Ostrich antithrombin III: kinetics and mechanism of inhibition of ostrich thrombin.
A kinetic investigation of ostrich thrombin specificity, its regulation and evolutionary development in comparison to those of other well-characterised species may contribute to the understanding of the structure-function relationships of thrombin. Antithrombin III (ATIII) was purified from ostrich plasma by heparin-Sepharose and Super Q-650S chromatography. It exhibited a M(r) of 59.2K and a pI in the range of 5.2-6.0. The ostrich N-terminal sequence was compared to those of other known species and showed the highest identity with rabbit ATIII (31%). Inhibition studies included the interaction of ostrich and human ATIII with bovine, human and ostrich thrombin. At a 2:1 molar ratio of ostrich ATIII to enzyme, 20 and 40% remaining activity was found for bovine and ostrich thrombin, respectively. Ostrich thrombin exhibited a pH and temperature optimum of 9.0 and 60 degrees C, respectively. Hydrolysis of seven peptide p-nitroanilide substrates by ostrich thrombin revealed D-Phe-Pip-Arg-pNA (k(cat)/K(m)=9.65 microM(-1)s(-1)) as the substrate with the highest catalytic efficiency. The effect of monovalent cations on ostrich thrombin catalysis revealed enhanced activity with Na(+). The calculated K(i) values for the complex formation between ostrich thrombin and ostrich (9.29 x 10(-11)M) and human (9.66 x 10(-11)M) ATIII are comparable to reported results. The results obtained from the present study confirmed that ostrich thrombin and ATIII are closely related to the corresponding molecules of other species in terms of physicochemical and kinetic properties.
Frost
Carminita L
CL
Department of Biochemistry and Microbiology, University of Port Elizabeth, P.O. Box 1600, South Africa. biomicro@upe.ac.za
Naudé
Ryno J
RJ
Muramoto
Koji
K
D000818:Animals; D000990:Antithrombin III / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D005075:Biological Evolution; D002414:Cations, Monovalent / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D016415:Sequence Alignment; D020310:Struthioniformes; D013696:Temperature; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D002414:Cations, Monovalent; D000990:Antithrombin III; D013917:Thrombin