Expression and tissue-specific assembly of human butyrylcholine esterase in microinjected Xenopus laevis oocytes.
Cholinesterases represent a ubiquitous, polymorphic family of acetylcholine hydrolyzing enzymes. The multileveled tissue-specific heterogeneity which characterizes these enzymes makes the cholinesterases an appropriate model for studying the mechanisms involved in regulating divergent pathways in protein biogenesis. For this purpose, a cDNA coding for human butyrylcholine esterase (BuChE) was subcloned into the SP 6 transcription vector. Synthetic mRNA transcribed from this construct was microninjected into Xenopus laevis oocytes alone, and in conjunction with poly(A)+ RNAs extracted from human brain or muscle. Injected BuChE-mRNA induced the biosynthesis of a protein exhibiting the catalytic activity, substrate specificity, and sensitivity to selective inhibitors characteristic of native human serum BuChE, and clearly distinct from the related enzyme acetylcholinesterase (AChE). The nascent BuChE was reproducibly distributed into low salt-soluble and detergent-extractable pools. Sucrose gradient analysis demonstrated that the nascent human enzyme was capable of limited subunit assembly, appearing as functional dimeric molecules in both of these fractions. Co-injection with brain or muscle-derived mRNAs facilitated higher order oligomeric assembly. Co-injected brain mRNA induced the appearance of tetramers while co-injected muscle mRNA induced the appearance of an array of heavy molecular forms, including a heavy 16 S form. These results indicate that the molecular determinants which distinguish BuChE from AChE are inherent to its primary amino acid sequence and that additional, tissue-specific protein(s) are involved in the modulation of subunit assembly within particular biological milieues.
Soreq
H
H
Department of Biological Chemistry, Hebrew University, Jerusalem, Israel.
Seidman
S
S
Dreyfus
P A
PA
Zevin-Sonkin
D
D
Zakut
H
H
D000818:Animals; D002091:Butyrylcholinesterase / Q000235:genetics* / Q000378:metabolism; D002802:Cholinesterases / Q000235:genetics*; D004247:DNA / Q000235:genetics; D005260:Female; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics*; D015183:Restriction Mapping; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic; D014982:Xenopus laevis
D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA; D002091:Butyrylcholinesterase; D002802:Cholinesterases