In the investigated 14 day old triticale seedlings a much higher GDH activity was observed in roots than in leaves. The enzyme from the roots was purified up to the state of homogeneity (about 400 fold). The purified enzyme showed a higher activity in the presence of reduced coenzyme forms (NAD(P)H) than their oxidated forms. In the presence of NAD(P)H the enzyme showed absolute specificity to 2-oxoglutarate and in cooperation with NAD(P) + to L-glutamate. The Km values determined for particular substrates indicate a high affinity of NADPH-GDH to ammonium ions.Optimum pH, temperature and thermostability of GDH depended on the type and form of the coenzyme. Molecular mass of purified enzyme was 257 kDa. It seems that native GDH is composed of six identical subunits of the molecular mass 42.5 kDa.
Glutamate dehydrogenase (GDH, EC 1.4.1.2-4) and glutamine synthetase (GS, EC 6.3.1.2) activities as well as protein content and dry matter in developing kernels of winter Triticale were determined. The relatively low level of GS activity compared to high level of NAD(P)H-dependent GDH activity during intensive filling of grains with storage compounds may indicate the participation of GDH in reductive amination of 2-oxoglutarate. The amination activity of this enzyme in all grain development phases exceeded the deaminating activity several fold. Moreover, the dynamics in the change of NAD(P)H-GDH and NAD(P)+-GDH activities were analysed in various tissues of the developing grains. The high amination activity of the enzyme in the seed coat, where the intensive protein synthesis occurs would also be an indication of the anabolic function of this enzyme.
A b s t r a c tThe influence of selected factors on the activity of highly purllied GDH in triticale roots was investigated in vitro. In the presence of 2-ME, NADH-GDH activity increased by 400 %, while NADPH-GDH activity rose by 500 %. No effect of reducing factors on NAD(P)+-GDH reaction was detected. The sulphydryl groups inhibitors, such as p-chloromercuribenzoate (p-CMB) and iodoacetamide, proved the strongest inhibitors of the aminative reaction. Metal-binding compounds: ethylenediaminetetraacetic acid disodium salt (EDTA) and Zinkov also considerably inhibited NAD(P)H-GDH activity, Diisopropylfluorophosphate (DFP) and pepstatin A, the inhibitors specific for -OH serine and CO0-aspartic acid groups respectively, caused a non-significant NAD(P)H-GDH activity decrease.Cd ,Co ~Hg ,Mg ,Pb andZn ions strongly inhibited the amination reaction, whereas their inhibiting effect upon NAD+-GDH activity was negligible. Among the applied ions, only Ca 2+ activated NADH-GDH.
List of abbreviations:p-CMB -p-chloromercuribenzoate; DFP -diisopropytfluorophosphate; EDTA -ethylenediaminetetraacetic acid disodium salt; 2-ME -2-mercaptoethanol
The [3-endoglucanase is one of the enzymes taking part in the degradation of the cell wall structural polysaccharides. The use of two Triticale varieties differing in their resistance to the preharvest sprouting allowed the comparison of that enzyme activity and changes in the internal structnre of the cell wall observed in the light and electron microscope. The most interesting observations seem to be the channels and even the holes in the walls of aleurone cells found mostly in the samples showing the elevated activities of [3-endoglucanase. As those sampies concern the grains of a lower resistance to pre-harvest sprouting, it might be suggested that the loosening of the wall structure may be one of the probable mechanisms in facilitating the enzyme, metabolite and water translocations through the grain tissues. Those changes can accelerate the water imput into the grain, the metabolic process and thus the increased susceptibility to sprouting.
3 M urea has been shown to cause considerable, and only partially reversible conformational changes of gluten molecules. Homogenization has proved to act mechanically, breaking down some molecular bonds. No structural changes could be observed during freeze drying gluten, as well as after brief heating of its acetic acid extracts.
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