The presence of free D-aspartic acid in rodents and man

Free D-aspartic acid is present in appreciable quantities in the brain and other tissues of rodents and in human blood. In the newborn rat, the highest concentration of D-aspartic acid was found in cerebral hemispheres, where, at 164 nmol/g (8.4% of the total aspartic acid), the level of D-aspartic acid exceeds that of many essential L-amino acids. The highest ratio of D- to total aspartic acid (38%) occurred in neonatal blood cells. In the adult rat, the highest concentration was present in the pituitary gland (127 nmol/g, 3.8%). Within the central nervous system marked regional differences are present and characteristic changes with development take place. In general, the levels of D-aspartic acid fall rapidly with increasing age. In cerebral hemispheres adult values (13 nmol/g, 0.43%) are approached within one week. D-aspartic acid concentrations may also be higher in young humans since fetal blood, taken from placental cord, contains 2.6 nmol/g (4.9%) of D-aspartic acid, a value five times that of adult human blood. These distributional patterns and developmental changes may be the result of differences in the ability of various tissues to dispose of an extraneous metabolite, or, reflect alterations in a specific functional requirement for D-aspartic acid.     

Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves

Leaf extracts of both Nicotiana tabacum and Nicotiana sylvestris contain multiple forms of catalase (H₂O₂:H₂O₂ oxidoreductase, EC 1.11.1.6) which are separable at different pH values by chromatofocusing columns. Marked changes in distribution of these catalases occur during seedling development and leaf maturation. The form of catalase eluting first (peak 1) was predominant during early seedling growth and present at all stages of development. Two more acidic forms (peaks 2 and 3) appeared later and comprised 29% of the total activity by 11 days postgermination. Mature leaves of N. tabacum contained peak 1 catalase, but peaks 2 and 3 represented 62% of the total activity. No interconversion of peaks 1, 2, and 3 was detected. The three forms of catalase differed in thermal stability with peak 1 > peak 2 peak 3. For N. sylvestris, t_½ at 55°C was 31.5 and 3.0 min for peaks 1 and 3, respectively, and for N. tabacum, t_½ was 41.5 and 3.2 min, respectively. All forms of catalase in tobacco show peroxidatic (measured as ethanol to acetaldehyde conversion) as well as catalatic activities. However, for both Nicotiana species the ratio peroxidatic/catalatic activity is at least 30-fold higher in peak 3 than in peaks 1 and 2. Chromatofocusing of extracts from spinach leaves separated at least four peaks of catalase activity, one of which had a 10-fold higher ratio of peroxidatic/catalatic activity than the others. Short-term growth (5 days) of tobacco seedlings under atmospheric conditions suppressing photorespiration (1% CO₂/21% O₂) reduced total catalase activity and caused a decline in peak 1 catalase and a substantial increase in the activity of peaks 2 and 3 relative to air-grown seedlings at the same stage.     

Effects of CO(2) and O(2) on Photosynthesis and Growth of Autotrophic Tobacco Callus

Gas exchange measurements were made on plants from two natural populations differing in salt tolerance of Andropogon glomeratus, a C₄ nonhalophyte, to examine the effect of salinity on components responsible for differences in photosynthetic capacity. Net CO₂ uptake and stomatal conductance decreased with increasing salinity in both populations, but to a greater extent in the inland (nontolerant) population. The intercellular CO₂ concentrations increased with increasing salinity in the inland population, but decreased in the marsh (tolerant) population. Water use efficiency decreased as salinity increased in the inland population, and remained unchanged in the marsh population. Carboxylation efficiency decreased and CO₂ compensation points increased with increasing salinity in both populations, but to a lesser extent in the marsh population. Carboxylation efficiencies were higher with 2% relative to 21% atmospheric O₂ in salt stressed plants, suggesting that a decrease in the carboxylation:oxygenation ratio of ribulose 1,5-bisphosphate carboxylase/oxygenase was partly responsible for the decrease in photosynthetic capacity. Populational differences in photosynthetic capacity were the result of greater salinity-induced changes in carboxylation efficiency in the inland population, and not due to differences in the stomatal limitation to CO₂ diffusion.     

Regulation of Catalase Activity in Leaves of Nicotiana sylvestris by High CO(2)

The effect of high CO₂ (1% CO₂/21% O₂) on the activity of specific forms of catalase (CAT-1, -2, and -3) (EA Havir, NA McHale [1987] Plant Physiol 84: 450-455) in seedling leaves of tobacco (Nicotiana sylvestris, Nicotlana tabacum) was examined. In high CO₂, total catalase activity decreased by 50% in the first 2 days, followed by a more gradual decline in the next 4 days. The loss of total activity resulted primarily from a decrease in CAT-1 catalase. In contrast, the activity of CAT-3 catalase, a form with enhanced peroxidatic activity, increased 3-fold in high CO₂ relative to air controls after 4 days. Short-term exposure to high CO₂ indicated that the 50% loss of total activity occurs in the first 12 hours. Catalase levels increased to normal within 12 hours after seedlings were returned to air. When seedlings were transferred to air after prolonged exposure to high CO₂ (13 days), the levels of CAT-1 catalase were partially restored while CAT-3 remained at its elevated level. Levels of superoxide dismutase activity and those of several peroxisomal enzymes were not affected by high CO₂. Total catalase levels did not decline when seedlings were exposed to atmospheres of 0.04% CO₂/5% O₂ or 0.04% CO₂/1% O₂, indicating that regulation of catalase in high CO₂ is not related directly to suppression of photorespiration. Antibodies prepared against CAT-1 catalase from N. tabacum reacted strongly against CAT-1 catalase from both N. sylvestris and N. tabacum but not against CAT-3 catalase from either species. This observation, along with the rapid changes in CAT-1 and the much slower changes in CAT-3 suggest that one form is not directly derived from the other.     

Studies of feedback suppression of bile salt synthesis in the bile-fistula rat

We have previously reported that intravenous infusion of taurocholate at 10 mumol (100 g.hr) into bile-fistula rats suppressed bile salt synthesis by 85% (Pries et al. 1983. J. Lipid Res. 24: 141-146). Recently, however, infusion rates twice this high have been reported not to suppress synthesis (Davis et al. 1984. Falk Symposium 42. MTP Press Ltd., Boston. 37-45). Because the only major difference in design of these two studies was supplementation with sodium bicarbonate to replace biliary losses induced by bile salt choleresis, we have repeated our studies with and without bicarbonate supplementation. Without bicarbonate, as before, we found suppression of synthesis during infusion of taurocholate at 10 mumol/(100 g.hr). With bicarbonate, no suppression of synthesis occurred at these infusion rates. These data indicate that bicarbonate supplementation is essential when testing physiological effects of infused bile salt in the bile-fistula rat.     

A Starchless Mutant of Nicotiana sylvestris Containing a Modified Plastid Phosphoglucomutase

A mutant (NS 458) of Nicotiana sylvestris (Spegazzini and Comes) unable to synthesize leaf starch was isolated in the M₂ generation following ethyl methanesulfonate mutagenesis by testing with iodine. Segregation ratios in reciprocal F₂ progenies showed that the starchless phenotype resulted from a recessive mutation in a single nuclear gene. DEAE-agarose chromatography showed that the mutant is grossly deficient in plastid phosphoglucomutase (EC 2.1.5.1) activity. The structure of the enzyme is changed, as evidenced by increased Michaelis constants and by the prolonged activation period (>40 minutes) observed when the enzyme is assayed in triethanolamine buffer rather than imidazole buffer. The activity of the wild-type enzyme with saturating glucose 6-P alone was 7% of the activity when saturating glucose 1,6-P₂ was also present. The results suggest that glucose 1,6-P₂ is both an effector and a dissociable reaction intermediate. The growth rate of mutant and wild-type plants were not significantly different in continuous light and on an 8-hour dark, 16-hour light cycle and the mutants grew normally under greenhouse conditions. The mutant supports growth during diurnal periods of darkness by vacuolar storage of sugars instead of chloroplast storage of starch. The simplification in metabolism achieved by blocking the diversion of plastid fructose-6-P to starch facilitates the induction of oscillations in CO₂ fixation.     

A Mutant of Nicotiana sylvestris Lacking Serine:Glyoxylate Aminotransferase: Substrate Specificity of the Enzyme and Fate of [2-C]Glycolate in Plants with Genetically Altered Enzyme Levels

The photorespiratory mutant of Nicotiana sylvestris, NS 349, lacking serine:glyoxylate aminotransferase (SGAT) grows in 1% CO₂ but not in normal air (NA McHale, EA Havir, I Zelitch 1988 Theor Appl Genet. In press). Alanine:hydroxypyruvate and asparagine:hydroxypyruvate aminotransferase activities were also lacking in the mutant, and plants heterozygous with respect to SGAT which grow in normal air had 50% of the activities present in homozygous plants. Therefore, all these activities are associated with the same enzyme. On feeding [2-¹⁴C]glycolate to leaf discs in the light, NS 349 showed reduced incorporation of radioactivity into the neutral and organic acid fractions and increased incorporation into the amino acid fraction, principally into serine. The effect of reducing SGAT by 50% in heterozygous plants produced little change in the metabolism of [2-¹⁴C]glycolate, showing there is a large excess of this enzyme in wild-type plants.