IDENTIFICATION OF BINDING-PROTEINS FOR PSP94 IN HUMAN PROSTATE ADENOCARCINOMA CELL-LINES LNCAP AND PC3

BACKGROUND. Prostatic secretory protein of 94 amino acids (PSP94) is o ne of the predominant proteins found in human seminal fluid. Limited i nformation is available regarding a physiological function for PSP94. An important step in the elucidation of this function is the determina tion of the mechanism of interaction of PSP94 with potential cellular targets. METHODS. Equilibrium binding assay was employed to demonstrat e specific binding of biotinylated-PSP94 to the LNCaP and PC-3 cell Li nes. Binding proteins were partially purified by PSP94 affinity-chroma tography from LNCaP, PC-3 cells, and prostate tissues. RESULTS. Bindin g of biotinylated-PSP94 to LNCaP and PC-3 cells was saturable and time and temperature dependent. The binding could be specifically competit ively inhibited by unlabelled PSP94. Two types of PSP94 binding sites with distinct affinity (K-d) and density (B-max) were determined by Sc atchard analysis for each of the two cell lines. For the LNCaP cells, these values were K-d 1 = 0.75 nM and B(max)1 = 300 fmol/mg protein an d K-d 2 = 4.5 nM, B(max)2 = 780 fmol/mg protein, respectively Similar affinity and density results were obtained for PC-3 cells: K-d 1 = 0.8 3 nM, B(max)1 = 250 fmol/mg protein, and K-d 2 = 5.0 nM, B(max)2 = 700 fmol/mg. The binding of biotinylated-PSP94 to the LNCaP cells was com petitively inhibited by the partially purified proteins. Analysis of t hese proteins SDS-PAGE showed three main bands and the molecular weigh ts of these three bands were approximately 180, 100 and 60 kD, respect ively. CONCLUSIONS. The data showed the presence of specific binding p roteins to the PSP94 in LNCaP, PC-3 cells, and prostate tissue. (C) 19 98 Wiley-Liss, Inc.