Antipeptide antibodies confirm the topology of the human norepinephrine transporter

M. Bruss, R. Hammermann, William Stephen Brimijoin, H. Bonisch

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

We have raised polyclonal antibodies (N6-28, L211-226, L371-384, and C590- 607) against peptides corresponding to hydrophilic sequences of the human norepinephrine transporter (hNET). The antisera immunoprecipitated the [35S]Met-labeled hNET. Antiserum L211-226, directed against a sequence of the putative second (large) extracellular loop of hNET, also immunoprecipitated the human dopamine transporter. Antisera N6-28 and C590- 607, raised against a hNET peptide region of the N and the C termini, respectively, recognized a 58-kDa protein from transfected COS-7 cells expressing the hNET. This 58-kDa species represents a functional, glycosylated form of the hNET and not a degradation product. Tunicamycin treatment of transfected COS-7 cells as well as peptide-N-glycosidase F digestion of the transporter converted the 58-kDa species to a 50-kDa form, indicating that the latter represents the hNET core protein. In indirect immunofluorescence studies, our antisera confirmed the originally proposed topology of hNET. Antisera N6-28 and C590-607 detected hNET only in permeabilized cells. In contrast, antisera L211-226 and L371-384 directed against peptide sequences of the second and fourth putative extracellular loop displayed fluorescence signals with the intact cells.

Original languageEnglish (US)
Pages (from-to)9197-9201
Number of pages5
JournalJournal of Biological Chemistry
Volume270
Issue number16
DOIs
StatePublished - 1995
Externally publishedYes

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Norepinephrine Plasma Membrane Transport Proteins
Topology
Antibodies
Immune Sera
COS Cells
Peptides
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Tunicamycin
Dopamine Plasma Membrane Transport Proteins
Indirect Fluorescent Antibody Technique
Digestion
Proteins
Fluorescence
Cells
Degradation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Antipeptide antibodies confirm the topology of the human norepinephrine transporter. / Bruss, M.; Hammermann, R.; Brimijoin, William Stephen; Bonisch, H.

In: Journal of Biological Chemistry, Vol. 270, No. 16, 1995, p. 9197-9201.

Research output: Contribution to journalArticle

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AU - Hammermann, R.

AU - Brimijoin, William Stephen

AU - Bonisch, H.

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