INTERACTION OF LIPOPROTEIN-LIPASE WITH HEPARIN FRAGMENTS AND WITH HEPARAN-SULFATE - STOICHIOMETRY, STABILIZATION, AND KINETICS

The interaction of lipoprotein lipase (LPL) with heparan sulfate and w ith size-fractionated fragments of heparin was characterized by severa l approaches (stabilization, sedimentation, surface plasmon resonance, circular dichroism, fluorescence). The results show that heparin deca saccharides form a 1:1 complex with dimeric LPL and that decasaccharid es are the shortest heparin fragments which can completely stabilized dimeric LPL, while shorter fragments (hexa- and tetrasaccharides) were less efficient. Binding of heparin did not induce major rearrangement s in the conformation of LPL, supporting the view that the heparin bin ding region is preformed in the native structure, Interaction of LPL w ith heparan sulfate, as studied by surface plasmon resonance, was foun d to be a fast exchange process characterized by a high value for the association rate constant, 1.7 x 10(8) M(-1) s(-1), a relatively high dissociation rate constant, 0.05 s(-1), and as a result a very low equ ilibrium dissociation constant equal to 0.3 nM at 0.15 M NaCl, The con tribution of electrostatics was estimated to be 44% for the binding of LPL to heparan sulfate, 49% for the binding of LPL to unfractionated heparin, and 60% for the binding of LPL to affinity-purified heparin d ecasaccharides at 0.15 M NaCl. The number of ionic interactions betwee n LPL and high affinity decasaccharides was estimated to be 10, We pro pose an essential role of electrostatic steering in the association, M onomeric LPL had 6000-fold lower affinity for heparin than dimeric LPL had, expressed as a ratio of equilibrium dissociation constants. A mo del for binding of LPL to heparan sulfate-covered surfaces is proposed . Due to the fast rebinding, LPL is concentrated to the close proximit y of the heparan sulfate surface, As the dissociation is also fast, th e enzyme exchanges rapidly between specific binding sites on the immob ilized heparan sulfate, without leaving the surface. This model may al so apply to LPL at the endothelium of blood vessels.