Hendrik Weiner

Subcellular Compartmentation of Pyrophosphate and Alkaline Pyrophosphatase in Leaves

Abstract The subcellular localisation of pyrophosphate and alkaline pyrophosphatase in leaves has been studied using non-aqueous density gradient centrifugation of spinach leaves, and membrane filtration of wheat mesophyll protoplasts. The pyrophosphate was measured in extracts prepared in trichloroacetic acid, and could be quantitatively recovered from the leaf material. It was located predominantly in the cytosol, with a concentration of 0.2–0.3 mM. In contrast, the alkaline pyrophosphatase was largely, if not, exclusively, located in the chloroplast. By comparing the pyrophosphate levels in the cytosol with previously published data on the cytosolic levels of phosphate and metabolic intermediates, it is shown that the reactions catalysed by pyrophosphate: fructose-6-phosphate phosphotransferase and UDP-glucose pyrophosphorylase are close to the thermodynamic equilibrium and, thus, freely reversible in vivo. Comparison of the pyrophosphate levels with the reported electrical and pH gradient across the tonoplast membrane shows the free energy released during pyrophosphate hydrolysis is similar to that required to move a proton across the tonoplast membrane. It is suggested that pyrophosphate could operate as a secondary energy donor in the cytosol of plant cells.

The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

Abstract. The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRAmax) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRAmax and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRAmax and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRAmax and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRAmax was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRAmax in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NRmax by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRAmax.

Inter- and intracellular distribution of amino acids and other metabolites in maize (Zea mays L.) leaves

In illuminated maize (Zea mays L.) leaves, the distribution of triose phosphates, 3-phosphoglycerate, malate and various amino acids between the chloroplastic and the extrachloroplastic compartments of mesophyll and bundle-sheath cells, and the total vacuolar fraction of the leaves, was determined by a combination of previously published methods, for separating mesophyll from bundle-sheath material, and for nonaqueous subcellular fractionation. The results show that the triose phosphate/3-phosphoglycerate ratio in the extrachloroplastic fraction of the mesophyll cells is about 20-fold higher than in the bundle-sheath cells, which is in accordance with a triose phosphate/phosphoglycerate shuttle postulated previously. Whereas the vacuolar compartment was shown to contain most of the cellular malate, amino acids were found to be almost absent from this compartment. The amino-acid pattern in the extrachloroplastic fraction of the bundle-sheath cells largely resembled the pattern in whole leaves. These results show that for future studies the analysis of amino-acid contents in whole maize leaves can be used as a measure for the amino-acid levels in the cytosol of bundle-sheath cells.

Binding to 14-3-3 proteins is not sufficient to inhibit nitrate reductase in spinach leaves.

To assess the role of 14‐3‐3 proteins in the magnesium‐dependent inhibition of nitrate reductase (NR) we tested the effect of magnesium on NR binding to 14‐3‐3s by coimmunoprecipitation and gel filtration. The stability of the 14‐3‐3 complex of NR was, unlike its activity, unaffected by magnesium. We therefore conclude that binding to 14‐3‐3s per se does not inhibit NR. Magnesium inhibited 14‐3‐3‐bound NR much more strongly than 14‐3‐3‐free NR. 14‐3‐3s possibly reinforce NR inhibition by magnesium.

Antibodies That Distinguish between the Serine-158 Phospho- and Dephospho-Form of Spinach Leaf Sucrose-Phosphate Synthase

Serum antibodies were raised against a synthetic peptide corresponding to the amino acid sequence surrounding the major inactivating phosphorylation site (serine-158) of spinach (Spinacia oleracea) leaf sucrose-phosphate synthase (SPS). The anti-peptide antibodies precipitated highly activated SPS preferentially to ATP-inactivated SPS and interacted only weakly with the sodium dodecyl sulfate-denatured enzyme bound to a membrane. The antibodies blocked phosphorylation but not dephosphorylation of SPS. Highly activated SPS was not entirely dephosphorylated and ATP-inactivated SPS was not completely phosphorylated on serine-158, as indicated by the sensitivities of immunopurified serine-158 phospho- and dephospho-SPS to inhibition by inorganic phosphate. The anti-peptide antibodies can be used to detect changes in the phosphorylation state of serine-158, and they are useful to purify and characterize distinct kinetic forms of SPS.