Author : Feihua Cao
Publisher :
ISBN 13 :
Total Pages : 386 pages
Book Rating : 4.:/5 (953 download)
Book Synopsis Effective Regulation of Peptide Hormones by : Feihua Cao
Download or read book Effective Regulation of Peptide Hormones written by Feihua Cao and published by . This book was released on 2012 with total page 386 pages. Available in PDF, EPUB and Kindle. Book excerpt: In the work described in this thesis, peptidylglycine a-amidating monooxygenase (PAM) was extracted from human H889 and DMS53 small cell lung cancer (SCLC) cells, PC-3 prostate cancer cells and SW1783 glioblastoma cells. Optimization of PAM assay conditions was carried out to maximize PAM activity. The extracted enzymes and optimized assay conditions provided a platform to assess substrate and inhibitor kinetic parameters and to compare the enzyme specificities of PAM from different human cancer sources, as well as differences between human PAM and commercial frog PAM. Potent and selective PAM inhibitors were then developed on the basis of structure-activity-relationships as well as the recognition by PAM of the peptide chain of natural substrates. Furthermore, a direct, rapid, efficient and sensitive HPLC method was developed in order to carry out whole cell inhibition studies and to directly monitor the inhibitory effects on natural hormone and prohormone levels. This method includes online extraction, separation, derivatization and fluorescence detection of peptide hormones, and in principle could be used for any peptide hormone with a free primary amine for fluorescamine tagging. Calcitonin (CT) and its prohormones were studied due to their high expression in DMS53 cells. This is the first time that the levels of CT and its prohormones, CTG, CTGK and CTGKK, have been separated and quantified separately. The detection limits of the present HPLC method were determined to be around 1 ng/mL of cell medium. This method was then applied to whole cell inhibition studies, and this is the first time that inhibitors of CT biosynthesis in human cancer cells were studied, allowing the direct observation of the effects on CT levels and its prohormones. As CPE and PAM are involved in the specific cleavage of C-terminal basic amino acids and the last step of peptide bioactivation, respectively, CPE was also targeted to moderate CT levels. The newly developed whole cell PAM inhibition assay allowed direct determination of the effects of PAM and CPE inhibition on CT production.