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1.Transaminase was cross-linked with various proteins after the addition of glutaraldehyde or ethyl chloroformate. The water insoluble conjugates were used for the purification of antitransaminase and antiapotransaminase. A method is described.2. Pig heart transaminase and ox heart transaminase react differently with anti-pig-heart transaminase and apotransaminase. Slight differences were also observed in the reactions between holoenzyme and apoenzyme with their homologous and heterologous antibodies.3. L-Aspartate and a-ketoglutarate protect transaminase from the inhibitory action of antitransaminase. Pyridoxal-5'-phosphate has no effect on the enzyme-antienzyme binding when added in the system in stoichiometric amounts. Higher concentrations of the coenzyme, however, activate the complex antigen-antibody by facilitating its solubilization. 4. The intact conformation of transaminase is not essential for enzyme-antienzyme binding. Peptides in the dialysate of a tryptic or a chymotryptic hydrolysate of transaminase were able to react with the antibody and form a precipitate.The preparation of water insoluble antigens and their use for the isolation of antibody has been recently reviewed [l].Glutamic-aspartic transaminase is one of the few pyridoxal enzymes which has been obtained in pure form and studied extensively [2-41. The enzyme has a molecular weight of approximately 90000 or less [5,6] contains two pyridoxal phosphate groups and two N-terminal alanyl residues in its molecule [7,8]. The amino acid sequence around the coenzyme was investigated by two different groups [9,10] and others suggested that an histidine residue might be critical to the catalytic activity of the enzyme [ 5 ] . Recent publications reveal that the transaminase molecule is composed of two identical subunits [Ill and that the complex formation between enzyme and substrate requires a t least two binding points [12].In the present report we describe the conjugation of transaminase with different proteins a t various pH by means of glutaraldehyde or ethyl chloroformate and the use of the insoluble complexes for the isolation of antitransaminase in pure form. Some of the properties of antitransaminase and the characteristics of the interaction between the pure forms of enzyme and antienzyme in absence or in presence of substrates and coenzyme are presented.Enzyme. Glutamic-aspartic transaminase or L-aspartate 2-oxoglutarate aminotransferase (EC 2.6.1.1). EXPERIMENTAL PROCEDURE MaterialsGlutamic-aspartic transaminase from pig hearts was prepared by the method of Jenkins et al. [7] using succinate buffer [ 131. Additional purification was achieved by fractionation on a DEAE-Sephadex A-50 column equilibrated and eluted with 0.02 M phosphate buffer pH 7.2. The enzyme preparations used had an absorption ratio (280 nm:360 nm) of 9.5 to 10.5 a t pH 9.0. All the experiments were performed with the native enzyme, no attempt being made to study individual fractions of transaminase separately [14]. Apotransaminase was prepared from transaminase with th...

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