Characterization of oxysterol-binding protein in rat embryo fibroblasts and variations as a function of the cell cycle.
A cell-free system assay involving cell freeze-thawing and protein fractionation by ammonium sulfate precipitation was developed to characterize a cytosol binding protein specific for oxysterols in rat embryo fibroblasts. This protein shared common characteristics with the oxysterol-binding protein described in L cells and in normal human lymphocytes: 8 S sedimentation coefficient, sterol-protein complex of Mr 160 600, saturability, high affinity (Kd in the range of 10(-9) M) and specificity for cholesterol derivatives oxidized on the side chain. These compounds were better inhibitors of DNA synthesis than the compounds oxidized on the nucleus, whereas no difference was found between sterols oxygenated either on the side chain or on the nucleus, as far as inhibition of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) was concerned. Macromolecular components capable of specifically binding 25-hydroxycholesterol were also detected in the fibroblast nucleus. The cytosol oxysterol-binding protein showed a sharp increase (5-fold) in the G2M phase of the cell cycle and in exponentially growing cells (maximal binding site number/cell: 43 500, versus 8850 in confluent cells). Neither the affinity nor the sedimentation coefficient of the protein changed in either situation. The quantitative (but not qualitative) variations of oxysterol-binding protein could be related to the inhibitory effect of 25-hydroxycholesterol on DNA synthesis, which becomes critical when this sterol is added in the G2M phase of the cell cycle.
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002453:Cell Cycle; D002460:Cell Line; D004261:DNA Replication / Q000187:drug effects; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006888:Hydroxycholesterols / Q000378:metabolism; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D007700:Kinetics; D060588:LIM Domain Proteins; D008970:Molecular Weight; D009124:Muscle Proteins; D051381:Rats; D013256:Steroids / Q000494:pharmacology; D013261:Sterols / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins
D002352:Carrier Proteins; D006888:Hydroxycholesterols; D006903:Hydroxymethylglutaryl CoA Reductases; D060588:LIM Domain Proteins; D009124:Muscle Proteins; D013256:Steroids; D013261:Sterols; C517184:Fhl1 protein, rat; C007997:25-hydroxycholesterol; C101149:FHL1 protein, human; D047908:Intracellular Signaling Peptides and Proteins