Extracts of cell cultures labelled with [3H]
leucine were incubated with human
alpha 2-macroglobulin (alpha 2M), a plasma
proteinase inhibitor. The
proteinase-alpha 2M complexes were then precipitated with immobilized
monoclonal antibodies to alpha 2M and analysed by SDS/
polyacrylamide-gel electrophoresis. Parallel experiments were done with
methylamine-inactivated alpha 2M to check for unspecific binding of cell
proteins to alpha 2M. Several 3H-labelled cell
proteins bound to active, but not to inactivated, alpha 2M. Such
proteins are likely to be
proteinases. Putative
endopeptidases of subunit Mr 112000, 78,000, 53,000, and in some experiments 88,000 and 16,000, were trapped by alpha 2M in supernatant fractions from IMR90 human fibroblasts, EBTr bovine fibroblasts and HeLa human
carcinoma cells. No additional
proteins were trapped in the presence of
ATP. The Mr-78,000
endopeptidase was identified as
calpain II by immunoblotting. At pH 5.3 putative
endopeptidases of subunit Mr 80,000, 53,000 and 28,000-32,000 were trapped from IMR90-fibroblast extracts. Immunoblotting showed that both
cathepsin B and
cathepsin D were present in the Mr-28,000-32,000 electrophoretic bands. The use of alpha 2M and immobilized antibody to alpha 2M thus allows a rapid enrichment of
endopeptidases from
cell extracts. Some potentials and limitations of the method are discussed.