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

Abstract

Lipoprotein lipase (LPL) is dependent on apolipoprotein CII (apoCII),and a slow off-rate with a dissociation rate constant k diss ‫؍‬ 1.2 ؋ 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. Lipoprotein lipase (LPL)1 is one of the central proteins in blood lipid metabolism (for reviews, see Refs. 1-3). The enzyme is bound to heparan sulfate proteoglycans at the vascular endothelium and hydrolyzes triacylglycerols and phospholipids in plasma lipoproteins so that lipolysis products can be taken up in adjacent cells for metabolic purposes. In addition and independent of catalysis, the LPL protein functions as a mediator for binding and uptake of lipoproteins by cells by bridging between the lipoproteins and heparan sulfate proteoglycans or receptors of the low density lipoprotein receptor family.LPL belongs to the family of mammalian triglyceride lipases together with pancreatic lipase, hepatic lipase, and endothelial lipase (2). Based on sequence homology with pancreatic lipase, models of the 55-kDa subunit of LPL have been created (4 -6). The active form of LPL is a noncovalent dimer of identical subunits (7,8) that are arranged in a head-to-tail fashion (5, 9). LPL can engage in a number of interactions with heparin/ heparan sulfate, receptors, lipid/water interfaces, the activator apolipoprotein CII (apoCII), and perhaps with other apolipoproteins. Fatty acids with long acyl chains have been shown to bind to LPL with high affinity (10) and to inhibit the catalytic activity of LPL as well as binding of LPL to lipid/water interfaces (lipoproteins, emulsion droplets) (11) and to heparin (12). Thus, fatty acids exert strong control over the activity of LPL. Their removal by uptake in tissues or by binding to albumin is essential to allow the action of the enzyme to proceed unabated.ApoCII, a 79-amino acid residue peptide belonging to the apoC family of exchangeable apolipoproteins, is a necessary activator for LPL in vivo (13,14). Deficiency of apoCII leads to massive accumulation of lipids in blood and symptoms similar to what is seen on LPL deficiency (15). Systematic mutagenesis of residues in the LPL binding domain of apoCII and studies of the three-dimensional structure of apoCII by NMR strongly suggest that the basis for the activation is formation of a complex between apoCII and LPL at the surface of a lipid droplet (16,17). Although the region of apoCII responsible for the interaction with LPL has been localized to the C-terminal part of the peptide (18), it is still unclear which part(s) of LPL interacts wit...

Adipogenic potential of skeletal muscle satellite cells

“…Nevertheless, as shown in Figure 4, a hypothetical model can be envisioned based on evidence gleaned from in vivo studies detailing the maturation of well-studied protein substrates, such as viral and some host proteins [72,106,107], and from in vitro studies monitoring the structure of folding intermediates occurring during the refolding of chemically denatured LPL [80,108,109]. In particular, LPL refolding experiments have demonstrated that folding of the smaller C-terminal domain happens quickly and completely, whereas folding of the N-terminal domain is much less efficient and occurs through folding intermediates exhibiting a degree of disordered tertiary structure [80].…”

Phosphorylation of hormone‐sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area

“…Previous work has shown that lipoprotein lipase (LPL) interacts strongly with, and in fact is inhibited by, the hydrophobic probe 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid (bis‐ANS) [12]. Another hydrophobic probe, i.e.…”

Identification of a potential hydrophobic peptide binding site in the C‐terminal arm of trigger factor

“…All measurements were made at 25°C. To obtain the dissociation constant ( K d ), fluorescence titration curves were analyzed using the equation: where F is the fluorescence intensity of the TF·bis‐ANS complex, [ TB ] is the concentration of the TF·bis‐ANS complex, k is a proportionality constant, [ T ] is the concentration of TF, and [ B ] is the concentration of free bis‐ANS at equilibrium (Lookene et al 2003). The data were also analyzed by Scatchard plot: where [ bound ] and [ free ] is the concentration of bound and free bis‐ANS at equilibrium, and n is the number of bis‐ANS molecules bound to TF.…”

“…The reactivation yield of GAPDH or lysozyme in the presence of TF without bis‐ANS were set as 100%. The parameter K i characterizing the inhibition efficiency was obtained from the equation: where A is the measured activity, A r is a calculated constant describing the activity of the TF‐bis‐ANS complex, K i is the inhibition constant, m is a proportionality constant, and C is the concentration of bis‐ANS (Lookene et al 2003).…”