Abstract
We have previously described an enzyme-linked immunosorbent assay for the quantification of C-1 inactivator-kallikrein complexes in plasma (Lewin, M. F., Kaplan, A. P., and Harpel, P. C. (1983) J. Biol. Chem. 258, 6415-6421). We have now developed an immunoimmobilization-enzyme assay for alpha 2-macroglobulin-kallikrein complexes. In this assay these complexes are removed from plasma by immunoabsorption with the IgG fraction of rabbit anti-alpha 2-macroglobulin antiserum coupled to an agarose gel. The immobilized alpha 2-macroglobulin-kallikrein complex hydrolyzes the fluorogenic substrate D-Ser-Pro-Phe-Arg-7-amino-4-trifluoromethyl coumarin, and this activity is proportional to the concentration of complexes in the plasma. Using these assays we have studied the distribution of plasma kallikrein between its inhibitors under several different experimental conditions. When kallikrein is added to plasma, about 57% binds to C-1 inactivator and 43% to alpha 2-macroglobulin. When prekallikrein is activated endogenously in plasma by the addition of kaolin or Hageman factor fragment, approximately 84% of kallikrein is now bound to C-1 inactivator and 16% to alpha 2-macroglobulin. Temperature dramatically affects the distribution of kallikrein. The binding of kallikrein to alpha 2-macroglobulin in plasma is inversely related to temperature, whereas the binding to C-1 inactivator is directly related: 85% of the kallikrein is bound to alpha 2-macroglobulin at 4 degrees C, whereas at 37 degrees C, only 33% is bound. The total amount of kallikrein bound to the two inhibitors is similar at each temperature. These studies thus provide new insight concerning kallikrein formation and regulation in plasma.
Highlights
We have reported on the development of an enzymenoimmobilization-enzymeassay for az-macroglobulin- linked immunosorbent assay (ELISA‘) for the quantification kallikrein complexes
When-kallikrein is ment or kaolin was added to plasma, the rate of formation added to plasma, about 57% binds to C1 inactivator and final amount of complexes generated were proportional and 43% to az-macroglobulin
No generation of inhibitor-kallikrein complexes occurred over the incubation period in the absence of prior studies have established the distributionof ls51-labeledkallikrein between C i inactivator and as-macroglobulin in plasma [8, 9], methods to assess the formationof these complexes in native plasma have not been previously available.Utilizing the two immunoassaysdetailedin this report,theformation of C i inactivator-kallikreinand a n macroglobulin-kallikreincomplexes in plasma was studied under three different conditions
Summary
Assay of Purified a z - ~ acrog ~ bulin - K a ~ ~ iCkormep~lnexes by the Immunoimmobi~izationEnzyme Assay-Complexes between ~*-macrogiobu~and plasma kal~kreinwere formed by adding increasing amounts of kallikrein to the inhibitor (Fig. 2). When kaolin was added to plasma congeni$lly deficient in Hageman factor or prekaliikrein, neither C 1 inactivator-k~likreinnor a,-macroglobulinkallikrein complexes were formed (Table I) When these two deficient plasmas were mixedin equal amounts andactivated with kaolin, approximately one-half the amount of total complexes obtained in normal plasma was formed documenting that both proteins are required for the formation of the a2-macroglobulin-kallikreincomplex. 6. Comparison of the hydrolysis of two fluorogenic This Complex-Since activation of the Hageman factor-kal- peptide substrates by immunoimmobilized a2-macroglobulinlikrein enzyme pathway in blood may activate plasminogen [24,25,26], we have examined the activitoyf a,-macroglobulin-plasmin complexes bound to the insolubilized anti-anmacroglobulin antibody against thefluorogenic plasma kallikrein substrate D-Ser-Pro-Phe-Arg-AFC. No generation of inhibitor-kallikrein complexes occurred over the incubation period in the absence of
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