Two step procedure for purification of enzymatically active prostate-specific antigen from seminal plasma

Bindukumar, B.; Kawinski, Elzbieta; Cherrin, Craig; Gambino, Leah M.; Nair, Madhavan; Schwartz, Stanley A.; Chadha, Kailash C.

doi:10.1016/j.jchromb.2004.09.014

Cited by 16 publications

(22 citation statements)

References 29 publications

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“…We reported previously that f-PSA isolated from seminal plasma by T-gel chromatography had a significant level of enzymatic activity [18]. Human prostate tissue and seminal plasma are known to have the highest concentration of zinc in the human body [28].…”

Section: Effect Ofzn 2+ and Other Trace Elements On The Enzymatic Actmentioning

confidence: 99%

“…f-PSA was purified from seminal plasma by a two step procedure described previously [18]. Briefly, thiophilic-gel chromatography was used as a first step in the purification procedure.…”

Section: Separation and Purification Of F-psa From Seminal Plasmamentioning

confidence: 99%

“…The blank was prepared containing the substrate in assay buffer. The details of the procedure are described elsewhere [18,26].…”

Section: Enzymatic Activity Of Psamentioning

confidence: 99%

“…In our earlier studies, we have shown that PSA and PSA complexes have strong affinity for different thiophilic gels (T-gels), and that T-gel affinity can be successfully applied for the purification of PSA and PSA-complexes from biological fluids, including serum and seminal plasma [16,17]. We have shown that f-PSA purified from seminal plasma by T-gel chromatography is enzymatically active [18]. In the present study, for the first time, the use of TIC technique has been applied for the isolation and subsequent characterization of T-PSA from freshly harvested human prostate cancer tissue.…”

Section: Introductionmentioning

confidence: 99%

“…In the present study, for the first time, the use of TIC technique has been applied for the isolation and subsequent characterization of T-PSA from freshly harvested human prostate cancer tissue. TIC was selected for isolation of T-PSA because we earlier have shown that T-gel has strong affinity for all molecular forms [free and complexed forms] of PSA [16,18]. Taking this approach, we were able to show that T-PSA, isolated from human prostate tumor tissue contains only the "free" form of PSA and that this PSA is enzymatically active.…”

Section: Introductionmentioning

confidence: 99%

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Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Babu

Vijayalakshmi

Smith

et al. 2008

Journal of Chromatography B

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Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelia 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 33kDA. 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 (10 nM to 50 μM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serineprotease, was affected differently. At higher concentrations of Zn 2+ (10 μM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn 2+ (5 μM and lower), the enzymatic activity was enhanced.

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Section: Effect Ofzn 2+ and Other Trace Elements On The Enzymatic Actmentioning

confidence: 99%

“…f-PSA was purified from seminal plasma by a two step procedure described previously [18]. Briefly, thiophilic-gel chromatography was used as a first step in the purification procedure.…”

Section: Separation and Purification Of F-psa From Seminal Plasmamentioning

confidence: 99%

“…The blank was prepared containing the substrate in assay buffer. The details of the procedure are described elsewhere [18,26].…”

Section: Enzymatic Activity Of Psamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Babu

Vijayalakshmi

Smith

et al. 2008

Journal of Chromatography B

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Proteomic approach for purification of seminal plasma proteins involved in tumor proliferation

Hassan

Kumar

Kashav

et al. 2007

J of Separation Science

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Human seminal plasma contains a large array of proteins required for the normal physiology and metabolism of spermatozoa. These are mainly secreted from prostate epithelium, testes, and seminal vesicles. Fortunately, many of these are found to be present at elevated concentration in seminal plasma and act as a biomarker of different carcinomas as their levels are also enhanced in serum and are found to be involved in tumor progression and metastasis apart from fertility. The proteins which were overexpressed in the seminal plasma of prostate carcinoma patients were identified by 2-DE and MALDI-TOF/MS. We have designed a strategy to purify these four proteins prostate specific antigen (PSA), prostatic acid phosphatase (PAP), Zinc alpha2-glycoprotein (ZAG), and progastricsin (PG), together in homogeneity by using simple chromatographic techniques. Acidic and basic fractions of human seminal plasma were separated by ion exchange chromatography and further purified by gel permeation chromatography. Our results form a new and valuable resource for those attempting structure-based drug designing for prostate and other cancers where the amount of proteins is required in plenty and in native form.

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Proteomic profiling of the effect of prostate‐specific antigen on prostate cancer cells

et al. 2008

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The relevance of the level of PSA in prostate tissue microenvironment and its relation to tumor progression has not been elucidated. PSA has been shown to down-regulate several proteins that are known to have involvement in tumor progression. This suggests that normal physiological levels of PSA in prostate tissue microenvironment may be promoting non-angiogenic environment and its down-regulation may promote tumor growth.

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Two step procedure for purification of enzymatically active prostate-specific antigen from seminal plasma

Cited by 16 publications

References 29 publications

Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Proteomic approach for purification of seminal plasma proteins involved in tumor proliferation

Proteomic profiling of the effect of prostate‐specific antigen on prostate cancer cells

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