Corn phosphoglycolate phosphatase: Modulation of activity by pyridine nucleotides and adenylate energy charge.

The activity of corn phosphoglycolate phosphatase (EC 3.1.3.18), a bundle sheath chloroplastic enzyme, is modulated, in vitro, both by NADP(H) and adenylate energy charge. The Vmax of the enzyme is increased by NADP (25%) and NADPH (16%) whatever the pH used, 7.0 or 7.9 respective pH of the stroma in the dark and in the light. At both pH, the adenylate energy charge alone has a positive effect with two peaks of activation, characteristics for this enzyme, at 0.2 and a maximum at 0.8 accentuated under nonsaturating concentration of phosphoglycolate. At low energy charge, NADP(H) increased the activation with an additive effect most particularly observed at pH 7.9 under saturating phosphoglycolate concentration; at high energy charge, NADP(H) had a positive or negative effect on the activation, depending on the pH value and the concentrations of substrate and NADP(H).The ferredoxin-thioredoxin system does not regulate the activity since i) DTT addition do not have any effect, ii) the light-reconstituted system containing ferredoxin, ferredoxin-thioredoxin reductase, thioredoxins and thylakoids is not effective either. However, light-dark experiments indicate that phosphophycolate phosphatase can be subjected to a fine tuning of its activity.All these data suggest that light cannot induce a modification of the protein but could exert a tight control of its activity by the intermediate of Mg(2+) and substrate concentrations and the levels of metabolites such as NADP(H), ATP, ADP, AMP. So, the regulation of the activity shown, in vitro, by energy charge and NADP(H) might be of physiological significance.

Corn phosphoglycolate phosphatase: purification and properties.

Phosphoglycolate phosphatase (EC 3.1.3.18), isolated from maize leaf bundle sheath, was purified 200-fold to a specific activity of about 99 µmol mg(-1) protein · min(-1). The purification procedure included Sephadex G-75 filtration, and diethylaminoethyl-cellulose and Phospho-Ultrogel A6R chromatography. This partially purified enzyme exhibited optimum activity over a broad pH range, from pH 6.3 to pH 8.0. It displayed a very high degree of specificity for phosphoglycolate and required a divalent cation to be active; Mg(2+) was the most effective activator. Saturation curves of the Michaelis-Menten type were observed both with phosphoglycolate (Km=0.57 mmol·l(-1)) and with Mg(2+) (Km=0.015 mmol·l(-1)). The activation constant for Mg(2+) was unchanged when the pH was raised from 7.0 to 8.0. These results indicate that variations of stromal pH and Mg(2+) during the darklight transition could not directly modifity the activity of the phosphoglycolate phosphatase in maize bundle-sheath chloroplasts. The undissociated protein showed a pI of 4.95, as determined by isoelectric focusing. For the native phosphatase a molecular mass of about 61 500 Da was estimated by polyacrylamide gradient gel electrophoresis. The subunit was found to have a relative molecular mass of 31 500 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is concluded that maize phosphoglycolate phosphatase is a dimer.

[Metabolism of γ-aminobutyrate in Agaricus bisporus Lge. : II: γ-aminobutyrate: γ-ketoglutarate aminotransferase]. / Metabolisme du γ-aminobutyrate chez Agaricus bisporus Lge. : II. La γ-aminobutyrate: α-cetoglutarate aminotransferase.

Transamination between γ-aminobutyrate and α-ketoglutarate provides a pathway for the utilization of γ-aminobutyrate in fruit-bodies of Agaricus bisporus Lge. This reaction leads to the formation of succinic semialdehyde, a metabolic intermediate in the metabolism of γ-aminobutyrate to succinate in the cell. γ-aminobutyrate: α-ketoglutarate aminotransferase (E.C. 2.6.1.19) was sonically extracted from the mitochondrial fraction and partially purified by DEAE-cellulose column chromatography. Aminotransferase had a pH optimum between 8.1 and 8.5 and did not require pyridoxal-phosphate in vitro; however, the enzyme was inhibited by carbonyl-trapping reagents such as pyridoxal-phosphate activated enzymes. The Km values for γ-aminobutyrate and α-ketoglutarate calculated from Lineweaver-Burk plots were 2.2×10(-4) M and 2.5×10(-3) M, respectively. The transaminase was specific for α-ketoglutarate but not for γ-aminobutyrate; aspartate, α-alanine and δ-aminovalerianate also functioned as amino-group donors. Activity of the enzyme was not influenced by the addition of carboxylic acids of the Krebs cycle. The reversal of the transamination reaction showed optimal rates at pH 9.0-9.3. Some considerations on the physiological significance of these results are given.