Isolation and characterization of a murine serum esterase which hydrolyzes a tumor promoter, 12-O-tetradecanoyl phorbol 13-acetate.
An enzyme which catalyzes the following esterase reaction was isolated from mouse serum: 12-O-tetradecanoyl phorbol 13-acetate (TPA) + H2O----phorbol 13-acetate + tetradecanoic acid. The recovery was 0.18% of total serum protein and 820-fold purification was achieved. The enzyme is composed of a single polypeptide chain with sugar moiety; its molecular weight was estimated to be 77,000. Its sugar content is 15%, the isoelectric point was 4.3, and the alpha-helix content was 15.3% . The activity is stable between pH 5 and 9 under 40 degrees C; it is insensitive to 2-mercaptoethanol and is not dependent on divalent cations. The optimal pH is around 7.5. The apparent Km for TPA is 6.6 X 10(-7)M. The hydrolysis of [3H]TPA is inhibited by phorbol diesters and phorbol 12-myristate, but not by phorbol and phorbol 13-acetate. The activity is inhibited to some extent by phosphatidylcholine, cholesterol, and lanosterol, but not by free fatty acids, fatty acid esters of glycerol, cholesterol esters, or cholestanol. The enzyme hydrolyzes ester linkages, but not peptide linkages of synthetic substrates. Esterase inhibitors and serine-reactive reagents affect the activity. Although sera from rodents displayed strong activity, such activity was not detected in human serum. Unlike lipoprotein lipase, the serum enzyme activity was not enhanced by treatment of the animal with heparin. These characteristics and the amino acid composition do not agree with any of the reported characteristics of known serum enzymes with esterase activity.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004950:Esterases / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D006868:Hydrolysis; D007122:Immunoelectrophoresis; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008970:Molecular Weight; D010704:Phorbols / Q000378:metabolism*; D013045:Species Specificity; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism*
D000596:Amino Acids; D010704:Phorbols; D004950:Esterases; D013755:Tetradecanoylphorbol Acetate