Distribution of plasma kallikrein between C-1 inactivator and alpha 2-macroglobulin in plasma utilizing a new assay for alpha 2-macroglobulin-kallikrein complexes.
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.
Harpel
P C
PC
Lewin
M F
MF
Kaplan
A P
AP
D003174:Complement C1 Inactivator Proteins / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized / Q000378:metabolism; D005174:Factor XII / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007610:Kallikreins / Q000097:blood*; D007616:Kaolin / Q000378:metabolism; D007700:Kinetics; D008722:Methods; D013696:Temperature; D000511:alpha-Macroglobulins / Q000032:analysis*
D003174:Complement C1 Inactivator Proteins; D004800:Enzymes, Immobilized; D000511:alpha-Macroglobulins; D007616:Kaolin; D005174:Factor XII; D007610:Kallikreins; D005341:Fibrinolysin