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Alternative splicing determines the binding of platelet-derived growth factor (PDGF-AA) to glycosaminoglycans.

Artikel i vetenskaplig tidskrift
Författare Florentina Lustig
Johan Hoebeke
Gunnel Östergren Lundén
F Velge-Roussel
Göran Bondjers
U Olsson
Ulla Rüetschi
G Fager
Publicerad i Biochemistry
Volym 35
Nummer/häfte 37
Sidor 12077-85
ISSN 0006-2960
Publiceringsår 1996
Publicerad vid Wallenberglaboratoriet
Institutionen för laboratoriemedicin, Avdelningen för klinisk kemi/transfusionsmedicin
Sidor 12077-85
Språk en
Länkar dx.doi.org/10.1021/bi960118l
Ämnesord Alternative Splicing, Amino Acid Sequence, Binding Sites, Cloning, Molecular, Dimerization, Escherichia coli, Genetic Variation, Glycosaminoglycans, chemistry, metabolism, Heparin, chemistry, metabolism, Heparitin Sulfate, chemistry, metabolism, Humans, Kinetics, Molecular Sequence Data, Peptide Fragments, chemical synthesis, chemistry, metabolism, Platelet-Derived Growth Factor, biosynthesis, chemistry, metabolism, Protein Conformation, Recombinant Proteins, biosynthesis, chemistry, metabolism, Thermodynamics
Ämneskategorier Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

Sammanfattning

We have shown previously that the platelet-derived growth factor (PDGF) and a synthetic oligopeptide, corresponding to the basic carboxyl-terminal amino acid extension of the long PDGF-A isoform, bind to heparin. Here, we have expressed the long (rA125) and the short (rA109) variants of PDGF A-chains in Escherichia coli and produced the functional homodimers. Surface plasmon resonance analyses showed that while the dimeric rA125 bound with high affinity to low molecular weight heparin, the rA109, lacking the basic extension, did not. This strongly indicated that high affinity binding is due to the carboxyl-terminal extension. Investigations of kinetics and thermodynamics suggested an allosteric binding mechanism. Thus, dimeric rA125 contains two equivalent binding sites. Following low affinity binding of heparin to one binding site, the dimer undergoes a conformational change, increasing the affinity for heparin about 40 times. This positive cooperativity requires the basic amino acid extension in both monomers of the dimeric PDGF molecule. Thermodynamics of the reaction, showing an entropy-driven endothermic process, suggest the involvement of hydrophobic interactions in this rearrangement. Three amino acids in the basic carboxyl-terminal extension were essential for the interaction: the basic residues Arg111 and Lys116, and the polar Thr125. We also found that other glycosaminoglycan species, corresponding to those produced by human arterial smooth muscle cells, bound to dimeric rA125 and that heparan sulfate showed the highest affinity.

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