Contamination of highly purified human serum cholinesterase by dipeptidyl peptidase IV causing hydrolysis of substance P.
The purification and kinetic characterization of cholinesterase from blood plasma (pseudocholinesterase; butyrylcholinesterase: EC 3.1.1.8) is described. The hydrolysis of the artificial peptide substrate Lys-Pro-p-nitroanilide served as a model of the second step in degradation of substance P by dipeptidyl peptidase IV. The substrate is hydrolyzed by a gel-electrophoretic homogeneous cholinesterase preparation with a reaction rate of 5.8 mumol/min X mg and a KM value of 0.12 mmol/l. The proteolytic reaction could not be affected with typical cholinesterase inhibitors NaF and dibucain. On the other hand Lys (pNO2-Z)-Pro and a specific suicide substrate (diacylhydroxylamine derivative) inhibit the activity in a manner analogous to dipeptidyl peptidase IV. Though these active site-directed inhibitors also influenced the benzoylcholine hydrolyzing activity of serum cholinesterase, we conclude from the data that dipeptidyl peptidase IV was the true Lys-Pro-p-nitroanilide cleaving activity. Furthermore, the conclusion can also be drawn that hydrolysis of substance P reported by Lockridge 1982 is caused by the contamination that cannot be completely separated from the esterase during the purification method used.
D002802:Cholinesterases / Q000097:blood* / Q000302:isolation & purification; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000097:blood*; D004340:Drug Contamination; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D013373:Substance P / Q000032:analysis*; D013379:Substrate Specificity
D013373:Substance P; D002802:Cholinesterases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4