Molecular cloning, structure, and expression of dopamine-beta-hydroxylase from bovine adrenal medulla.
Dopamine-beta-hydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine to norepinephrine, remains the topic of many unanswered questions. We isolated DBH cDNA clones from a bovine adrenal medulla cDNA library in the vector lambda gt10. The longest cDNA had an open reading frame encoding an entire mature DBH 578 amino acid (64,808 dalton) polypeptide chain, though lacking a portion of the signal peptide. Additional 5' clones, obtained by the polymerase chain reaction, established the sequence of a 19 amino acid signal peptide. The mature protein sequence was 84% homologous to that of human pheochromocytoma DBH, including preservation of four potential copper ligand sites (HH or HXH) and substrate binding domains. There were no hydrophobic (putative membrane spanning) domains, other than the signal peptide. All available DBH peptide and protein sequence data can be accounted for by the cDNA-deduced 578 amino acid mature protein primary structure. Prokaryotic DBH expression yielded a 65-kilodalton DBH-immunoreactive peptide that differed from eukaryotic adrenal DBH only in N-linked, endoglycosidase F-sensitive glycosylation in the latter. Southern analysis suggested one DBH gene, whereas Northern analysis suggested a single 2.6 kbp tissue-specific DBH transcript. Comparison of the DBH primary structure with other reported sequences [Protein Identification Resource (PIR), New Atlas (NEWAT)] did not indicate that DBH is a member of any known gene family. The results suggest that a single DBH gene encodes a message specifying a single DBH polypeptide chain.
Department of Medicine, University of California, San Diego.
H J
HJ
Wu
R J
RJ
Parmer
A H
AH
Koop
D J
DJ
Rozansky
D T
DT
O'Connor
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D004299:Dopamine beta-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D012313:RNA / Q000032:analysis
D012313:RNA; D004247:DNA; D004299:Dopamine beta-Hydroxylase