Pro-protein convertase-2 (PC2) and carboxypeptidase-E (CPE) proteins are two major members of the pro-protein convertases that involve in the maturation of protein precursor. By using PC2 activity, immunocytochemistry (ICC) and Western blot method, PC2, CPE and preproNPY protein expression levels were compared among mature retina tissue, RGC-5 cells and its differentiated cells, or brain cortex tissue, NS20Y tumor cells and its differentiated cells, or mature breast tissue, breast tumor cell RM1 and breast adenocarcinoma tissue. The experimental results indicated that the differentiated cells or tissues had higher or highest PC2 activity. In the comparative experiments, more PC2 protein expression in the mature tissues and more CPE and preproNPY protein expression in the tumor cells or tumor tissue were observed, but no expression of preproNPY protein was observed in the mature tissues. Compared with NS20Y or RGC-5 undifferentiated cells, its differentiated cells showed less proPC2, more proCPE and more preproNPY protein expressions. The results demonstrated that the mature tissues showed stronger PC2/CPE-mediated pro-protein processing ability than the tumor cells or tissue. The results also showed that the artificial differentiation of RGC-5 or NS20Y cells was different from maturation of its corresponding normal tissue.

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Activity and half-life play key roles in the application of GHRH analogues. The GHRH monomers produced in a solid synthesizer were incubated, respectively, in NH4OH solution and lyophilized to obtain their dimers. The activities, specificities, and receptor affinities of the GHRH dimers were evaluated in rGH release/inhibition, rACTH/LH/PRL release, pituitary homogenate binding, and fluorescent staining. Compared to hGHRH(1-44)NH2 (S), PP-hGHRH(1-44)-GGC-CGG-hGHRH(44-1)-PP (2D), P-hGHRH(1-44)-GGC-CGG-hGHRH(44-1)-P (2E), (1)P-hGHRH(2-44)-GGC-CGG-hGHRH(44-2)-(1)P (2F), or hGHRH(1-44)-GGC-CGG-hGHRH(44-1) (2Y) had potency of 104 ± 16.7%, 94 ± 32.6%, 114 ± 16.6%, or 122 ± 14.5% and similar specificities. The inhibition effect of GHIH on rGH stimulated by GHRH dimer was in dose-/time-dependent manner. The staining of FITC-labeled dimer showed cytomembrane distribution and the binding ranking was 2F>2D>2Y>2E>S. 2F presents the strongest activity and the highest affinity to pituitary cells. The dimer with (1)Pro-GHRH stimulates stronger rGH release than that with (1)Tyr-GHRH and the N-terminal single cyclic amino acid is required for the stimulation.

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