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1.
K K Arora P Cortis P A Bleakley J F Williams 《The International journal of biochemistry》1985,17(12):1329-1337
The enzyme D-glycero D-ido octulose 1,8-bisphosphate:D-altro-heptulose 7-phosphotransferase (abbreviated to phosphotransferase, PT) catalyses the transfer of the phosphate ester group at C-1 between altro-heptulose (sedoheptulose) and octulose phosphate intermediates of the L-type pentose pathway. Using synthetically prepared and 14C-labelled octulose mono- and bisphosphates, two methods are described for the measurement of the catalytic capacity of the PT reaction operating in both the "forward" and "reverse" modes of L-type pentose pathway operation. PT activity was found in normal, regenerating and foetal rat liver, rat heart, rat epididymal fat pad, rat kidney, brain and skeletal muscle, extracts of C. fusca, pea leaf and a variety of tumour tissues. The highest activity of the enzyme was found in the neoplasms. The Michaelian kinetic constants, temperature and pH optima for the reaction of the enzyme from rat liver together with an assortment of its substrate specificities have been determined. Vanadate anion was found to inhibit the enzyme and the pattern of inhibition suggests that the PT may act by a sequential mechanism. Neither arabinose 5-phosphate nor inorganic phosphate showed any effect on the catalytic activity of the PT enzyme in liver. 相似文献
2.
M. H. Sawyer P. Baumann L. Baumann S. M. Berman J. L. Cánovas R. H. Berman 《Archives of microbiology》1977,112(1):49-55
Cell-free extracts of d-fructose grown cells of Pseudomonas putida, P. fluorescens, P. aeruginosa, P. stutzeri, P. mendocina, P. acidovorans and P. maltophila catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose and contained 1-P-fructokinase activity suggesting that in these species fructuse-1-P and fructose-1,6-P2 were intermediates of d-fructose catabolism. Neither the 1-P-fructokinase nor the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose was present in significant amounts in succinate-grown cells indicating that both activities were inducible. Cell-free extracts also contained activities of fructose-1,6-P2 aldolase, fructose-1,6-P2 phosphatase, and P-hexose isomerase which could convert fructose-1,6-P2 to intermediates of either the Embden-Meyerhof pathway or Entner-Doudoroff pathway. Radiolabeling experiments with 1-14C-d-fructose suggested that in P. putida, P. aeruginosa, P. stutzeri, and P. acidovorans most of the alanine was made via the Entner-Doudoroff pathway with a minor portion being made via the Embden-meyerhof pathway. An edd
- mutant of P. putida which lacked a functional Entner-Doudoroff pathway but was able to grow on d-fructose appeared to make alanine solely via the Embden-Meyerhof pathway.Non-Standard Abbreviations cpm
counts per min
-
edd
-
mutant lacking Entner-Doudoroff dehydrase (6-PGA dehydrase)
- EDP
Entner-Doudoroff pathway
- EMP
Embden-Meyerhof pathway
- FDP
fructose-1,6-P2
- FDPase
FDP phosphatase
- F-1-P
fructose-1-P
- F-6-P
fructose-6-P
- FPTs
PEP: d-fructose phosphotransferase system
- G-6-P
glucose-6-P
- KDPG
2-keto-3-deoxy-6-P-gluconate
- PEP
P-enolpyruvate
- 1-PFK
1-P-fructokinase
- 6-PFK
6-P-fructokinase
- 6-PGA
6-P-gluconate 相似文献
3.
Werner M. Kaiser 《Planta》1979,145(4):377-382
Hydrogen peroxide (6x10-4 M) causes a 90% inhibition of CO2-fixation in isolated intact chloroplasts. The inhibition is reversed by adding catalase (2500 U/ml) or DTT (10 mM). If hydrogen peroxide is added to a suspension of intact chloroplasts in the light, the incorporation of carbon into hexose- and heptulose bisphosphates and into pentose monophosphates is significantly increased, whereas; carbon incorporation into hexose monophosphates and ribulose 1,5-bisphosphate is decreased. At the same time formation of 6-phosphogluconate is dramatically stimulated, and the level of ATP is increased. All these changes induced by hydrogen peroxide are reversed by addition of catalase or DTT. Additionally, the conversion of [14C]glucose-6-phosphate into different metabolites by lysed chloroplasts in the dark has been studied. In presence of hydrogen peroxide, formation of ribulose-1,5-bisphosphate is inhibited, whereas formation of other bisphosphates,of triose phosphates, and pentose monophosphates is stimulated. Again, DTT has the opposite effect. The release of 14CO2 from added [14C]glucose-6-phosphate by the soluble fraction of lysed chloroplasts via the reactions of oxidative pentose phosphate cycle is completely inhibited by DTT (0.5 mM) and re-activated by comparable concentrations of hydrogen peroxide. These results indicate that hydrogen peroxide interacts with reduced sulfhydryl groups which are involved in the light activation of enzymes of the Calvin cycle at the site of fructose- and sedoheptulose bisphophatase, of phosphoribulokinase, as well as in light-inactivation of oxidative pentose phosphate cycle at the site of glucose-6-phosphate dehydrogenase.Abbreviations ADPG
ADP-glucose
- DHAP
dihydroxyacetone phosphate
- DTT
dithiothreitol
- FBP
fructose-1,6-bisphosphate
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- HMP
hexose monophosphates (fructose-6-phosphate, glucose-6-phosphate, glucose-1-phosphate)
- 6-PGI
6-phosphogluconate
- PMP
pentose monophosphates (xylulose-5-phosphate, ribose-5-phosphate, ribulose-5-phosphate)
- RuBP
ribulose-1,5-bisphosphate
- S7P
sedoheptulose-7-phosphate
- SBP
sedoheptulose-1,7-bisphosphate
Dedicated to Prof. Dr. W. Simonis on the occasion of his 70th birthday 相似文献
4.
Glucose was required for the transport of arabinose into Bifidobacterium breve. The non-metabolisable glucose analogue 2-deoxy-d-glucose (2-DG) did not facilitate assimilation of arabinose. Studies using d-[U-14C]-labelled arabinose showed that it was fermented to pyruvate, formate, lactate and acetate, whereas the principal metabolic products of d-[U-14C]-labelled glucose were acetate and formate. In contrast to glucose, arabinose was not incorporated into cellular macromolecules. A variety of metabolic inhibitors and inhibitors of sugar transport (proton ionophores, metal ionophores, compounds associated with electron transport) were used to investigate the mechanisms of sugar uptake. Only NaF, an inhibitor of substrate level phosphorylation, and 2-DG inhibited glucose assimilation. 2-DG had no effect on arabinose uptake, but NaF was stimulatory. High levels of phosphorylation of glucose and 2-DG by PEP and to a lesser degree, ATP were seen in phosphoenolpyruvate: phosphotransferase (PEP:PTS) assays. These data together with strong inhibition of glucose uptake by NaF suggest a role for phosphorylation in the transport process. Arabinose uptake in B. breve was not directly dependent on phosphorylation or any other energy-linked form of transport but may be assimilated by glucose-dependent facilitated diffusion.Abbreviations (2,4-DNP)
2,4-dinitrophenol
- (2,4-DNP)
carbonylcyanide m-chlorophenylhydrazone
- (CCCP)
(phosphoenolpyruvate phosphotransferase system)
- PEP: PTS
trichloroacetic acid
- (TCA)
2-deoxy-d-glucose
- (2-DG)
2-deoxy-d-glucose 相似文献
5.
K K Arora J G Collins J K MacLeod J F Williams 《Biological chemistry Hoppe-Seyler》1988,369(7):549-557
Methods for the synthesis of carbon-13 enriched substrates, intermediates and products of the pentose-phosphate pathway, viz. ribose, arabinose, xylulose and ribulose 5-phosphates, sedoheptulose mono- and bisphosphates, octulose (both the ido- and altro-epimers) mono- and bisphosphates, are described. The procedure of the classical Kiliani synthesis was adopted for the preparation of the two starting compounds, [1-13C]ribose and [1-13C]arabinose 5-phosphates. Using these initial reactants and enzymic methods involving the group-transferring enzymes, transketolase, aldolase and transaldolase, a variety of specifically 13C-labelled five-, six-, seven- and eight-carbon sugar phosphates were synthesized in high yield and purity. The isolation and authenticity of each of the 13C-labelled sugars were established by column, paper and thin layer chromatographic methods and specific enzymic assays. The purity and positional isotopic analysis of these sugar-P's were confirmed by 13C-NMR spectroscopy. These specifically 13C-enriched compounds are required for enzymatic, mechanistic and quantitative investigations of pentose-pathway reactions in animal, plant and tumour tissues in vitro and in vivo. 相似文献
6.
Rabbit muscle aldolase was used to synthesize d-glycero-d-altro-octulose 1,8-bisphosphate and d-glycero-d-ido-octulose 1,8-bisphosphate. The products, isolated by ion-exchange chromatography, were characterized with the cysteine-sulfuric acid reaction and shown to be 90–95% pure by analysis for organic phosphorus and for dihydroxyacetone phosphate formed on cleavage with aldolase. The kinetic constants for synthesis and cleavage of these octulose bisphosphates with muscle and liver aldolases were determined. In the direction of cleavage both octulose bisphosphates were excellent substrates for liver aldolase, comparable to fructose 1,6-bisphosphate with respect to both V and Km. With muscle aldolase the rate of cleavage was 1–5% of that with fructose bisphosphate and comparable to that with fructose 1-phosphate. In the direction of synthesis, ribose 5-phosphate was a better substrate than arabinose 5-phosphate for both the liver and muscle enzymes, although for both pentose phosphates the values of Km fell in the range between 5 and 25 mm. It is concluded that reactions catalyzed by aldolase might account for the reported presence of these eight-carbon sugar phosphate esters in liver and in red cells. 相似文献
7.
K K Arora J P Longenecker J F Williams 《The International journal of biochemistry》1987,19(2):133-146
An investigation of the mechanism and quantitative contribution of the pentose phosphate pathway in the glucose metabolism of Morris Hepatoma 5123C is reported. Morris Hepatoma 5123C has an active non-oxidative segment of pentose pathway as judged by its ability to convert ribose 5-P to hexose 6-P in a standard assay. Based on compliance with qualitative and quantitative criteria, the cells exhibit the L-type pentose pathway reaction sequence rather than the F-type pathway. This compliance included the formation of intermediates characteristic of the L-type pathway, namely arabinose 5-P, octulose mono- and bisphosphates and sedoheptulose 1,7-bisphosphate, during the dissimilation of ribose 5-P to hexose 6-P. The intermediary role of arabinose 5-P was suggested by the incorporation of its carbon into various intermediates and products of the pentose pathway. Intermediary roles for ido octulose mono- and bisphosphates were supported by their participation in the reaction catalyzed by the phosphotransferase enzyme of the L-type pentose pathway. Presence of L-type PP reactions was further affirmed by 14C-prediction labelling experiments using [5-14C]- and [2-14C]glucose as specifically labelled substrates. Using two methods of measurement, the F-type pentose cycle made a negligibly small contribution to glucose metabolism, while the measured value of the L-type pentose pathway accounted for 30% (approx.) of the total glucose metabolism of these cells, a value consistent with the high activity of the enzymes of the L-type pentose pathway in Morris Hepatoma 5123C cells and the very high activity of the non-oxidative segment of the pathway in vitro. The findings validate the proposal that the L-type pentose pathway reactions constitute the non-oxidative segment of the pathway in Morris Hepatoma 5123C cells. Reasons involving pyruvate recycling reactions show why there is low incorporation of 14C-isotope in C-1 of glucose 6-P, when [4,5,6-14C]glucose and [6-14C]glucose are L-type PP test substrates in intact cells. 相似文献
8.
The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with 14C-labelled substrates led to 14CO2 production, that was dependent upon leucoplast intactness, from [U-14C]glucose 6-phosphate, [U-14C]glucose 1-phosphate, [U-14C] fructose 6-phosphate and [U-14C]glucose+ATP, but not from [U-14C]fructose-1,6-bisphosphate or [U-14C]triose phosphate. The yield from [U-14C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-14C]-, [2-14C]-, [3,4-14C]-, and [6-14C]glucose 6-phosphate were supplied to leucoplasts significant 14CO2 production that was dependent upon leucoplast intactness was found only for [1-14C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship. 相似文献
9.
Muller AP Rotta LN Kawano C Leszczinski DN Schweigert ID Londero LG Gravina FS da Silveira CK de Souza CG Battu CE Gonçalves CA de Souza DO Perry ML 《Neurochemical research》2006,31(3):417-422
We studied the effect of different concentrations of 2-deoxy-d-glucose on the l-[U-14C]leucine, l-[1-14C]leucine and [1-14C]glycine metabolism in slices of cerebral cortex of 10-day-old rats. 2-deoxy-d-glucose since 0.5 mM concentration has inhibited significantly the protein synthesis from l-[U-14C]leucine and from [1-14C]glycine in relation to the medium containing only Krebs Ringer bicarbonate. Potassium 8.0 mM in incubation medium did not stimulate the protein synthesis compared to the medium containing 2.7 mM, and at 50 mM diminishes more than 2.5 times the protein synthesis compared to the other concentration. Only at the concentration of 5.0 mM, 2-deoxy-d-glucose inhibited the CO2 production and lipid synthesis from l-[U-14C] leucine. This compound did not inhibit either CO2 production, or lipid synthesis from [1-14C]glycine. Lactate at 10 mM and glucose 5.0 mM did not revert the inhibitory effect of 2-deoxy-d-glucose on the protein synthesis from l-[U-14C]leucine. 2-deoxy-d-glucose at 2.0 mM did not show any effect either on CO2 production, or on lipid synthesis from l-[U-14C]lactate 10 mM and glucose 5.0 mM. 相似文献
10.
A model of the C
3 photosynthetic system is developed which describes the sensitivity of the steadystate rate of carbon dioxide assimilation to changes in the activity of several enzymes of the system. The model requires measurements of the steady-state rate of carbon dioxide assimilation, the concentrations of several intermediates in the photosynthetic system, and the concentration of the active site of ribulose 1,5-bisphosphate carboxyalse/oxygenase (Rubisco). It is shown that in sunflowers (Helianthus annuus L.) at photon flux densities that are largely saturating for the rate of photosynthesis, the steady-stete rate of carbon dioxide assimilation is most sensitive to Rubisco activity and, to a lesser degree, to the activities of the stromal fructose, 6-bisphosphatase and the enzymes catalysing sucrose synthesis. The activities of sedoheptulose 1,7-bisphosphatase, ribulose 5-phosphate kinase, ATP synthase and the ADP-glucose pyrophosphorylase are calculated to have a negligible effect on the flux under the high-light conditions. The utility of this analysis in developing simpler models of photosynthesis is also discussed.Abbreviations
c
i
intercellular CO2 concentration
-
C
infP
supJ
control coefficient for enzyme P with respect to flux J
- DHAP
dihydroxyacetonephosphate
- E4P
erythrose 4-phosphate
- F6P
fructose 6-phosphate
- FBP
fructose 1,6-bisphosphate
- FBPase
fructose 1,6-bisphosphatase
- G3P
glyceraldehyde 3-phosphate
- G1P
glucose 1-phosphate
- G6P
glucose 6-phosphate
- Pi
inorganic phosphate
- PCR
photosynthetic carbon reduction
- PGA
3-phosphoglyceric acid
- PPFD
photosynthetically active photon flux density
-
R
n
J
response coefficient for effector n with respect to flux J
- R5P
ribose 5-phosphate
- Rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- Ru5P
ribulose 5-phosphate
- RuBP
ribulose 1,5-bisphosphate
- S7P
sedoheptulose 7-phosphate
- SBP
sedoheptulose 1,7-bisphosphate
- SBPase
sedoheptulose 1,7-bisphosphatase
- SPS
sucrose-phosphate synthase
- Xu5P
xylulose 5-phosphate
-
n
P
elasticity coefficient for effector n with respect to the catalytic velocity of enzyme P
This research was funded by an Australian Research Council grant to I.E.W. and was undertaken during a visity by K.A.M. to the James Cook University of North Queensland. The expert help of Glenys Hanley and Mick Kelly is greatly appreciated. 相似文献
11.
d-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and d-mannitol dehydrogenase (EC 1.1.1.67) were estimated in a cell-free extract of the unicellular alga Platymonas subcordiformis Hazen (Prasinophyceae), d-Mannitol dehydrogenase had two activity maxima at pH 7.0 and 9.5, and a substrate specifity for d-fructose and NADH or for d-mannitol and NAD+. The K
m values were 43 mM for d-fructose and 10 mM for d-mannitol. d-Mannitol-1-phosphate dehydrogenase had a maximum activity at pH 7.5 and was specific for d-fructose 6-phosphate and NADH. The K
m value for d-fructose 6-phosphate was 5.5 mM. The reverse reaction with d-mannitol 1-phosphate as substrate could not be detected in the extract. After the addition of NaCl (up to 800 mM) to the enzyme assay, the activity of d-mannitol dehydrogenase was strongly inhibited while the activity of d-mannitol-1-phosphate dehydrogenase was enhanced. Under salt stress the K
m values of the d-mannitol dehydrogenase were shifted to higher values. The K
m value for d-fructose 6-phosphate as substrate for d-mannitol-1-phosphate dehydrogenase remained constant. Hence, it is concluded that in Platymonas the d-mannitol pool is derectly regulated via alternative pathways with different activities dependent on the osmotic pressure.Abbreviations Fru6P
d-fructose 6-phosphate
- Mes
2-(N-morpholino)ethanesulfonic acid
- MT-DH
d-mannitol-dehydrogenase
- MT1P-DH
d-mannitol-1-phosphate dehydrogenase
- Pipes
1,4-piperazinediethanesulfonic acid
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
12.
K. Hager A. Hazama H. M. Kwon D. D. F. Loo J. S. Handler E. M. Wright 《The Journal of membrane biology》1995,143(2):103-113
The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na+/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V
m
), the external myo-inositol concentration and the external Na+ concentration, yielding the kinetic parameters: K
0.5
MI
, K
0.5
Na
, and the Hill coefficient n. At 100 mM NaCl, K
0.5
MI
was about 50 m and was independent of V
m
. At 0.5 mm
myo-inositol, K
0.5
Na
ranged from 76 mm at V
m
=–50 mV to 40 mm at V
m
=–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na+ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K
I
of 64 m at V
m
=–50 mV and 130 m at V
m
= –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V
m
=–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V
m
=–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na+-dependent uptake of 3H-d-glucose and 3H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na+ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V
m
. The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model.
Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812
Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554. 相似文献
13.
The aim of this study was to examine whether flux through the pathways of carbohydrate oxidation is accurately reflected in the pattern of 14CO2 release from positionally labelled [14C]substrates in conventional radiolabel feeding studies. Heterotrophic cell suspension cultures of Arabidopsis thaliana were used for this work. The presence of an alkaline trap to capture metabolically generated 14CO2 had no significant effect on the ratio of 14CO2 release from specifically labelled [14C]substrates, or on the metabolism of [U-14C]glucose by the cells. Although the amount of 14CO2 captured in a conventional time-course study was only about half of that released from a sample acidified at an equivalent time point, the ratios of 14CO2 released from different positionally labelled [14C]glucose and [1-14C]gluconate were the same in untreated and acidified samples. Less than 5% of radioactivity supplied to the growth medium as [14C]bicarbonate was incorporated into acid-stable compounds, and there was no evidence for appreciable reassimilation of 14CO2 generated intracellularly during oxidation of [1-14C]gluconate by the cells. It is concluded that the ratio of label captured from specifically labelled [14C]glucose is a valid and convenient measure of the relative rates of oxidation of the different positional carbon atoms within the supplied respiratory substrate. However, it is argued that failure to compensate for the incomplete absorption of 14CO2 by an alkaline trap may distort estimates of respiration that rely on an absolute measure of the amount of 14CO2 generated by metabolism. 相似文献
14.
The specific binding of (3H)ethylketocyclazocine to frog brain membrane preparation was enhanced in the presence of sodium ions administered as NaCl, both at 0 °C and at room temperature. The optimal NaCl concentration was 25 mM at 0 °C and 50 mM at 24 °C. MgCl2 inhibited the [3H]ethylketocyclazocine binding. Two binding sites (high and low affinity) were established with [3H]ethylketocyclazocine as ligand by equilibrium binding studies. Addition of NaCl increased the Bmax of the low-affinity site more than that of the high-affinity site at both temperatures. Affinities were higher at 0 °C than at 24 °C. TheK
D values were not significantly influenced by sodium ions. The dissimilarities between the rat and frog brain opioid receptors in [3H]ethylketocyclazocine binding are attributed to the different lipid composition of the two membranes.Abbreviations used DAGO
D-Ala2-(Me)Phe4-Gly-ol5-enkephalin
- DALE
d-Ala2-l-Leu5-enkephalin
- DADLE
d-Ala2-d-Leu5-enkephalin
- EKC
Ethylketocyclazocine
- DHM
Dihydromorphine
- BIT
2-(p-ethoxybenzyl)1-diethylaminoethyl-5-isothiocyanobenzimidazole isothiocyanate
- FIT
Fentanyl isothiocyanate 相似文献
15.
M. R. Williams G. Duncan P. C. Croghan R. Riach S. F. Webb 《The Journal of membrane biology》1992,129(2):179-187
Summary The intracellular pH (pH
i
) of tissue-cultured bovine lens epithelial cells was measured in small groups of 6 to 10 cells using the trapped fluorescent dye 2,7-bis-(2-,carboxyethyl)-5 (and 6)carboxyfluorescein (BCECF). When perifused at 35°C with artificial aqueous humour solution (AAH) containing 16 mM HCO
3
-
and 5% CO2, pH 7.25, pH
i
was 7.19±0.02 (sem, n = 95). On removing HCO
3
-
and CO2 there was an initial transient alkalinization followed by a fall in pH to a steady value of 6.97±0.03 (sem, n = 54). Addition of 0.25 mM 4,4-diisothiocyanatostilbene2, 2-disulfonic acid (DIDS) to AAH containing HCO
3
-
and CO2 led to a rapid and pronounced fall in pH. Exposure to Na+-free AAH again led to a marked fall in pH
i
, but in this case the addition of DIDS did not produce a further fall. Substitution of the impermeant anion gluconate for Cl– in the presence of HCO
3
-
led to a rise in pH
i
, while substitution in the absence of HCO
3
-
led to a fall in pH
i
. The above data indicate a significant role for a sodium-dependent Cl–-HCO
3
-
exchange mechanism in the regulation of pH
i
. Addition of 1 mM amiloride to control AAH in both the presence and absence of HCO
3
-
led to a marked fall in pH
i
, indicating that a Na+/H+ exchange mechanism also has a significant role in the regulation of pH
i
. There is evidence for a lactic acid transport mechanism in bovine lens cells, as addition of lactate to the external medium produced a rapid fall in pH
i
. Larger changes in pH
i
were observed in control compared to HCO
3
-
-free AAH and in the latter case a pronounced alkalinizing overshoot was obtained on removing external lactate. Tissue-cultured bovine lens cells thus possess at least three membrane transport mechanisms that are involved in pH regulation. The buffering capacity of the lens cells was measured by perturbing pH
i
with either NH
4
+
or procaine. The values obtained were similar in both cases and the intrinsic buffering capacity measured in the absence of external HCO
3
-
was 5 mm/pH unit (procaine). However, in the presence of HCO
3
-
and CO2 the buffer capacity increases approximately fourfold, indicating that HCO
3
-
is the principal intracellular buffer.We acknowledge financial support from the Wellcome Trust and the Humane Research Trust for this project. M.R. Williams was in receipt of a Science & Engineering Research Council studentship. 相似文献
16.
Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences.
Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal
plots revealed K
m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N
3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate. 相似文献
17.
Birichevskaya LL Kvach SV Sivets GG Kalinichenko EN Zinchenko AI Mikhailopulo IA 《Biotechnology letters》2007,29(4):585-591
Enzymatic 5′-monophosphorylation and 5′-phosphatidylation of a number of β-l- and β-d-nucleosides was investigated. The first reaction, catalyzed by nucleoside phosphotransferase (NPT) from Erwinia herbicola, consisted of the transfer of the phosphate residue from p-nitrophenylphosphate (p-NPP) to the 5′-hydroxyl group of nucleoside; the second was the phospholipase d (PLD)-catalyzed transphosphatidylation of l-α-lecithin with a series of β-l- and β-d-nucleosides as the phosphatidyl acceptor resulted in the formation of the respective phospholipid-nucleoside conjugates.
Some β-l-nucleosides displayed similar or even higher substrate activity compared to the β-d-enantiomers. 相似文献
18.
The relationship between the gas-exchange characteristics of attached leaves of Amaranthus edulis L. and the contents of photosynthetic intermediates was examined in response to changing irradiance and intercellular partial pressure of CO2. After determination of the rate of CO2 assimilation at known intercellular CO2 pressure and irradiance, the leaf was freeze-clamped and the contents of ribulose-1,5-bisphosphate, glycerate-3-phosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, fructose-6-phosphate, triose phosphates, phosphoenolpyruvate, pyruvate, oxaloacetate, aspartate, alanine, malate and glutamate were measured. A comparison between the sizes of metabolite pools and theoretical calculations of metabolite gradients required for transport between the mesophyll and the bundle-sheath cells showed that aspartate, alanine, glycerate-3-phosphate and triose phosphates were present in sufficient quantities to support transport by diffusion, whereas pyruvate and oxaloacetate were not likely to contribute appreciably to the flux of carbon between the two cell types. The amounts of ribulose-1,5-bisphosphate were high at low intercellular partial pressures of CO2, and fell rapidly as the CO2-assimilation rate increased with increasing intercellular partial pressures of CO2, indicating that bundle-sheath CO2 concentrations fell at low intercellular partial pressures of CO2. In contrast, the amount of phosphoenolpyruvate and of C4-cycle intermediates declined at low intercellular partial pressures of CO2. This behaviour is discussed in relation to the co-ordination of carbon assimilation between the Calvin and C4 cycles.Abbreviations PEP
phosphoenolpyruvate
- PGA
glycerate-3-phosphate
-
p
i
intercellular CO2 pressure
- RuBP
ribulose-1,5-bisphosphate
- triose-P
triose phosphates 相似文献
19.
Activities of enzymes of photosynthesis and photorespiration have been measured in extracts of vegetative cells and heterocysts from the filamentous cyanobacterium Anabaena cylindrica. Phosphoribulokinase, d-ribulose 1,5-bisphosphate carboxylase/oxygenase, phosphoglycollate phosphatase and glycollate dehydrogenase activities were readily measured in vegetative cell extracts, but were undetectable or negligible in heterocyst preparations. The data help to explain why heterocysts are unable to perform photosynthetic CO2 fixation. They also exemplify the co-ordinate compartmentation of enzymes of photosynthesis and photorespiration which occur in a differentiated phototrophic prokaryote.Abbreviations Ru5P
d-ribulose 5-phosphate
- RuBP
d-ribulose 1,5-bisphosphate
- DCPIP
2,6-dichlorophenolindophenol
- TES
N-tris(hydroxymethyl)methyl-2-aminoethanesulphonate 相似文献
20.
Recombinant plasmids encoding 3-deoxy-d-manno-oct-2-ulosonate-8-phosphate (Kdo-8-P) synthase (KdsA; EC 4.1.2.16) were identified from a cDNA library of Pisum sativum L. (pea) by complementing a temperature-sensitive kdsA
ts
mutant of the Gram-negative bacterium Salmonella enterica. Sequence analysis of several inserts revealed a central open reading frame encoding a protein of 290 amino acids with a
high degree of amino acid sequence similarity to bacterial KdsA. The cDNA was confirmed by amplifying a 1,812-bp DNA fragment
from the chromosome of pea that encoded four exons around the 5′-end of kdsA. The recombinant enzyme was subcloned, overexpressed and characterized to synthesize Kdo-8-P from d-arabinose-5-phosphate and phosphoenolpyruvate. The pH optimum was 6.1 and the activity of the enzyme was neither stimulated by the addition of divalent cations
nor inhibited by EDTA. The cDNA of kdsA could not complement Escherichia coli K-12 strain AB3257, which is defective in all three isoenzymes (AroFGH) of 3-deoxy-d-arabino-hept-2-ulosonate-7-phosphate (Dha-7-P) synthase (EC 4.1.2.15), and neither d-erythrose-4-phosphate nor d-ribose-5-phosphate could substitute for d-arabinose-5-phosphate in vitro. Thus, plant cells possess a specific enzyme for the biosynthesis of Kdo-8-P with remarkable structural and functional similarities to bacterial KdsA proteins.
Received: 14 July 2000 / Accepted: 30 August 2000 相似文献