A filter paper assay for transamidating enzymes using radioactive amine substrates

A filter paper assay was developed for measuring the activity of transamidating enzymes by the incorporation of radioactive amine substrates into proteins. The method is analogous to those generally employed for monitoring the enzymic coupling of isotopic groups to proteins, such as in the ATP-dependent phosphorylation of casein or histone. Use of minimal quantities of reagents, as well as simplicity of handling a large number of test samples, are distinct advantages of this technique. The guinea pig liver transglutaminase-catalyzed incorporation of C14-histamine and C14-putrescine into β-lactoglobulin and casein were studied. The apparent Michaelis kinetics of the reactions permitted evaluation of Km,app's for both isotopic substrates and competitive inhibition constants for nonradioactive amines were also obtained. In contrast to transglutaminase, the fibrinoligase—generated in human plasma from Factor XIII by the addition of thrombin—could not utilize β-lactoglobulin to an appreciable extent. Hence experiments had to be restricted to casein as acceptor protein. Apparent Michaelis constants for the isotopic substrates and inhibition constants for nonradioactive amines could be readily measured also in the complex plasma system. Finally, details of a rapid filter paper assay are described for measuring Factor XIII levels in human plasma by measuring the incorporation of C14-putrescine, at close to saturating concentrations, into casein.