PubMed · 11956514
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Lewis B Sheiner, Nicholas H G Holford. 2002. Determination of maximum effect.. https://doi.org/10.1067/mcp.2002.122277
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K5 lyase of coliphage K5A degrades the K5 polysaccharide of encapsulated E. coli strains expressing the K5 antigen thereby contributing to virus binding and infection. We have investigated the affinities of the recombinant enzyme for different GAG ligands by isothermal fluorescence titrations and correlated them with substrate processing and protein structural changes. Chondroitin sulfate (CS) and heparan sulfate (HS) bound to K5 lyase with a Kd of 0.5 microM whereas heparin exhibited a Kd=1.1 microM. The natural substrate K5 polysaccharide displayed a similar apparent affinity as CS and HS but was the only ligand of the enzyme which induced a large structural rearrangement of the protein as detected by far-UV CD spectroscopy. Since significant enzymatic degradation was only found for the K5 polysaccharide peaking at 44 degrees C, but binding was also detected for heparin, we propose that the K5 lyase is able to discriminate between specific (acetylated/non-sulfated) and unspecific (acetylated/sulfated) ligands by its heparin binding motif in the C-terminus. This is proposed to be the origin for the enzyme's residual HS degrading activity.
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In this investigation, new biodegradable brush-like amphiphilic copolymers were synthesized by ring opening polymerization. Poly(L-lactide) (PLLA) was grafted onto chondroitin sulfate (CS), which is one of the physiologically significant specific glycosaminoglycans (GAGs), using a tin octanoate [Sn(Oct)2] catalyst in DMSO. The hydroxyl groups of the chondroitin sulfate were used as initiating groups. These functional groups enable specific mucoadhesion or receptor recognition. The degree of substitution (DS), the degree of polymerization (DP) and the chondroitin sulfate content (from 1.1 to 15.4%) were analyzed by 1H NMR. The characteristics of these grafted copolymers, including the structure, the thermal properties and biodegradability, etc., were examined with respect to CS content. Meanwhile, the amphiphilic core (PLLA)-corona (CS) nanoparticles, with size smaller than 200 nm, was examined by dynamic light scattering (DLS). Zeta potential analysis exhibited the value in the range -18.3 to -49.4 mV. The morphologies of the nanoparticles were observed by field-emission scanning electron microscopy (FE-SEM). The nanoparticles with lower cytotoxicity were examined by MTT assay. Furthermore, the in vitro BSA release kinetics of those CSn-PLLA nanoparticles was also determined in this study.