Abstract
Several proteins from bovine platelet lysate bound to type I collagen immobilized to the beads of formyl derivatives of cellulose. Among these proteins, a protein of about 100,000 daltons was purified to homogeneity by two additional affinity chromatographies, an organomercurial-agarose and a lentil lectin-agarose. This protein consisted of a single polypeptide chain which contains carbohydrate moiety and many intrapolypeptide disulfide bridges. In addition to platelets, this protein was present in plasma and cultured endothelial cells but not in red blood cells, leukocytes, and smooth muscle cells. Furthermore, it was released from platelets upon stimulation by various agonists. The purified 100-kDa protein was labeled with 125I to quantitate its binding to fibrillar type I collagen. The protein specifically bound to fibrillar collagen with the apparent dissociation constant of 5.6 x 10(-8) M for the high affinity site and 5.5 x 10(-7) M for the low affinity site. Analyses of amino acid sequences of both intact and tryptic fragments of this protein revealed that it had strong homology to the propolypeptide of human von Willebrand factor, which is also known as von Willebrand antigen II. Various properties of this protein listed above also strongly suggest that it was indeed the propolypeptide of bovine von Willebrand factor.
Highlights
Several proteins from bovine platelet lysate bound to type I collagen immobilized to the beads of formyl derivatives of cellulose
Formyl-Cellulofineand lentil lectin-agarose were obtained from Seikagaku Kogyo Co. (Tokyo, Japan), organomercurial-agarose (AffiGel501)was from Bio-Rad, and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG was from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD)
Several proteins from bovine platelet lysate bound to the collagen-Cellulofine gel and were eluted by raising the NaCl concentration in thebuffer (Fig. lA).The fraction, which did not bind to thecolumn, lacks somespecific proteins (Fig. l B, compare lanes 1 and 2)
Summary
Several proteins from bovine platelet lysate bound to the collagen-Cellulofine gel and were eluted by raising the NaCl concentration in thebuffer (Fig. lA).The fraction, which did not bind to thecolumn, lacks somespecific proteins (Fig. l B , compare lanes 1 and 2). They have relative molecular weights of 100,000, 93,000, 85,000, 68,000, 54,000, 40,000, and 36,000 under reducing conditions.The 85- and 54-kDa proteins were platelet factor XI11 and actin, respectively. Some of them seemed to bind more tenaciously than others(compare lane 3 with 5). The fraction eluted with 0.2 M NaCl (lane 4 ) was somewhat enriched with a 100-kDa proteiInn. order to purify this 100-kDa protein from the fraction eluted with 0.2 M NaCl, two additional affinitychromatographies were em-
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