Glycosylation and membrane insertion of newly synthesized rat dopamine β-hydroxylase in a cell-free system without signal cleavage

Zhehui Feng, Ruth Hogue Angeletti, Barry E. Levin, Esther L. Sabban

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Dopamine β-hydroxylase (DBH, EC 1.14.17.1) is present in both membrane-bound and soluble forms in neurosecretory vesicles. This study was designed to investigate the differences between membrane-bound and soluble DBH and how they may arise from translation of a single mRNA. Antisera to a peptide corresponding to the carboxyl terminus of rat DBH was found to specifically immunoprecipitate the 77- and 73-kDa subunits of newly synthesized DBH in rat brain. Thus, both soluble and membrane-bound forms contain the same carboxyl terminus. To investigate differences at the amino terminus, full-length rat DBH mRNA, translated in a cell-free system, produced a 66-kDa peptide. An additional higher molecular mass product was synthesized upon co-translational addition of microsomal membranes. This product was glycosylated since it bound to concanavalin A-Sepharose and reverted to the 66-kDa polypeptide after treatment with endoglycosidase H. This glycosylated product was resistant to protease digestion and fractionated with microsomal membranes on sucrose gradients, indicating that it is incorporated into the microsomal membranes. Amino-terminal sequencing of the glycosylated translation product indicated that the amino-terminal "signal" sequence was not cleaved. The results indicate that in the cell-free system newly synthesized DBH undergoes glycosylation and incorporation into microsomal membranes without cleavage of the NH2-terminal signal sequence.

Original languageEnglish (US)
Pages (from-to)21808-21815
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number30
StatePublished - Oct 25 1992

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Glycosylation
Signal systems
Cell-Free System
Mixed Function Oxygenases
Rats
Dopamine
Membranes
Protein Sorting Signals
Peptides
Dopamine beta-Hydroxylase
Messenger RNA
Glycoside Hydrolases
Molecular mass
Sucrose
Immune Sera
Digestion
Brain
Peptide Hydrolases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Glycosylation and membrane insertion of newly synthesized rat dopamine β-hydroxylase in a cell-free system without signal cleavage. / Feng, Zhehui; Angeletti, Ruth Hogue; Levin, Barry E.; Sabban, Esther L.

In: Journal of Biological Chemistry, Vol. 267, No. 30, 25.10.1992, p. 21808-21815.

Research output: Contribution to journalArticle

Feng, Zhehui ; Angeletti, Ruth Hogue ; Levin, Barry E. ; Sabban, Esther L. / Glycosylation and membrane insertion of newly synthesized rat dopamine β-hydroxylase in a cell-free system without signal cleavage. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 30. pp. 21808-21815.
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