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

Li, Choh Hao; Bewley, Thomas A.

doi:10.1073/pnas.73.5.1476

Cited by 69 publications

(32 citation statements)

References 18 publications

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“…It is possible that substitutions of Aspl'3 and Ile'79 by His and Met, respectively, in the hCS structure prevent the generation of full biological activity. These results demonstrate that the three-di- (2,8).…”

Section: Resultssupporting

confidence: 57%

“…It is evident that recombinant I had 50% of the potency of hGH, whereas recombinant II was considerably less active. (2,4) in that the COGH-terminal fragment was first dissolved in 1 M NH40H to give a clear solution before mixing with the NH2-terminal fragment. This gives rise to a better yield of purified recombinant product.…”

Section: Resultsmentioning

confidence: 99%

“…The NH2-terminal 134-amino-acid fragment of the reduced carbamoylmethylated (Cam) human somatotropin (human growth hormone, hGH)molecule has been shown to react noncovalently with the natural or synthetic COOH-terminal 51-amino-acid fragment to restore full biological and immunological activity (2)(3)(4). Because the primary structures of human chorionic somatomammotropin (hCS) (5) and hGH (6) are nearly identical (see Fig.…”

mentioning

confidence: 99%

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Noncovalent interaction of the NH2-terminal fragment of human somatotropin with the COOH-terminal fragment of human choriomammotropin to generate growth-promoting activity.

Li¹

1978

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

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Complementations of the plasmin fragments of reduced-carbamoy methylated (Cam) human somatotropin (hGH) with those of reduced-carbamoylmethylated human chorionic somatomammotropin (hCS) have been investigated. It was found that the recombinant obtained by noncovalent interaction of with exhibits 50% growth-promoting activity and nearly full immunoreactivity. Complementation of tCys-(Cam)3]hCS-1-133) with the COOH-terminal fragment of hGH generated lower growth-promoting and immunological activities.The NH2-terminal 134-amino-acid fragment of the reduced carbamoylmethylated (Cam) human somatotropin (human growth hormone, hGH)molecule has been shown to react noncovalently with the natural or synthetic COOH-terminal 51-amino-acid fragment to restore full biological and immunological activity (2-4). Because the primary structures of human chorionic somatomammotropin (hCS) (5) and hGH (6) are nearly identical (see Fig. 1), it seems of interest to investigate the possibility of generating biological activities by complementation of plasmin fragments of hGH with those of hCS. MATERIALS AND METHODSPlasmin fragments of reduced-carbamoylmethylated hGH and hCS were obtained as previously described (7,8). Exclusion chromatography of the reaction mixture was performed at 22°o n a Sephadex G-100 column (1.5 X 60 cm) in 0.01 M NH4HCO3 at pH 8.4. For radioimmunoassay, the doubleantibody procedure (9) using a guinea pig antiserum to native hGH was followed. The growth-promoting activity was determined by the rat tibia test (10). and 30% for recombinant II with a relative elution volume Ve/Vo of 1.85. This is exactly the elution position of native hGH or hCS under the same experimental conditions. RESULTS ComplementationThe growth-promoting activity of purified recombinants I and II are summarized in Table 1. It is evident that recombinant I had 50% of the potency of hGH, whereas recombinant II was considerably less active. Fig. 3 presents the radioimmunoassay results with purified recombinants using guinea pig antiserum to hGH. It may be noted that recombinant I at low concentrations gave an inhibition nearly identical to that of hGH, but the inhibition curve was not parallel with that for hGH. Although recombinant II gave a parallel inhibition curve, it was much less effective than hGH, requiring at least 30 times higher antigen concentrations to give the same degree of inhibition. DISCUSSIONComplementation reactions have been slightly modified from earlier experiments (2, 4) in that the COGH-terminal fragment was first dissolved in 1 M NH40H to give a clear solution before mixing with the NH2-terminal fragment. This gives rise to a better yield of purified recombinant product. For recombination of the hGH fragments, the yield has been increased from 25% (2) to nearly 50% (data not shown).As shown in Table 1 and Fig. 3

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Section: Resultssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Noncovalent interaction of the NH2-terminal fragment of human somatotropin with the COOH-terminal fragment of human choriomammotropin to generate growth-promoting activity.

Li¹

1978

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

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“…The (9). The complementation reactions were carried out by published procedures (2,10). Briefly, 3 (9).…”

Section: Methodsmentioning

confidence: 99%

“…1), [Carbamoylmethylcys- give a recombinant protein with the full biological activities (2,3) of the natural hormone. Subsequently, it was found that complementation of the natural NH2-terminal fragment with the synthetic COOH-terminal fragment or its synthetic analogs gave rise to fully active recombinants (4,5).…”

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confidence: 99%

Semisynthesis of human somatotropin analogs.

Li¹,

Blake²

1979

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

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Complementation of the natural NHrterminal 134amino acid fragment of the reduced, carbamoylmethylated human somatotropin with synthetic analogs of COOH-terminal fragments of 57, 42, or 38 amino acids of reduced, carbamoylmethylated human somatotropin has been investigated. It was found that synthetic fragments of 57 and 42 amino acids gave recombinants with full growth-promoting activity, whereas attempts to obtain a recombinant with the synthetic 38-amino acid fragment were unsuccessful. The synthesis of two analogs of the COOH-terminal fragment of human somatotropin, human [Nlel70, Alal65,i82 1891 somatotropin(150-191) and human [Nlel7O, Ala's65'82,, is herein described.It has been shown that the two plasmic fragments (1) of human somatotropin (HGH) (see Fig. 1 give a recombinant protein with the full biological activities (2, 3) of the natural hormone. Subsequently, it was found that complementation of the natural NH2-terminal fragment with the synthetic COOH-terminal fragment or its synthetic analogs gave rise to fully active recombinants (4, 5). This paper reports complementation reactions of the natural NH2-terminal 134-amino acid (AA) fragment with synthetic COGH-terminal fragment analogs of different chain lengths to obtain full growth-promoting activity. MATERIALS AND METHODSHuman somatotropin was isolated from fresh-frozen pituitary glands as described (6). Plasmic digests of HGH and the isolation of the NH2-terminal 134-AA and COOH-terminal 51-AA fragments were obtained as described (1) except that the NH2-terminal fragment was further purified by gel filtration on Sephadex G-100 in 0.01 M NH4HCO3 at pH 8.2 (7). Analogs of the COGH-terminal fragment were synthesized by the solid-phase method (8). The synthesis of one of the analogs, [Lysl35,136,138 7Glul37,139, Nle170, Alal65,182,, has already been described (9). The complementation reactions were carried out by published procedures (2, 10). Briefly, 3.0 mg (0.20 ,umol) of the NH2-terminal fragment in 1.5 ml of 0.1 M Tris-HCl, pH 8.4/5% (vol/vol) n-butanol was added to 1.3 mg (0.21 ,mol) of COGH-terminal fragment or synthetic fragment in 0.1 ml of 1 M NH40H. Both solutions were clear before mixing and became turbid after mixing. The mixtures were stored at 20C for 10 days. After removal of the insoluble material, the clear supernatants were purified by exclusion chromatography on a Sephadex G-100 column (1.5 X 60 cm) in 0.01 M NH4HCO3 (pH 8.2) at 22°C. The growth-promoting activity was estimated by the rat tibia test (11).Peptide Synthesis. The peptides [Nle170, Alal65'82,189]- and [Nle'70, were synthesized as described for the corresponding analogs and HGH-(145-191) (9). t-Butoxycarbonylphenylalanine (Boc-Phe) resin was alternately deblocked with 55% trifluoroacetic acid/methylene chloride, neutralized with N-methylmorpholine or diisopropylethylamine, and coupled with the symmetrical anhydride (12) of the Boc-amino acid. N-Methylmorpholine was used for the bulk of the neutralization for the aspartyl-containing peptide to minimize base-cat...

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In vivo association of protein fragments giving active AraC

Eustance

Schleif

1996

Proteins

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

Cited by 69 publications

References 18 publications

Noncovalent interaction of the NH2-terminal fragment of human somatotropin with the COOH-terminal fragment of human choriomammotropin to generate growth-promoting activity.

Noncovalent interaction of the NH2-terminal fragment of human somatotropin with the COOH-terminal fragment of human choriomammotropin to generate growth-promoting activity.

Semisynthesis of human somatotropin analogs.

In vivo association of protein fragments giving active AraC

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