Purification and characterization of an alpha-macroglobulin proteinase inhibitor from the mollusc Octopus vulgaris.
The cell-free haemolymph of the mollusc Octopus vulgaris inhibited the proteolytic activity of the thermolysin against the high-molecular-mass substrate hide powder azure. The purified inhibitor was a glycoprotein composed of two identical 180 kDa disulphide-linked subunits. In addition to the inhibition of the metalloproteinase thermolysin, the protein inhibited the serine proteinases human neutrophil elastase, pig pancreatic elastase, bovine chymotrypsin, bovine trypsin and the cysteine proteinase papain. A fraction of the proteinase-inhibitor complex resisted dissociation after denaturation indicating that some of the proteinase molecules became covalently bound. The nucleophile beta-aminopropionitrile decreased the covalent binding of proteinases to the Octopus vulgaris protein, suggesting that this interaction is mediated by an internal thiol ester; the reactivity and the amino acid sequence flanking the reactive residues of the putative thiol ester were consistent with this hypothesis. Bound trypsin remained active against the low-molecular-mass chromatogenic substrate H-D-Pro-Phe-Arg p-nitroanilide and was protected from inhibition by active-site-directed protein inhibitors of trypsin; however, the bound trypsin was readily inhibited by small synthetic inhibitors. This indicates that the inhibition of proteinases is accomplished by steric hindrance. The proteinase-inhibitory activity of this protein is characteristic of inhibition by mammalian alpha-macroglobulins and the presence of a putative thiol ester suggests that the Octopus vulgaris proteinase inhibitor is a homologue of human alpha 2-macroglobulin.
Thøgersen
I B
IB
Duke University Medical Center, Durham, NC 27710.
Salvesen
G
G
Brucato
F H
FH
Pizzo
S V
SV
Enghild
J J
JJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000629:Aminopropionitrile / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000378:metabolism; D008969:Molecular Sequence Data; D049831:Octopodiformes / Q000378:metabolism*; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; D000629:Aminopropionitrile