| Budget Amount *help |
¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1990: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1989: ¥3,500,000 (Direct Cost: ¥3,500,000)
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| Research Abstract |
Most of peptide hormones are initially synthesized as propeptides that require post-translational processing to produce biologically active peptides. Generally these steps include tryptic cleavage at paired basic residues, their subsequent removal by carboxypeptidase H, and formation of a carboxyl terminal amide moiety via the action of peptidyl-glycyl alpha-amidating monooxygenase. By this last step, amidation of their carboxyl terminus most of peptide hormones express their full biological activity. Thus, peptide alpha-amidation capability is a critical function of endocrine cells that secrete amidsted peptides. However, some endocrine cells such as anterior pituitary somatotroph cells produce peptide hormones that are not amidsted. We undertook these studies, therefore, to investigate whither the potential to express the amidsting enzyme is a characteristic common to all endocrine cells 'or a feature restricted to cells that secrete amidsted hormones. For our studies we used a serie
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s of cell lines, including those that originated from non-endocrine cells (psi2 cells derived from NIH3T3 fibroblasts and BHK cells derived ftom baby hamster kidney), from endocrine cells that secrete amidsted peptides (PC-l 2 cells derived from a rat pheochromocytoma, AtT20 cells derived from a mouse anterior pituitary tumor and RINm5F cells derived from a rat insulinoma), and endocrine cells not known to secrete amidsted peptides (GH3 cells derived from a rat anterior pituitary tumor). Amidation activity in cell homogenates was assayed directly by measuring the chemical conversion of a synthetic substrate D-Tyr-Val-Glyto D-Tyr-Val-NH2. Although all of the endocrine cell lines demonstrated measurable activity, the non-endocrine cells did not. For further confirmation of these findings, we transformed the various cell lines using a retroviral expression vector pZip-Neo-SV (X) which permits the simultaneous expression of a cDNA insert and a neomycine resistant gene. For expression, we utilized a human pancreatic polypeptide (PP) CDNA insert. Since the PP precursor contains an amidation site, we could measure the production of amidsted PP from the precursor by radioimmunoassay using region specific antisera S1 1 (which recognizes the amidated carboxyl terminus of PP) and S3 (which recognizes the midportion of PP). Although both non-endocrine cell lines expressed the PP precursor, no evidence of carboxylterminal amidation was detected. In contrast, all four endocrine cell lines produced amidated molecular forms of PP, the principal one co-eluted with synthetic PP on Sephadex G-50 chromatography. Our data suggest that peptide a-amidating activity is not a property common to all cell types but is a fundamental characteristic of endocrine cells, regardless of their natural peptide products. Less
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