Localization of the cross-linking site of GPRVVERHK in the gamma-chain of human fibrinogen.
The peptide alpha 17-24-Lys (GPRVVERHK) corresponding to the N-terminus of the alpha chain of fibrin was synthesized and used to localize its binding site in the fibrinogen molecule. The peptide was radioiodinated, incubated with fibrinogen, cross-linked with a bifunctional reagent disuccinimidyl suberate and the resulting product was analyzed in several ways, including plasmin digestion. The binding of the radioactive peptide was mainly to the gamma-chain and was inhibited by unlabelled GPRVVERHK and GPRP. After plasmin digestion, the radioactivity was present in fragment D1 and also in its gamma-chain remnant, but not in fragments D2, D3 or E3. Fragment D1 cross-linked with iodinated GPRVVERHK was purified by affinity chromatography on immobilized anti-fragment D IgG, further digested with plasmin in the presence of EGTA and the peptides were fractionated by reverse-phase HPLC. The amino acid sequence analysis of the radioactive peak revealed the presence of two peptides, gamma 357-373 and GPRVVERHK. It was concluded that the binding site for GPRVVERHK is in the sequence gamma 357-373 which is present in fragment D1 but absent in fragments D2 and D3.
Cierniewski
C S
CS
Department of Biophysics, Medical School of Lodz, Poland.
Budzynski
A Z
AZ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D005340:Fibrinogen