Construction and activity of a novel GHRH analog, Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys.
OBJECTIVE
To construct another growth hormone releasing hormone (GHRH) analog, Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys peptide and to compare its activity with that of Pro-Pro-hGHRH(1-44)OH peptide.


METHODS
The pro-pro-hGHRH(1-44)-gly-gly-cys DNA fragment was synthesized by polymerase chain reaction. The recombinant protein was expressed to high levels in Escherichia coli BL21 (DE3). The Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys peptide was purified to homogeneity by cell disruption, washing, ethanol precipitation, acid hydrolysis, and SP-Sephadex C-25 and Sephadex G-25 column chromatography. The peptide molecular mass was determined by electrospray ionization mass spectroscopy (ESI-MS). Its purity was determined by SDS-PAGE. The concentration of growth hormone (GH) stimulated by GHRH and its analogs was determined with antiserum kit against human GH. The other human hormones as hTSH, hFSH, hLH, and hPRL were determined with a paramagnetic particle chemiluminescent immunoassay kit.


RESULTS
The molecular weight of Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys was 5455.4 kDa which was coincident with the theoretical calculations. All the three peptides at 5 mg/L stimulated GH release from the human fetal pituitary but only the difference between Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys group and blank group was significant (P<0.05). Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys 0.01, 0.1, and 1 mg/L and Pro-Pro-hGHRH (1-44)OH 0.1 and 1 mg/L stimulated GH release from rat pituitary in a concentration-dependent manner (P<0.05, P<0.01). At the same concentration Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys stimulated more GH release than Pro-Pro-hGHRH (1-44)OH. Pro-Pro-hGHRH (1-44)OH 0.01 mg/L and hGHRH (1-40)OH 2 mg/L did not stimulate GH release from rat pituitary (P>0.05). Pro-Pro-hGHRH (1-44)OH and Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys peptides at 5 mg/L did not stimulate hTSH, hFSH, hLH, and hPRL release from human fetal pituitary in vitro.


CONCLUSIONS
Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys had a better activity than that of Pro-Pro-hGHRH(1-44)OH. Variation at C-terminus of GHRH could modulate its GH releasing activity.
Tang
Song-shan
SS
Laboratory of Minigene Pharmacy, Biopharmaceutical College, Jiangsu Key Laboratory of Drug Delivery System, China Pharmaceutical University, Nanjing, China.
Zhang
Juan-hui
JH
Du
Ming-hua
MH
Wu
Jie
J
Liu
Jing-jing
JJ
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005333:Fetus; D043373:Follicle Stimulating Hormone, Human / Q000378:metabolism; D013006:Growth Hormone / Q000378:metabolism*; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D010902:Pituitary Gland / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013972:Thyrotropin / Q000378:metabolism
D043373:Follicle Stimulating Hormone, Human; D010446:Peptide Fragments; C497663:Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys; D011993:Recombinant Fusion Proteins; D013972:Thyrotropin; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone