Reaction of proteinases with alpha 2-macroglobulin from the American horseshoe crab, Limulus.
The products generated by the reaction of Limulus alpha 2-macroglobulin with trypsin were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Unreacted Limulus alpha 2-macroglobulin had a subunit molecular mass of 185 kDa. Trypsin-reacted samples contained two prominent peptides smaller (85 and 100 kDa) and three peptides larger (200, 250, and 300-350 kDa) than the unreacted subunit. Reaction of methylamine-treated Limulus alpha 2-macroglobulin with trypsin resulted in the same two prominent reaction products smaller than 185 kDa, but all of the reaction products larger than 185 kDa were absent. The covalent binding of biotinylated trypsin with Limulus alpha 2-macroglobulin was detected by probing Western blots with horseradish peroxidase-avidin. Surprisingly, the only reaction products that contained trypsin were bands at 100 and 120 kDa. The staining of these bands with horseradish peroxidase-avidin was weak: most of the biotinylated trypsin that remained associated with alpha 2-macroglobulin during gel filtration chromatography was located at the dye front following reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The reaction products larger than 185 kDa did not contain trypsin. Methylamine-reacted Limulus alpha 2-macroglobulin failed to bind any biotinylated trypsin. In contrast to the reaction of trypsin with Limulus alpha 2-macroglobulin, all high molecular mass bands generated by the reaction of human alpha 2-macroglobulin with biotinylated trypsin stained intensely with horseradish peroxidase-avidin. Thus, Limulus alpha 2-macroglobulin forms thiol ester-dependent, high molecular mass products involving isopeptide bonding between trypsin-generated fragments, without the incorporation of trypsin into the complexes. Most of the alpha 2-macroglobulin-associated trypsin is non-covalently trapped rather than covalently cross-linked.
Quigley
J P
JP
Department of Pathology, State University of New York, Stony Brook 11794-8691.
Ikai
A
A
Arakawa
H
H
Osada
T
T
Armstrong
P B
PB
D000818:Animals; D001710:Biotin / Q000737:chemistry; D015153:Blotting, Western; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D006737:Horseshoe Crabs / Q000201:enzymology*; D014357:Trypsin / Q000737:chemistry; D000511:alpha-Macroglobulins / Q000378:metabolism*
D000511:alpha-Macroglobulins; D001710:Biotin; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin