OPH present in prostate cell lysates appears as a single MW weight species in SDS PAGE Westerns Since we observed multiple sellekchem OPH bands in prostate cell lysates with n PAGE, we next wanted to determine if SDS PAGE gels similarly had multiple bands or a single OPH polypeptide with the known MW of the OPH monomer. Western blots of non tumorigenic RWPE 1 and tumorigenic LNCaP, DU 145, and PC3 cell lysates were found to be a single 81 kDa band. Densi tometry analysis showed significant differences in OPH ex pression levels among the four cell lines. LNCaP cell lysates contained approximately 40% more OPH than RWPE 1, while DU 145 and PC3 lysates contained less OPH than RWPE 1. Mammalian OPH has been primarily reported as a homotetramer with each OPH subunit being active within the tetramer.

It has been shown that citraco nylation of the amino groups of purified OPH tetramer reversibly dissociates the quaternary structure of OPH. When acylated with citraconic anhydride, OPH separated by n PAGE forms multiple OPH bands. Interestingly, our prostate cell lysates produced four uniformly dis tributed activity bands when separated by n PAGE. Some explanations for the multiple OPH activity bands are OPH multi mers, isoforms, degradation products, protein interactions, and post translational modifications. OPH isoforms and degradation products appear to be unlikely causes for the multiple bands. Isoforms and degredation products typically result in multiple bands when separated by SDS PAGE, however, western blots of the prostate lysates reveal a single 80kD OPH band.

The interaction of native OPH with other proteins is plausible. There is evidence that under conditions of oxidative stress OPH translocates to the cell membrane of eryth rocytes and degrades oxidized proteins. Similarly, OPH was found to translocate to the aggresome when the proteasome was inhibited in COS 7 cells. High levels of oxidative stress are known to oxidize proteins resulting in protein aggregations that can inhibit the proteasome. LNCaP, DU 145, and PC3 cell lines are reported to have significantly higher free radical production compared to RWPE 1, which might induce OPH to interact with aggresomal or membrane proteins. Our mass spectrometry analysis of the OPH bands revealed several proteins known to be associated with aggresomes and were consistent with previously published data.

We are actively pursuing an ex planation for the multiple OPH bands. The higher expression of OPH protein in LNCaP cell lysates is reflected by a higher activity towards N acetyl alanyl p nitroanilide N acetyl alanyl p nitroanilide is a specific OPH substrate and is routinely used to measure OPH activity and follow OPH selleck chemicals Calcitriol purification from tissue homogenates. The product p nitroaniline is released upon hydrolysis of AcApNA and its absorbance measured at 405 nm. We found that non tumorigenic and tumorigenic cell lysates incubated with AcApNA released p NA at differential rates.