Isolation of a novel 45 kDa plasminogen receptor from human endothelial cells.
We have previously identified an endothelial cell membrane protein of M(r) 45 kDa that binds plasminogen in a kringle-dependent, specific and reversible manner (Dudani et. al. (1991) Mol. Cell. Biochem. 108: 133-139). In this study, we have developed and optimized a protocol for the isolation of the 45 kDa plasminogen receptor from venous endothelial cells using a four step procedure consisting of lysis and detergent extraction followed by ligand affinity chromatography and preparative polyacrylamide gel electrophoresis. Control experiments were carried out using BSA-Sepharose instead of plasminogen-Sepharose as the affinity matrix. No plasminogen binding proteins were recovered from the former columns. However, a 45 kDa protein was recovered from lysine eluates of plasminogen-Sepharose. This material was then purified to homogeneity using preoperative electrophoresis. Analyses of proteins at various steps in the purification by SDS-PAGE showed enrichment of a band of 45 kDa which superimposed with the observed binding activity of plasminogen in ligand blots. The above binding could be inhibited by excess lysine. The 45 kDa protein could be distinguished from alpha-enolase which also binds plasminogen by: (i) significant differences in the profile of retention times of CNBr-degradation fragments on reversed phase HPLC; and (ii) partial peptide sequencing of one of the CNBr-degradation fragments of the 45 kDa protein. Moreover, the derived sequence did not show any significant homology to any protein in the Swiss Prot (release 20) database. We thus propose that the 45 kDa protein represents a novel plasminogen receptor on human venous endothelial cells.
Dudani
A K
AK
Department of Medicine, University of Ottawa, Ontario, Canada.
Cummings
C
C
Hashemi
S
S
Ganz
P R
PR
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003902:Detergents / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000648:ultrastructure*; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D055293:Receptors, Urokinase Plasminogen Activator; D012995:Solubility
D003902:Detergents; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D010958:Plasminogen; D010751:Phosphopyruvate Hydratase