A functional, thioester-containing alpha 2-macroglobulin homologue isolated from the hemolymph of the American lobster (Homarus americanus).
An alpha 2-macroglobulin-like protease inhibitor was isolated from the cell-free hemolymph of the american lobster (Homarus americanus) by ion-exchange chromatography and gel filtration. Whereas the undissociated molecule has a molecular weight of 342,000 as determined by ultracentrifugation studies, under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a subunit molecular weight of 180,000. On the basis of this and other evidence, we conclude that the lobster protein is a dimer consisting of two disulfide-bonded monomers. The purified protein inhibits proteolytic enzymes but protects the esterolytic activity of trypsin toward low molecular weight substrates from inactivation by soybean trypsin inhibitor. The methylamine sensitivity of this activity suggests the presence of an internal thioester bond. This was confirmed by the covalent incorporation of [14C]methylamine, by the formation of Mr 55,000 and 125,000 autolytic cleavage fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and, more directly, by the amino acid sequence of a tryptic peptide containing the putative thioester region. Whereas the N-terminal amino acid sequence (22 residues) of the protein revealed an overall identity of only 18% when compared with the human protein, the sequence of the thioester-containing peptide was highly conserved, both with respect to human alpha 2-macroglobulin and to other proteins having a thioester bond. The protein showed the "slow to fast" conformational change typical in alpha 2-macroglobulins in nondenaturing gel electrophoresis after treatment with trypsin, but not after incubation with methylamine.
D000595:Amino Acid Sequence; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002364:Caseins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000032:analysis*; D008744:Methylamines / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008121:Nephropidae; D011487:Protein Conformation; D014357:Trypsin / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000032:analysis / Q000302:isolation & purification* / Q000494:pharmacology
D002241:Carbohydrates; D002364:Caseins; D008744:Methylamines; D000511:alpha-Macroglobulins; C027451:methylamine; D014357:Trypsin