Part of purified human plasma kallikrein removed together with a remaining IgG fraction--immunoblot experiments and functional tests.
In the present study it is shown that a preparation of highly purified plasma kallikrein (specific activity 81 S-2302 U/mg) still contained small amounts of an IgG fraction. Amidase assays of the fresh enzyme with four peptide substrates (S-2302, Bz-Pro-Phe-Arg-pNA, S-2366, S-2222) did not reveal any inhomogeneity, and immunoblot experiments with antibodies against prekallikrein yielded only an 85 kD double band. After a storage period (2 years at -70 degrees C), S-2366 and S-2222 amidase activities not reflecting the initial kallikrein appeared. Immunoblot studies with Fc-specific antibodies against IgG showed a 170 kD band, and immunoblots with a monoclonal antibody against prekallikrein demonstrated that, in addition to the 85 kD band, a band with a mol weight of about 152 kD could also be detected. Immunoblots showed that only 85 kD kallikrein was recovered in the eluate from Protein G columns, and amidase assays based on S-2302 and Bz-Pro-Phe-Arg-pNA showed a recovery of about 70%. The other part of the kallikrein (30%) was removed along with the additional S-2366 and S-2222 activities and the IgG3 fraction present. The theory is advanced that the additional activity reflects a kallikrein fraction with a mol weight of about 152 kD, and is present in the fresh enzyme preparation in inactive complex with IgG. Storage of the kallikrein preparation led to some weakening of this complex and the appearance of functional activities of the kallikrein fraction involved.
Briseid
K
K
Department of Pharmacology, Institute of Pharmacy, University of Oslo, Blindern, Norway.
Johannesen
S
S
D000581:Amidohydrolases / Q000378:metabolism; D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005172:Factor XI / Q000302:isolation & purification; D005174:Factor XII / Q000302:isolation & purification; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood* / Q000276:immunology; D007610:Kallikreins / Q000097:blood* / Q000276:immunology
D007074:Immunoglobulin G; D005174:Factor XII; D005172:Factor XI; D007610:Kallikreins; D000581:Amidohydrolases; C019665:amidase