PMID: 3755135Jul 5, 1986Paper

The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction.

The Journal of Biological Chemistry
S KrishnaswamyM E Nesheim

Abstract

The activation of bovine prothrombin by prothrombinase (Factor Xa, Factor Va, synthetic phospholipid vesicles, and calcium ion) was studied in the presence of the fluorescent, reversible thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA). Recordings of fluorescence intensity during prothrombin activation exhibited maxima that decreased to stable limiting values. These data suggested the transient appearance of the meizothrombin-DAPA complex, which exhibits fluorescence with 1.5-fold greater intensity than the thrombin-DAPA complex. At substrate concentrations well below Km, progress curves could be fitted by equations describing an ordered, sequential conversion of prothrombin to thrombin through the intermediate meizothrombin via two pseudo-first order steps. The pseudo-first order rate constants for both steps varied linearly with enzyme concentration, indicating that both steps are catalyzed by prothrombinase. The progress of the reaction was also monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry analyses of aliquots removed at intervals spanning the reaction. These analyses confirmed both the existence of meizothrombin and its time course as predicted from the eq...Continue Reading

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