A SIMPLE ASSAY FOR INTRACELLULAR LIPID-BINDING PROTEINS USING DISPLACEMENT OF 1-ANILINONAPHTHALENE 8-SULFONIC ACID

The fluorescent probe 1-anilinonaphthalene 8-sulfonic acid (1,8-ANS) h as been used to characterize a general assay for members of the intrac ellular lipid-binding protein (ALBP) multigene family. The adipocyte l ipid-binding protein (ALBP), the keratinocyte lipid-binding protein (K LBP), the cellular retinol-binding protein (CRBP), and the cellular re tinoic acid-binding protein I (CRABPI) have been characterized as to t heir ligand binding activities using 1,8-ANS. ALBP and KLBP exhibited the highest affinity probe binding with apparent dissociation constant s (K-d) Of 410 and 530 nM, respectively, while CRBP and CRABPI bound 1 ,8-ANS with apparent dissociation constants of 7.7 and 25 mu M, respec tively. Ln order to quantitate the fatty acid and retinoid binding spe cificity and affinity of ALBP, KLBP, and CRBP, a competition assay was developed to monitor the ability of various lipid molecules to displa ce bound 1,8-ANS from the binding cavity. Oleic acid and arachidonic a cid displaced bound 1,8-ANS from ALBP, both with apparent inhibitor co nstants (K-i) of 134 nM, while all-trans-retinoic acid exhibited a sev enfold lower K-i (870 nM). The short chain fatty acid octanoic acid an d all-trans-retinol did not displace the fluorophore from ALBP to any measurable extent. In comparison, the displacement assay revealed that KLBP bound oleic acid and arachidonic acid with high affinity (K-i = 420 and 400 nM, respectively) but bound all-trans-retinoic acid with a markedly reduced affinity (K-i = 3.6 mu M). Like that for ALBP, neith er octanoic acid nor all-trans-retinol were bound by KLBP. Displacemen t of 1,8-ANS from CRBP by all-trans-retinal and all-trans-retinoic aci d yielded K-i values of 1.7 and 5.3 mu M, respectively. These results indicate the utility of the assay for characterizing the ligand bindin g characteristics of members of the iLBP family and suggests that this technique may be used to characterize the ligand binding properties o f other hydrophobic ligand binding proteins. (C) 1996 Academic Press, Inc.