Isolation and characterization of cancer procoagulant: a cysteine proteinase from malignant tissue.
Cancer procoagulant, a proteolytic procoagulant enzyme, has been purified from rabbit V2 carcinoma extracts by two procedures. In the first, the protein was purified by benzamidine--Sepharose affinity chromatography, gel filtration chromatography, and phenyl-Sepharose hydrophobic chromatography. Antiserum was raised against the purified protein and was used to prepare an immunoadsorbent column. In the second, tumor extracts were purified by immunoaffinity chromatography followed by p-(chloromercuri)benzoate affinity chromatography. The second procedure was substantially quicker and easier. The final product of both procedures was homogeneous on the basis of analytical sodium dodecyl sulfate--polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weight was 68 000 and the isoelectric point 4.8. The proteinase activity of cancer procoagulant directly activated factor X, in the absence of factor VII, and was inhibited by 1 mM iodoacetamide and 0.1 mM mercury which are classic cysteine proteinase inhibitors. A carbohydrate analysis showed less than 1 mol of hexose or sialic acid/mol of protein. The amino acid analysis showed that serine (19.1%), glycine (18.77%), and glutamic acid (12.5%) were the prevalent amino acids. The amino acid composition of cancer procoagulant was substantially different than other known factor X activating proteinases or other cysteine proteinases including cathepsin B.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000936:Antigen-Antibody Complex; D001779:Blood Coagulation Factors / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007106:Immune Sera; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D009363:Neoplasm Proteins; D009374:Neoplasms, Experimental / Q000201:enzymology*; D011817:Rabbits
D000596:Amino Acids; D000936:Antigen-Antibody Complex; D001779:Blood Coagulation Factors; D007106:Immune Sera; D009363:Neoplasm Proteins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant