Abstract
Platelet-derived growth factor (PDGF)-BB is a dimeric protein held together by two disulfide bonds involving the 2nd and 4th cysteine residues from the NH2 terminus. To localize the three intrachain disulfide bonds in PDGF, a method was devised that made it possible to cleave PDGF at specific sites. A set of PDGF derivatives in which specific amino acids were mutated to methionine residues was generated. The recombinant proteins, immunoprecipitated from metabolically labeled transfected COS cells, were then subjected to CNBr cleavage and analyzed by SDS-gel electrophoresis under nonreducing conditions. Based on whether the mutated proteins remained in one piece or fell apart after CNBr cleavage, it was possible to deduce the disulfide bond arrangement in the PDGF B-chain; one bond involves the 1st and 6th cysteine residues, another the 3rd and 7th, and the last the 5th and 8th. The latter disulfide bond was found to be dispensable for receptor binding, whereas the former two were found to be essential for the correct folding or stability of the PDGF B-chain.
Highlights
Platelet-derived growth factor (PDGF) with retained receptor binding activity [12]
Platelet-derived growth factor (PDGF)’ is a potentmitogen for connective tissue cells and occurs as homo- or heterodimers of disulfide-bonded A- and B-polypeptide chains(reviewed in Refs. 1 and 2)
Receptor dimerization leads to autophosphorylation of the receptor molecules, which occurs in tram in the receptor complex [6]
Summary
S csc c eluted by the addition of200 pl of nonreducing sample buffer and incubation a t 95 "Cfor 3 min. Half of the eluted material was reduced by the addition of dithiothreitol to a final concentration of 10 mM,. +s csc c followed by incubation at 95 "C for 2 min, and was alkylated by the addition of iodoacetamide to a final concentrationof 50 mM.
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