ABSTRACT
Aim To provide practical method for screening human carbonic anhydrase Ⅱ(hCA Ⅱ) inhibitors in drug discovery.Methods hCA Ⅱ protein was obtained from induced BL21(DE3) E.coli containing plasmid pET-28b-hCA Ⅱ.The hCA Ⅱ activity was detected under pH 7.6 and 25℃ by its esterase activity which could decompose PNPA to increase the photoabsorption at 348 nm. After the assay conditions were finally selected, 35 new compounds were tested.Results A practical method for screening hCA Ⅱ inhibitors was successfully constituted by using recombinant hCA Ⅱ protein expressed in E.coli as the source of hCA Ⅱ enzyme.10 new compounds had better inhibitory effect and 9 new compounds had the same inhibitory effect on hCA Ⅱ compared with acetazolamide.Conclusions The hCA II inhibitor screening technique constituted in this work possesses advantages of being reliable, rapid, and practical. 19 new compounds are worth further research for developing high efficiency and low side effect drugs used for high-altitude illness.
ABSTRACT
Objective To study protein variation between PtenL/LMEFs and Pten△/△MEFs cells.Methods Two-dimensional electrophoresis(2-DE) was employed to compare the differential expression proteins between PtenL/LMEFs and Pten△/△MEFs cells.Six differential expression proteins were digested in gel by enzyme and the mass of generated peptides was measured by matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS).The data obtained from peptide mass fingerprinting(PMF) were searched using the Internet available database and five proteins were identified.Results Compared with that of PtenL/LMEFs,expression level of proteins including phosphoglycerate mutase 1(PGAM1) and peptidyl-prolyl cis-trans isomerase C(PPIC) was up-regulated,whereas expression level of Transgelin 2 was down-regulated in Pten△/△MEFs cells.B and F proteins were both identified to be peroxiredoxin-6.They had similar molecular weight but different PI which might be caused by post-translation modification.B protein was only expressed in Pten△/△MEFs cells.Conclusion The protein profile of Pten△/△MEFs cells displayed obvious difference compared to that of PtenL/LMEFs cells.The results implied that various distinct different proteins might lead to cancer.