The use of mRNA translation in vitro and in ovo followed by crossed immunoelectrophoretic autoradiography to study the biosynthesis of human cholinesterases.
The synthesis of various cholinesterases in different fetal human tissues was studied using in vitro and in ovo translation of poly(A)+ RNA, followed by crossed immunoelectrophoretic autoradiography. When unfractionated poly(A)+ mRNA from fetal brain, muscle, or liver was translated in vitro, in the reticulocyte lysate cell-free system, polypeptides were synthesized which reacted with antibodies against either "true" acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) or "pseudo", butyrylcholinesterase (acylcholine acylhydrolase; EC 3.1.1.8). The two nascent cholinesterases could be separated by crossed immunoelectrophoresis followed by autoradiography, suggesting that acetylcholinesterase and butyrylcholinesterase are produced in all three tissues from nascent polypeptides containing different immunological domains. To examine whether the biosynthesis of cholinesterases includes posttranslational processing events, Xenopus oocytes were microinjected with mRNA from these tissues. Immunoelectrophoretic analysis of oocyte intracellular homogenates and incubation medium revealed various precipitation arcs, reflecting the synthesis and posttranslational processing of multiple forms of tissue-specific exported and intracellular acetylcholinesterase and butyrylcholinesterase. These findings demonstrate that polymorphic cholinesterases are produced from nascent polypeptide products which undergo further posttranslational processing events in a tissue-specific manner before they become mature compartmentalized cholinesterases.
D000110:Acetylcholinesterase / Q000096:biosynthesis*; D000818:Animals; D001345:Autoradiography; D002091:Butyrylcholinesterase / Q000096:biosynthesis*; D002474:Cell-Free System; D002802:Cholinesterases / Q000096:biosynthesis*; D005260:Female; D005333:Fetus / Q000378:metabolism*; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D066298:In Vitro Techniques; D009865:Oocytes / Q000378:metabolism; D009928:Organ Specificity; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D014982:Xenopus laevis
D012333:RNA, Messenger; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase; D002802:Cholinesterases