W. Terry Jenkins

[7] l-Alanine aminotransferase (pig heart)

Publisher Summary This chapter discusses the assay, purification, and properties of L-alanine aminotransferase. The assay method is based on the fact that pyruvate and other compounds with a CH3CO-grouping form a red color when incubated with strongly alkaline solutions of salicylaldehyde. One of the difficulties encountered in the purification procedure is the marked instability of the enzyme beyond a certain stage in its purification. This may be counteracted by 5 mM EDTA together with 10 mM mercaptoethanol. This finding indicates that the cause of instability is the oxidation of sensitive thiol groups. The purified enzyme is very specific for L isomers of alanine and glutamate. Effective inhibitors of the enzyme include aspartate, aminoadipate, and monocarboxylic acids. The enzyme is, particularly sensitive to L-cycloserine, aminoxyacetate, and cysteine. The optimum pH for L-alanine aminotransferase is 8.0. In context to purity, two bands, both with enzymatic activity, upon starch gel electrophoresis are obtained. The second minor band may be an aggregation product formed upon aging. This purification is essentially an extended modification of the original procedure of Lenard and Straub.

[110] Branched-chain amino acid aminotransferase (pig heart, soluble)

Publisher Summary This chapter presents the assay, purification, and properties of aminotransferase from pig heart. When leucine is used as the substrate, the 2, 4-dinitrophenylhydrazone of α- ketoisocaproate may be selectively extracted by cyclo hexane from acidic solutions containing an excess of α -ketoglutarate 2, 4-dinitrophenylhydrazone. The enzyme is sensitive to sulfhydryl reagents. The loss of activity upon storage at high pH values may be recovered by treatment with thiols. Just as other transaminases, the enzyme is inhibited by carboxylic acid substrate analogs and by a variety of carbonyl reagents, especially phenylhydrazine and hydroxylamine. The calculated sedimentation constant for zero protein concentration is 5.1 S. With 6.7 m M substrate concentrations, the assay shows a sharp optimum at about pH 8.3. Further analysis of this pH activity curve shows that the decrease on the acid side of the optimum is due to both a decrease in maximum velocity and an increase in the K m for leucine. The decrease on the basic side of the optimum, which is due to an increase in the K m for ketoglutarate, does not occur when transamination from leucine to α -ketoisovalerate is studied.

BINARY COMPLEXES INVOLVED IN ENZYMIC TRANSAMINATION

SUMMARY (1) The unsatisfactory nature of the evidence for the participation of ternary complexes in transamination is discussed. (2) A method is presented for the analysis of the different possible kinds of binary complexes occurring in steady-state transamination systems. This analysis was applied to the pig heart glutamic alanine aminotransferase where it was shown that the contribution of the abortive complexes was negligible. (3) It was shown that this analysis indicates that none of the intermediary complexes is the conjugate base of any other observable complex. Such a hypothesis is also supported by the lack of Vm variation with pH and current experiments with substrate analogs. (4) Binary complexes which have been identified as being of the intermediary type have maxima at about 325 mμ, 360 mμ and 492 mμ. (5) It is suggested that substrate analogs may play an important role in the elucidation of the mechanism of transamination.