Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions.
Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelial cells and prostate cancer cells. We explored "thiophilic-interaction chromatography" (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the "free" form having a molecular weight of 33 kDa. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T-PSA over a wide range of concentrations of Zn(2+) ions (10nM to 50 microM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serine-protease, was affected differently. At higher concentrations of Zn(2+) (10 microM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn(2+) (5 microM and lower), the enzymatic activity was enhanced.
Satheesh Babu
A K
AK
Centre for Bio-Separation Technology, VIT University, Vellore, India.
Vijayalakshmi
M A
MA
Smith
Gary J
GJ
Chadha
Kailash C
KC
D015153:Blotting, Western; D002384:Catalysis / Q000187:drug effects; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008297:Male; D017430:Prostate-Specific Antigen / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology*
D002918:Chymotrypsin; D017430:Prostate-Specific Antigen; D015032:Zinc