Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms.

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP+/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, Km for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg2+ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots.

Anaemia and serum protein alteration in patients with pressure ulcers.

The presence of anaemia and serum protein alteration frequently makes the treatment of pressure ulcers more difficult. Several haemato-chemical parameters were observed in 40 patients with sacral pressure ulcers in order to determine the pathogenesis of these complications. All of the patients showed mild-moderate anaemia with low serum iron and normal or increased ferritin and hypoproteinemia with hypoalbuminemia. Our results suggest that both anaemia and serum protein alteration depend on the chronic inflammatory state due to the presence of pressure ulcers. Both anaemia and hypoproteinemia disappeared after pressure ulcer healing. A correct diagnosis is important for the treatment. Iron therapy is useless and potentially dangerous (iatrogenic haemochromatosis) since anaemia is the result of the inability to use iron stores and not iron deficiency. The treatment of serum protein alterations should be based on a dietary therapy rich in protein and calories; the administration of albumin should be reduced, since albumin is low in essential amino-acids and too expensive; albumin administration should be limited to cases with severe hypoproteinemia and oedema.

Ammonium assimilation by young plants of Hordeum vulgare in light and darkness: effects on respiratory oxygen consumption by roots.

Barley plants (Hordeum vutgare L.) grown for 10 d in nitrogen-free hydroponic culture, after a rapid initial phase absorbed supplied NH4 (+) at a constant rate of 15.1 ±1.2 µ mol h(-1) g(-1) f. wt in the light, arid at a rate of 13.81 ± 1.6 µ mol h(-1) g(-1) f. wt in darkness. Ammonium-grown plants assimilated NH4 (+) at a rate of 7.5 ± 0.33 µmol h-1 g(-1) f. wt and at a 50% lower rate in darkness. Nitrogen-free grown plants showed low concentrations of free amino acids in both root and shoot tissues. Supplying NH4 (+) caused an immediate increase in the concentration of free amino in the root tissues of both illuminated and darkened plants over a 120 mm period. The increase in concentration of glutamine then exhibited a lag period of 120 min, after which it resumed, but to a very small extent. Glutamine also accumulated in shoot tissue of illuminated plants at increasing rates, attaining a concentration which, 8 h after NH4 (+) supply, was 1.61-fold greater than that attained in the roots. In shoots of darkened plants, by contrast, the concentration of glutamine increased slowly and was always smaller than that in the root tissue. Overall formation of glutamine (in shoots and roots) occurred at decreasing rates during the first 4 h, and then at increasing rates. The increase was more pronounced in illuminated plants than in darkened plants, liven 24 h after NH4 (+) was supplied, glutamine content in root tissue was lower than that in shoot tissue. However, 48 h later, the concentrations of glutamine in root and shoot were similar, attaining values that were almost 47-fold (in root) and 134-fold (in shoot) greater than initial values. Significant levels of asparagine were detected in the root and in the shoot 24 h after adding NH4 (+) . These increased further during the succeeding period. Ammonium supply caused a transitory drop in the concentration of ATP in root tissue, along with noticeable transitory variations in glucose-6-P concentration. A permanent decrease in free glucose concentration was also detected. Addition of NH4 (+) caused 2- and 1.43-fold increases in respiratory oxygen consumption by roots of illuminated and darkened plants, respectively. Both in the light and in the dark, the root tissue accumulated methylammonium up to a concentration of 55-67 µmol h(-1) g(-1) f. wt. Methylammonium was never found in shoot tissue of either illuminated or darkened plants. Methylammonium stimulated respiration of root barley plants by a factor of 1.2. Regulatory aspects of NH4 (+) metabolism are discussed.

Depression of nitrate reductase in the presence of excess ammonium in a unicellular alga growing under conditions of phosphate limitation.

Chemostat cultures of the unicellular alga Cyanidium caldarium have shown that under conditions of phosphate limitation nitrate reductase is completely derepressed even in cells growing in a large excess of ammonium, but that it occurs mainly in a catalytically inactive form. It is hypothesized that phosphate limitation contributes to maintaining intracellular level of glutamine suitable to stimulate inactivation but not repression of nitrate reductase. It is not excluded that in addition to variations in the intracellular level of glutamine, there are other metabolic events of the cell by which repression and inactivation of nitrate reductase could be differently influenced.

Active and inactive nitrate reductase. Effects of mild treatment with denaturing agents of protein.

Nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) of the unicellular alga Cyanidium caldarium can exist in two interconvertible forms; one catalytically active and one inactive. The inactive nitrate reductase can be activated by mild treatment with denaturing agents of protein. By treatment with urea or mersalyl, activation of both the NADPH and benzyl viologen activities can be realized under mild conditions, whereas by treatment with heat, the activation of benzyl viologen activity is concomitant with loss of the NADPH activity. On the other hand, both activities are activated and destroyed concomitantly by ethylene glycol. In the present of FAD, either activation of benzyl viologen activity or loss of NADPH activity upon heating occur only at higher temperatures. The existence of a controlling region in the nitrate reductase molecule is postulated.