Purification and immunochemical studies of the protein-bound J substance from cattle serum

J active substance associated with protein was purified from cattle J^cs serum extracted with lipid solvents. Serum residue, after extraction, was deproteinized first by heat coagulation and then by extraction with Genetron. Fractionation of the resulting deproteinized J active material was performed on DEAE-Sephadex, and the most active fraction eluted from this column with 0.15 M NaCl, was finally purified by preparative polycrylamide gel electrophoresis. Purified J active material exhibited a single band on analytical polyacrylamide gel, and was composed of protein, carbohydrate, long-chain base, phosphorus, glycerol and fatty acid. This purified protein-associated J substance is eight fold less active than the previously described J lipid when tested by hemolysis-inhibition assay with the J system. The decrease in activity parallels the decrease in phosphosphingoglycolipid content in the protein-associated J substance. The capacity to inhibit hemolysis (J system) was lost when the substance was subjected to mild alkaline methanolysis, periodate-oxidation or action by phospholipase C. Enzymatic desialytion did not affect the activity of the protein-bound J substance. Neither the J active lipid nor the protein-associated substance inhibited agglutination in the human A system.