The peptidase activity of human serum butyrylcholinesterase: studies using monoclonal antibodies and characterization of the peptidase.
Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90-95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyrylcholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. The pH optimum of the peptidase was in the range of 7.5-9.5 and the divalent cations Co2+, Mn2+, and Zn2+ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co2+, Mn2+, and Zn2+ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited alpha-chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.
Rao
R V
RV
Department of Neurological Sciences, Christian Medical College and Hospital Vellore, India.
Balasubramanian
A S
AS
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D002091:Butyrylcholinesterase / Q000097:blood* / Q000276:immunology; D002614:Chelating Agents / Q000494:pharmacology; D002802:Cholinesterases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D010450:Endopeptidases / Q000097:blood*; D004743:Enkephalin, Leucine / Q000737:chemistry; D004789:Enzyme Activation; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011480:Protease Inhibitors / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D008670:Metals; D011480:Protease Inhibitors; D013438:Sulfhydryl Compounds; D006639:Histidine; D004743:Enkephalin, Leucine; D002091:Butyrylcholinesterase; D002802:Cholinesterases; D010450:Endopeptidases; D000581:Amidohydrolases; C019732:aryl acylamidase