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

Abstract

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

Semisynthesis of human somatotropin analogs.

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

Ovine chorionic somatomammotropin and foetal growth

“…The ability of hPL to increase IGF-I content and its release from foetal pancreatic explants has been shown recently by Swenne et al (1987), and hPL might participate at least in foetal pancreatic development. (Li, 1978) elicits 50 % growth activity as compared to a homologous hGH recombinant (1 -134) (141 -191 ) (Li et al, 1977). The reduced activity could be attributed to the substitution, in the last third of this molecule of aspartic acid in position 153 and isoleucine in position 179 for hGH, and of histidine and methionine respectively for hPL (Fig.…”