Identification, purification, and characterization of a non-heme lactoperoxidase in bovine milk.
A purification procedure for a protein related to lactoperoxidase devoid of the heme prosthetic group under conditions also yielding enzymatically active lactoperoxidase is described. These two forms were separated from bovine milk according to their respective behaviors on cation exchange. Lactoperoxidase and non-heme lactoperoxidase had the same apparent molecular weight in the denatured (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and native form (velocity sedimentation on sucrose gradient) about 85,000; but unlike lactoperoxidase, non-heme lactoperoxidase was devoid of light absorption properties in the Soret region and of enzyme activity. Lactoperoxidase and non-heme lactoperoxidase contained a similar amount of carbohydrate and gave very similar peptide maps after limited proteolysis by subtilisin or trypsin. The two forms appeared to be immunologically related since they gave a single line in immunodiffusion using anti-lactoperoxidase antibodies and since 125I-labeled non-heme lactoperoxidase and 125I-labeled lactoperoxidase reacted with anti-lactoperoxidase antibodies in radioimmunoassay. Lactoperoxidase and nonheme lactoperoxidase were compared in their ability to interact with diiodotyrosine and tubulin (Rousset, B., and Wolff, J. (1980) J. Biol. Chem. 255, 2514-2523). 125I-labeled diiodotyrosine bound specifically to lactoperoxidase. No detectable binding has been observed with nonheme lactoperoxidase. In contrast, lactoperoxidase and non-heme lactoperoxidase coupled to an insoluble matrix were able to bind rat brain tubulin, indicating that both forms of lactoperoxidase can be used for an affinity chromatography purification procedure of brain tubulin. Non-heme lactoperoxidase was found in milk from several origins, cow, goat, sheep, and human. In bovine milk, lactoperoxidase and non-heme lactoperoxidase were found in comparable amounts.
D000818:Animals; D002417:Cattle; D004105:Diiodotyrosine / Q000378:metabolism; D005260:Female; D006418:Heme; D005779:Immunodiffusion; D007784:Lactoperoxidase / Q000032:analysis*; D008892:Milk / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010544:Peroxidases / Q000032:analysis*; D013053:Spectrophotometry; D013381:Subtilisins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D006418:Heme; D004105:Diiodotyrosine; D007784:Lactoperoxidase; D010544:Peroxidases; D013381:Subtilisins; D014357:Trypsin