Phosphorylation of human fibrinogen in vitro with protein kinase C: characterization of the phosphorylated sites |
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| Authors: | P Heldin E Humble |
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| Affiliation: | 1. Ghent University, Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, Salisburylaan 133, B-9820 Merelbeke, Belgium;2. Institute of Agricultural and Fisheries Research (ILVO), Animal Sciences Unit, Scheldeweg 68, B-9090 Melle, Belgium;3. KU Leuven University, Science, Engineering and Technology Group, Department of Biosystems (BIOSYST), Division of Gene Technology, Kasteelpark Arenberg 21, B-3001 Heverlee, Belgium;1. Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan;2. Department of Biomedical Laboratory Sciences, Shinshu University School of Medicine, Matsumoto, Japan;3. Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan;4. Department of General Pediatrics, Nagano Children''s Hospital, Azumino, Japan;5. Faculty of Health and Medical Sciences, Department of Medical Technology and Clinical Engineering, Hokuriku University, Kanazawa, Japan;1. Institute of Biochemistry and Clinical Biochemistry, Catholic University of the Sacred Heart, L.go F. Vito 1, 00168 Rome, Italy;2. Department of Laboratory Medicine, "Policlinico Gemelli" Foundation, L.go A. Gemelli 1, 00168 Rome, Italy;3. PICU, Department of Anesthesiology and Critical Care, Catholic University of the Sacred Heart, L.go F. Vito 1, 00168 Rome, Italy;4. APHP, South Paris University Hospitals, Medical Center "A. Beclere", Paris, France;5. Institute of Chemistry of Molecular Recognition - CNR, c/o Catholic University of the Sacred Heart, L.go F. Vito 1, 00168 Rome, Italy;3. From the Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews KY16 9ST, Scotland,;4. the School of Medicine, University of St. Andrews, St. Andrews KY16 9TF, Scotland, and;5. The Advanced Centre for Biochemical Engineering, University College London, London WC1E 7JE, United Kingdom |
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| Abstract: | Phosphorylation of human fibrinogen in vitro by incubation with [gamma-32P]ATP and protein kinase C purified from pig spleen, led to incorporation of [32P]phosphate at serine residues located in the A alpha-chain. In order to identify the residues that were phosphorylated, the A alpha-chain of fibrinogen was isolated and subjected to consecutive cleavage by cyanogen bromide, trypsin, and chymotrypsin. The resulting radioactive phosphopeptides were purified by gel chromatography and high-performance liquid chromatography using a reversed-phase column. Subsequent amino acid analysis and manual Edman degradation of the purified phosphopeptides revealed that Ser557, Ser558, Ser559, and Ser599 were phosphorylated. These serine residues are located in the carboxy-terminal part of the A alpha-chain. This region also contains lysine residues participating in the cross-linking of fibrin and, possibly, a site involved in the binding of fibrinogen to receptors on platelets. In addition, peptides derived from the middle section of the polypeptide chain were found to contain [32P]phosphate; in these cases, however, the exact localization of the phosphate could not be determined, due to the low yield of radioactivity. Two glutamine residues, Gln328 and Gln366, in this portion of the A alpha-chain take part in the cross-linking of fibrin. |
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