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Synthesis and properties of 5-azido-UDP-glucose. Development of photoaffinity probes for nucleotide diphosphate sugar binding sites

A new active site directed photoaffinity probe, which is a model compound for studying nucleotide diphosphate sugar binding proteins, has been synthesized by coupling 5-azido-UTP and [32P]Glc-1-P using yeast UDP-glucose pyrophosphorylase to produce [beta-32P]5-azidouridine 5'-diphosphoglucose (5N3UDP-Glc). This probe has photochemical properties similar to that of 5-azidoUTP (Evans, R. K., and Haley, B. E. (1987) Biochemistry 26, 269-276). The efficacy of 5N3UDP-Glc as an active site directed probe was demonstrated using yeast UDP-Glc pyrophosphorylase. Saturation effects of photoinsertion were observed with an apparent Kd of 51 microM and the natural substrate, UDP-Glc, prevented photoinsertion of [beta-32P]5N3UDP-Glc with an apparent Kd of 87 microM. Prevention of photoinsertion was also seen with UTP and pyrophosphate with apparent Kd values less than 200 microM. UMP, UDP, ATP, and GTP were much less effective competitors. Selective photoinsertion was observed with several partially purified enzymes including UDP-Glc dehydrogenase, UDP-Gal-4-epimerase, Gal-1-P uridyltransferase, and phosphorylase a. The absence of nonselective photoinsertion into bulk proteins was demonstrated with crude homogenates of rabbit liver as well as with several UDP-Glc binding proteins. Of the six purified enzymes tested, only phosphoglucomutase has been shown to incorporate radiolabel from the photoprobe in the absence of UV irradiation. These results and a discussion of the utility of 5N3UDP-Glc for detecting UDP-Glc binding proteins and isolating active site peptides are presented.     

Effect of CO2 on the fermentation capacities of the acetogen Peptostreptococcus productus U-1.

The fermentative capacities of the acetogenic bacterium Peptostreptococcus productus U-1 (ATCC 35244) were examined. Although acetate was formed from all the substrates tested, additional products were produced in response to CO2 limitation. Under CO2-limited conditions, fructose-dependent growth yielded high levels of lactate as a reduced end product; lactate was also produced under CO2-enriched conditions when fructose concentrations were elevated. In the absence of supplemental CO2, xylose-dependent growth yielded lactate and succinate as major reduced end products. Although supplemental CO2 and acetogenesis stimulated cell yields on fructose, xylose-dependent cell yields were decreased in response to CO2 and acetogenesis. In contrast, glycerol-dependent growth yielded high levels of ethanol in the absence of supplemental CO2, and pyruvate was subject to only acetogenic utilization independent of CO2. CO2 pulsing during the growth of CO2-limited fructose cultures stopped lactate synthesis immediately, indicating that CO2-limited cells were nonetheless metabolically poised to respond quickly to exogenous CO2. Resting cells that were cultivated at the expense of fructose without supplemental CO2 readily consumed fructose in the absence of exogenous CO2 and formed only lactate. Although the specific activity of lactate dehydrogenase was not appreciably influenced by supplemental C02 during cultivation, cells cultivated on fructose under CO2-enriched conditions displayed minimal capacities to consume fructose in the absence of exogenous CO2. These results demonstrate that the utilization of alternative fermentations for the conservation of energy and growth of P. productus U-1 is augmented by the relative availability of CO2 and growth substrate.     

Direct Inhibition of Plant Mitochondrial Respiration by Elevated CO2

Doubling the concentration of atmospheric CO2 often inhibits plant respiration, but the mechanistic basis of this effect is unknown. We investigated the direct effects of increasing the concentration of CO2 by 360 [mu]L L-1 above ambient on O2 uptake in isolated mitochondria from soybean (Glycine max L. cv Ransom) cotyledons. Increasing the CO2 concentration inhibited the oxidation of succinate, external NADH, and succinate and external NADH combined. The inhibition was greater when mitochondria were preincubated for 10 min in the presence of the elevated CO2 concentration prior to the measurement of O2 uptake. Elevated CO2 concentration inhibited the salicylhydroxamic acid-resistant cytochrome pathway, but had no direct effect on the cyanide-resistant alternative pathway. We also investigated the direct effects of elevated CO2 concentration on the activities of cytochrome c oxidase and succinate dehydrogenase (SDH) and found that the activity of both enzymes was inhibited. The kinetics of inhibition of cytochrome c oxidase were time-dependent. The level of SDH inhibition depended on the concentration of succinate in the reaction mixture. Direct inhibition of respiration by elevated CO2 in plants and intact tissues may be due at least in part to the inhibition of cytochrome c oxidase and SDH.     

Peptide crystal growth via sulfonate salt formation: The structure of bis-glycylglycine-1,5-naphthalenedisulfonate hydrate

Summary Continuing a line of investigations on methods for formation and growth of high-quality crystals of peptides, the glycylglycine sequence has been crystallized by evaporation methods as a salt with 1,5-naphthalenedisulfonic acid. The structure of the peptide is highly extended, and is conformationally quite similar to the structures which have been characterized for other zwitterionic and salt forms of this sequence. Thus, crystallization as a salt with this sulfonic acid has imposed no undue influence upon the molecular conformation. These results offer further indication that the preparation of peptide sulfonate salts, particularly with arene templates, may have broad general utility for crystallization of interesting sequences which until now have not been approachable in their zwitterionic forms.     

beta]-Glucan Synthesis in the Cotton Fiber (III. Identification of UDP-Glucose-Binding Subunits of [beta]-Glucan Synthases by Photoaffinity Labeling with [[beta]-32P]5[prime]-N3-UDP-Glucose

Using differential product entrapment and photolabeling under specifying conditions, we identifIed a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Gossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring [beta]-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of [beta]-1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring [beta]-1,3-glucan synthesis, is coincident with [beta]-1,3-glucan synthase activity, and is calcium dependent. The possible involvement of other polypeptides in the synthesis of [beta]-1,3-glucan is discussed.     

Interactions between C3 and C4 salt marsh plant species during four years of exposure to elevated atmospheric CO2

Elevated atmospheric CO2 is known to stimulate photosynthesis and growth of plants with the C₃ pathway but less of plants with the C₄ pathway. An increase in the CO₂ concentration can therefore be expected to change the competitive interactions between C₃ and C₄ species. The effect of long term exposure to elevated CO₂ (ambient CO₂ concentration +340 µmol CO₂ mol^-1) on a salt marsh vegetation with both C₃ and C₄ species was investigated. Elevated CO₂ increased the biomass of the C₃ sedgeScirpus olneyi growing in a pure stand, while the biomass of the C₄ grassSpartina patens in a monospecific community was not affected. In the mixed C₃/C₄ community the C₃ sedge showed a very large relative increase in biomass in elevated CO₂ while the biomass of the C₄ species declined.The C₄ grassSpartina patens dominated the higher areas of the salt marsh, while the C₃ sedgeScirpus olneyi was most abundant at the lower elevations, and the mixed community occupied intermediate elevations.Scirpus growth may have been restricted by drought and salt stress at the higher elevations, whileSpartina growth at the lower elevations may be affected by the higher frequency of flooding. Elevated CO₂ may affect the species distribution in the salt marsh if it allowsScirpus to grow at higher elevations where it in turn may affect the growth ofSpartina.     

Direct and indirect effects of elevated CO2 on transpiration from Quercus myrtifolia in a scrub-oak ecosystem

Elevated atmospheric carbon dioxide (C_a) usually reduces stomatal conductance, but the effects on plant transpiration in the field are not well understood. Using constant‐power sap flow gauges, we measured transpiration from Quercus myrtifolia Willd., the dominant species of the Florida scrub‐oak ecosystem, which had been exposed in situ to elevated C_a (350 µmol mol ^{? 1} above ambient) in open‐top chambers since May 1996. Elevated C_a reduced average transpiration per unit leaf area by 37%, 48% and 49% in March, May and October 2000, respectively. Temporarily reversing the C_a treatments showed that at least part of the reduction in transpiration was an immediate, reversible response to elevated C_a. However, there was also an apparent indirect effect of C_a on transpiration: when transpiration in all plants was measured under common C_a, transpiration in elevated C_a‐grown plants was lower than that in plants grown in normal ambient C_a. Results from measurements of stomatal conductance (g_s), leaf area index (LAI), canopy light interception and correlation between light and g_s indicated that the direct, reversible C_a effect on transpiration was due to changes in g_s caused by C_a, and the indirect effect was caused mainly by greater self‐shading resulting from enhanced LAI, not from stomatal acclimation. By reducing light penetration through the canopy, the enhanced self‐shading at elevated C_a decreased stomatal conductance and transpiration of leaves at the middle and bottom of canopy. This self‐shading mechanism is likely to be important in ecosystems where LAI increases in response to elevated C_a.