Regulation of the activity of intracellular alanylaminopeptidase synthesized by Pseudomonas sp.

Activity of purified alanylaminopeptidase of Pseudomonas sp. measured in the presence of the alanine derivative of 2-naphthoic acid (NA-Ala) is inhibited by 1,10-phenanthroline, EDTA, bestatin and amastatin; this finding supports the conclusion that this enzyme is a metallo-aminopeptidase. A decrease of its activity in the presence of iodoacetamide and its activation by thiols points to the significant role of -SH groups in the regulation of its activity. Co2+, Ca2+ and Mg2+ ions increased the enzyme activity while Zn2+, Cd2+ and Pb2+ markedly inhibited the enzyme even at low concentrations. A high thermal stability of alanylaminopeptidase depended on the presence of 1 mmol/L Co2+ and of 1 mmol/L L-cysteine in the incubation mixture.

Production, purification and characterization of intracellular alanylaminopeptidase of Pseudomonas sp.

The soil bacterium Pseudomonas sp. was found to synthesize an aminopeptidase that prefers Ala-beta-naphtylamide as substrate. The enzyme was purified 660-fold by ammonium sulfate fractionation, preparative electrophoresis, ion exchange chromatography on Protein-Pak Q 8 HR and molecular sieving chromatography on Zorbax SE-250. When purified to homogeneity, the enzyme was shown to be a monomeric protein with a molar mass of 65 kDa; it showed a maximum activity at pH 7.5 and 45 degrees C.

Purification and partial characterization of glutamine synthetase isoforms from Triticale seedlings.

Two isoforms of glutamine synthetase (EC 6.3.1.2), cytoplasmic (GS1) and chloroplastic (GS2) were isolated from shoots of 14-day-old Triticale seedlings, and purified 260-fold and 248-fold, respectively. Specific activities of the two preparations were 35.1 and 33.5 mumol x min-1 per mg of protein, respectively. Both crude extracts and homogeneous GS1 and GS2 preparations required divalent metal ions (Mg2+, Mn2+, Co2+) for their activities. Mg2+ was the most effective activator, the highest activity of GS1 being reached at 5 mM, and that of GS2 at 20 mM MgCl2. The optimum pH for the two isoforms showed large differences, dependent also on the kind of divalent ion. Molecular masses of GS1 and GS2 were 305000 Da and 385200 Da, respectively. It seems that native protein of GS1 is built from eight identical subunits of Mm 38000 Da and that of GS2 of the same number of subunits but of Mm of about 48000 Da. Proteins of GS isoforms differed significantly in their amino-acid composition.

Reduced and oxidised glutathione and glutathione-reductase activity in tissues of Pisum sativum.

In three-week-old pea plants (Pisum sativum L., cv. Little Marvel) grown in the light, total glutathione levels were highest in apex and expanding leaves (1.5 µmol·(g FW)(-1)), and lower (0.4-0.6 µmol·(g FW)(-1)) in older leaves and roots. In the light period, levels in expanded leaves increased by about 40%, compared with dark values, with lesser increases in roots and apex. In illuminated plants the proportion in the reduced form was 86-88% in leaves and 80% in roots, and these values fell during the dark period (to 82% and 69%, respectively). Reduced glutathione makes up 65-70% of the low-molecular-weight thiol in leaves, but over 95% in roots. Chloroplasts contained about 10% of the leaf glutathione, at a concentration of 1-2 mM; total glutathione in the chloroplasts, and the proportion of oxidised form (GSSG) increased in light, while NADP(+)/NADPH ratios decreased, indicating both synthesis and active oxidation of glutathione in light. Chloroplasts contained 52% (young leaf) to 75% (mature leaf) of the GSSG-reductase (EC 1.6.4.2) activity in the leaves. In roots, over 30% of the GSSG reductase was in the plastid fraction. Very little GSSG-reductase activity was associated with mitochondria in leaf or root.

Photosynthesis in detached rye leaves at normal and low oxygen concentration. I. Incorporation of 14CO2 into 2-oxo acids.

In the leaves of rye plants at the milk maturity stage of grains, the following eight 2-oxo acids were detected by catalytic hydrogenolysis of the respective dinitrophenylhydrazone derivatives: 2-oxoglutarate, pyruvate, oxaloacetate, glyoxylate, hydroxypyruvate, 2-oxoisovalerate, 2-oxo-6-aminocaproate and 2-oxo-5-guanidinovalerate. Lowering of O2 concentration decreased incorporation of 14CO2 not only into the intermediates of the glycolate pathway, i.e. glyoxylate and hydroxypyruvate but also into other 2-oxo acids. At the same time, the level of these 2-oxo acids was much higher than in the leaves photosynthesizing at normal O2 concentration. On transfer to the dark for 6 h, the level of most of 2-oxo acids was lowered and the differences in specific radioactivity persisted. It seems that, under normal conditions, formation of pyruvate, oxaloacetate, glyoxylate and hydroxypyruvate in the light is closely related to CO2 assimilation.

Photosynthesis in detached rye leaves at normal and low oxygen concentration. II. Incorporation of 14CO2 into amino acids.

The pools of Arg, Gly, His, Ile, Ser, Glu, Leu, Val and Asp were lower during photosynthesis at 1% O2 concentration. At the same time specific radioactivity of a number of amino acids--following 14CO2 incorporation--was different than that at atmospheric O2 concentration. This is probably due to the inhibition of the photorespiratory nitrogen cycle and the resulting NH+4 deficiency. The pattern of response to O2 concentration suggests that in rye plants in the light, all Gly and a part of Ser are synthesized by an O2-sensitive pathway, i.e. via the glycolate pathway, but most of Ser is probably formed from 3-phosphoglycerate (3-PGA) via the O2-insensitive pathway. Changes in the pool size and specific radioactivity after 6 h incubation in darkness suggest that synthesis of Gly is strongly light-dependent, whereas synthesis of Ser was substantial also in darkness. The specific radioactivity of Ala, Asp, Ser and Glu indicates that in darkness those amino acids are formed from a common precursor, i.e. glycolytic 3-PGA, and undergo rapid metabolic turnover.