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1.
(H+ + K+)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg2+-ATPase and p-nitrophenylphosphatase activities (68 ± 9 μmol Pi and 2.9 ± 0.6 μmol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K+ (up to 0.69 ± 0.09 nmol Pi incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1–2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 × g × 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K+-stimulated ATPase and p-nitrophenylphosphatase activities and K+-sensitive phosphorylation: Mg2+-ATPase (μmol Pi/mg protein per h) 32 ± 9 (basal) and 86 ± 20 (K+-stimulated); Mg2+-p-nitrophenylphosphatase (μmol p-nitrophenol/mg protein per h) 2.6 ± 0.5 (basal) and 22.2 ± 3.2 (K+-stimulated); Mg2+-phosphorylation (nmol Pi/mg protein) 0.214 ± 0.041 (basal) and 0.057 ± 0.004 (in the presence of K+). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H+ + K+)-ATPase in a still active structure. 相似文献
2.
Han Y Randell E Vasdev S Gill V Gadag V Newhook LA Grant M Hagerty D 《Molecular and cellular biochemistry》2007,305(1-2):123-131
The reactive aldehydes methylglyoxal and glyoxal, arise from enzymatic and non-enzymatic degradation of glucose, lipid and
protein catabolism, and lipid peroxidation. In Type 1 diabetes mellitus (T1DM) where hyperglycemia, oxidative stress, and
lipid peroxidation are common, these aldehydes may be elevated. These aldehydes form advanced glycation end products (AGEs)
with proteins that are implicated in diabetic complications. We measured plasma methylglyoxal and glyoxal in young, complication-free
T1DM patients and assessed activity of the ubiquitous membrane enzyme, Na+/K+ ATPase. A total of 56 patients with TIDM (DM group), 6–22 years, and 18 non-diabetics (ND group), 6–21 years, were enrolled.
Mean plasma A1C (%) was higher in the DM group (8.5 ± 1.3) as compared to the ND group (5.0 ± 0.3). Using a novel liquid chromatography-mass
spectrophotometry method, we found that mean plasma methylglyoxal (nmol/l) and glyoxal levels (nmol/l), respectively, were
higher in the DM group (841.7 ± 237.7, 1051.8 ± 515.2) versus the ND group (439.2 ± 90.1, 328.2 ± 207.5). Erythrocyte membrane
Na+/K+ ATPase activity (nmol NADH oxidized/min/mg protein) was elevated in the DM group (4.47 ± 0.98) compared to the ND group (2.16 ± 0.59).
A1C correlated with plasma methylglyoxal and glyoxal, and both aldehydes correlated with each other. A high correlation of
A1C with Na+/K+ ATPase activity, and a regression analysis showing A1C as a good predictor of activity of this enzyme, point to a role for
glucose in membrane alteration. In complication-free patients, increased plasma methylglyoxal, plasma glyoxal, and erythrocyte
Na+/K+ ATPase activity may foretell future diabetic complications, and emphasize a need for aggressive management. 相似文献
3.
Ibuprofen, one of the nonsteroidal anti-inflammatory drugs, inhibited arylamine N-acetyltransferase activity of Klebsiella pneumoniae both in vitro and in vivo. The NAT activities of Klebsiella pneumoniae were inhibited by ibuprofen in a dose-dependent manner both in vitro and in vivo. In vitro, the NAT activity was 0.675 ±
0.028 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was
0.506 ± 0.002 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In vivo, the NAT activity was 0.279 ± 0.016 nmol/min/1010 colony forming units (CFU) for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was
0.228 ± 0.008 nmol/min/1010 CFU for the acetylation of 2-aminofluorene. The inhibition of NAT activity by ibuprofen was shown to persist for at least
4 h. For in vitro examination, the values of apparent K
m
and V
max were 1.08 ± 0.05 mM and 9.17 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen
was added to the reaction mixtures, the values of apparent K
m
and V
max were 1.19 ± 0.01 mM and 6.67 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. For in vivo examination, the
values of apparent K
m
and V
max were 1.24 ± 0.48 mM and 4.18 ± 1.06 nmol/min/10 × 1010 CFU, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the culture, the values of apparent
K
m
and V
max were 0.95 ± 0.29 mM and 2.77 ± 0.37 nmol/min/mg protein, respectively, for 2-aminofluorene, respectively. This report is
the first finding of ibuprofen inhibition of arylamine N-acetyltransferase activity in a strain of Klebsiella pneumoniae.
Received: 28 January 1997 / Accepted: 12 February 1997 相似文献
4.
5.
Y. C. Yen-Chow S. Y. Chow W. S. S. Jee D. M. Woodbury 《In vitro cellular & developmental biology. Plant》1984,20(9):677-684
Summary The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was −10.2±0.20 mV (n=390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal
preparations, such as dibutyryladenosine 3′,5′-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10−5 to 10−4
M), hydrocortisone (10−7 to 10−6
M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na+, K+, and Cl− concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein
contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined
by [14C] dimethyloxazolidine-2, 4-dione, and3H2O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activiities of Na+, K+-, HCO3
−-, and Ca++, Mg++-ATPases in these cultured cells were 19.0±2.1, 13.6±2.1, and 6.6±1.2 nmol pi/mg protein per minute, respectively, and the
carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte
concentrations observed in this study indicate that Na+, K+, Cl−, and H+ are not distributed according to their electrochemical gradients across the cell membrane. Na+, Cl−, and H+ are actively transported out of the cells and K+ into the cells.
This study was supported by Grant AM20935 from the NIAMDD, NIH, Bethesda, Maryland, and National Aeronautics & Space Administration
NASA-Ames Grant NAG 2-108 and U.S. Department of Energy Contract DE-AC02-76-EV-00119. D. M. W. is the recipient of a Research
Career Award (5-K6-NB-13838), NINCDS, NIH. 相似文献
6.
Using GSH-PGA1 as substrate for determination of enzyme activity a pI 4.8 form of rabbit kidney prostaglandin 9-keto-reductase has been purified 95 times to a specific activity of 1755 nmol/min per mg protein. The purification procedures involve ion-exchange chromatography, gel-filtration and affinity chromatography. The latter procedure comprises Blue Sepharose affinity chromatography, and GSH-PGA1-Sepharose affinity chromatography.The purified enzyme preparation also showed a weak NADP+-dependent 15-hydroxyprostaglandin dehydrogenase activity, 20 nmol/min per mg protein with PGE1 as substrate. Km(PGE1) for the dehydrogenase is 142.6 ± 45.1 μM (S.E., n=7). 相似文献
7.
Sealed, inside-out human red cell membrane vesicles, prepared by a modified method of Steck (Steck T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.), Vol 2, pp. 245–281, Plenum Press, New York), accomplish an ATP and Mg2+-dependent uphill calcium uptake with a reproducible maximum rate of 12–15 nmol/mg vesicle protein per min under physiological conditions. This maximum rate is increased by about 60–70% in the presence of a heatstable cytoplasmic activator protein (calmodulin) obtained from red cells. Calcium efflux from inside-out vesicles is smaller than 0.01 nmol/mg vesicle protein per min at intravesicular calcium concentrations between 0.1 and 20.0 mM.In the presence of Mg2+, active calcium uptake is supported by ATP, ITP, or UTP, but not by ADP, AMP, or p-nitrophenyl phosphate. The optimum pH for the process is 7.4–7.6, and the activation energy is 19–20 kcal/mol, irrespective of the presence or absence of calmodulin. Calcium uptake in inside-out vesicles is unaffected by ouabain or oligomycin, but blocked by low concentrations of lanthanum, ruthenium red, quercetin and phloretin. K+ and Na+, when compared to choline+ or Li+, significantly increase active calcium uptake. This stimulation by K+ and Na+ is independent of that by calmodulin.Concentrated red cell cytoplasm activates calcium uptake at low soluble protein:membrane protein ratios, while a ‘deactivation’ of the transport occurs at high cytoplasm: membrane protein ratios. A heat-labile cytoplasmic protein fraction antagonizing calmodulin activation, can be separated by DEAE-Sephadex chromatography. Based on these findings the regulation of active calcium transport in human red cells is discussed. 相似文献
8.
Summary The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150mm KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639±84 and 619±65 nmol/min×mg protein, respectively, giving an H+/ATP ratio of 1.03±0.7. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (<10 nmol/min×mg protein) was exhibited by vesicles in 150mm NaCl at pH 6.1 despite ATP hydrolysis of 187±46 nmol/min×mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H+/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K+ greater than 10mm. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H+/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K++H+)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach. 相似文献
9.
N-Acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene as substrates were determined in isolates of the bacterium Escherichia coli. The N-acetyltransferase activity was determined by an acetyl CoA recycling assay and high pressure liquid chromatography.
The N-acetyltransferase activities from a number of E. coli isolates were found to be 0.67 ± 0.04 nmole/min/mg protein for 2-aminofluorene, and 0.46 ± 0.02 nmole/min/mg protein for
p-aminobenzoic acid. The apparent K
m
and V
max values obtained were 2.85 ± 0.65 mM and 7.51 ± 0.86 nmol/min/mg protein, respectively, for 2-aminofluorene, and 2.35 ± 0.39
mM and 9.43 ± 0.78 nmol/min/mg protein, respectively, for p-aminobenzoic acid. The optimal pH value for the enzyme activity was 7.0 for both substrates tested. The optimal temperature
for enzyme activity was 37°C for both substrates. The N-acetyltransferase activity was inhibited by iodoacetamide: at 0.25
mM iodoacetamide, activity was reduced 50%, and at 1.0 mM, more than 90%. Among a series of divalent cations and salts, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. This report is the first demonstration of acetyl CoA:arylamine N-acetyltransferase
activity in E. coli.
Received: 29 April 1997 / Accepted: 2 July 1997 相似文献
10.
L. Adamec 《Biologia Plantarum》1997,39(4):599-606
The uptake of K+ by the leafy shoots of four submersed higher aquatic plants (Elodea canadensis, Ranunculus aquatilis, R. trichophyllus, and
Callitriche hamulata) with different HCO3
- affinity was measured in successive 2-h periods under the conditions of high or low photosynthetic rates (i.e. at pH 7.5
or 10). At pH 7.5 the uptake of K+ by species with the higher HCO3
- affinity (E. canadensis, R. trichophyllus) was significantly faster than that by species with a lower HCO3
- affinity (R. aquatilis, C. hamulata). In the former group of species, the K+ uptake rate at pH 7.5 was 1.7 - 3.5 times higher than at pH 10. At pH 10, the soft-water species, R. aquatilis, had the lowest
net photosynthetic rate (PN) of the three HCO3
- users but, in contrast to the relative hard-water species, R. trichophyllus, showed a small K+ efflux (47 nmol kg-1 s-1). Thus, K+ uptake by shoots was not strictly correlated with PN. A significant K+ efflux (73 - 86 nmol kg-1 s-1) occurred from all HCO3
- users in darkness. The relatively low K+ uptake by the strict CO2 user, C. hamulata, was quite independent of PN and light or darkness. It may be suggested that uptake of K+ by shoots of submersed plants depends on their HCO3
- affinity.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
11.
Human brain cortical homogenate derived from surgical operations exhibited Na+, K+-ATPase and K-p-nitrophenylphosphatase activity values of 2.12 ± 0.08 μmol Pi/mg protein/15 min and 0.38 ± 0.01 μmol p-nitrophenol/mg protein/15 min, respectively which is in the range of those observed in rat brain cortical homogenates. Vanadate concentration dependently inhibited the activity of both enzymes. Noradrenaline, dopamine and isoprenaline reversed the inhibitory effect of vanadate in the presence of EDTA (0.2 mM). When Mg2+ concentration was enhanced from 4 to 24 mM, the inhibitory effect of vanadate (1 μM) was significantly potentiated. Evidence has been obtained that the effect of catecholamines is not a receptor mediated process; antagnoists such as phentolamine, phenoxybenzamine, propranolol, haloperidol failed to prevent the effect of adrenoceptor agonists. It is concluded that there is an interaction between vanadate and noradrenaline on human brain Na, K-ATPase. 相似文献
12.
The ability of trout to metabolize aniline in vitro in the presence of some divalent metal ions was investigated in the liver microsomes of rainbow trout, Salmo gairdneri. Trout liver microsomes were highly capable of catalyzing aniline hydroxylation to p-aminophenol with a specific activity of 0.068 nmoles/min per mg of microsomal protein in potassium phosphate buffer, pH 7.4 at 25°C. The activity of the aniline hydroxylase system was competitively inhibited by Hg+2, Ni+2, Cd+2, and Zn+2, while Cu+2 and Fe+3 seemed to inhibit the activity noncompetitively at 1 mM aniline concentrations. IC50 values at fixed aniline concentration were estimated to be 0.45 mM for Hg+2, Ni+2, and Cd+2, 1.8 mM for Zn+2 and Fe+3, and 1.3 mM for Cu+2. Eadie-Hofstee plots gave identical Vmax values of approximately 0.046 nmol/min per mg of protein while Km values were increased in the presence of Hg+2, Ni+2, CD+2, and Zn+2, indicating competitive inhibition. Both Km and Vmax values were affected by Fe+3 and Cu+2, suggesting noncompetitive inhibition. Ki values extracted from the Dixon plots were determined t be 0.23, 0.43, and 0.65 mM for Hg+2, Ni+2, and Cd+2, respectively, providing the most effective inhibition on the aniline hydroxylase system among studied metal ions. The Ki values were much higher in the presence of others. The results indicate a selective inhibition of the aniline hydroxylase system of trout liver microsomes by divalent metal ions. © 1997 John Wiley & Sons, Inc. 相似文献
13.
Influx of [3H]-l-proline into renal OK cells revealed that basal transport was mediated by the transporter SIT1. When cells were submitted
for 8 h to amino acid deprivation, uptake of l-proline was now dominated by a low-affinity system with an apparent K
m of 4.4 ± 0.6 mM and a V
max of 10.2 ± 0.6 nmol/mg of protein/min operating in addition to the high-affinity SIT1 system with a K
m of 0.12 ± 0.01 mM and a V
max of 0.28 ± 0.04 nmol/mg of protein/min. The low- and high-affinity proline transporting systems were sensitive to inhibitors
of JNK and PI-3 kinases, whereas a GSK-3 inhibitor affected only the upregulated transport system. Ion-replacement studies
and experiments assessing substrate specificities for both systems provided strong evidence that SNAT2, that showed two- to
threefold increased mRNA levels, is the responsible transporter mediating the increased proline influx under conditions of
amino acid deprivation. 相似文献
14.
Harinder S. Hundal Peter M. Taylor Nicholas M. Willhoft Bryan Mackenzie Sylvia Y. Low Martyn R. Ward Michael J. Rennie 《生物化学与生物物理学报:疾病的分子基础》1992,1180(2):137-146
We wished to examine the effects of diabetes on muscle glutamine kinetics. Accordingly, female Wistar rats (200 g) were made diabetic by a single injection of streptozotocin (85 mg/kg) and studied 4 days later; control rats received saline. In diabetic rats, glutamine concentration of gastrocnemius muscle was 33% less than in control rats: 2.60 ± 0.06 μmol/g vs. 3.84 ± 0.13 μmol/g (P < 0.001). In gastrocnemius muscle, glutamine synthetase activity (Vmax) was unaltered by diabetes (approx. 235 nmol/min per g) but glutaminase Vmax increased from 146 ± 29 to 401 ± 94 nmol/min per g; substrate Km values of neither enzyme were affected by diabetes. Net glutamine efflux (AZ concentration difference × blood flow) from hindlimbs of diabetic rats in vivo was greater than control values (?30.0 ± 3.2 vs. ?1.9 ± 2.6 nmol/min per g (P < 0.001) and hindlimb NH3 uptake was concomitantly greater (about 27 nmol/min per g). The glutamine transport capacity (Vmax) of the Na-dependent System Nm in perfused hindlimb muscle was 29% lower in diabetic rats than in controls (820 ± 50 vs. 1160 ± 80 nmol/min per g (P < 0.01)), but transporter Km was the same in both groups (9.2 ± 0.5 nM). The difference between inward and net glutamine fluxes indicated that glutamine efflux in perfused hindlimbs was stimulated in diabetes at physiological perfusate glutamine (0.5 mM); ammonia (1 mM in perfusate) had little effect on net glutamine flux in control and diabetic muscles. In Intramuscular Na+ was 26% greater in diabetic (13.2 μmol/g) than control muscle, but muscle K+ (100 μmol/g) was similar. The accelerated rate of glutamine release from skeletal muscle and the lower muscle free glutamine concentration observed in diabetes may result from a combination of; (i), a diminished Na+ electrochemical gradient (i.e., the net driving force for glutamine accrual in muscle falls); (ii), a faster turnover of glutamine in muscle and (iii), an increased Vmax/Km for sarcolemmal glutamine efflux. 相似文献
15.
Harinder S. Hundal Philip Babij Peter M. Taylor Peter W. Watt Michael J. Rennie 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1991,1092(3):376-383
Intramuscular glutamine falls with injury and disease in circumstances associated with increases in blood corticosteroids. We have investigated the effects of corticosteroid administration (0.44 mg/kg dexamethasone daily for 8 days, 200 g female rats) on intramuscular glutamine and Na+, muscle glutamine metabolism and sarcolemmal glutamine transport in the perfused hindlimb. After dexamethasone treatment intramuscular glutamine fell by 45% and Na+ rose by 25% (the respective muscle/plasma distribution ratios changed from 8.6 to 4.5 and 0.12 to 0.15); glutamine synthetase and glutaminase activities were unchanged at 475 ± 75 and 60 ± 19 nmol/g muscle per min. Glutamine output by the hindlimb of anaesthetized rats was increased from 31 to 85 nmol/g per min. Sarcolemmal glutamine transport was studied by paired-tracer dilution in the perfused hindlimb: the maximal capacity (Vmax) for glutamine transport into muscle (by Na+-glutamine symport) fell from 1058 ± 310 to 395 ± 110 nmol/g muscle per min after dexamethasone treatment, accompanied by a decrease in the Km (from 8.1 ± 1.9 to 2.1 ± 0.4 mM glutamine). At physiological plasma glutamine concentration (0.75 mM) dexamethasone appeared to cause a proportional increase in sarcolemmal glutamine efflux over influx. Addition of dexamethasone (200 nM) to the perfusate of control rat hindlimbs caused acute changes in Vmax and Km of glutamine transport similar to those resulting from 8-day dexamethasone treatment. The reduction in muscle glutamine concentration after dexamethasone treatment may be primarily due to a reduction in the driving force for intramuscular glutamine accumulation, i.e., in the Na+ electrochemical gradient. The prolonged increase in muscle glutamine output after dexamethasone treatment (which occurs despite a reduction in the size of the intramuscular glutamine pool) appears to be due to a combination of (a) accelerated sarcolemmal glutamine efflux and (b) increased intramuscular synthesis of glutamine. 相似文献
16.
Ole M. Sejersted 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,586(2):330-340
It is not known whether ouabain injected into the kidney in vivo is bound exclusively to the (Na+ + K+)-ATPase and whether the reduction of sodium pumping capacity is large enough to account for the reduction in sodium reabsorption. In the present study on dogs the total amount of parenchymal ouabain was therefore estimated and the specific renal binding compared to the reduction in (Na+ + K+)-ATPase activity. Ouabain, 120 nmol/kg body weight, was injected into the renal artery in vivo reducing the (Na+ + K+)-ATPase activity by 3lmost 80%. After nephrectomy, tissue ouabain could be quantified by radioimmunoassay after heating the homogenate to 70°C for 30 min; negligible amounts were detectable without heating. No correlation between ouabain binding and tissue volume, protein content, DNA content or Mg2+-ATPase content could be found when comparing the following four fractions of the kidney: outer cortex, inner cortex, outer medulla and papilla. For the whole kidney, mean parenchymal tissue concentration of ouabain equalled 0.58 ± 0.03 μmol/100 g wet tissue. Only 21.3 ± 1.2% of the ouabain was confined to the outer medulla corresponding to 54 ± 4 nmol giving a tissue concentration of 1.08 ± 0.05 μmol/100 g wet tissue. The renal ouabain concentrations were highly correlated to the reduction in (Na+ + K+)-ATPase activity, giving a ratio between the reduction in hydrolysis rate and bound ouabain (turnover number) of 6105 min?1 which is close to the value of 7180 min?1 found by in vitro Scatchard analysis. No ouabain seems to be bound to other tissue components than the (Na+ + K+)-ATPase and the present method is therefore a simple way of measuring the number of inhibited (Na+ + K+)-ATPase molecules after in vivo injection of ouabain. 相似文献
17.
Volodymyr Ivanov Saeid Rezaeinejad Jian Chu 《Journal of bioenergetics and biomembranes》2013,45(5):505-510
It is considered that all growing cells, for exception of acidophilic bacteria, have negatively charged inside cytoplasmic membrane (Δψ? - cells). Here we show that growing populations of microbial cells contain a small portion of cells with positively charged inside cytoplasmic membrane (Δψ+ - cells). These cells were detected after simultaneous application of the fluorescent probes for positive membrane potential (anionic dye DIBAC-) and membrane integrity (propidium iodide, PI). We found in exponentially growing cell populations of Escherichia coli and Saccharomyces cerevisiae that the content of live Δψ- - cells was 93.6?±?1.8 % for bacteria and 90.4?±?4.0 % for yeasts and the content of live Δψ+ - cells was 0.9?±?0.3 % for bacteria and 2.4?±?0.7 % for yeasts. Hypothetically, existence of Δψ+ - cells could be due to short-term, about 1 min for bacteria and 5 min for yeasts, change of membrane potential from negative to positive value during the cell cycle. This change has been shown by the reversions of K+, Na+, and Ca2+ ions fluxes across the cell membrane during synchronous yeast culture. The transformation of Δψ-- cells to Δψ+ - cells can be explained by slow influx of K+ ions into Δψ-- cell to the trigger level of K+ concentration (“compression of potassium spring”), which is forming “alternative” Δψ+-cell for a short period, following with fast efflux of K+ ions out of Δψ+-cell (“release of potassium spring”) returning cell to normal Δψ- state. We anticipate our results to be a starting point to reveal the biological role of cell dualism in form of Δψ- - and Δψ+ - cells. 相似文献
18.
Sampathkumar R Balasubramanyam M Tara C Rema M Mohan V 《Molecular and cellular biochemistry》2006,282(1-2):169-176
Objective: Although recent studies link altered cellular redox state to protein dysfunction in various disease-states, such associations
are least studied in clinical diabetes. Therefore, this study assessed the levels of reduced glutathione (GSH) and Na+/K+ ATPase activities in type 2 diabetic patients with and without microangiopathy. Methods: The study group comprised of a total of 160 subjects, which included non-diabetic healthy controls (n = 40) and type 2 diabetic patients without (n = 60) and with microangiopathy (n = 60), defined as presence of retinopathy with or without nephropathy. Erythrocyte Na+/K+ ATPase activity and GSH levels were estimated spectrophotometrically and fluorometry was used to determine the plasma thiobarbituric
acid reactive substances (TBARS) and serum advanced glycation end products (AGEs). Results: GSH levels in diabetic subjects without (4.8± 0.15 μmol/g Hb) and with microangiopathy (5.2± 0.14 μmol/g Hb) were significantly
lower (p < 0.001) compared to control subjects (6.3± 0.14 μmol/g Hb). Erythrocyte Na+/K+ ATPase activity was significantly reduced (p < 0.001) in diabetes subjects with (272± 7 nmol Pi/mg protein/h) and without microangiopathy (304 ± 8) compared to control
(374 ± 6) subjects. TBARS were significantly higher (p < 0.001) in diabetes subjects with (10.65± 0.81 nM/ml) and without microangiopathy (9.90± 0.5 nM/ml) compared to control
subjects (5.18± 0.18 nM/ml). Advanced glycation end product levels were also significantly (p < 0.001) elevated in diabetic subjects with microangiopathy (8.2± 1.8 AU) when compared to diabetes subjects without microangiopathy
(7.0± 2.0 AU) and control subjects (4.6± 1.9 AU). On multivariate regression analysis, GSH levels showed a positive association
with the Na+/K+ ATPase activity and negative association with TBARS and AGE levels. Conclusion: Hypoglutathionemia and increased oxidative stress appears to be early biochemical aberrations in diabetes, and through protein
alterations, oxidative stress and redox modifications may contribute to pathogenesis of diabetic microangiopathy. 相似文献
19.
P. GOMES M.P SERRO M.A VIEIRA-COELHO P. SOARES-DA-SILVA 《Cell biology international》1997,21(4):249-255
The present work was aimed at studying the kinetics and nature of the l-DOPA transporter in opossum kidney (OK) cells. Saturation experiments were performed in OK cells incubated for 6 min with increasing concentrations of l-DOPA (10 to 2500 μm); non-linear analysis of the saturation curve revealed for l-DOPA aKmof 129 μm (114, 145) and aVmaxof 30.0±0.4 nmol mg protein?16 min?1The uptake of l-DOPA (250 μm) was inhibited in a concentration-dependent manner by cyanine 863, an organic cation inhibitor, with aKivalue of 638 (430, 947) μmthe organic anion inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS), was devoid of effect upon the uptake of l-DOPA. The uptake of l-DOPA (250 μm) was significantly (P<0.02) decreased (25% reduction) when cells were incubated in the presence of 137 mm K+plus 5 mm Na+when compared with the control condition (137 mm Na+plus 5 mm K+); substitution of NaCl by choline chloride (137 mm) did not affect l-DOPA uptake. Similarly, inwardly or outwardly directed proton gradients of 0.5 pH units (7.9, 7.4, 6.9, 6.4 and 5.9) were found not to change l-DOPA uptake. In conclusion, the l-DOPA uptake system in OK cells has the characteristics of an organic cation potential-dependent and proton-independent transporter. 相似文献
20.
Abstract: Rats were given LiCl in their diet (40 mmol/kg dry weight) for at least 3 months to elucidate the regional and subcellular localization of Li+ in the brain as well as the effect of chronic lithium administration on the distribution of other cations. At steady-state the mean concentrations of Li+ were 0.66 mmol/kg wet weight in the whole brain and 0.52 mM in plasma. The tissue/plasma concentration ratio exceeded unity in all anatomical regions. No region showed excessive accumulation of Li+. Whole brain or regional contents of Na+ or K+ were unaffected by lithium treatment. Subcellular Li+ localization was demonstrated in nuclear, crude mitochondrial, and microsomal fractions of whole brain homogenate. Subfractionation of the crude mitochondrial fraction revealed energy-independent intrasynaptosomal and intramitochondrial Li+ and K+ localization at 0–4°C. Li+ administered in vivo disappeared within 10 min from synaptosomes incubated at 37°C. Li+ added in vitro at 1 mM attained a synaptosomal steady-state concentration within 30 min at 37°C. In control rats, synaptosomal concentrations and synaptosomal/medium concentration gradients of cations paralleled their respective in vivo concentrations and gradients. Lithium treatment caused synaptosomal depletion of K+ and Mg2+ and hence probably partial membrane depolarization. Addition of 1 mM Li+ in vitro also caused synaptosomal Mg2+ depletion. The results indicate that Li+ is “accumulated” in brain sediments and synaptosomes following its long-term treatment. The estimated intracellular and intrasynaptosomal Li+ concentrations are lower than predicted by passive distribution according to the Nernst equation, evidencing active extrusion of Li+. 相似文献