The preparation and catalytic properties of recombinant human prostate-specific antigen (rPSA)

被引:20
作者
Hsieh, MC [1 ]
Cooperman, BS [1 ]
机构
[1] Univ Penn, Dept Chem, Philadelphia, PA 19104 USA
来源
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY | 2000年 / 1481卷 / 01期
关键词
recombinant PSA; refolding; Zn2+ inhibition; polyamine inhibition; Na+ activation; glycerol activation;
D O I
10.1016/S0167-4838(00)00116-3
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The serine proteinase prostate-specific antigen (PSA), and its complex with the serine proteinase inhibitor alpha(1)-antichymotrypsin (ACT), have been used as markers for the diagnosis of prostate cancer. PSA prepared from seminal fluid is typically contaminated with the trypsin-like glandular kallikrein (hK2). Here we describe a convenient and reproducible preparation of catalytically active recombinant PSA (rPSA) and demonstrate an overall similarity in the properties of cloned and refolded rPSA to PSA purified from seminal fluid. We also present results that are relevant for increasing the sensitivity of assays of PSA activity in biological fluids, for the putative role of PSA activity in physiologically important processes, including prostate cancer metastasis, and for the design of PSA inhibitors. Specifically, we find that added salts, in particular NaCl, give rise to dramatic increases in rPSA catalytic activity, as does added glycerol. On the other hand, Zn2+, spermine, and spermidine, each a major component of seminal and prostatic fluid, strongly inhibit rPSA activity, with Zn2+ being a non-competitive inhibitor while spermine is a competitive inhibitor. Citrate, also a major component of seminal and prostatic fluid, spermine, and spermidine each protect rPSA from Zn2+ inhibition, presumably via Zn2+ sequestration. Finally, rPSA efficiently proteolyzes several protein substrates. (C) 2000 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:75 / 87
页数:13
相关论文
共 57 条
[1]   Inactivation of human lung tryptase: Evidence for a re-activatable tetrameric intermediate and active monomers [J].
Addington, AK ;
Johnson, DA .
BIOCHEMISTRY, 1996, 35 (42) :13511-13518
[2]  
[Anonymous], 1964, STABILITY CONSTANTS
[3]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[4]   Use of the percentage of free prostate-specific antigen to enhance differentiation of prostate cancer from benign prostatic disease - A prospective multicenter clinical trial [J].
Catalona, WJ ;
Partin, AW ;
Slawin, KM ;
Brawer, MK ;
Flanigan, RC ;
Patel, A ;
Richie, JP ;
deKernion, JB ;
Walsh, PC ;
Scardino, PT ;
Lange, PH ;
Subong, ENP ;
Parson, RE ;
Gasior, GH ;
Loveland, KG ;
Southwick, PC .
JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION, 1998, 279 (19) :1542-1547
[5]   ENZYMATIC-ACTIVITY OF PROSTATE-SPECIFIC ANTIGEN AND ITS REACTIONS WITH EXTRACELLULAR SERINE PROTEINASE-INHIBITORS [J].
CHRISTENSSON, A ;
LAURELL, CB ;
LILJA, H .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1990, 194 (03) :755-763
[6]   BIOLOGICAL EFFECTS OF PROSTATE-SPECIFIC ANTIGEN AS AN INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-3 PROTEASE [J].
COHEN, P ;
PEEHL, DM ;
GRAVES, HCB ;
ROSENFELD, RG .
JOURNAL OF ENDOCRINOLOGY, 1994, 142 (03) :407-415
[7]   Evidence for a zinc uptake transporter in human prostate cancer cells which is regulated by prolactin and testosterone [J].
Costello, LC ;
Liu, YY ;
Zou, J ;
Franklin, RB .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (25) :17499-17504
[8]   Zinc inhibition of mitochondrial aconitase and its importance in citrate metabolism of prostate epithelial cells [J].
Costello, LC ;
Liu, YY ;
Franklin, RB ;
Kennedy, MC .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (46) :28875-28881
[9]  
Costello LC, 1998, PROSTATE, V35, P285, DOI 10.1002/(SICI)1097-0045(19980601)35:4<285::AID-PROS8>3.0.CO
[10]  
2-F