[Various properties and kinetics of interaction of high and low molecular weight human kininogens with human plasma kallikrein].
A relatively simple procedure for isolation and purification of human blood plasma kallikrein (HPK) by QAE-Sephadex A-50 SP-Sephadex C-50 and affinity chromatography on Sepharose 4B with immobilized soybean trypsin inhibitor with the activity yield of about 40% has been developed. The method allows for simultaneous isolation of low (LMW) and high molecular weight (HMW) kininogens from the same HPK sample. HPK preparations are homogeneous upon 7.5% polyacrylamide gel electrophoresis in the presence of 0.1% SDS; its Mr is 90,000. After treatment with beta-mercaptoethanol, HPK dissociates into two fragments with Mr of 43,000 and 37,000. HPK preparations have high specific activities of esterase (31 microM/min), amidase (78 microM/min), and kininogenase (420 micrograms equiv. bradikinin/min). The high degree of protein purification was demonstrated by titration of active centers with 4-methylumbelliferylguanidine benzoate. The values of equilibrium dissociation constants for the HPK complex with aprotinin (Ki) equal to 1 X 10(-8) M (ethyl ester of N-alpha-benzoyl-L-arginine) and 1,5 X 10(-9) M (HMW) were determined. The kinetics of HPK-induced liberation of bradikinin from purified preparations of HMW and LMW was studied. The kinetic parameters (Km, kcat and kcat/Km) of this reaction suggest a high affinity of HPK for HMW, but not for LMW. LMW does not compete with HMW for the enzyme active center. It is assumed that LMW is not a physiological substrate for HPK.
D001920:Bradykinin / Q000097:blood; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007610:Kallikreins / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D007704:Kininogens / Q000097:blood* / Q000302:isolation & purification; D008970:Molecular Weight
D007704:Kininogens; D007610:Kallikreins; D001920:Bradykinin
Nekotorye svoĭstva i kinetika vzaimodeĭstviia s vysoko- i nizkomolekuliarnymi kininogenami cheloveka kallikreina plazmy krovi cheloveka.