Identification of binding proteins for PSP94 in human prostate adenocarcinoma cell lines LNCaP and PC-3.
BACKGROUND
Prostatic secretory protein of 94 amino acids (PSP94) is one of the predominant proteins found in human seminal fluid. Limited information is available regarding a physiological function for PSP94. An important step in the elucidation of this function is the determination of the mechanism of interaction of PSP94 with potential cellular targets.


METHODS
Equilibrium binding assay was employed to demonstrate specific binding of biotinylated-PSP94 to the LNCaP and PC-3 cell lines. Binding proteins were partially purified by PSP94 affinity-chromatography from LNCaP, PC-3 cells, and prostate tissues.


RESULTS
Binding of biotinylated-PSP94 to LNCaP and PC-3 cells was saturable and time and temperature dependent. The binding could be specifically competitively inhibited by unlabelled PSP94. Two types of PSP94 binding sites with distinct affinity (Kd) and density (Bmax) were determined by Scatchard analysis for each of the two cell lines. For the LNCaP cells, these values were Kd 1 = 0.75 nM and Bmax1 = 300 fmol/mg protein and Kd 2 = 4.5 nM, Bmax2 = 780 fmol/mg protein, respectively. Similar affinity and density results were obtained for PC-3 cells: Kd 1 = 0.83 nM, Bmax1 = 250 fmol/mg protein, and Kd 2 = 5.0 nM, Bmax2 = 700 fmol/mg. The binding of biotinylated-PSP94 to the LNCaP cells was competitively inhibited by the partially purified proteins. Analysis of these proteins SDS-PAGE showed three main bands and the molecular weights of these three bands were approximately 180, 100 and 60 kD, respectively.


CONCLUSIONS
The data showed the presence of specific binding proteins to the PSP94 in LNCaP, PC-3 cells, and prostate tissue.
Yang
J P
JP
Department of Microbiology and Immunology, University of Western Ontario, London, Canada. jyang@julian.uwo.ca
Baijal-Gupta
M
M
Garde
S V
SV
Fraser
J E
JE
Finkelman
M A
MA
Clarke
M W
MW
D000230:Adenocarcinoma / Q000737:chemistry*; D001667:Binding, Competitive; D019921:Biotinylation; D002352:Carrier Proteins / Q000032:analysis*; D002452:Cell Count; D002846:Chromatography, Affinity; D006801:Humans; D007700:Kinetics; D008297:Male; D011471:Prostatic Neoplasms / Q000737:chemistry*; D029584:Prostatic Secretory Proteins; D011506:Proteins / Q000378:metabolism*; D029589:Seminal Plasma Proteins; D013696:Temperature; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D029584:Prostatic Secretory Proteins; D011506:Proteins; D029589:Seminal Plasma Proteins; C043076:beta-microseminoprotein