Cell-free synthesis of rat and human catechol O-methyltransferase. Insertion of the membrane-bound form into microsomal membranes in vitro.
The protein-coding capacities of rat and human catechol O-methyltransferase (COMT) DNA clones were analysed by in vitro transcription and translation using bacteriophage RNA polymerase and rabbit reticulocyte lysate. Two types of clones corresponding to the structures of human placental cDNA clones were used. The shorter clones, containing the 663-residue open reading frame for the soluble COMT (S-COMT), produced 24-kDa (rat) and 26-kDa (human) polypeptides. Translation of the longer clones, containing 43 (rat) or 50 (human) amino acid amino-terminal extensions to the S-COMT polypeptides, yielded 28-kDa (rat) and 30-kDa (human) putative membrane-bound COMT (MB-COMT) polypeptides as the main products. These clones also yielded low amounts of the S-COMT polypeptides. Labelling time or ionic conditions during translation did not eliminate the shorter products, suggesting translation initiation from the second S-COMT AUG codon. In accordance with this postulation, the relative amount of S-COMT could be affected by changing the translation initiation contexts preceding the first AUG codon. The 28-kDa and 30-kDa products, but not the 24-kDa and 26-kDa products, associated with microsomal membranes cotranslationally, indicating that the amino-terminal extensions were functional signal sequences. However, the presence of membranes did not affect the mobilities of the proteins in SDS/polyacrylamide gels. The MB-COMT polypeptides could not be released from the microsomes by treatments with phospholipase C or alkali and were not protected by the microsomes against proteinase K digestion. These results indicate that MB-COMT synthesized in vitro is an integral membrane protein having an amino-terminal signal-anchor sequence.
Ulmanen
I
I
Orion Corporation, Laboratory of Molecular Genetics, Helsinki, Finland.
Lundström
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002394:Catechol O-Methyltransferase / Q000096:biosynthesis / Q000235:genetics*; D002474:Cell-Free System; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004285:Dogs; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D014176:Protein Biosynthesis; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D004247:DNA; D002394:Catechol O-Methyltransferase