To further characterize esterase activity in human http://www.selleckchem.com/products/17-AAG(Geldanamycin).html prostate cells, we next examined cell lysates of several human prostate cell lines for esterase activity using 6% n PAGE followed by activity staining with naphthyl acetate, or chiral ester substrates R ANAA, or S ANAA. By performing 6% n PAGE electrophoresis, the higher MW proteins were better separated and esterase bands gener ally more defined. The n PAGE esterase activity profiles Parallel gels stained with S ANAA or R ANAA show two prominent and sharp esterase bands at 216 kDa and 198 kDa native protein marker points. Densitometry ana lysis of the 216 kDa band showed significantly higher ester ase activity in the LNCaP cell lysate stained with R ANAA and S ANAA compared to all other cell lysates.

Moreover, the 216 kDa band for LNCaP cells showed higher activity with the S ANAA substrate compared to the R ANAA substrate. The degree of chiral substrate selectivity was more apparent in the esterase activity of the 198 kDa band. Densitometry of the 198 kDa band showed a significant preference for S ANAA sub strate in all of the cell lines except DU 145. The LNCaP lysates contained 40 50% higher esterase activity in both bands with S ANAA substrate than with the R isomer and a 40 60% higher activity compared to RWPE 1 lysate. RWPE 2 and PC3 lysates had similar staining profiles to RWPE 1, while DU 145 showed less activity compared to RWPE 1. The n PAGE esterase profiles obtained with S or R AANA showed fewer and more distinct bands than with the naphthyl acetate. We, therefore, focused on deter mining the identity of the protein in the more active 198 kDa band.

This band was excised, trypsinized, and the resulting peptides were subjected to mass spectrom etry analysis to identify the esterase responsible for the activity. As shown in Table 1, we identified oxidized protein hydrolase, also called N acylaminoacyl peptide hydrolase, or acylamino acid releasing enzyme in the 198 kDa band. OPH is a serine esterase protease with a well characterized exopeptidase activity for removing N terminally acetylated residues from peptides. These LC MS MS results as well as the hydrolysis of the ANAA substrates by the 198 kDa band are consistent obtained with naphthyl acetate showed diffuse bands in the 720 to 1048 kDa native protein marker range for LNCaP, DU145 and PC3 cell lysates that were faintly present in the RWPE 1 or RWPE 2 cell lysates.

The staining intensity with naphthyl acetate in the 720 to 1048 kDa region was greater for the LNCaP, DU145 and PC3 cell lysates compared to the RWPE 1 or RWPE 2 cell lysates. with the known activity of OPH to remove N terminally acetylated alanine residues. LNCaP lysates HTS showed significantly higher levels of esterase activity with naphthyl acetate and ANAA substrates compared to non tumorigenic RWPE 1 and tumorigenic RWPE 2, DU145 and PC3 cell lysates.