Purification and characterization of a tetrameric alpha-macroglobulin proteinase inhibitor from the gastropod mollusc Biomphalaria glabrata.
The alpha-macroglobulin proteinase inhibitors (alpha Ms) are a family of proteins with the unique ability to inhibit a broad spectrum of proteinases. Whereas monomeric, dimeric and tetrameric alpha Ms have been identified in vertebrates, all invertebrate alpha Ms characterized so far have been dimeric. This paper reports the isolation and characterization of a tetrameric alpha M from the tropical planorbid snail Biomphalaria glabrata. The sequence of 18 amino acids at the N-terminus indicates homology with other alpha Ms. The subunit mass of approx. 200 kDa was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and SDS/PAGE. The quaternary structure was determined by sedimentation equilibrium centrifugation and native pore-limit electrophoresis. Evidence for a thioester is provided by the fact that methylamine treatment prevents the autolytic cleavage of the snail alpha M subunit and results in the release of 4 mol of thiols per mol of snail alpha M. The snail alpha M inhibited the serine proteinase trypsin, the cysteine proteinase bromelain and the metalloproteinase thermolysin. The spectrum of proteinases inhibited, together with the demonstration of steric protection of the proteinase active site and a "slow to fast' conformational change after reacting with trypsin, all suggest that the inhibitory mechanism of the snail alpha M is similar to the "trap mechanism' of human alpha 2-macroglobulin.
Bender
R C
RC
Department of Zoology, Oregon State University, Corvallis 97331-2914, USA.
Bayne
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D001700:Biomphalaria; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism; D014714:Vertebrates; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000378:metabolism
D046911:Macromolecular Substances; D009844:Oligosaccharides; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; D014357:Trypsin