Characterization of the 4 S polycyclic aromatic hydrocarbon-binding protein in human liver and cells.
The 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein (PBP) is a soluble protein that binds PAHs with high affinity in mouse, rat, and rabbit. Until now, this protein had not been detected in human placenta or human cells in culture by cytosol labeling and gradient centrifugation assay. Thanks to a preliminary fractionation of cytosol by sedimentation on sucrose gradient or/and gel permeation chromatography, we found that PBP was present in liver, MCF-7 cell line, and hepatocytes of human. To accurately quantitate PBP binding and determine specific binding parameters, a reduction in the amount of charcoal used to adsorb nonspecifically bound benzo[a]pyrene was required. By saturation analysis, the concentration of specific binding sites for [3H]BP in PBP fraction from human liver was 4.6 pmol/mg of protein compared with 14.7 +/- 1.4 pmol/mg in the same fraction from DBA/2J mouse liver. Kinetic studies analyzed by Scatchard and Woolf plots indicate that human liver and MCF-7 cells contain a low-affinity PBP form: the Kd derived from Woolf plot analysis were 14.2 +/- 1.4 and 26.2 +/- 1.8 nM, respectively. DBA/2J mouse possesses a higher-affinity PBP form, the same analysis indicating a Kd of 6.1 +/- 0.3 nM. These data demonstrate that, by comparison to the mouse liver, a lower-affinity form of PBP is present in reduced concentration in human liver, explaining the impossibility of detecting this protein by sedimentation of human cytosol in sucrose gradient.
Peryt
B
B
Department of Biochemistry, Institute of Biopharmacy, Warsaw, Poland.
Maurel
P
P
Lesca
P
P
D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism*; D001665:Binding Sites; D001943:Breast Neoplasms; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D005260:Female; D050938:Glycine N-Methyltransferase; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008780:Methyltransferases; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D000072317:Polychlorinated Dibenzodioxins; D011725:Pyridines; D001564:Benzo(a)pyrene; C028474:1,4-bis(2-(3,5-dichloropyridyloxy))benzene; D008780:Methyltransferases; D050938:Glycine N-Methyltransferase