Alternative pathway activities in aged potato tuber slices measured by three methods

To find a simple and reliable oxygen electrode-based method to estimate the values of alternative pathway activity (V _alt) and its contribution to total respiration V _alt/V _t) in aged potato (Solanum tuberosum L.) tuber slices, we compared conventional hydroxamate-inhibiting method, improved hydroxamate-inhibiting method with 2,6-dichlorophenol indophenol (DCPIP), and the oxygen isotope discrimination (OID) method. The values of V _alt and V _alt/V _t obtained with an improved hydroxamate-inhibiting method with DCPIP in 12-h- and 24-h-aged slices were about twice higher than those with the conventional hydroxamate-inhibiting method. Only a relatively small difference in the values of V _alt and V _alt/V _t obtained by the OID method and the improved hydroxamate-inhibiting method with DCPIP in 12-h and 24-h-aged slices was observed. These results indicated that the improved hydroxamate-inhibiting method with DCPIP could be considered as a new, simple, and reliable technique for the noninvasive assay of the AP activity.From Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 311–315.Original English Text Copyright © 2005 by Hou, Zhou, Kong, Liang, Zhang.This article was submitted by the authors in English.This revised version was published online in April 2005 with a corrected cover date.     

Residual backbone and side-chain <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of the intrinsic transmembrane light-harvesting 2 protein complex by solid-state Magic Angle Spinning NMR spectroscopy

This study reports the sequence specific chemical shifts assignments for 76 residues of the 94 residues containing monomeric unit of the photosynthetic light-harvesting 2 transmembrane protein complex from Rhodopseudomonas acidophila strain 10050, using Magic Angle Spinning (MAS) NMR in combination with extensive and selective biosynthetic isotope labeling methods. The sequence specific chemical shifts assignment is an essential step for structure determination by MAS NMR. Assignments have been performed on the basis of 2-dimensional proton-driven spin diffusion ¹³C–¹³C correlation experiments with mixing times of 20 and 500 ms and band selective ¹³C–¹⁵N correlation spectroscopy on a series of site-specific biosynthetically labeled samples. The decreased line width and the reduced number of correlation signals of the selectively labeled samples with respect to the uniformly labeled samples enable to resolve the narrowly distributed correlation signals of the backbone carbons and nitrogens involved in the long -helical transmembrane segments. Inter-space correlations between nearby residues and between residues and the labeled BChl a cofactors, provided by the ¹³C–¹³C correlation experiments using a 500 ms spin diffusion period, are used to arrive at sequence specific chemical shift assignments for many residues in the protein complex. In this way it is demonstrated that MAS NMR methods combined with site-specific biosynthetic isotope labeling can be used for sequence specific assignment of the NMR response of transmembrane proteins.     

Inorganic nitrogen source utilization byFagus crenata on different soil types

The nitrogen source utilization by Fagus crenata distributed on soils with different forms of inorganic nitrogen in a cool-temperate deciduous forest in central Japan was determined by measuring foliar ¹⁵N. Two soil habitat types along a slope were delineated based on nitrogen transformation patterns, i.e., soils with high net nitrification rates and with no or low net nitrification, respectively. Despite differences in soil types, the study species, F. crenata, was distributed along the entire slope. The foliar ¹⁵N value of F. crenata from the lower slope area was significantly lower than that from the upper slope. Given the finding of a previous study that the ¹⁵N of NO₃^– was lower than that of NH₄⁺, our results indicate that reliance on NO₃^– as a nitrogen source was greater in the lower slope area than in the upper slope area. Differences in the values of foliar ¹⁵N were about 1, which is far less than the 10 ¹⁵N value of soil inorganic N reported in the previous study. This discrepancy might suggest that the study species utilized NO₃^– even in the upper site where net nitrification had not been detected. Measurements of nitrate reductase activity, an index of NO₃^– uptake, also supported this interpretation. Nitrate reduction occurred in leaves and roots at both the lower and the upper sites. Thus, the study species may be able to use NO₃^– even in soils with no net nitrification, a factor that could allow the distribution of F. crenata along the entire length of the slope.     

Water uptake and transport in lianas and co-occurring trees of a seasonally dry tropical forest

Water uptake and transport were studied in eight liana species in a seasonally dry tropical forest on Barro Colorado Island, Panama. Stable hydrogen isotope composition (D) of xylem and soil water, soil volumetric water content (_v), and basal sap flow were measured during the 1997 and 1998 dry seasons. Sap flow of several neighboring trees was measured to assess differences between lianas and trees in magnitudes and patterns of daily sap flow. Little seasonal change in _v was observed at 90–120 cm depth in both years. Mean soil water D during the dry season was –19 at 0–30 cm, –34 at 30–60 cm, and –50 at 90–120 cm. Average values of xylem D among the liana species ranged from –28 to –44 during the middle of the dry season, suggesting that water uptake was restricted to intermediate soil layers (30–60 cm). By the end of the dry season, all species exhibited more negative xylem D values (–41 to –62), suggesting that they shifted to deeper water sources. Maximum sap flux density in co-occurring lianas and trees were comparable at similar stem diameter (DBH). Furthermore, lianas and trees conformed to the same linear relationship between daily sap flow and DBH. Our observations that lianas tap shallow sources of soil water at the beginning of the dry season and that sap flow is similar in lianas and trees of equivalent stem diameter do not support the common assumptions that lianas rely primarily on deep soil water and that they have higher rates of sap flow than co-occurring trees of similar stem size.     

Carbon isotope composition of canopy leaves in a tropical forest in Panama throughout a seasonal cycle

The seasonal variation in ¹³C values was measured in leaves from 17 upper canopy, five mid- canopy and in four gap tree species, as well as in five epiphyte and five vine species, in a seasonally dry lowland tropical forest at Parque Natural Metropolitano near Panama City, Republic of Panama. No seasonal variation was detected in the ¹³C values of mature exposed leaves from either the upper or mid- canopy. However, canopy position did influence the ¹³C value. The mean isotopic composition of leaves from the mid- canopy was more negative than that of the upper canopy throughout the year. The ¹³C value was also influenced by leaf development, with juvenile leaves on average 1.5 less negative than mature leaves. The five epiphyte species exhibited ¹³C values that were typical of crassulacean acid metabolism (CAM). Codonanthe uleana, with isotopic values of –19.9 to –22.1, is only the second species in the Gesneriaceae reported to express CAM, whereas values between –14.6 and –22.0 indicate that Peperomia macrostachya can exhibit different degrees of CAM. The isotopic composition of exposed mature leaves from the vines showed little interspecific variation and was similar to the upper-canopy leaves of the trees.     

Stable carbon isotope ratios for the gray whale (<Emphasis Type="Italic">Eschrichtius robustus</Emphasis>) in the breeding grounds of Baja California Sur,Mexico

This work investigated the feeding ecology and behaviour of gray whales in Bahía Magdalena. Underwater observations of bottom feeding were made (n=4). Skin biopsies of the gray whale had a carbon isotope value of –16.5 ± 0.1 (range from –16.4 to –16.7, n=7). Prey in Bahía Magdalena had a carbon isotope value of –18.4. Dietary enrichment from prey in Bahía Magdalena would correspond to 2 ± 0.1, whereas previously published results for prey in Alaska would result in an enrichment of 3, which suggests that whales were more likely feeding on prey from Bahía Magdalena. Carbon isotopic oscillation along the baleen plate of a stranded 1-year-old whale showed a variation in diet during the year, which suggests continual feeding during this time and corresponding to dietary sample measurements from Bahía Magdalena in winter and Alaska in summer.     

The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-[(2)H]indole

The bacterial tryptophan synthase alpha(2)beta(2) complex catalyzes the final reactions in the biosynthesis of L-tryptophan. Indole is produced at the active site of the alpha-subunit and is transferred through a 25-30 A tunnel to the beta-active site, where it reacts with an aminoacrylate intermediate. Lane and Kirschner proposed a two-step nucleophilic addition-tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-[(2)H]indole reacts with the aminoacrylate intermediate. We have now observed a KIE of 1.4-2.0 in the reaction of 3-[(2)H]indole with the aminoacrylate intermediate in the presence of monovalent cations, but not when an alpha-subunit ligand, disodium alpha-glycerophosphate (Na(2)GP), is present. Rapid-scanning stopped flow kinetic studies were performed of the reaction of indole and 3-[(2)H]indole with tryptophan synthase preincubated with L-serine, following the decay of the aminoacrylate intermediate at 350 nm, the formation of the quinonoid intermediate at 476 nm, and the formation of the L-Trp external aldimine at 423 nm. The addition of Na(2)GP dramatically slows the rate of reaction of indole with the alpha-aminoacrylate intermediate. A primary KIE is not observed in the reaction of 3-[(2)H]indole with the aminoacrylate complex of tryptophan synthase in the presence of Na(2)GP, suggesting binding of indole with tryptophan synthase is rate limiting under these conditions. The reaction of 2-methylindole does not show a KIE, either in the presence of Na(+) or Na(2)GP. These results support the previously proposed mechanism for the beta-reaction of tryptophan synthase, but suggest that the rate limiting step in quinonoid intermediate formation from indole and the aminoacrylate intermediate is deprotonation.     

Coordinated changes in photosynthesis,water relations and leaf nutritional traits of canopy trees along a precipitation gradient in lowland tropical forest

We investigated leaf physiological traits of dominant canopy trees in four lowland Panamanian forests with contrasting mean annual precipitation (1,800, 2,300, 3,100 and 3,500 mm). There was near complete turn-over of dominant canopy tree species among sites, resulting in greater dominance of evergreen species with long-lived leaves as precipitation increased. Mean structural and physiological traits changed along this gradient as predicted by cost–benefit theories of leaf life span. Nitrogen content per unit mass (N_mass) and light- and CO₂-saturated photosynthetic rates per unit mass (P_mass) of upper canopy leaves decreased with annual precipitation, and these changes were partially explained by increasing leaf thickness and decreasing specific leaf area (SLA). Comparison of 1,800 mm and 3,100 mm sites, where canopy access was available through the use of construction cranes, revealed an association among extended leaf longevity, greater structural defense, higher midday leaf water potential, and lower P_mass, N_mass, and SLA at wetter sites. Shorter leaf life spans and more enriched foliar ¹⁵N values in drier sites suggest greater resorption and re-metabolism of leaf N in drier forest. Greater dominance of short-lived leaves with relatively high P_mass in drier sites reflects a strategy to maximize photosynthesis when water is available and to minimize water loss and respiration costs during rainless periods. Overall, our study links coordinated change in leaf functional traits that affect productivity and nutrient cycling to seasonality in lowland tropical forests.     

Differentially isotope-coded N-terminal protein sulphonation: combining protein identification and quantification

Most proteomic labelling technologies intend to improve protein quantification and/or facilitate (de novo) peptide sequencing. We present here a novel stable-isotope labelling method to simultaneously identify and quantify protein components in complex mixtures by specifically derivatizing the N-terminus of proteins with 4-sulphophenyl isothiocyanate (SPITC). Our approach combines protein identification with quantification through differential isotope-coded labelling at the protein N-terminus prior to digestion. The isotope spacing of 6 Da (unlabelled vs. six-fold 13C-labelled tag) between derivatized peptide pairs enables the detection on different MS platforms (MALDI and ESI). Optimisation of the reaction conditions using SPITC was performed on three model proteins. Improved detection of the N-terminally derivatized peptide compared to the native analogue was observed in negative-ion MALDI-MS. Simpler fragmentation patterns compared to native peptides facilitated protein identification. The 13C-labelled SPITC resulted in convenient peptide pair spacing without isotopic overlap and hence facilitated relative quantification by MALDI-TOF/TOF and LC-ESI-MS/MS. The combination of facilitated identification and quantification achieved by differentially isotope-coded N-terminal protein tagging with light/heavy SPITC represents, to our knowledge, a new approach to quantitative proteomics.     

Quantitation of protein phosphorylation in pregnant rat uteri using stable isotope dimethyl labeling coupled with IMAC

Quantitative analysis of protein phosphorylation provides important insights into molecular signaling mechanisms and a better understanding of many cellular processes. In this study, we coupled stable isotope dimethyl labeling with immobilized metal affinity chromatography (IMAC) enrichment to quantify protein phosphorylation at MS-determined phosphorylation sites. The proposed method was first characterized using alpha- and beta-casein as two model phosphoproteins, and further applied to the analysis of pregnant rat uteri with and without treatment with 8-bromo-cGMP. Dimethyl labeling has several significant advantages: global, fast (within 5 min) and complete (near 100%). Our results indicate that the labeling has no adverse effect on the IMAC enrichment for tryptic peptides having single and multiple phosphorylation sites. Moreover, the enhanced a1 signal and the complete reaction by dimethyl labeling provide unequivocal identification of both the N-terminal amino acid and the number of the labeling site. Using these two criteria in data validation, which is particularly important for identifying phosphoproteins, we found that the confidence in interpreting dimethyl-labeled peptides had greatly increased. In the analysis of late gestation rat uteri, the abundance ratio between treated and un-treated phosphopeptide signals ranged from 0.51 to 1.69 with an average of around 1.01 +/- 0.25. The obtained ratio of the phosphorylation levels at Ser 15 of HSP27 was further confirmed by the consistent results obtained from Western blot analyses. Based on the analysis of the results, it is interesting to note that the activated cGMP dependent protein kinase G (PKG) seems to affect the phosphorylation of proteins associated with the inhibition of cell migration and proliferation, redistribution of actin-associated proteins, and the increase of protein synthesis in late-gestation uteri. These observations provide important evidence suggesting that activated PKG may play a critical role in the shift of pregnant uteri from proliferative to hypertrophic states.