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

Mills, John; Kostyo, Jack L.; Reagan, Charles R.; Wagner, S. A.; Moseley, Martha H.; Wilhelmi, Alfred E.

doi:10.1210/endo-107-2-391

Cited by 41 publications

(28 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thrombin-like activity has also been detected in the rat pituitary gland (37,38) and in several other endocrine tissues (39). Both GH (20,21) and IGF-binding proteins (32)(33)(34) are cleaved by thrombin. In fact, increased breakdown of IGFBP-5 by thrombin during term pregnancy, diabetes, and cancer, which is accompanied by a loss of its capacity to bind to IGF-1, is believed to play a role in controlling its bioavailability.…”

Section: Discussionmentioning

confidence: 97%

“…Each value is a mean Ϯ SEM of triplicate determinations. mones and growth factors (20,21,(32)(33)(34), we reasoned that thrombin might be a promising candidate for cleaving PRL.…”

Section: Discussionmentioning

confidence: 99%

“…Assuming that cleavage of the hormone might occur at the target cell, thrombin, a proteolytic enzyme that is central to endothelial cell biology, was considered. Indeed, thrombin is active at a pH 7.4, is enriched at the endothelial cell surface (19), and has been reported to cleave human GH (20,21), a structural and functional homologue of human PRL (1).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Proteolysis of Human Prolactin: Resistance to Cathepsin D and Formation of a Nonangiostatic, C-Terminal 16K Fragment by Thrombin

Khurana¹

1999

Endocrinology

View full text Add to dashboard Cite

The N-terminal 16K fragments of rat and human PRLs possess angiostatic activity. 16K PRL has also been detected in vivo in both humans and rats. Based on an in vitro study, cathepsin D, an acid protease, has been implicated in the generation of rat 16K PRL. However, the proteolytic cleavage of human PRL has not been demonstrated. Our objective was to identify an enzyme that is capable of forming an angiostatic human 16K PRL. To confirm the angiostatic action of rat 16K PRL, the fragment was generated by incubating 23K PRL with rat mammary microsomal fraction at pH 3.2. Upon incubation with human umbilical vein endothelial cells (HUVEC), rat 16K PRL, but not 23K PRL, inhibited basal-and basic fibroblast growth factor-stimulated cell proliferation. Intact rat and human PRLs were then incubated with cathepsin D or acidified microsomal pellets of MCF-7 human breast cancer cells. Analysis by SDS-PAGE showed cleavage of rat, but not human, PRL. Next, hormones were incubated with thrombin at pH 7.4. As shown by SDS-PAGE, digestion of both human and rat PRL by thrombin resulted in the formation of 16K fragments. PRL contained within human amniotic fluid was also cleaved by thrombin. Enzyme specificity was supported by prevention of cleavage by the thrombin inhibitor hirudin. When tested with HUVEC, the human 16K PRL was devoid of angiostatic activity. The activity of this fragment in the Nb2 lymphoma bioassay was 10-to 15-fold lower than that of 23K PRL. Mass spectrometry revealed that the fragment has a mass of 16,878.30 Ϯ 15.8 Daltons. Subsequent N-terminal sequencing showed that the thrombin cleavage occurred between amino acid residues 53 (Lys) and 54 (Ala), resulting in the formation of a C-terminal, not an N-terminal, 16K fragment. We conclude that, unlike rat PRL, human PRL is resistant to cleavage by cathepsin D. Thrombin at a physiological pH can generate a C-terminal 16K fragment of human PRL that is not angiostatic and retains little mitogenic activity. We suggest that the precise nature of endogenous 16K PRL fragments that are present in human tissues and body fluids should be carefully examined. (Endocrinology 140: [4127][4128][4129][4130][4131][4132] 1999) H UMAN PRL is a single 23-kDa polypeptide hormone composed of four antiparallel ␣-helices with three intrachain disulfide loops between residues 4 -11, 58 -174, and 191-199 (1). Although the position of the disulfide bonds is homologous between PRLs from different species, their primary amino acid sequences may vary considerably. PRL can undergo several posttranslational modifications (2), including cleavage (3, 4), glycosylation (5, 6), and phosphorylation (7, 8) that have been implicated in its functional diversity. However, the N-terminal 16K fragment of PRL is the only well characterized isoform with a unique biological activity as an antiangiogenic agent (9 -11).Angiogenesis, or formation of new blood vessels, is an integral part of tumorigenesis and metastasis and often results from a shift in the balance between angiogenic and angiosta...

show abstract

Section: Discussionmentioning

confidence: 97%

“…Each value is a mean Ϯ SEM of triplicate determinations. mones and growth factors (20,21,(32)(33)(34), we reasoned that thrombin might be a promising candidate for cleaving PRL.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Proteolysis of Human Prolactin: Resistance to Cathepsin D and Formation of a Nonangiostatic, C-Terminal 16K Fragment by Thrombin

Khurana¹

1999

Endocrinology

View full text Add to dashboard Cite

show abstract

“…In the present work, we took advantage of the exclusive specificity of thrombin towards the Arg'-Thr'3 peptide bond of hGH (9,10) Samples (10 ug) of each fraction were subjected to NaDodSO4 gel electrophoresis, and all of the patterns are shown.…”

Section: Discussionmentioning

confidence: 99%

“…1) has been known to be highly susceptible to different kinds ofproteinases such as plasmin (7,8), thrombin (9,10), and subtilisin (11). Of the proteinases cleaving the peptide chain of hGH in this region, however, only the action of thrombin is restricted to a single peptide bond, Arg'34-Thr'35 (9,10). We report here that the same peptide bond can be resynthesized with thrombin by using the noncovalent recombinant of the thrombin fragments of reduced and carbamidomethylated hGH (RA-hGH) (12) as a substrate.…”

mentioning

confidence: 99%

Human somatotropin: covalent reconstitution of two polypeptide contiguous fragments with thrombin.

Gráf¹,

Li²

1981

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

View full text Add to dashboard Cite

Thrombin has been shown to resynthesize the Argl34-Thrl"s peptide bond between the 134-and 57-residue thrombin fragments of reduced and carbamidomethylated human somatotropin at pH 6.0 in 90% (voVvol) glycerol. The maximal amount ofsynthesis was about 20% as estimated by sodium dodecyl sulfate gel electrophoresis and radioreceptor assay. The resynthesized polypeptide was isolated and shown to be indistinguishable from the reduced and carbamidomethylated hormone when tested by two receptor-binding assays, radioimmunoassay, and rat tibia test.

show abstract

The Metabolic Actions of Growth Hormone

Goodman

2001

Comprehensive Physiology

View full text Add to dashboard Cite

show abstract

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

Cited by 41 publications

References 18 publications

Proteolysis of Human Prolactin: Resistance to Cathepsin D and Formation of a Nonangiostatic, C-Terminal 16K Fragment by Thrombin

Proteolysis of Human Prolactin: Resistance to Cathepsin D and Formation of a Nonangiostatic, C-Terminal 16K Fragment by Thrombin

Human somatotropin: covalent reconstitution of two polypeptide contiguous fragments with thrombin.

The Metabolic Actions of Growth Hormone

Contact Info

Product

Resources

About