Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.
Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.
Yamamoto
T
T
Department of Allergy, Kumamoto University Medical School, Japan.
Shibuya
Y
Y
Nishino
N
N
Okabe
H
H
Kambara
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D016264:Dextran Sulfate; D003911:Dextrans / Q000494:pharmacology; D004563:Electrochemistry; D005174:Factor XII / Q000378:metabolism*; D015956:Factor XIIa / Q000378:metabolism*; D006801:Humans; D007610:Kallikreins / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013997:Time Factors
D001426:Bacterial Proteins; D003911:Dextrans; D005174:Factor XII; D016264:Dextran Sulfate; D007610:Kallikreins; C038495:Pseudomonas serine proteinase; D012697:Serine Endopeptidases; D015956:Factor XIIa; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa