Mechanisms for dissociating proteoglycan aggregates.

K Kimata,V C Hascall,J H Kimura

doi:10.1016/s0021-9258(18)34856-7

Abstract

Preformed Cs2SO4 zonal gradients were used to purify aggregates from proteoglycan preparations derived from associative extracts of the Swarm rat chondrosarcoma. Zonal gradients were used in solvents with different concentrations of guanidine HCl and different solvent pH values to study the mechanisms for dissociating the aggregates. Aggregates are stable in concentrations of guanidine HCl up to 1.5 M at pH 6.6. At 2 M guanidine HCl, partial dissociation occurs over 20 h in which a link protein is completely dissociated for every monomer proteoglycan dissociated from the aggregate structure. This suggests that in this solvent disaggregation occurs concurrent with complete separation of link protein from monomer. At solvent pH 2.7 to 3.3 in ionic conditions which normally promote aggregation, dissociation occurs by a mechanism in which the link protein remains associated with monomer. Thus, link protein-monomer complexes dissociate as bimolecular units from hyaluronic acid; such complexes then exhibit physical properties indistinguishable from pure monomers. The link protein-monomer complexes reassociate with hyaluronic acid to form link-stabilized aggregates when the solvent pH is raised to pH 7, i.e. to associative conditions. The study provides additional evidence for the role that link protein-monomer interactions have in proteoglycan aggregate structures.

Highlights

Preformed Cs2S04zonal gradients were used to pu- proteoglycan monomers, link proteins, and hyaluronic acid rify aggregates fromproteoglycanpreparationsde
The results indicate that, while increasing concentrations of guanidine HC1cause separation of allthree components of the aggregate, low solvent pH dissociates a stable complex of proteoglycan monomer-link protein as a unit from hyaluronic acid
The experiments described in this report utilized preformed Cs2S04zonal gradients in different solvents to studydissociation of purified proteoglycan aggregates

Summary

Mechanisms for Dissociating Proteoglycan Aggregates*

(Received for publication, September 14, 1981, and in revised form, December 1, 1981). Preformed Cs2S04zonal gradients were used to pu- proteoglycan monomers, link proteins, and hyaluronic acid rify aggregates fromproteoglycanpreparationsde-. At 2 M guanidine HC1, partial dissociation occurs over 20 h in which a link protein is completely dissociated for every monomer proteoglycan dissociatedfrom the aggregate structure. The results indicate that, while increasing concentrations of guanidine HC1cause separation of allthree components of the aggregate, low solvent pH dissociates a stable complex of proteoglycan monomer-link protein as a unit from hyaluronic acid. For some of the experiments described in provides additional evidence for the role that link prot-his paper, the gradients were prepared in the presence of different tein-monomer interactionshave in proteoglycanaggre- guanidine HC1 concentrations and different solvent pH values. Analysis for Proteoglycan-For aliquots from gradients which did not contain guanidine HC1, hexuronic acid concentrations were measured directly with an automated carbazole procedure as described in the preceding paper [1]to estimate proteoglycan contents. I dients with guanidine HCl, the proteoglycans were precipitated with ethanol as described above and redissolved in 0.2 M sodium acetate, pH 7.2, before analysis for hexuronic acid content

RESULTS

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Findings

DISCUSSION