Secreted Protein Acidic and Risch in Cysteine (SPARC) is an extracellular protein that functions as a counteradhesive protein and as a potential regulator of angiogenesis. The hallmark activity attributed to SPARC is that its presence prevents the spreading of freshly-plated endothelial cells in vitro. This metallo- and glycoprotein binds to endothelial cells (Kd - 10-9M) through a complex mechanism that is Ca+2-dependent and involves the cooperative interaction of residues from the Cys and His-rich, cationic 2.1 domain with residues fromthe C-terminal Ca+2-binding EF-hand 4.2 domain. The known molecular response is the increased phosphorylation of Tyr residues of the focal adhesion proteins p130CRS and paxillin. The end result consists of disintegration of focal adhesion positive cells that are positive for au-containing integrin complexes, while the F-actin cytoskeleton is redistributed and vinculin is lostfrom plaques. A SPARC-affinity resin has been used to isolate a 60-64 kDa protein from bovine aortic endothelial cell membranes. A determination of the protein content of this sample withbicinchoninic acid indicated a yield of 12 micrograms from 3 x 108 cells. This protein(s) appeared as a doublet in SDS-PAGE. After electrotransfer of this protein(s) to a polyvinylidene fluoride membrane, we subjected the sample to Edman degradation. The results of this sequencing attempt indicated a blocked N-terminus. Significant amounts of this protein(s) were found to remain in the polyacrylamide gel after transfer. We have conservatively estimated that 3 micrograms (50 picomoles) are currently contained within the gel, as visualized by staining withCoomassie Brilliant Blue R250. We have submitted this gel to your facility for characterization by mass spectrometry because (1) we do not have suffiecient amounts of this sample to digest these samples with proteinases and to resequence by Edman degradation, and (2) we do not have the tools in our laboratory for sensitive peptide chromatography (e.g. microbore HPLC columns). It is our hope that the UCSF Mass Spectrometry Facility will assist our research goals in identifying this sample so that we can (1) design oligonucleotide primers for the cloning of cDNAs by the polymerase chain reaction, and (2) generate antibodies specific for this protein(s) for developmental characterization in chicken and mouse embryos.
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