Effect of alpha2M on earthworm fibrinolytic enzyme III-1 from Lumbricus rubellus.
Though it is known that human alpha2-macroglobulin (alpha2M) inhibits most proteases, the effect of alpha2M has not been investigated on earthworm fibrinolytic enzymes (EFEs) from Lumbricus rubellus, which could be transported from intestine epithelium into blood as an intact molecule (Fan et al., Biochim. Biophys. Acta 1526 (2001) 286). The activity of earthworm fibrinolytic III-1 (EFE-III-1) decreased to 65% when incubated with alpha2M, while it decreased to 30% in plasma under the same conditions. The first order rate of the inactivation of EFE-III-1 with alpha2M was similar to that of fast phase with plasma, indicating that alpha2M may be the inhibitor initially binding to the enzyme in blood. SDS-PAGE showed that incubation of EFE-III-1 with alpha2M a released fragment ( approximately 90 kDa), followed by formation of a high molecular weight complex (approximately 700 kDa). There was a linear relationship between the apparent inhibition rate constant (k1) and [alpha2M], by double reciprocal plot. It was suggested, as described by Tsou (Acta Biochem. Biophys. Sinica 5 (1965) 398) and Tian (Biochem. J. 21 (1982) 1028), that the mechanism of alpha2M/EFE-III-1 interaction could be coincided with a complexing irreversible inhibition. Experiments in both the inactivation and the intrinsic fluorescence showed that alpha2M bound to the enzyme mole by mole equivalently. The intrinsic fluorescence of alpha2M was enhanced with an observable blue shift in emission maxima, suggesting that alpha2M was one of the important inhibitors to EFEs when it absorbed into blood.
Wu
Cen
C
Lab of Visual Information Processing, Centre for Brain and Cognitive Science, Institute of Biophysics, Baiao Pharmaceuticals Beijing C.L., The Chinese Academy of Sciences, 15 Da Tun Road, Chaoyang District, 100101, Beijing, PR China.
Li
Li
L
Zhao
Jing
J
Fan
Qiao
Q
Tian
Wei-Xi
WX
He
Rong-Qiao
RQ
D000818:Animals; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009835:Oligochaeta / Q000201:enzymology*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D000511:alpha-Macroglobulins / Q000737:chemistry*
D000511:alpha-Macroglobulins; D010450:Endopeptidases; C083421:lumbrokinase