The Estimation of Dehydrogenases in Plant Tissue.

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Abstract
This paper is concerned with the opt. conditions for the extraction and quantitative determination of succinic and malic dehydrogenases, and of succinic, malic and alpha-ketoglutaric oxidases. Particles containing the enzymes were prepared by fractional centrifugation from Avena coleoptiles and from the apical internodes of etiolated Pisum seedlings. For the dehydrogenases (determined by dye reduction), water was the best extracting medium, but the activity was increased by the presence of a protein preparation, or asparagine, or to some extent by Sequestrene, during the dehydro-genation; crystalline bovine serum albumin was found very effective. Malic dehydrogenase requires coenzyme I, but succinic dehydrogenase does not; neither enzyme responds to the addition of sucrose or phosphate. As hydrogen acceptor, 2,6-dichloro-phenol-indophenol was much superior. to methylene blue, which is toxic. Succinic dehydrogenase is present only in the particles, but malic dehydrogenase is present also in soluble form, the soluble enzyme accounting for 3/4 of the activity of the homogenate. For the oxidases (detd. by O2 uptake), sucrose 0.2 [image] with phosphate 0.02 - 0.05.[image] is the opt. extraction medium, and the addition of protein increases activity only in purified preparations. The influence of sucrose is probably exerted on the availability of cytochrome c in the particles. The activity of all three enzymes is promoted by magnesium. Details of pH and phosphate optima for the oxidase reaction are given. Malic and alpha-ketoglutaric oxidase require coenzyme I; but in the latter case, ATP can be substituted. With succinic oxidase an increased oxygen uptake in presence of ATP is shown to be due to the promotion of oxidation processes beyond the stage of fumarate. In the absence of cofactors this does not occur appreciably. The limitations on the quantitative recovery of malic and alpha-keto-glutaric oxidases are discussed, and it is shown that malonate cannot be used to isolate the latter. The dehydrogenases, and succinic oxidase, are recovered essentially quantitatively by the methods used. Problems involved in comparing the enzyme contents with the respiration of the intact tissue are discussed.