lacks diabetogenic and insulin-like activities, biosynthetic 20-kDa hGH was found to have substantial diabetogenic activity when administered chronically to ob/ob mice and to possess approximately 20% the in vitro insulin-like activity of biosynthetic 22-kDa hGH on isolated epididymal adipose tissue of hypophysectomized rats. The diabetogenic and insulin-like activities of biosynthetic 20-kDa hGH cannot be ascribed to contamination of the hormone preparation with the 22-kDa form of hGH or with other diabetogenic or insulin-like pituitary peptides. Therefore, the results strongly suggest that diabetogenic and insulin-like activities are also intrinsic properties of the 20-kDa variant of hGH.A 20-kilodalton variant of human growth hormone (20-kDa hGH) has been isolated from human pituitary glands (1) and has been found to differ from the predominant 22-kilodalton form of hGH (22-kDa hGH) in that residues 32-46 are deleted (2, 3). The variant is produced in the human pituitary, because the nucleotide sequence encoding residues 32-46 is sometimes excised during the processing of hGH pre-mRNA to hGH mRNA (4). As a consequence, approximately 5-10%1 of the hGH present in the human pituitary is 20-kDa hGH (1). Physicochemical studies of 20-kDa hGH (3) suggest that its conformation is similar but not identical to that of 22-kDa hGH.The 20-kDa variant of hGH has been reported (1, 5) to have growth-promoting activity in the hypophysectomized rat equivalent to that of 22-kDa hGH. Moreover, its lactogenic activity in the pigeon crop sac assay equals that of 22-kDa hGH (1). On the other hand, Lewis et al. (5) reported that 20-kDa hGH lacked diabetogenic activity, in that it failed to produce hyperglycemia and glucose intolerance in dogs, when administered 10 hr prior to a glucose tolerance test. Also, this same group reported (6) that 20-kDa hGH lacked the acute insulin-like property of 22-kDa hGH, since the 20-kDa hGH variant failed to produce hypoglycemia and increase plasma free fatty acids when administered to hypophysectomized rats and did not increase glucose uptake or oxidation to CO2 by isolated rat adipose tissue wh...

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“…Later, Yadley and Chrambach (3) described in detail that digestion of HGH with plasmin leads to a progressive transformation of the hormone to various components of higher electrophoretic mobility and increased prolactin activity. Reagan et al (4) and Mills et al (5) reported that hydrolysis of HGH with plasmin did not impair metabolic effects in the rat or in man. A detailed chemical and physical characterization of the cleaved products of HGH by plasmin digestion was not given in any of these studies.…”

mentioning

confidence: 99%

Human pituitary growth hormone: restoration of full biological activity by noncovalent interaction of two fragments of the hormone.

Li¹,

Bewley²

1976

Proc. Natl. Acad. Sci. U.S.A.

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The NH2-terminal 134 amino-acid fragment of the reduced-carbamidomethylated human somatotropin molecule is found to react noncovalently with the COOS-terminal 51 amino-adid fragment in solutions of pH 8.4 at 20 to restore full biological activity as evidenced by the rat tibia and pigeon crop-sac assays. In addition, circular dichroism spectra of the recombinant show the conformation to be completely repaired in comparison with that of the native hormone.The use of plasmin for the partial digestion of somatotropins has been described by Ellis et al. (1) Li and Graif (6) reported the isolation and characterization of two biologically active fragments from plasmin digests of HGH. It was shown that these two fragments, Cys(Cam)53-HGH-(1-134) and Cys(Cam) '65,182,, (Cam is carbamidomethyl) were derived from cleavage of the Arg-Thr (positions 134-135) and the Lys-Gln (positions 140-141) bonds of the hormone molecule (see Fig. 1 Circular dichroism (CD) spectra were obtained on a Cary model 60 spectropolarimeter equipped with a model 6002 circular dichroism attachment according to procedures previously described (11). The concentrations of all solutions were determined spectrophotometrically using the relation A01%1cm,277nm = 0.931. For simplicity this procedure was used regardless of whether the sample contained intact HGH modified by limited digestion with human plasmin (PL-HGH) or was a mixture of PL-HGH and its fragments. Spectra were calculated as mean residue ellipticities using 115 as the mean residue molecular weight in all cases. The contents of a-helix were estimated as previously described (12).The growth-promoting activity was determined by the rat tibia test (13) and the prolactin activity by the local crop-sac assay in the pigeon (14,15). The fragment mixtute was submitted to exclusion chromatography on Sephadex G-100 and the elution pattern is shown in Fig. 2. Approximately 25% of the protein appeared in a distinct peak (designated fraction IV), with a Ve/Vo ratio of 2.12. This is precisely the elution position of intact PL-HGH on this same column (18). It is especially noteworthy that no detectable protein was found at a Ve/Vo ratio of 2.9-3.1, the elution position expected for the free COOHterminal fragment (16).1476

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

Cited by 35 publications

References 32 publications

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

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

Biosynthetic 20-kilodalton methionyl-human growth hormone has diabetogenic and insulin-like activities.

Human pituitary growth hormone: restoration of full biological activity by noncovalent interaction of two fragments of the hormone.

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