Intracellular Localization and Properties of NADH-Glutamate Dehydrogenase fromVitis viniferaL.: Purification and Characterization of the Major Leaf Isoenzyme

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Abstract
Glutamate dehydrogenase was partially purified from grapevine (Vitis vinifera L. cv. Soultanina) tissues and its activity and isoenzymic pattern were studied. Seven anodal migrating isoenzymes were revealed after PAGE. Leaf protoplasts were isolated from in vitro-grown axenic shoot cultures and used to study the intracellular localization of GDH. Results revealed that the enzyme was associated with the mitochondrial fraction. The isoenzyme with the lowest electrophoretic mobility, which accounted for 35 to 40% of total activity, was purified 2050-fold to homogeneity from leaves. The purification method included ammonium sulphate fractionation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and NAD-sepharose affinity chromatography. The molecular weight of the native enzyme was estimated to be 252 kDa and it consisted of identical 42.5 kDa subunits. pH optimum for the aminating reaction was 8.0 and for the deaminating reaction 9.3. At optimum pH conditions the apparent Km values for ammonium, as ammonium chloride and ammonium sulphate, α-ketoglutarate, NADH, glutamate, and NAD+ were 45.0, 13.0, 2.1, 0.069, 18.0, and 0.195 mM, respectively. The amination reaction of GDH was fully activated with about 100 μM Ca2+ while the deamination reaction was not affected by the addition of Ca2+. The isoenzymes of GDH showed different magnitude of their activating response to calcium ions.