PMID- 9230048
OWN - NLM
STAT- MEDLINE
DCOM- 19970808
LR  - 20191210
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 36
IP  - 30
DP  - 1997 Jul 29
TI  - Inhibition of preprotein translocation and reversion of the membrane inserted 
      state of SecA by a carboxyl terminus binding mAb.
PG  - 9159-68
AB  - SecA is the peripheral subunit of the preprotein translocase of Escherichia coli. 
      SecA consists of two independently folding domains, i.e., the N-domain bearing 
      the high-affinity nucleotide binding site (NBS-I) and the C-domain that harbors 
      the low-affinity NBS-II. ATP induces SecA insertion into the membrane during 
      preprotein translocation. Domain-specific monoclonal antibodies (mAbs) were 
      developed to analyze the functions of the SecA domains in preprotein 
      translocation. The antigen binding sites of the obtained mAbs were confined to 
      five epitopes. One of the mAbs, i.e., mAb 300-1K5, recognizes an epitope in the 
      C-domain in a region that has been implicated in membrane insertion. This mAb, 
      either as IgG or as Fab, completely inhibits in vitro proOmpA translocation and 
      SecA translocation ATPase activity. It prevents SecA membrane insertion and, more 
      strikingly, reverses membrane insertion and promotes the release of SecA from the 
      membrane. Surface plasmon resonance measurements demonstrate that the mAb 
      recognizes the ADP- and the AMP-PNP-bound state of SecA either free in solution 
      or bound at the membrane at the SecYEG protein. It is concluded that the mAb 
      actively reverses a conformation essential for membrane insertion of SecA. The 
      other mAbs directed to various epitopes in the N-domain were found to be without 
      effect, although all bind the native SecA. These results demonstrate that the 
      C-domain plays an important role in the SecA membrane insertion, providing 
      further evidence that this process is needed for preprotein translocation.
FAU - den Blaauwen, T
AU  - den Blaauwen T
AD  - Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology 
      Institute, University of Groningen, The Netherlands.
FAU - de Wit, J G
AU  - de Wit JG
FAU - Gosker, H
AU  - Gosker H
FAU - van der Does, C
AU  - van der Does C
FAU - Breukink, E J
AU  - Breukink EJ
FAU - de Leij, L
AU  - de Leij L
FAU - Driessen, A J
AU  - Driessen AJ
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Bacterial Proteins)
RN  - 0 (Escherichia coli Proteins)
RN  - 0 (Membrane Proteins)
RN  - 0 (Membrane Transport Proteins)
RN  - 0 (Protein Precursors)
RN  - 0 (SEC Translocation Channels)
RN  - EC 3.6.1.- (Adenosine Triphosphatases)
RN  - EC 7.4.2.4 (SecA Proteins)
SB  - IM
MH  - Adenosine Triphosphatases/*antagonists & inhibitors/immunology/metabolism
MH  - Antibodies, Monoclonal/biosynthesis/metabolism/*pharmacology
MH  - Bacterial Proteins/*antagonists & inhibitors/immunology/metabolism
MH  - *Binding Sites, Antibody
MH  - Biological Transport/immunology
MH  - Biosensing Techniques
MH  - Epitope Mapping
MH  - Escherichia coli/enzymology
MH  - *Escherichia coli Proteins
MH  - Membrane Proteins/*antagonists & inhibitors/immunology/metabolism
MH  - *Membrane Transport Proteins
MH  - Protein Conformation
MH  - Protein Precursors/*antagonists & inhibitors/immunology/metabolism
MH  - SEC Translocation Channels
MH  - SecA Proteins
MH  - Solubility
EDAT- 1997/07/29 00:00
MHDA- 1997/07/29 00:01
CRDT- 1997/07/29 00:00
PHST- 1997/07/29 00:00 [pubmed]
PHST- 1997/07/29 00:01 [medline]
PHST- 1997/07/29 00:00 [entrez]
AID - bi970344a [pii]
AID - 10.1021/bi970344a [doi]
PST - ppublish
SO  - Biochemistry. 1997 Jul 29;36(30):9159-68. doi: 10.1021/bi970344a.