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Isolation and purification of N-formylmethionine aminopeptidase from rat intestine
R M Sherriff1, M F Broom, V S Chadwick
1Department of Experimental Medicine, Wellcome Medical Research Institute, University of Otago Medical School, Dunedin, New Zealand.
Abstract:
The intestinal mucosal epithelium is exposed to products of intestinal bacteria including potent inflammatory N-formylmethionyl oligopeptides. An N-formylmethionine aminopeptidase has been purified 2300-fold from rat intestine and was shown to degrade natural fMet oligopeptides from Escherichia coli culture supernatants with loss of bioactivity (release of specific granule constituents from human polymorphonuclear leucocytes) and immuno-reactivity (assessed using a polyclonal anti-fMet-Leu-Phe antiserum). The enzyme which was specific for N-terminal acyl-methionine residues had a native Mr of 340,000 and comprised four sub-units of Mr 82,000. The presence of this enzyme in intestinal mucosa could prevent absorption of intact bioactive fMet peptides produced by commensal bacteria in the gut lumen.
Insights
Researchers purified an N-formylmethionine aminopeptidase from rat intestine that degrades bacterial peptides. This enzyme
Area of Science:
- Gastroenterology
- Microbiology
- Enzymology
Background:
- Intestinal mucosal epithelium encounters bacterial products, including inflammatory N-formylmethionyl oligopeptides.
- These peptides, derived from bacterial sources like Escherichia coli, can trigger immune responses.
Purpose of the Study:
- To purify and characterize an enzyme from rat intestine capable of degrading N-formylmethionyl oligopeptides.
- To investigate the enzyme's role in mitigating the bioactivity and immunogenicity of bacterial peptides in the gut.
Main Methods:
- Purification of N-formylmethionine aminopeptidase from rat intestinal mucosa.
- Assay of enzyme activity using natural fMet oligopeptides from E. coli.
- Assessment of bioactivity loss by measuring release of granule constituents from human polymorphonuclear leucocytes.
- Evaluation of immuno-reactivity using a polyclonal anti-fMet-Leu-Phe antiserum.
- Determination of enzyme subunit composition and molecular weight.
Main Results:
- An N-formylmethionine aminopeptidase was purified 2300-fold from rat intestine.
- The purified enzyme degraded natural fMet oligopeptides from E. coli, reducing their bioactivity and immuno-reactivity.
- The enzyme is specific for N-terminal acyl-methionine residues.
- The enzyme has a native molecular weight of 340,000 Da and consists of four 82,000 Da subunits.
Conclusions:
- The presence of N-formylmethionine aminopeptidase in the intestinal mucosa can prevent the absorption of intact bioactive N-formylmethionyl peptides produced by gut bacteria.
- This enzyme plays a crucial role in modulating the host-microbe interaction within the intestinal lumen.