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1.
Mühling J Nickolaus KA Halabi M Fuchs M Krüll M Engel J Wolff M Matejec R Langefeld TW Welters ID Menges T Dehne MG Sablotzki A Hempelmann G 《Amino acids》2005,29(3):289-300
Summary. The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine,
arginine or taurine on polymorphonuclear neutrophil (PMN) free α-keto acid profiles and, in a parallel study, on PMN immune
functions. Exogenous L-alanyl-L-glutamine significantly increased PMN α-ketoglutarate, pyruvate PMN superoxide anion (O2−) generation, hydrogen peroxide (H2O2) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in α-ketoglutarate, pyruvate,
MPO release and H2O2 generation. Formation of O2− on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and α-ketobutyrate
levels, decreased O2− and H2O2 formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-α-keto-acid
profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition
that considerably modulates the immunological competence of PMN. 相似文献
2.
Eugenia Reynoso R. Daniel Cacciari Carlos A. Suchetti Hernn A. Montejano M. Alicia Biasutti 《Luminescence》2019,34(3):324-333
Photosensitized oxidation of bovine serum albumin (BSA), by using perinaphtenone as a sensitizer, has been studied at pH 7.4 and 11. The selected sensitizer does not present ground‐state complexation with BSA and ensures that the mechanism is mediated by O2(1△g). Strong dependence between BSA–O2(1△g) photo‐oxidation and the pH of the medium has been found. The relative oxygen uptake rate (v? △ O2 ) and the total quenching rate constant (kt) values are higher at pH 11 than pH 7.4. The enhancement in the alkaline condition is due to conformational changes in the protein and the reactivity of tyrosinate anion with O2(1△g). Even when the tendency with the pH in the presence of sodium dodecyl sulfate (SDS) micelles is similar to that observed in homogeneous media, an increment on the kt value is detected. This effect may be attributable to the strong interaction of BSA–SDS, which leads to the protein unfolding and could leave more exposed photo‐oxidizable amino acids. A protective effect against the O2(1△g)‐mediated photo‐oxidation was observed in reverse micelles (RMs) of sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) by comparing the kt values obtained at W = 10 with respect to the one obtain in homogeneous media. The latter could be mainly explained by the modification in the solvent polarity. Also, another important observation was found, the internal pH inside RMs of AOT sensed through tyrosine absorption was independent of the one used for the formation of the water pool. Hence, the kt values observed at both pH, are quite similar. 相似文献
3.
Summary The effect of the substitution pattern on the kinetics of the Type II (O2(1g)-mediated) dye-sensitized photooxidation of a series of nine tyrosine derivatives was investigated. Overall (kt) and reactive (kr) rate constants for the interaction of the excited oxygen species with the amino acid derivatives were determined. A parallel study on solvent and pH effects was carried out.The presence of different substituents in nuclear positions or in the amino acid side chain greatly affect the photooxidation rates.An upper limit for photooxidation quantum yield, calculated from the kinetic data, varies from 0.03 to 0.25, being the higher for halogenated tyrosines and the lower for esterified tyrosines and for the nitro-derivative.The variation of solvent polarity and pH of the reaction medium confirm that the presence of the ionized phenolate group in tyrosine, clearly dominates the quenching process. As already postulated for generic phenolic derivatives, it proceeds through a polar intermediate complex which posses some component of charge-transfer character.Esterification of the carboxilic acid of tyrosine selectively decreases the contribution of the reactive step to the overall process of O2(1g) quenching. An amide group in the same position does not produce noticiable changes in this sense. The presence of a highly deactivating nitro group in nuclear positions greatly diminishes the magnitude of both overall and reactive interactions.For all three, o-, m- and p-tyrosine the values of photooxidation quantum yields show an excellent parallelism with the rates of consumption of the — NH2 group of the amino acid chain, upon sensitized irradiation. It could react, in the cases of 0- and m-tyrosine in a secondary, non photochemical, step.Abbreviations
O
2(3
g
–
)
ground state triplet oxygen
-
O
2(1g)
singlet molecular oxygen
- Tyr
L tyrosine
- TyrD
tyrosine derivatives
- Eos
eosine
- RB
rose bengal
- FFA
furfuryl alcohol
- DMA
9, 10-dimethyl anthracene 相似文献
4.
Leaf carbon isotope discrimination (Δ13C) varies with the balance between net photosynthesis (A) and stomatal conductance (g
s
). Inferences that can be made with Δ13C are limited, as changes could reflect variation in A and/or g
s
. Investigators have suggested that leaf δ18O enrichment above source water (Δ18O) may enable differentiation between sources of variation in Δ13C, as leaf Δ18O varies with transpiration rate (E), which is closely correlated with g
s
when leaves experience similar leaf to air vapor pressure differences. We examined leaf gas exchange of Salix arctica at eight sites with similar air temperatures and relative humidities but divergent soil temperatures and soil water contents
near Pituffik, Greenland (76°N, 38°W). We found negative correlations at the site level between g
s
and Δ18O in bulk leaf tissue (r
2 = 0.62, slope = −17.9‰/mol H2O m−2 s−1, P = 0.02) and leaf α-cellulose (r
2 = 0.83, slope = −11.5‰ mol H2O m−2 s−1, P < 0.01), consistent with the notion that leaf water enrichment declines with increasing E. We also found negative correlations at the site-level between intrinsic water-use efficiency (iWUE) and Δ13C in bulk leaf tissue (r
2 = 0.65, slope = −0.08‰/μmol CO2 /mol H2O, P = 0.02) and leaf α-cellulose (r
2 = 0.50, slope = −0.05 ‰/[μmol CO2 /mol H2O], P = 0.05). When increasing Δ13C was driven by increasing g
s
alone, we found negative slopes between Δ13C and Δ18O for bulk leaf tissue (−0.664) and leaf α-cellulose (−1.135). When both g
s
and A
max increased, we found steeper negative slopes between Δ13C and Δ18O for bulk leaf tissue (−2.307) and leaf α-cellulose (−1.296). Our results suggest that the dual isotope approach is capable
of revealing the qualitative contributions of g
s
and A
max to Δ13C at the site level. In our study, bulk leaf tissue was a better medium than leaf α-cellulose for application of the dual
isotope approach. 相似文献
5.
The physiological properties of transgenic tobacco plants (Nicotiana tabacum L.) with decreased or increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were
compared in order to investigate the extent to which the TPT controls metabolic fluxes in wild-type tobacco. For this purpose,
tobacco lines with an antisense repression of the endogenous TPT (αTPT) and tobacco lines overexpressing the TPT gene isolated
from the C4 plant Flaveria trinervia (FtTPT) were used. The F. trinervia TPT expressed in yeast cells exhibited transport characteristics identical to the TPT from C3 plants. Neither antisense TPT plants nor FtTPT overexpressors showed a phenotype when grown in a greenhouse in air. Contents
of starch and soluble sugars in upper source leaves were similar in TPT underexpressors and FtTPT overexpressors compared
to the wild type at the end of the photoperiod. The FtTPT overexpressors incorporated more 14CO2 in sucrose than the wild type, indicating that the TPT limits sucrose biosynthesis in the wild type. There were only small
effects on labelling of amino acids and organic acids. The mobilisation of starch was enhanced in αTPT lines but decreased
in FtTPT overexpressors compared to the wild type. Enzymes involved in starch mobilisation or utilisation, such as α-amylase
or hexokinase were increased in αTPT plants and, in the case of amylases, decreased in FtTPT overexpressors. Moreover, α-amylase
activity exhibited a pronounced diurnal variation in αTPT lines with a maximum activity after 8 h in the light. These changes
in starch hydrolytic activities were confirmed by activity staining of native gels. Activities of glucan phosphorylases were
unaffected by either a decrease or an increase in TPT activity. There were also effects of TPT activities on steady-state
levels of phosphorylated intermediates as well as total amino acids and malate. In air, there was no or little effect of altered
TPT transport activity on either rates of photosynthetic electron transport and/or CO2 assimilation. However, in elevated CO2 (1500 μl · l−1) and low O2 (2%) the rate of CO2 assimilation was decreased in the αTPT lines and was slightly higher in FtTPT lines. This shows that the TPT limits maximum
rates of photosynthesis in the wild type.
Received: 26 March 1999 / Accepted: 21 August 1999 相似文献
6.
Marjon J. H. van Haandel I. M. C. M. Rietjens Ans E. M. F. Soffers Cees Veeger Jacques Vervoort Sandeep Modi Madhu S. Mondal Prasanta K. Patel Digambar V. Behere 《Journal of biological inorganic chemistry》1996,1(5):460-467
The second-order rate constants for the oxidation of a series of phenol derivatives by horseradish peroxidase compound II
were compared to computer-calculated chemical parameters characteristic for this reaction step. The phenol derivatives studied
were phenol, 4-chlorophenol, 3-hydroxyphenol, 3-methylphenol, 4-methylphenol, 4-hydroxybenzoate, 4-methoxyphenol and 4-hydroxybenzaldehyde.
Assuming a reaction of the phenolic substrates in their non-dissociated, uncharged forms, clear correlations (r = 0.977 and r = 0.905) were obtained between the natural logarithm of the second-order rate constants (ln k
app and ln k
2 respectively) for their oxidation by compound II and their calculated ionisation potential, i.e. minus the energy of their
highest occupied molecular orbital [E(HOMO)]. In addition to this first approach in which the quantitative structure-activity
relationship (QSAR) was based on a calculated frontier orbital parameter of the substrate, in a second and third approach
the relative heat of formation (ΔΔHF) calculated for the process of one-electron abstraction and H• abstraction from the phenol derivatives was used as a parameter. Plots of the natural logarithms of the second-order rate
constants (k
app and k
2) for the reaction and the calculated ΔΔHF values for the process of one-electron abstraction also provide clear QSARs with
correlation coefficients of –0.968 and –0.926 respectively. Plots of the natural logarithms of the second-order rate constants
(k
app and k
2) for the reaction and the calculated ΔΔHF values for the process of H• abstraction provide QSARs with correlation coefficients of –0.989 and –0.922 respectively. Since both mechanisms considered,
i.e. initial electron abstraction versus initial H• abstraction, provided clear QSARs, the results could not be used to discriminate between these two possible mechanisms for
phenol oxidation by horseradish peroxidase compound II. The computer calculation-based QSARs thus obtained for the oxidation
of the various phenol derivatives by compound II from horseradish peroxidase indicate the validity of the approaches investigated,
i.e. both the frontier orbital approach and the approach in which the process is described by calculated relative heats of
formation. The results also indicate that outcomes from computer calculations on relatively unrelated phenol derivatives can
be reliably compared to one another. Furthermore, as the actual oxidation of peroxidase substrates by compound II is known
to be the rate-limiting step in the overall catalysis by horseradish peroxidase, the QSARs of the present study may have implications
for the differences in the overall rate of substrate oxidation of the phenol derivatives by horseradish peroxidase.
Received: 29 March 1996 / Accepted: 17 July 1996 相似文献
7.
Summary The type II singlet molecular oxygen [O2(1
g
)]-mediated photo-oxidation of the tripeptide gly-tyr-ala was studied. It has two non-oxidizable amino-acids (gly and ala) bonded to the oxidizable one, tyr. Overall (k
t) and reactive (k
r) rate constants for the interaction were determined by time-resolved methods (IR emission of O2(1
g
)) and stationary photolysis, in water at pH 11.5 as well as in alkaline non-aqueous etOH-MeCN (80:20, v/v, 10 mM in KOH) solutions. An important solvent polarity effect onk
t was detected; the rate constant increasing one order of magnitude in going from the organic mixture to water (k
t H2O = 2 × 109 M–1 s–1). Nevertheless,k
r does not parallel this trend; gly-tyr-ala being less photooxidizable in a more polar environment. The effective quantum yield (
r
) forTPE photooxidation is much higher in etOH-MeCN (
r
= 0.056) than in water (
r
= 0.023). Results are discussed on the basis of the formation of an exciplex with polar character between the TPE and O2(1
g
).Two remarkable points should be taken into account: a) the rate costants for the interaction of O2(1
g
) with gly-tyr-ala are practically the same as for free tyr. b) New -NH2 groups are generated upon sensitized irradiation. Both findings indicate that the peptide bonds in the TPE break as a result of the photooxidation. A thorough analysis with data for tyrosine and related dipeptides is undertaken.Abbreviations
O
2(1g)
singlet molecular oxygen
-
AA
amino-acid
-
TPE
tripeptide
-
gly-tyr-ala
glycyl-L-tyrosine-alanine
-
tyr-gly
L-tyrosyl-glycine
-
FFA
furfuryl alcohol
-
tyr
L-tyrosine
-
gly-tyr
glycyl-L-tyrosine
-
DPE
dipeptide
-
NaN
3
sodium azide
-
RB
rose bengal
-
ZnTPP
Zinc tetraphenyl porphyrine
-
MeCN
acetonitrile
-
DMA
9,10-dimethyl anthracene
-
etOH
ethanol
-
TRPD
time-resolved phosphorescence detection
-
NH
2 loss
loss of primary amine reactivity 相似文献
8.
D. P. Heil T. R. Derrick S. Whittlesey 《European journal of applied physiology and occupational physiology》1997,75(2):160-165
This study was designed to determine how changes in oxygen uptake (O2) and heart rate (HR) during submaximal cycle ergometry were determined by changes in cycle geometry and/or lower-limb kinematics.
Fourteen trained cyclists [Mean (SD): age, 25.5 (6.4) years; body mass 74.4 (8.8) kg; peak O2, 4.76 (0.79) l. min−1 peak] were tested at three seat-tube angles (70°, 80°, 90°) at each of three trunk angles (10°, 20°, 30°) using a modified
Monark cycle ergometer. All conditions were tested at a power output corresponding to 95% of the O2 at each subject's ventilatory threshold while pedalling at 90 rpm and using aerodynamic handlebars. Sagittal-view kinematics
for the hip, knee, and ankle joints were also recorded for all conditions and for the subjects' preferred positioning on their
own bicycles. No combination of seat-tube and trunk angle could be considered optimal since many of the nine conditions elicited
statistically similar mean O2 and HR values. Mean hip angle (HA) was the only kinematic variable that changed consistently across conditions. A regression
relationship was not observed between mean O2 or HR and mean hip angle values (P > 0.45). Significant curvilinear relationships were observed, however, between ΔO2 (O2 − minimum O2) and ΔHA (mean HA − preferred HA) using the data from all subjects (R = 0.45, SEE = 0.13 l . min−1) and using group mean values (R = 0.93, SEE = 0.03 l . min−1). In both cases ΔO2 minimized at ΔHA = 0, which corresponded to the subjects' preferred HA from their own bicycles. Thus, subjects optimized
their O2 cost at cycle geometries that elicited similar lower-limb kinematics as the preferred geometries from their own bicycles.
Accepted: 3 July 1996 相似文献
9.
Transgenic tobacco (Nicotiana tabacum L.) plants with decreased and increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT)
were used to study the control the TPT exerts on the flux of starch and sucrose biosynthesis, as well as CO2 assimilation, respiration and photosynthetic electron transport. For this purpose, tobacco lines with an antisense repression
of the endogenous TPT (αTPT) and tobacco lines overexpressing a TPT gene from Flaveria trinervia (FtTPT) were used. In ambient CO2, there was no or little effect of altered TPT transport activities on either rates of photosynthetic electron transport and/or
CO2 assimilation. However, in elevated CO2 (1500 μl · l−1) and low O2 (2%) the TPT exerted strong control on the rate of CO2 assimilation (control coefficient for the wild type; CJA
TPT=0.30) in saturating light. Similarly, the incorporation of 14C into starch in high CO2 was increased in tobacco plants with decreased TPT activity, but was reduced in plants overexpressing the TPT from F. trinervia. Thus, the TPT exerted negative control on the rate of starch biosynthesis with a CJStarch
TPT=−0.19 in the wild type estimated from a hyperbolic curve fitted to the data points. This was less than the positive control
strength on the rate of sucrose biosynthesis (CJSuc
TPT=0.35 in the wild type). Theoretically, the positive control exerted on sucrose biosynthesis should be numerically identical
to the negative control on starch biosynthesis unless additional metabolic pathways are affected. The rate of dark respiration
showed some correlation with the TPT activity in that it increased in FtTPT overexpressors, but decreased in αTPT plants with
an apparent control coefficient of CJRes
TPT=0.24. If the control on sucrose biosynthesis is referred to as “gain of carbon” (positive control) and the control on starch
biosynthesis as well as dark respiration as a “loss of carbon” (negative control) for sucrose biosynthesis and subsequent
export, the sum of the control coefficients on dark respiration and starch biosynthesis would be numerically similar to the
control coefficient on the rate of sucrose biosynthesis. There was also some control on the rate of photosynthetic electron
transport, but only at high light and in elevated CO2 combined with low O2. The control coefficient for the rate of photosynthetic electron transport was CJETR
TPT=0.16 in the wild type. Control coefficients were also calculated for plants with elevated and lowered TPT activity. Furthermore,
the extent to which starch degradation/glucose utilisation compensates for the lack of triose phosphate export was assessed.
The TPT also exerted control on metabolite contents in air.
Received: 26 March 1999 / Accepted: 21 August 1999 相似文献
10.
S. C. J. Meskers C. Dennison Gerard W. Canters Harry P. J. M. Dekkers 《Journal of biological inorganic chemistry》1998,3(6):663-670
The dynamic quenching of the luminescence of racemic Eu(III)(pyridine-2,6-dicarboxylate=dpa)3
3– by the title proteins is investigated and the enantioselectivity of the proteins in the quenching of the Δ and Λ enantiomers
of Eu(dpa)3
3– is determined. The two diastereomeric quenching rate constants pertaining to azurin (k
q
Δ=3.3×106, k
q
Λ=2.7×106 M–1 s–1, pH 7.2, ionic strength I=22 mM) are lower than for its Met→44Lys mutant (k
q
Δ=1.9×107, k
q
Λ=1.4×107 M–1 s–1, same pH and I), indicating that energy transfer occurs from Eu(dpa)3
3– to the Cu(II) centre when the luminophore is bound to the hydrophobic patch of the protein near residue 44. The enantioselectivity
remains unaltered by the mutation: k
q
Δ/k
q
Λ=1.27±0.04, so Lys44 is probably not in direct contact with the Eu chelate. The I and pH dependence of k
q indicate that the lysine residue interacts electrostatically with Eu(dpa)3
3–. For plastocyanin the quenching rates are of the order of 106 M–1 s–1; for amicyanin they are two orders of magnitude larger (k
q
Δ=12×107, k
q
Λ=11×107 M–1 s–1, pH 7.2, I=22 mM). The variation of k
q is attributed to differences in the charge distribution on the proteins, which influences the binding of the luminophore
to the protein surface. For amicyanin the anion binding site near Lys59 and Lys60 may be involved in the energy transfer.
Received: 16 June 1998 / Accepted: 18 September 1998 相似文献
11.
Maize (Zea mays L.) cell cultures incorporated radioactivity from [14C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the
CoA pool had lost its 14C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a
pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age.
In young (1–3 d) cultures, polysaccharide-bound [14C]feruloyl- and [14C]diferuloyl residues were both detectable within 1 min of [14C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least
the first 2.3 h after [14C]cinnamate feeding, polysaccharide-bound [14C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [14C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter,
and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H2O2 (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In
older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [14C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [14C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H2O2 induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H2O2-limited. In both 2- and 4-d-old cultures, polysaccharide-bound 14C-trimers and larger coupling products exceeded [14C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually
quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides
in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely
to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response
of cell walls to an oxidative burst are discussed.
Received: 19 January 2000 / Accepted: 13 April 2000 相似文献
12.
The role of cyclic electron transport has been re-examined in leaves of C3 plants because the bioenergetics of chloroplasts (H+/e = 3 in the presence of a Q-cycle; H+/ATP = 4 of ATP synthesis) had suggested that cyclic electron flow has no function in C3 photosynthesis. After light activation of pea leaves, the dark reduction of P700 (the donor pigment of PSI) following far-red
oxidation was much accelerated. This corresponded to loss of sensitivity of P700 to oxidation by far-red light and a large
increase in the number of electrons available to reduce P700+ in the dark. At low CO2 and O2 molar ratios, far-red light was capable of decreasing the activity of photosystem II (measured as the ratio of variable to
maximal chlorophyll fluorescence, Fv/Fm) and of increasing light scattering at 535 nm and zeaxanthin synthesis, indicating formation of a transthylakoid pH gradient.
Both the light-induced increase in the number of electrons capable of reducing far-red-oxidised P700 and the decline in Fv/Fm brought about by far-red in leaves were prevented by methyl viologen. Antimycin A inhibited CO2-dependent O2 evolution of pea leaves at saturating but not under limiting light; in its presence, far-red light failed to decrease Fv/Fm. The results indicate that cyclic electron flow regulates the quantum yield of photosystem II by decreasing the intrathylakoid
pH when there is a reduction in the availability of electron acceptors at the PSI level (e.g. during drought or cold stresses).
It also provides ATP for the carbon-reduction cycle under high light. Under these conditions, the Q-cycle is not able to maintain
a H+/e ratio of 3 for ATP synthesis: we suggest that the ratio is flexible, not obligatory.
Received: 23 February 1999 / Accepted: 19 August 1999 相似文献
13.
A microsomal preparation from suspension-cultured potato stem cells (Solanum tuberosum L. cv. AZY) was incubated with [14C]acetyl-CoA resulting in a precipitable radiolabeled product. Analysis of the product revealed that it consisted mostly of
acetylated proteins and cell wall polysaccharides, including xyloglucan, homogalacturonan and rhamnogalacturonan I. Thus,
acetyl-CoA is a donor-substrate for the O-acetylation of wall polysaccharides. A rhamnogalacturonan acetylesterase was used to develop an assay to measure and characterize
rhamnogalacturonan O-acetyl transferase activity in the microsomal preparation. Using this assay, it was shown that the transferase activity was
highest during the linear growth phase of the cells, had a pH-optimum at pH 7.0, a temperature optimum at 30 °C, an apparent
K
m of 35 μM and an apparent V
max of 0.9 pkat per mg protein. Further analysis of the radiolabeled acetylated product revealed that it had a molecular mass
>500 kDa.
Received: 3 July 1999; Accepted: 27 September 1999 相似文献
14.
Structure-Function Relationship of the Ion Channel Formed by Diphtheria Toxin in Vero Cell Membranes
Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed
by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane
of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming
transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel,
and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin,
Δg
m
, was initially determined. DT-AB induced a ∼10-fold lower Δg
m
than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Δg
m
, whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Δg
m
. Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches.
No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B
D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability
and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is
reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode.
Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane
domain (T-domain) strongly affect the overall channel activity expressed as Δg
m
and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero
cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel.
Received: 20 June 1996/Revised: 8 November 1996 相似文献
15.
Summary. A concise preparation of N
α-Fmoc-N
ɛ-(Boc, methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide is described.
N
α-Fmoc-N
ɛ-(Boc, methyl)-lysine is obtained, via consecutive reductive benzylation and reductive methylation in a one-pot reaction,
followed by debenzylation through catalytic hydrogenolysis and Boc protection in another one-pot reaction. A peptide containing
monomethylated lysine is successfully synthesized by incorporating N
α-Fmoc-N
ɛ-(Boc, methyl)-lysine as a building block via solid-phase peptide synthesis. 相似文献
16.
A nonphotochemical-quenching-deficient mutant of Arabidopsis thaliana possessing normal pigment composition and xanthophyll-cycle activity 总被引:1,自引:0,他引:1
Higher-plant chloroplasts alter the distribution of absorbed radiant energy between photosynthesis and heat formation in
response to changing illumination level or environmental stress. Fluorescence imaging was used to screen 62 yellow-green T-DNA
insertion mutant lines of Arabidopsis thaliana (L.) Heynh. for reduced photoprotective nonphotochemical quenching (NPQ) capacity. Pulse-modulation fluorometry was employed
to characterize one line (denoted Lsr1−) that exhibited an approximately 50% reduction in NPQ compared to the wild type (WT). The loss in NPQ capacity was associated
with the ΔpH-dependent phase of quenching (qE). Under the growth conditions employed, pigment composition and levels of the
six photosystem-II light-harvesting chlorophyll a/b proteins were identical in mutant and WT. Changes in the in-vivo levels of the xanthophyll pigments violaxanthin, antheraxanthin,
and zeaxanthin in excess light were the same for mutant and WT. However, use of the violaxanthin de-epoxidase inhibitor dithiothreitol
indicated that a zeaxanthin-dependent component of NPQ was specifically reduced in the mutant. The mutant exhibited diminished
suppression of minimum fluorescence yield (F
o
) in intense light suggesting an altered threshold in the mechanism of response to light stress in the mutant. The NPQ-deficient
phenotype was meiotically transmissible as a semidominant trait and mapped near marker T27K12 on chromosome 1. The results
suggest that the mutant is defective in sensing the transthylakoid ΔpH that reports exposure to excessive illumination.
Received: 26 May 1999 / Accepted: 17 June 1999 相似文献
17.
Photosynthetic and stomatal responses of two tropical and two temperate trees to atmospheric humidity 总被引:4,自引:0,他引:4
The effects of leaf to air vapour pressure differences (ΔW) on net photosynthetic rate (PN) and stomatal conductance (gs) were examined in the leaves of two tropical rain forest trees, Eugenia grandis and Pongamia pinnata, and two temperate evergreen
trees, Viburnum awabuki and Daphniphyllum macropodum. A single leaf was set inside a small chamber and ΔW was varied from
7 to 24 mmol mol-1 at 25 and 500 μmol m-2 s-1 of photon flux density. PN and gs of the two tropical rain forest trees decreased with increasing ΔW, while the two temperate evergreen trees were not highly
responsive to ΔW. P. pinnata was more sensitive to ΔW in its stomatal response, and had a higher stomatal density and higher
stomatal index than did the two temperate trees and another tropical tree. Significant reductions i n gs and intercellular CO2 concentrations in the two tropical trees at high ΔW suggest that the decline of PN was due to the decrease in gs. The responses of PN and gs indicated that the tropical trees were more sensitive to ΔW than were the temperate ones.
This revised version was published online in September 2006 with corrections to the Cover Date. 相似文献
18.
Summary The kinetics of the eosin-sensitized photooxidation ([O2(1g)]-mediated) of the protein lysozyme (Lyso) was investigated under two different pH conditions (pH 7 and pH 11). Rates of oxygen consumption and the fade in the protein fluorescence spectrum upon sensitized irradiation were monitored. Parallel studies on both denatured Lyso (absence of the four-S-S- bridges in the protein) and different mixtures of the photooxidizable amino acids of Lyso were also carried out. The mixtures maintained the same molar ratio as in the native protein, and were selected just in order to throw into relief the preferential amino acids that were being photooxidized at both pH values.Under work conditions Lyso was only photooxidizable at pH 7, whereas the opposite accounted for the denatured protein: only measurable oxygen consumption was detected at pH 11. Nevertheless, Lyso at pH 11, evidenced an important physical quenching of O2(1g) due to the Tyr and Trp residues.The results for the native protein were interpreted on the basis of a previously described dark complex Eosin-Lyso, which selectively favours the photooxidation of the bounded protein. The Trp residues were the main reactive entities in the native protein. The photodinamic effect in denatured Lyso was characterized by the prevalence of Tyr residues as photooxidizable targets.In the discussion of the results, a comparisson with the photooxidation kinetics of the mixtures of free amino acids was made.Abbreviations O2(3g
–)
ground state triplet oxygen
- O2(1g)
singlet molecular oxygen
- Lyso
lysozyme
- LysoD
denatured lysozyme
- Eos
eosin
- FFA
furfuryl alcohol
- Trp
tryptophan
- Tyr
tyrosine
- Cys
cysteine
- Cis
cystine
- Met
methionine
- His
histidine
- AA
amino acid
- a.u.
arbitrary units 相似文献
19.
Summary. In continuation of our previous work dedicated to the detection of the oxidation products of aminoethylcysteine ketimine
dimer by oxygen reactive species, we give here data for the identification of the α, β unsaturated sulfoxide as the main product of interaction of the dimer with H2O2. Identification has been done on the basis of mass spectrometry and NMR analyses of the product isolated by preparative chromatography.
Received March 24, 1998, Accepted October 20, 1998 相似文献
20.
We used 31P NMR to investigate the temperature-dependence of intracellular pH (pH
i
) in isolated frog skeletal muscles. We found that ln[H+
i
] is a linear function of 1/T
abs paralleling those of neutral water (i.e., H+= OH−) and of a solution containing the fixed pH buffers of frog muscle cytosol. This classical van't Hoff relationship was unaffected
by inhibition of glycolysis and was not dependent upon the pH or [Na+] in the bathing solution. Insulin stimulation of Na+-H+ exchange shifted the intercept in the alkaline direction but had no effect on the slope. Acid loading followed by washout
resulted in an amiloride-sensitive return to the (temperature dependent) basal pH
i
.
These results show that the temperature dependence of activation of Na+-H+ exchange is similar to that of the intracellular buffers, and suggest that constancy of [H+]/[OH−] with changing temperature is achieved in the short term by intracellular buffering and in the long term by the set-point
of the Na+-H+ exchanger. Proton activation of the exchanger has an apparent standard enthalpy change (ΔH°) under both control and insulin-stimulated
conditions that is similar to the ΔH° of the intracellular buffers and approximately half of the ΔH° for the dissociation
of water. Thus, the temperature-dependent component of the standard free-energy change (ΔF°) is unaffected by insulin stimulation,
suggesting that changes in Arrhenius activation energy (E
a
) may not be a part of the mechanism of hormone stimulation.
Received: 12 February 1997/Revised: 1 October 1997 相似文献