Abstract
In cartilage proteoglycan aggregates, link protein stabilizes the binding of proteoglycan monomers to hyaluronate by binding simultaneously to hyaluronate and to the G1 globular domain of proteoglycan monomer core protein. Studies reported here involving metal chelate affinity chromatography demonstrate that link protein is a metalloprotein that binds Zn2+, Ni2+, and Co2+. Zn2+ and Ni2+ decrease the solubility of link protein and result in its precipitation. However, link protein is readily soluble and functional in low ionic strength solvents from which divalent cations have been removed with Chelex 100. These observations make it possible to study the biochemical properties of link protein in low ionic strength, physiologic solvents. Studies were carried out to define the oligomeric state of link protein alone in physiologic solvents, and the transformation in oligomeric state that occurs when link protein binds hyaluronate. Sedimentation equilibrium studies demonstrate that in 0.15 M NaCl, 5 mM EDTA, 50 mM Tris, pH 7, link protein exists as a monomer-hexamer equilibrium controlled by a formation constant of 2 x 10(27) M-5, yielding a delta G' of -36 kcal/mol for the formation of the hexamer from six monomers. On binding hyaluronate oligosaccharides (HA10 or HA12), link protein dissociates to dimer. Link protein hexamer is rendered insoluble by Zn2+. Greater than 90% of the protein is precipitated by 2 mol of Zn2+/mol of link protein monomer. The binding of hyaluronate oligosaccharide by link protein strongly inhibits the precipitation of link protein by Zn2+. The link protein/hyaluronate oligosaccharide complex is completely soluble in the presence of 2 mol of Zn2+/mol of link protein. At higher molar ratios of Zn2+/link protein, the inhibitory effect of hyaluronate oligosaccharide on the precipitation of link protein is gradually overcome. Hyaluronate oligosaccharide is not dissociated from link protein by Zn2+. Hyaluronate remains bound to the link protein which is precipitated by Zn2+, or to the link protein which binds to Zn2(+)-charged iminodiacetate-Sepharose columns. Hyaluronate oligosaccharides and Zn2+ bind to different sites on link protein.
Highlights
Cartilage proteoglycan aggregates are assembled from prostabilizes the binding of proteoglycanmonomersto teoglycan monomers, hyaluronate, and link protein [1,2,3,4]
Sedimentation equilibrium studies demonstrate that in 0.15 M NaCl, 5 mM EDTA, 50 mM Tris, pH 7,link protein exists as a monomer-hexamer equilibrium controlled by a formation constant of 2 X lo’? ””, yielding a AG‘ of -36 kcallmol for the formation of the hexamer from six monomers
On binding hyaluronate oligosaccharides (HAloor HAIS),link proteindissociates to dimer.Link protein hexamer is rendereindsoluble byZn2+.Greater than 90%of the protein is precipitated by 2 mol of vents [4, 11,12,13]
Summary
Link protein stabilizes the binding of proteoglycan monomertohyaluronate [8,9,10] by bindingsimultaneouslyto hyaluronate and to the G1’ globular domain of proteoglycan monomer core protein. Studies of the biochemical and functional propertieosf link ionic strengthsolventsfromwhichdivalentcations protein have been hampered by the apparent insolubility of have been removed with Chelex 100. These observa- link proteins in low ionic strength associativesolvents. 5 mM EDTA, 50 mM Tris, pH 7,link protein exists as a monomer-hexamer equilibrium controlled by a formation constant of 2 X lo’? Link protein is readily soluble and functional in associative solvents from which divalent cations have beenremoved
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