1. Partially purified preparations of rat brain 4-aminobutyrate aminotransferase were inhibited in a time-dependent manner by ethanolamine O-sulphate. The inhibition was not reversed by dialysis. 2. The inhibitor formed an initial reversible complex with the enzyme (K(i)=4.4x10(-4)m) and the rate of inactivation followed pseudo-first-order kinetics (k=7.15x10(-4)s(-1)). The inclusion of 4-aminobutyrate markedly slowed the rate of inactivation. 3. Ethanolamine O-sulphate did not inhibit glutamate decarboxylase, alanine aminotransferase or aspartate aminotransferase. 4. Intracisternal injection of ethanolamine O-sulphate into rats led to rapid inactivation of 4-aminobutyrate aminotransferase in vivo.

Rat liver ornithine aminotransferase is found to exchange the pro-S hydrogen on the 5-carbon atom of ornithine exclusively, thus showing that the mammalian enzyme transfers the 3-amino group and not the a-amino group as has been demonstrated with the plant enzyme [Mestichelli, Gupta & Spenser (1979) J. Biol. Chem. 254, The enzyme also transfers the a-amino group of glutamate and the kinetics of the half reactions between the enzyme and both amino acids are compared. Rate and dissociation constants for both half reactions are determined.Accounts of the metabolism of ornithine in standard textbooks (e.g. Mahler & Cordes, 1966;Lehninger, 1975) indicate that transamination of this diamino acid entails loss of the 3-amino group and not the a-amino group. The enzyme responsible, ornithine 5-aminotransferase (L-ornithine: 2-oxoacid aminotransferase, EC 2.6.1.13), also interconverts 2-oxoglutarate and glutamate efficiently in the second half of the complete Ping-Pong mechanism. It seemed curious that although ornithine has an a-amino group configurationally identical with that of L-glutamate the enzyme should act exclusively on the 3-amino group even though at pH values near neutrality the a-amino group (pK = 8.7) is more reactive than the 3-amino group (pK = 10.8).Mestichelli et al. (1979) provided evidence that the metabolic route from ornithine to proline in plants occurs via loss of the a-amino group, not the 3-amino group, and they pointed out that much of the evidence indicating 3-amino group transfer depended on the occurrence of a reaction between the product and 2-aminobenzaldehyde to give a yellow colour. The product of transamination of either group is a cyclic internal imine; Al-pyrroline-5-carboxylate if the 3-amino group is transferred and Al-pyrroline-2-carboxylate if the a-amino group is transferred. As pointed out by Mestichelli et al. (1979), both compounds react similarly with aminobenzaldehyde.Because transamination proceeds via labilization of a proton on the relevant carbon atom, the amino group transferred may be identified by determining which proton is exchanged by the enzyme in deuterated solvent. The present paper identifies the exchangeable ornithine proton. In addition the equilibria and kinetics of the separate half reactions with ornithine and glutamate are compared. Experimental Enzyme preparationOrnithine aminotransferase was prepared by a method based on that described by Peraino et al. (1969) but with the following modifications.(1) The amount of the enzyme present in rat liver is increased several-fold when the animals are fed on a high-protein diet. Because of the high cost of the commercial 60% casein laboratory diet used by Peraino et al. (1969) the rats in the present study were fed for four days on dry textured soya bean protein (Temptein Soya Protein Meat-Like Chunks, Brooke Bond Oxo Catering Services Division, Croydon, Surrey, U.K.).(2) The method of Peraino et al. (1969) includes a step in which the preparation is heated to 55 0C. We experienced occasional severe losses at ...

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Ornithine aminotransferase is shown to bind 1 mol of amino[14C]hexynoate per mol of coenzyme in the ‘suicide’ inactivation process. At the same time the coenzyme pyridoxal phosphate becomes irreversibly bound to the enzyme protein. Apart from the inactivation, the labelled enzyme is indistinguishable from native ornithine aminotransferase by several separation techniques. Because the rate of degradation of the labelled enzyme is the same as that of the normal enzyme it is concluded that loss of coenzyme does not initiate turnover. Free aminohexynoate is rapidly eliminated from the liver, and 70% of the compound is excreted unchanged in 7.5 h. Inactivated ornithine aminotransferase accounts for 11% of the total labelled liver protein and significant amounts of label are found in aspartate aminotransferase which is also extensively inactivated. The rate of return of enzyme activity is determined and found to be more rapid than expected for a process in which the enzyme is synthesized at a constant rate and degraded in a single, first-order process.

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