The use of mRNA translationin vitro andin ovo followed by crossed immunoelectrophoretic autoradiography to study the biosynthesis of human cholinesterases

Abstract

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.

Biochemical and molecular studies using human autopsy brain tissue

“…Such translations are highly dependent on the integrity of the 5¢-terminal cap structure and the 3¢ untranslated region (Guhaniyogi and Brewer 2002;Mitchell and Tollervey 2002). Microinjection of Xenopus oocytes has been used successfully to translate mRNA transcripts extracted from human brain (Soreq et al 1986). Polysomes from autopsied frontal cortex and cerebellum retain physical and molecular integrity on sucrose-density-gradient analysis Langstrom et al 1989).…”

How Important Are Brain Banks for Alcohol Research?

“…Specific mRNAs from frozen autopsy tissue retain sufficient integrity to be used as templates for protein synthesis in vitro. Microinjection of Xenopus oocytes has been used successfully to translate mRNA transcripts extracted from human brain (Soreq et al, 1986). Polysomes from autopsied frontal cortex and cerebellum retain physical and molecular integrity, and neither their size nor translational efficiency depends on age, PMI, or agonal state (Langstrom et al, 1989;Marotta et al, 1981).…”

Entwicklungen der Pharmakogenetik — Ein Rückblick zum 75. Geburtstag von Hans Herken

“…Es stellt sich heraus, dab die ,,esteratic site" der Plasma-Cholinesterase und der neurogenen Azetylcholinesterase fast identisch sind [18]; die beiden Cholinesterasen sind weitgehend homolog, selbst wenn sie vom Menschen und vom elektrischen Aal kommen. Wahrscheinlich sind Plasmacholinesterase und Azetylcholinesterase verschiedene Schnittstticke aus dem gleichen, groBen Vorl/iuferProtein [20]. Die atypische Variante der Plasmacholinesterase muB auf einer Mutation in der ,, anionic site" beruhen, die aber noch nicht identifiziert ist.…”