Abstract
A novel protein has been purified from fetal calf serum and from serum-free bovine aortic endothelial cell conditioned culture medium. This protein consists of a single polypeptide chain of reduced Mr 70,000 (70K protein) and was separated from bovine serum albumin and other proteins by ion-exchange chromatography and immunoabsorption on Sepharose-coupled anti-70K protein antiserum. The 70K protein was shown to be structurally and immunologically distinct from bovine serum albumin, alpha-fetoprotein, and vitronectin by one- and two-dimensional peptide mapping, amino acid analysis, and enzyme-linked immunosorbent assay and/or immunoblotting. The 70K protein was located in endothelial cell cytoplasmic granules of irregular size and distribution. Metabolic radiolabeling studies showed that the 70K protein was not a biosynthetic product of these cells; its cytoplasmic location was due to a selective uptake from the fetal calf serum in which the cells were initially grown. After subconfluent cultures of endothelial cells were shifted to serum-free medium, nearly 80% of the total 70K protein that was measurable in the medium was released between 0 and 20 min. Moreover, sparse, rapidly proliferating cells released approximately 18-fold more 70K protein within 2 min as compared to dense, nonproliferating cultures. The concentration of 70K protein in fetal calf serum was estimated to be 400-600 micrograms/ml. Proliferating bovine aortic endothelial cells, 24 h after plating at an intermediate density, released approximately 250 pg of 70K protein/cell within the first 20 min after exposure to serum-free conditions. The data provide evidence for a novel protein in serum which is selectively internalized by endothelial cells in vitro and which in turn is released rapidly under conditions such as osmotic imbalance due to serum removal, or during periods of cellular proliferation, conditions which we term "culture shock."
Highlights
A novel protein has been purified from fetal calf designed to maintain the several morphologicallyand biosynserum and from serum-freebovine aortic endothelial thetically unique phenotypes during i n vitro propagation
We propose that theselective uptake and rapid release of this novel 70K protein from BAE cells in vitro is indicative of culture shock and concomitant cellular injury
When dialyzed BAE cell-conditioned medium was passed over this column, the major qpecies that bound was neither 43K protein nor bovine serum albumin (BSA), but a protein of apparent reduced M, 70,000 that exhibitedaslightly lower mobility than BSA (Mr67,000-68,000) on SDS-PAGE
Summary
A novel protein has been purified from fetal calf designed to maintain the several morphologicallyand biosynserum and from serum-freebovine aortic endothelial thetically unique phenotypes during i n vitro propagation That certain of a single polypeptide chain of reduced M, 70,000 metabolic properties characteristicof the endothelium become (70K protein) and was separated from bovine serum significantly altered asa result of subcultivation [3]. Quantitativemeasurement of the endocytotic of the total 70K protein that was measurable in the rate in bovine aortic endothelial (BAEl) cells in vitro showed medium was released between 0 and 20 min. Significant increases in growing as compared to quiescent sparse, rapidly proliferating cells released approximately 18-fold more 70Kproteinwithin 2 min as compared to dense, nonproliferating cultures
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