Kinetic and electrophoretic characterization of NADP dependent dehydrogenases from root tissues of Norway spruce [Picea abies (L.) Karst.] employing a rapid one-step extraction procedure

Guttenberger, Martin; Schaeffer, Christoph; Hampp, Rüdiger

doi:10.1007/bf00196846

Cited by 18 publications

(8 citation statements)

References 38 publications

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“…Visualization of SuSy enzyme activity was by using nondenaturating mini-slab-PAGE (35´28´0.5 mm; Laemmli 1970; Guttenberger et al 1994). Two microlitres of extract was applied per lane.…”

Section: Methodsmentioning

confidence: 99%

Extractable activities and protein content of sucrose-phosphate synthase, sucrose synthase and neutral invertase in trunk tissues of Robinia pseudoacacia L. are related to cambial wood production and heartwood formation

Hauch

Magel

1998

Planta

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The presence of sucrose synthesizing and degrading enzymes and the correlation of their enzyme activity with cambial growth and heartwood formation are demonstrated in trunks of Robinia pseudoacacia L., black locust. Sucrose is formed by sucrose-phosphate synthase (SPS; EC 2.4.1.14), predominantly in the storage part of the sapwood. In the cambial dierentiation zone and the sapwood-heartwood transition zone, both of which constitute carbohydrate sinks, sucrose is primarily cleaved by sucrose synthase (SuSy; EC 2.4.1.13) and a neutral invertase (NI; EC 3.2.1.26). In spring, enhanced activities of SuSy and NI were found in the dierentiating xylem tissues. This coincided with elevated SPS rates at the sites of starch mobilization. Heartwood formation in autumn, a period of intense accumulation of phenolics in the innermost living wood tissues, was accompanied by high activities of SuSy and NI. Increased SPS and NI activities in all tissues of winter samples could be correlated with cold acclimation. Probing of SPS and SuSy protein from black locust with heterologous antibodies revealed a subunit size of 130 kDa for SPS and of 89 kDa for SuSy. Both SPS and SuSy exhibited a linear correlation between catalytic activity and amount of enzyme protein with respect to the radial pro®le from bark to inner core and with respect to the seasonal course. The highest amounts of SuSy-speci®c mRNA were detected in dierentiating xylem in summer and the sapwood-heartwood transition zone in autumn. These data are taken as evidence for a pivotal role of SuSy in supplying carbon skeletons for the biosynthesis of secondary substances in woody axes.

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“…Visualization of SuSy enzyme activity was by using nondenaturating mini-slab-PAGE (35´28´0.5 mm; Laemmli 1970; Guttenberger et al 1994). Two microlitres of extract was applied per lane.…”

Section: Methodsmentioning

confidence: 99%

Extractable activities and protein content of sucrose-phosphate synthase, sucrose synthase and neutral invertase in trunk tissues of Robinia pseudoacacia L. are related to cambial wood production and heartwood formation

Hauch

Magel

1998

Planta

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“…Extracts of protein and enzymes were prepared in a buffer system containing Tris/borate (300 mM/ 100 mM, pH 7-6, 4 °C, Guttenberger, Schaeffer & Hampp, 1994) supplemented with 1 mM /?-mercaptoethanol, 0-85% BSA, 5% polyvinylpyrrolidone (PVP; sucrose phosphate synthase, SPS), 1 mM /^-mercaptoethanol, 3 % PVP (sucrose synthase), 3% PVP (acid invertase), 13% PVPP (alkaline invertase, ADP-glucose pyrophosphorylase (AGPase), and starch phosphorylase). Extractable activities of sucrose phosphate synthase, sucrose synthase, acid (pH 3-8) and alkaline invertase (pH 6-8) were measured using a microplate photometer according to Egger & Hampp (1993).…”

Section: Methodsmentioning

confidence: 99%

Ozone‐induced accumulation of carbohydrates changes enzyme activities of carbohydrate metabolism in birch leaves

et al. 1997

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SUMMARYBirch cuttings {Betula pendula Roth) were grown in a sand-culture system with two concentrations (0-05, HF and 0-005%, LF) of fertilizer containing macronutrients and micronutrients, and were exposed to 90/40 nll"^ O3 (day/night) and < 3 nl T^ O3 (control) for one growing season in the field fumigation chambers at Birmensdorf (Switzerland). Leaves of different ages were analysed for gas exchange, contents of chlorophyll, protein, and for metabolites as well as enzyme activities of carbohydrate metabolism.Ozone reduced net photosynthesis and chlorophyll contents in mature leaves of both fertilization treatments, whereas that of protein was only reduced in high-fertilized plants (HF). However, net photosynthesis, chlorophyll, and protein increased in young leaves of low'-fertilized plants (LF). The effects of ozone on enzyme activities of carbohydrate metabolism were most pronounced in leaves of LF plants. Specific activities of the sucrose-cleaving enzymes, sucrose synthase and alkaline invertase, were induced, whereas acid invertase was unchanged. Extractable activity of sucrose phosphate synthase, which is a key enzyme of sucrose synthesis, was reduced. Levels of fructose 2,6-bisphosphate, an inhibitor of sucrose synthesis, were increased in leaves of O3/LF plants, but reduced in O3/HF plants. In addition, activities of enzymes involved in starch metabolism, ADP-glucose pyrophosphorylase and starch phosphorylase, were lowered in ozone-treated samples and the ratio of ATP: ADP was increased.It is concluded that chronic ozone exposure leads to an inhibition of sucrose synthesis and favours sucrose degradation. This effect is modulated by the nutrient status of the plants, indicating higher O3 tolerance in HF plants. Furthermore, as the metabolic responses in the ozone-treated samples resemble very closely those observed under end-product inhibition of photosynthesis, we assume that the O3 effect is mainly due to reduced photosynthate export.

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“…The extracts did not contain measurable activities of UDPase and PEP-phosphatase (data not shown), which would have interfered with the assay. Borate, an essential ingredient of the extraction buffer (Guttenberger et al, 1994), inhibited SPS activity by only 10% at 30 mM compared with 1 mM assay concentration (the latter was achieved by concentration and subsequent dilution of the crude extract). Because this inhibition showed no interaction with the allosteric effector Pi, it could be ignored (data not shown).…”

Section: Metabolic Fine Control Of Spsmentioning

confidence: 99%

Coarse and Fine Control and Annual Changes of Sucrose-Phosphate Synthase in Norway Spruce Needles

1996

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The control of Suc synthesis constitutes an important factor in the regulation of Suc export, photosynthesis, and starch formation. One of the key enzymes in the control of Suc synthesis is SPS, and its activity can be regulated via metabolic fine control, coarse control by covalent modification, and by the amount of enzyme protein. Metabolic fine control is exerted by the allosteric effectors Glc-6-P and Pi. Glc-6-P, an intermediate of Suc synthesis, activates, whereas Pi inhibits SPS activity in most tissues (Doehlert and Huber, 1983; Huber et al., 198910;Reimholz et al., 1994). Some species exhibit a light-dependent covalent modification of SPS. Accordingly, the enzymes can be classified into three different types (for review, see Huber and Huber, 1992). Organisms such as maize and other monocotyledons show a light-induced increase in V , , , under nonlimiting substrate conditions. In other plants (e.g. spinach), V,,, is not influenced by a dark + light transition; in contrast, an increase in SPS activity in the presence of limiting substrate * The work was supported by grants from the Projekt Europaisches Forschungszentrum fiir Massnahmen zur Luftreinhaltung and the Deutsche Forschungsgemeinschaft (Graduiertenkolleg: Organismische Interaktionen in Waldokosystemen).

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Kinetic and electrophoretic characterization of NADP dependent dehydrogenases from root tissues of Norway spruce [Picea abies (L.) Karst.] employing a rapid one-step extraction procedure

Cited by 18 publications

References 38 publications

Extractable activities and protein content of sucrose-phosphate synthase, sucrose synthase and neutral invertase in trunk tissues of Robinia pseudoacacia L. are related to cambial wood production and heartwood formation

Extractable activities and protein content of sucrose-phosphate synthase, sucrose synthase and neutral invertase in trunk tissues of Robinia pseudoacacia L. are related to cambial wood production and heartwood formation

Ozone‐induced accumulation of carbohydrates changes enzyme activities of carbohydrate metabolism in birch leaves

Coarse and Fine Control and Annual Changes of Sucrose-Phosphate Synthase in Norway Spruce Needles

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