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J. Biol. Chem., Vol. 278, Issue 39, 37183-37194, September 26, 2003

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1,1'-Bis(anilino)-4-,4'-bis(naphtalene)-8,8'-disulfonate Acts as an Inhibitor of Lipoprotein Lipase and Competes for Binding with Apolipoprotein CII^*

Aivar Lookene   ¶, Liyan Zhang , Vello Tougu || and Gunilla Olivecrona  **

From the Department of Medical Biosciences, Physiological Chemistry, Umeå University, Umeå, Sweden SE-901 87, Department of Gene Technology, Tallinn Technical University, Tallinn 19086, Estonia, and ^||National Institutes of Chemical Physics and Biophysics, Tallinn 12618, Estonia

Lipoprotein lipase (LPL) is dependent on apolipoprotein CII (apoCII), a component of plasma lipoproteins, for function in vivo. The hydrophobic fluorescent probe 1,1'-bis(anilino)-4,4'-bis(naphthalene)-8,8'-disulfonate (bis-ANS) was found to be a potent inhibitor of LPL. ApoCII prevented the inhibition by bis-ANS, and was also able to restore the activity of inhibited LPL in a competitive manner, but only with triacylglycerols with acyl chains longer than three carbons. Studies of fluorescence and surface plasmon resonance indicated that LPL has an exposed hydrophobic site for binding of bis-ANS. The high affinity interaction was characterized by an equilibrium constant K_d of 0.10–0.26 µM and by a relatively high on rate constant k_ass = 2.0 x 10⁴ M^–1 s^–1 and a slow off-rate with a dissociation rate constant k_diss = 1.2 x 10^–4 s^–1. The high affinity binding of bis-ANS did not influence interaction of LPL with heparin or with lipid/water interfaces and did not dissociate the active LPL dimer into monomers. Analysis of fragments of LPL after photoincorporation of bis-ANS indicated that the high affinity binding site was located in the middle part of the N-terminal folding domain. We propose that bis-ANS binds to an exposed hydrophobic area that is located close to the active site. This area may be the binding site for individual substrate molecules and also for apoCII.

Received for publication, April 14, 2003 , and in revised form, July 4, 2003.

^* This study was funded by Swedish Medical Research Council Grant 12203 and by the Estonian Science Foundation Grant 4925. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a scholarship from the Swedish Royal Academy of Sciences.

^** To whom correspondence should be addressed: Dept. of Medical Biosciences, Physiological Chemistry, Umeå University, Bldg. 6M, 3rd floor, SE-901 87 Umeå, Sweden. Tel.: 46-90-785-4491; Fax: 46-90-785-4496; E-mail: Gunilla.Olivecrona{at}medbio.umu.se.

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