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Neuroactive drugs inhibit trypsin and outer membrane protein processing in Escherichia coli K-12
Abstract:
Previous studies demonstrated that a cloned 2-megadalton (MDal) fragment of Escherichia coli DNA contained the structural gene for major outer membrane protein a (also known as 3b or M2 (40 kDal). The present study demonstrates that M2 is synthesized from a 42-kDal precursor that also is present in the outer membrane. The conversion of the 42-kDal precursor to M2 is inhibited by a number of different local anesthetics (procaine, piperocaine, lidocaine, cocaine), by the neuroactive drug atropine, and by the classical trypsin inhibitors N alpha-tosyllysine chloromethyl ketone (TLCK) and benzamidine. Our kinetic studies demonstrate that the amidase action of pure trypsin is inhibited competitively by the local anesthetics tested (excluding lidocaine) as well as by atropine and neostigmine. A mechanism of action for local anesthetics as well as atropine in E. coli may to be inhibit trypsinlike proteases, in a competitive manner, in the region of the outer membrane. The mechanism of action of these compounds in regulating nerve conduction in man have certain features in common with the mechanism proposed in E. coli.
Insights
Local anesthetics and atropine inhibit the conversion of a major outer membrane protein precursor to its mature form in Escherichia coli. This suggests a shared mechanism of action involving trypsin-like proteases in both bacteria and human nerve conduction.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Escherichia coli outer membrane protein a (M2) is essential.
- M2 is synthesized from a precursor protein.
Purpose of the Study:
- Investigate the mechanism of M2 maturation.
- Determine if local anesthetics and other drugs affect M2 synthesis.
- Explore potential shared mechanisms between bacterial and human cellular processes.
Main Methods:
- In vitro studies using cloned DNA fragments.
- Analysis of protein precursor conversion.
- Kinetic studies of enzyme inhibition.
- Competitive inhibition assays with trypsin.
Main Results:
- M2 is synthesized from a 42-kDa precursor in the outer membrane.
- Local anesthetics (procaine, piperocaine, cocaine), atropine, TLCK, and benzamidine inhibit precursor conversion.
- Trypsin-like amidase activity is competitively inhibited by local anesthetics (excluding lidocaine), atropine, and neostigmine.
Conclusions:
- Local anesthetics and atropine likely inhibit trypsin-like proteases in the E. coli outer membrane.
- This inhibition affects M2 maturation.
- The mechanism may share similarities with how these drugs affect nerve conduction in humans.