Characterization of a family of high-molecular-weight plasminogen activators secreted by a lung tumor cell line.
In an earlier publication (Harvey, et al. (1982) J. Biol. Chem., 257, 5645-5651) the discovery of a family of unusually large molecules with plasminogen activator activity in the conditioned medium of a human lung cancer cell line was reported. These molecules are related to urokinase (uPA) by functional and immunological criteria. We have now purified two representatives of this glycoprotein family of Mr 900,000 (PA900) and Mr 660,000 (PA660). While these could be fractionated into subspecies exhibiting size and charge differences, reduction yielded in all cases two predominant chains of 70 and 40 kDa, respectively. Since the amino acid composition of the subfractions was identical, we conclude that the heterogeneity is due to demonstrated differences in glycosylation. The amino acid composition of the unreduced species and of the major reduced chains differed from that of 55 kDa uPA. These enzymes are active toward the substrate, plasminogen, as well as toward the uPA-specific synthetic substrate, Spectrozyme UK, and these activities are inhibitable by diisopropylphosphorofluoridate (DFP). Treatment of PA660 with [3H]DFP resulted in the incorporation of 1.4 mol of DFP into 1 mol of enzyme, suggesting the presence of a single active site. The label was quantitatively recovered in a 21 kDa fragment in a reduction experiment. This fragment also demonstrated immunological reactivity with antiurokinase. It is postulated that PA660 is composed of five or six pairs of the 70 and 40 kDa chains, and of a single uPA-like entity. All of these chains are linked by disfulfide bonds. Whether larger portions of uPA are also present in this molecule, is not yet clear. By electron microscopy, PA900 shows a filamentous structure, while PA660 is predominantly globular. The occurrence of large uPA-like activators in extracts of human colon carcinomas that crossreact with monospecific antibody against uPA, is discussed.
Harvey
S R
SR
Department of Experimental Biology, New York State Department of Health, Roswell Park Cancer Institute, Buffalo 14263.
Loftus
J R
JR
Hurley
E L
EL
Ross
M E
ME
Markus
G
G
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D046911:Macromolecular Substances; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000276:immunology
D000596:Amino Acids; D046911:Macromolecular Substances; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator