PMID- 9116013
OWN - NLM
STAT- MEDLINE
DCOM- 19970421
LR  - 20131121
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 36
IP  - 11
DP  - 1997 Mar 18
TI  - The photoactivatable inhibitor 7-azido-8-iodoketanserin labels the N terminus of 
      the vesicular monoamine transporter from bovine chromaffin granules.
PG  - 3345-52
AB  - In monoaminergic cells, the hormone or neurotransmitter is concentrated into 
      secretory vesicles by a tetrabenazine- and reserpine-sensitive vesicular 
      monoamine transporter (VMAT), catalyzing a H+/monoamine antiport. Ketanserin is 
      another powerful inhibitor of VMAT that binds to the tetrabenazine binding site. 
      A photoactivatable derivative, 7-azido-8-iodoketanserin (AZIK), labels covalently 
      the transporter from bovine chromaffin granules, VMAT-2. Digestion with 
      endoproteinases V8 or Lys-C, which cleave peptide bonds at acidic or lysine 
      residues, respectively, revealed that the AZIK label is located in a 7 kDa 
      segment of the VMAT-2 polypeptide. The photolabeled chromaffin granule 
      transporter was purified by DEAE and WGA chromatography followed by selective 
      aggregation and size-exclusion HPLC. After treatment by V8 or Lys-C, digestion 
      products were separated by electrophoresis in SDS and sequenced. For both 
      enzymes, the material comigrating with the labeled peptide produced a sequence 
      matching the N terminus of VMAT-2. A K55E mutant of the bovine VMAT-2 cDNA was 
      constructed and expressed in COS-7 cells. The mutant protein exhibited a full 
      VMAT activity and could be labeled by AZIK. However, the formation of the 7 kDa 
      labeled peptide upon Lys-C proteolysis was prevented in the mutant, with a 
      redistribution of the label in higher-molecular mass digestion products. The 
      localization of the label upstream of lysine 55 was confirmed by an immunological 
      and enzymatic analysis. We conclude that the segment 2-55 of bovine VMAT-2, which 
      encompasses the cytosolic N terminus and the first transmembrane segment in the 
      current topological model of the transporter, contains residues involved in the 
      binding of ketanserin and, possibly, tetrabenazine.
FAU - Sagne, C
AU  - Sagne C
AD  - CNRS UPR 9071, Institut de Biologie Physico-Chimique, Paris, France.
FAU - Isambert, M F
AU  - Isambert MF
FAU - Vandekerckhove, J
AU  - Vandekerckhove J
FAU - Henry, J P
AU  - Henry JP
FAU - Gasnier, B
AU  - Gasnier B
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Affinity Labels)
RN  - 0 (Azides)
RN  - 0 (DNA Primers)
RN  - 0 (Iodine Radioisotopes)
RN  - 0 (Membrane Glycoproteins)
RN  - 0 (Membrane Transport Proteins)
RN  - 0 (Neuropeptides)
RN  - 0 (Neurotransmitter Agents)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Vesicular Biogenic Amine Transport Proteins)
RN  - 0 (Vesicular Monoamine Transport Proteins)
RN  - 136769-36-3 (azidoiodoketanserin)
RN  - 97F9DE4CT4 (Ketanserin)
SB  - IM
MH  - Adrenal Medulla/*metabolism
MH  - Affinity Labels
MH  - Amino Acid Sequence
MH  - Animals
MH  - Azides/*metabolism
MH  - Binding Sites
MH  - COS Cells
MH  - Cattle
MH  - Chromaffin Granules/*metabolism
MH  - Cloning, Molecular
MH  - DNA Primers
MH  - Intracellular Membranes/metabolism
MH  - Iodine Radioisotopes
MH  - Ketanserin/*analogs & derivatives/metabolism
MH  - Membrane Glycoproteins/chemistry/isolation & purification/*metabolism
MH  - *Membrane Transport Proteins
MH  - Mutagenesis, Site-Directed
MH  - *Neuropeptides
MH  - Neurotransmitter Agents/metabolism
MH  - Polymerase Chain Reaction
MH  - Recombinant Proteins/chemistry/isolation & purification/metabolism
MH  - Transfection
MH  - Vesicular Biogenic Amine Transport Proteins
MH  - Vesicular Monoamine Transport Proteins
EDAT- 1997/03/18 00:00
MHDA- 1997/03/18 00:01
CRDT- 1997/03/18 00:00
PHST- 1997/03/18 00:00 [pubmed]
PHST- 1997/03/18 00:01 [medline]
PHST- 1997/03/18 00:00 [entrez]
AID - bi9623439 [pii]
AID - 10.1021/bi9623439 [doi]
PST - ppublish
SO  - Biochemistry. 1997 Mar 18;36(11):3345-52. doi: 10.1021/bi9623439.