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1.
Summary We have measured the intracellular potassium activity, [K+]i and the mechanisms of transcellular K+ transport in reabsorptive sweat duct (RSD) using intracellular ion-sensitive microelectrodes (ISMEs). The mean value of [K+]i in RSD is 79.8±4.1mm (n=39). Under conditions of microperfusion, the [K+]i is above equilibrium across both the basolateral membrane, BLM (5.5 times) and the apical membrane, APM (7.8 times). The Na+/K+ pump inhibitor ouabain reduced [K+]i towards passive distribution across the BLM. However, the [K+]i is insensitive to the Na+/K+/2 Cl– cotransport inhibitor bumetanide in the bath. Cl– substitution in the lumen had no effect on [K+]i. In contrast, Cl– substitution in the bath (basolateral side) depolarized BLM from –26.0±2.6 mV to –4.7*±2.4 mV (n=3;* indicates significant difference) and decreased [K+]i from 76.0±15.2mm to 57.7* ±12.7mm (n=3). Removal of K+ in the bath decreased [K+]i from 76.3±15.0mm to 32.3*±7.6mm (n=4) while depolarizing the BLM from –32.5±4.1 mV to –28.3*±3.0 mV (n=4). Raising the [K+] in the bath by 10-fold increased [K+]i from 81.7±9.0mm to 95.0*±13.5mm and depolarized the BLM from –25.7±2.4 mV to –21.3*±2.9 mV (n=4). The K+ conductance inhibitor, Ba2+, in the bath also increased [K+]i from 85.8±6.7mm to 107.0*±11.5mm (n=4) and depolarized BLM from –25.8±2.2 mV to –17.0*±3.1 mV (n=4). Amiloride at 10–6
m increased [K+]i from 77.5±18.8mm to 98.8*±21.6mm (n=4) and hyperpolarized both the BLM (from –35.5±2.6 mV to –47.8*±4.3 mV) and the APM (from –27.5±1.4 mV to –46.0* ±3.5 mV,n=4). However, amiloride at 10–4
m decreased [K+]i from 64.5±0.9mm to 36.0*±9.9mm and hyperpolarized both the BLM (from –24.7±1.4 mV to –43.5*±4.2 mV) and APM (from –18.3±0.9 mV to –43.5*±4.2 mV,n=6). In contrast to the observations at the BLM, substitution of K+ or application of Ba2+ in the lumen had no effect on the [K+]i or the electrical properties of RSD, indicating the absence of a K+ conductance in the APM. Our results indicate that (i) [K+]i is above equilibrium due to the Na+/K+ pump; (ii) only the BLM has a K+ conductance; (iii) [K+]i is subject to modulation by transport status; (iv) K+ is probably not involved in carrier-mediated ion transport across the cell membranes; and (v) the RSD does not secrete K+ into the lumen. 相似文献
2.
Docosahexaenoic acid is found to be bound to three equivalent sites on albumin with the same affinities as palmitic acid
at 0–38°C, which demonstrates that ethene-1,2-diyl- and methylene-groups contribute equally to the affinity. The equilibrium
dissociation constants (K
dm
s) for red cell membrane binding sites of linoleic- and docosahexaenoic acid at pH 7.3 are determined at temperatures between
0 and 37°C. The temperature-independent capacities for binding are 12 ± 1 and 25.4 ± 3.0 nmoles g−1 ghosts respectively. Double isotope binding experiments reveal that the unsaturated fatty acids: arachidonic-, linoleic-,
docosahexaenoic-, and oleic acid have partially shared capacities in ratios approximately 1:2:4:5, in contrast to the noncompetitive
binding of palmitic acid. The observations suggest a two-tier binding limitation. One is the number of protein sites binding
fatty acid anions electrostatically and the other is the number of suitable annular lipids adaptively selected among membrane
lipids by the hydrocarbon chain. These competition conditions are confirmed by measurements of the tracer exchange efflux
at near 0°C from albumin-free and albumin-filled ghosts of linoleic- and docosahexaenoic acid, either alone or in the presence
of arachidonic- and palmitic acid. Under equilibrium conditions, the calculated ratios of inside to outside membrane binding
is below 0.5 for four unsaturated fatty acids. The unidirectional rate constants of translocation between the inside and the
outside correlate with the number of double bonds in these fatty acids, which are also correlated with the dissociation rate
constants of the complexes with albumin. The membrane permeation occurs presumably by binding of the anionic unsaturated fatty
acids to an integral protein followed by channeling of the neutral form between opposite binding sites of the protein through
annular lipids encircling the protein.
Received: 30 June 1997/Revised: 23 February 1998 相似文献
3.
Summary Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20°C in the presence of [3H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na+ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 m and at 37°C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes,K
m
=0.12, 0.11 and 0.10 m andJ
max=0.01, 0.02 and 0.03 pmol·l–1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts,K
m
=0.16 and 0.51 m andJ
max=0.01 and 0.04 pmol·l–1 intracellular water after 15 and 30 min incubation times, respectively. At 20°C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence ofd-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein-(probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN– showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations. 相似文献
4.
P. Fong Nicholas P. Illsley J. H. Widdicombe A. S. Verkman 《The Journal of membrane biology》1988,104(3):233-239
Summary Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J
Cl) was increased significantly from 0.32±0.12 nmol · sec–1 · mg protein–1 (mean±sem) to 0.50±0.07 nmol · sec–1 · mg protein–1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ
Cl from a control value of 0.37±0.03 nmol · sec–1 · mg protein–1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ
Cl significantly (J
Cl values gives as percent change from control): 50mm
cis Na (–1±5%), 0.1mm furosemide (–3±4%), 0.1mm furosemide in the presence of 50mm
cis Na (–5±2%), 0.1mm H2DIDS (–18±9%), a 1.5 pH unit inwardly directed H gradient (–7±7%), and 0.1mm H2DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J
Br andJ
1, respectively) were not significantly different fromJ
Cl.J
Cl was a saturable function of Cl concentration with apparentK
d
of 24mm and apparentV
max of 0.54 nmol · sec–1 · mg protein–1. Measurement of the temperature dependence ofJ
Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance. 相似文献
5.
Summary Ascorbic acid is essential for the formation of bone by osteoblasts, but the mechanism by which osteoblasts transport ascorbate has not been investigated previously. We examined the uptake ofl-[14C]ascorbate by a rat osteoblast-like cell line (ROS 17/2.8) and by primary cultures of rat calvaria cells. In both systems, cells accumulatedl-[14C]ascorbate during incubations of 1–30 min at 37°C. Unlike propionic acid, which diffuses across membranes in protonated form, ascorbic acid did not markedly alter cytosolic pH. Initial ascorbate uptake rate saturated with increasing substrate concentration, reflecting a high-affinity interaction that could be described by Michaelis-Menten kinetics (apparentK
m
=30±2 m andV
max=1460±140 nmol ascorbate/g protein/min in ROS 17/2.8 cells incubated with 138mm extracellular Na+). Consistent with a stereoselective carrier-mediated mechanism, unlabeledl-ascorbate was a more potent inhibitor (IC50=30±5 m) ofl-[14C]ascorbate transport than wasd-isoascorbate (IC50=380±55 m). Uptake was dependent on both temperature and Na+, since it was inhibited by cooling to 4°C and by substitution of K+, Li+ or N-methyl-d-glucamine for extracellular Na+. Decreasing the external Na+ concentration lowered both the affinity of the transporter for ascorbate and the apparent maximum velocity of transport. We conclude that osteoblasts possess a stereoselective, high-affinity, Na+-dependent transport system for ascorbate. This system may play a role in the regulation of bone formation. 相似文献
6.
Friedhelm Schroeder 《The Journal of membrane biology》1982,68(1):1-10
Summary The fluorescent fatty acids,trans-parimaric andcis-parinaric acid, were used as analogs of saturated and unsaturated fatty acids in order to evaluate binding of fatty acids to liver plasma membranes isolated from normal fed rats. Insulin (10–8 to 10–6
m) decreasedtrans-parinaric acid binding 7 to 26% whilecis-parinaric acid binding was unaffected. Glucagon (10–6
m) increasedtrans-parinaric acid binding 44%. The fluorescence polarization oftrans-parinarate,cis-parinarate and 1,6-diphenyl-1,3,5-hexatriene was used to investigate effects of triiodothyronine, insulin and glucagon on the structure of liver plasma membranes from normal fed rats or from rats treated with triiodothyronine or propylthiouracil. The fluorescence polarization oftrans-parinarate,cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene was 0.300±0.004, 0.251±0.003, and 0.302±0.003, respectively, in liver plasma membranes from control rats and 0.316±0.003, 0.276±0.003 and 0.316±0.003, respectively, in liver plasma membranes from hyperthyroid rats (p<0.025,n=5). Propylthiouracil treatment did not significantly alter the fluorescence polarization of these probe molecules in the liver plasma membranes. Thus, liver plasma membranes from hyperthyroid animals appear to be more rigid than those of control animals. The effects of triiodothyronine, insulin and glucagon addedin vitro to isolated liver plasma membrane preparations were also evaluated as follows: insulin (10–10
m) and triiodothyronine (10–10
m) increased fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene in liver plasma membranes while glucagon (10–10
m) had no effects. These hormonal effects on probe fluorescence polarization in liver plasma membranes were abolished by pretreatment of the rats for 7 days with triiodothyronine. Administration of triiodothyronine (10–10
m)in vitro increased the fluorescence polarization of trans-parinaric acid in liver plasma membranes from propylthiouracil-treated rats. Thus, hyperthyroidism appeared to abolish thein vitro increase in polarization of probe molecules in the liver plasma membranes. Temperature dependencies in Arrhenius plots of absorption-corrected fluorescence and fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene were noted near 25°C in liver plasma membranes from triiodothyronine-treated rats and near 18°C in liver plasma membranes from propylthiouracil-treated rats. In summary, hormones such as triiodothyronine, insulin and glucagon may at least in part exert their biological effects on metabolism by altering the structure of the liver plasma membranes. 相似文献
7.
Summary The objectives of the title were accomplished by a four-step experimental procedure followed by a simple graphical and mathematical analysis. Platelets are (i) overloaded with the indicator quin2 to cytoplasmic concentrations of 2.9mm and (ii) are exposed to 2mm external Ca2+ and 1.0 m ionomycin to rapidly achieve cytoplasmic Ca2+ ([Ca2+]cyt) of ca. 1.5 m. (iii) The external Ca2+ is removed by EGTA addition, and (iv) the active Ca2+ extrusion process is then monitored as a function of time. Control experiments show that the ionophore shunts dense tubular uptake and does not contribute to the Ca2+ efflux process during phases iii–iv and that the extrusion process is sensitive to metabolic inhibitors.The progress curves for the decline of quin2 fluorescence (resulting from active Ca2+ extrusion) were analyzed as a function of [Ca2+]cyt using a mathematical model involving the probability that an exported Ca2+ was removed from a quin2 complex (vs. a cytoplasmic binding element). The observed rates of decline of quin2 fluorescence at a particular [Ca2+]cyt are dependent upon (i) the absolute rate of the extrusion system (a function of itsK
m, Vm and Hill coefficient (n)), (ii) the intrinsic Ca2+ buffer capacity of the cytoplasm (a function of the total site concentration ([B]
T
) and itsK
d) and (iii) the buffer capacity of the intracytoplasmic quin2 (a function of its concentration andK
d). The contribution of (iii) was known and varied and was used to determine (ii) and (i) as a function of [Ca2+]cyt.The Ca2+ binding data were verified by45Ca2+ experimentation. The data fit a single binding site ([B]
T
=730±200 m) with an averageK
d of 140±10n
m. This can be accounted for by platelet-associated calmodulin. The rate of the Ca2+ extrusionvs. [Ca2+]cyt curve can be described by two components: A saturable one withV
m=2.3±0.3 nmol min–1 mg-membrane–1,K
m=80±10 andn=1.7±0.3 (probably identified with a Ca2+-ATPase pump) and a linear one (probably identified with a Na+/Ca2+ exchanger). 相似文献
8.
Summary The effects of several sulfamoyl benzoic acid derivatives on Na–K–Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC50 values) and order of potency of these derivatives were benzmetanide, 5×10–8
m> bumetanide 3×10–7
m>piretanide 3×10–6
m>furosemide 7×10–6
m> amino piretanide 1×10–5 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [3H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forK
d
and number of binding sites (n) were: bovine kidney,K
d
=1.6×10–7,n=10.5 pmol/mg protein; winter flounder intestine,K
d
1.2×10–7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,K
d
=5.3×10–7,n=20.4 pmol/mg protein. The estimatedK
d
for bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10–7
m. The similarity in magnitudes of IC50 andK
d
for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mm concentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mm Cl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mm Cl concentrations showed that bothK
d
andn were lower at the higher Cl concentration (5mm Cl:K
d
=5.29×10–7
m,n=20.4 pmol/mg protein; 100mm Cl:K
d
=2.3×10–7
m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent with a competitive interaction with Cl. 相似文献
9.
Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels 总被引:4,自引:0,他引:4
Daniel Wolff Ximena Cecchi Anda Spalvins Mitzy Canessa 《The Journal of membrane biology》1988,106(3):243-252
Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K+ (Ca–K) channel of human red cells (RBC). Cytosolic Ca2+ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na+ media containing CaCl2. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca2+ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K+ efflux (90 mmol/liter cell K+) into Na+ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca2+ was raised, with aK
m of 0.37 m and saturating between 2 and 10 m Ca2+. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC500.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K+ efflux into 75mm K+ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K+ channel in Na+ media. CTX did not block Valinomycin-activated K+ efflux into Na+ or K+ medium and therefore it does not inhibit K+ movement coupled to anion conductive permeability.TheV
max, but not theK
m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V
max was 9.36±3.0 FU (mean ±sd,n=17). TheV
max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV
max of Ca–K+ efflux.Estimation of the number of CTX-sensitive Ca-activated K+ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K+ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway. 相似文献
10.
We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (V
m), intracellular K+, Cl-, and Na+ activities (a
i
k, a
Cl
i
and a
Na
i
), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 m
l-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mm tetramethylammonium ion (TMA+) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA+ activity measured with the K+ electrode. Hepatocyte control V
m was -33±1 mV. l-alanine uptake first depolarized V
m by 2±0.2 mV and then hyperpolarized V
m by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, a
Na
i
increased by 30% from 19±2 to 25±3 mm (P < 0.01), and a
K
i
did not change significantly from 83±3 mm. However, with added ouabain (1 mm) l-alanine caused only a 2-mV increase in V
m, but now a
K
i
decreased from 61±3 to 54±5 mm (P < 0.05). Hyperpolarization of V
m by l-alanine uptake also resulted in a 38% decrease of a
Cl
i
from 20±2 to 12±3 mm (P < 0.001). Changes in V
m and V
Cl — V
m voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl– with the V
m in hepatocytes. Added Ba2+ abolished the l-alanineinduced hyperpolarization, and a
Cl
i
remained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte a
K
i
is regulated by increased Na+-K+ pump activity during l-alanine uptake in spite of cell swelling and increased V
m due to increased K+ conductance. The hyperpolarization of V
m during l-alanine uptake provides electromotive force to decrease a
Cl
i
. The latter may contribute to hepatocyte volume regulation during organic solute transport.This work was supported by grant AA-08867 from the Alcohol, Drug Abuse, and Mental Health Association. 相似文献
11.
Summary The alpha2-adrenergic antagonist yohimbine (YOH) and the closely related isomers corynanthine (COR) and rauwolscine (RAU) caused brief interruptions in current characteristic of a fast blocker Ca2+-activated K+ channels in cultured medullary thick ascending limb (MTAL) cells. The apparent dissociation constants (K
app), for COR, YOH, and RAU, respectively, at the intracellular face of the channel in the presence of 200mm K+ are 45±1, 98±2, and 310±33 m. TheK
app for COR on the extracellular side also in the presence of 200mm. K+ was much greater at 1.6±0.17mm. Increasing K+ on the same side as the blocker relieves the blocking reaction. TheK
app for the alkaloids varies with K+ in a manner quantitatively consistent with K+ and the alkaloids competing for a common binding site. Finally, blocking by the charged form of these alkaloids is voltage dependent with changes inK
app of 86±7 and 94±6 m pere-fold change in voltage for blockers applied either from the inside or outside. The alkaloids block at an electrical distance similar to tetraethylammonium, suggesting that the site within the channel pore of these molecules may be similar. 相似文献
12.
Divalent cation (Mn2+, Ca2+) entry into rat parotid acinar cells is stimulated by the release of Ca2+ from the internal agonist-sensitive Ca2+ pool via a mechanism which is not yet defined. This study examines the effect of temperature on Mn2+ influx into internal Ca2+ pool-depleted acini (depl-acini, as a result of carbachol stimulation of acini in a Ca2+-free medium for 10 min) and passive 45Ca2+ influx in basolateral membrane vesicles (BLMV). Mn2+ entry into deplacini was decreased when the incubation temperature was lowered from 37 to 4°C. At 4°C, Mn2+ entry appeared to be inactivated since it was not increased by raising extracellular [Mn2+] from 50 m up to 1 mm. The Arrhenius plot of depletion-activated Mn2+ entry between 37 and 8°C was nonlinear, with a change in the slope at about 21°C. The activation energy (Ea) increased from 10 kcal/mol (Q10=1.7) at 21–37°C to 25 kcal/mol (Q10=3.0) at 21-8°C. Under the same conditions, Mn2+ entry into basal (unstimulated) cells and ionomycin (5 m) permeabilized depl-acini exhibit a linear decrease, with E
a
of 7.8 kcal/mol (Q10=1.5) and 6.2 kcal/mol (Q10 < 1.5), respectively. These data suggest that depletion-activated Mn2+ entry into parotid acini is regulated by a mechanism which is strongly temperature dependent and distinct from Mn2+ entry into unstimulated acini.As in intact acini, Ca2+ influx into BLMV was decreased (by 40%) when the temperature of the reaction medium was lowered from 37 to 4°C. Kinetic analysis of the initial rates of Ca2+ influx in BLMV at 37°C demonstrated the presence of two Ca2+ influx components: a saturable component, with K
Ca
=279 ± 43 m, Vmax = 3.38 ± 0.4 nmol Ca2+/mg protein/min, and an apparently unsaturable component. At 4°C, there was no significant change in the affinity of the saturable component, but Vmax decreased by 61% to 1.3 ± 0.4 nmol Ca2+/mg protein/min. There was no detectable change in the unsaturable component. When BLMV were treated with DCCD (5 mm) or trypsin (1100, enzyme to membrane) for 30 min at 37°C there was a 40% decrease in Ca2+ influx. When BLMV were treated with DCCD or trypsin at 4°C and subsequently assayed for Ca2+ uptake at 37°C there was no significant loss of Ca2+ influx. These data suggest that the temperature sensitive high affinity Ca2+ flux component in BLMV is mediated by a protein which undergoes a modification at low temperatures, resulting in decreased Ca2+ transport.We thank Dr. Bruce Baum, Dr. Yukiharu Hiramatsu, Dr. Ofer Eidelman, and our other colleagues for their support during this work. 相似文献
13.
Rochelle Carpenter Livermore Jerffrey R. Hazel 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1988,158(3):363-368
Summary Acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT) (EC 2.3.1.23) activity was assayed in liver microsomes from rainbow trout,Salmo gairdneri, acclimated to 5°C and 20°C to assess its contribution to the temperature-induced restructuring of phospholipid acyl chain composition. The synthesis of phosphatidylcholine (PC) (from lyso-PC) was threefold the synthesis of phosphatidylethanolamine (PE) (from lyso-PE) under similar assay conditions. LPCAT activity (i) displayed an absolute requirement for lysophosphatidylcholine (LPC) and was enhanced by the presence of ATP, MgCl2 and CoA (which reduced the impact of endogenous acyl-CoA hydrolase activity by regenerating the acyl-CoA substrate) in the assay medium; (ii) remained linear with time up to 30 min; and (iii) increased linearly with microsomal protein concentration up to 0.2 mg/ml for the 20°C assay and 0.4 mg/ml for the 5°C assay. There was no difference in Km or Vmax values due to the acclimation history of the fish, but there were obvious differences due to assay temperature. The apparent Km values for LPC were 58.54±7.24 M and 12.26±2.14 M when assayed at 5°C and 20°C respectively; values for oleoyl-CoA were 9.11±0.78 M and 1.23±0.25 M under the same assay conditions. Activity was 1.99±0.31 nmol min–1 mg protein–1 when assayed at 5°C, and 3.8±0.45 nmol min–1 mg protein–1 when assayed at 20°C. These findings indicate that adjustments in the activity of LPCAT play no significant role in the temperature-induced restructuring of PC molecular species composition. However, the marked temperature dependence of the Km values for LPC and oleoyl CoA suggest that patterns of fatty acid incorporation (i.e. substrate preference) may vary with assay temperature, and in this way LPCAT could contribute to the restructuring response.Abbreviations
PC
phosphatidylcholine
-
PE
phosphatidylethanolamine
-
LPCAT
acyl-CoA: lysophosphatidylcholine acyltransferase
-
LPEAT
acyl-CoA: lysophosphatidylethanolamine acyltransferase
-
LPC
1-palmitoyl,2-lysophosphatidylcholine 相似文献
14.
P. G. Kostyuk S. L. Mironov P. A. Doroshenko V. N. Ponomarev 《The Journal of membrane biology》1982,70(3):171-179
Summary The shifts of current-voltage characteristics of sodium and calcium inward currents produced by changes in the concentration of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+) and in pH of the extracellular solution have been measured on isolated neurons of the molluscHelix pomatia intracellularly perfused with potassium-free solutions. On the basis of these shifts and using Stern's theory (O. Stern, 1924.Z. Electrochem.
30508–516), the binding constants for the ions to charged groups of the outer side of the somatic membrane and the density of the surface charges produced by these groups have been calculated. For groups located in the vicinity of sodium channels we obtainedK
Ca=90±10,K
Sr=60±10,K
Ba=25±5 andK
Mg=16±5m
–1 at pH=7.7 and for groups located in the vicinity of calcium channelsK
Ca=67±10,K
Sr=20±5 andK
Ba=19±5m
–1 at pH=7.0. The same groups bind H+ ions with apparent pK=6.2±0.2 that corresponds toK
H=1.6×106
m
–1. The density of fixed charges near the sodium channels is 0.17±0.05 e/nm2 (pH=7.7) and near the calcium channels is 0.23±0.05 electrons/nm2 (pH=7.0). From the comparison of the obtained values with the data about binding constants of the same ions to different negatively charged phospholipids, a suggestion is made that just the phophatidylserine is responsible for the surface potential of the outer side of the somatic membrane. It was also shown that the presence of this potential results in a change in the concentration of carrier ions near the membrane which affects the maximal values of the corresponding transmembrane currents. 相似文献
15.
Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK
1=0.67±0.16 m,k
2=1.6±0.7 sec–1,k
–2=0.17±0.09 sec–1,K
1=6.3±1.7 m,k
2=9±4 sec–1 andk
–2=7±3 sec–1. The apparent dissociation constants of chloride from band 3,K
Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3. 相似文献
16.
Edit A. Zylber Catalina A. Rotunno Marcelino Cereijido 《The Journal of membrane biology》1973,13(1):199-216
Summary Isolated epithelial cells were obtained from abdominal skin of the frogLeptodactylus ocellatus by a trypsination-dissection method. As estimated by nigrosin staining, the amount of damaged cells is only 6.6±0.7 per cent. When washed briefly after incubation the ionic concentrations in these cells were (mm): K+ 14.20±4.0; Na+ 15.8±1.8; and Cl– 57.2±5.3. If they are not washed, the concentration of K+ remains essentially the same (131.2±1.4mm) but the Na+ concentration is much higher (38.5±0.9mm). It is shown that a large fraction of Na+ is contained in a compartment that is freely connected with the bathing solution. Ouabain (10–4
m) elicits a marked decrease of K+, a slight decrease of Cl–, and an increase of Na+ content. In an equal period, low temperature (3°C) produces a similar effect, although less marked than ouabain. 相似文献
17.
L. Felipe Barros J. Carlos Bustamante David L. Yudilevich Simon M. Jarvis 《The Journal of membrane biology》1991,119(2):151-161
Summary The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparentK
m
approx. 150 m) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparentK
d
0.98±0.21nm). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparentK
i
values were 0.23±0.012, 0.36±0.035, 0.78±0.1, 0.70±0.12 (mm), and 0.12 and 4.2±1.4 (nm). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brushborder membrane vesicles (apparentK
d
1.05±0.13nM and apparentK
i
values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14±0.045, 0.54±0.046, 1.26±0.20, 1.09±0.18mm and 0.14 and 3.7±0.5nm, respectively). Exposure of both membrane vesicles to UV light in the presence of [3H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparentM
r
on SDS gel electropherograms of 77,000–45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surface of the human placental syncytiotrophoblast posses broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters. 相似文献
18.
The aim of this work was to examine the effects of changes in external K+ concentration (K
o
) around its physiological value, of various K+ channels blockers, including internal Cs+, of vacuolar H+-ATPase inhibitors and of the protonophore CCCP on the resting potential and the voltage-dependent K+ current of differentiated neuroblastoma x glioma hybrid NG108-15 cells using the whole-cell patch-clamp technique. The results are as follows: (i) under standard conditions (K
o
=5 mm) the membrane potential was –60±1 mV. It was unchanged when K
o
was decreased to 1 mm and was depolarized by 4±1 mV when Ko was increased to 10 mm. (ii) Internal Cs+ depolarized the membrane by 21±3 mV. (iii) The internal application of the vacuolar H+-ATPase inhibitors N-ethylmaleimide (NEM), NO
3
–
and bafilomycin A1 (BFA) depolarized the membrane by 15±2, 18±2 and 16±2 mV, respectively, (iv) When NEM or BFA were added to the internal medium containing Cs+, the membrane was depolarized by 45±1 and 42±2 mV, respectively. (v) The external application of CCCP induced a transient depolarization followed by a prolonged hyperpolarization. This hyperpolarization was absent in BFA-treated cells. The voltage-dependent K+ current was increased at negative voltages and decreased at positive voltages by NEM, BFA and CCCP. Taken together, these results suggest that under physiological conditions, the resting potential of NG108-15 neuroblastoma cells is maintained at negative values by both voltage-dependent K+ channels and an electrogenic vacuolar type H+-ATPase.This work was supported by a grant from INSERM (CRE 91 0906). 相似文献
19.
Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula 总被引:1,自引:0,他引:1
Masahiro Nakao Masami Harada Yukiko Kodama Toru Nakayama Yuji Shibano Teruo Amachi 《Applied microbiology and biotechnology》1994,40(5):657-663
We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K
m=0.75 mm and molecular activity (k
cat)= 63.1 s–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K
m=0.04 mm
k
cat= 55.8 s–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl2) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl2). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose.
Correspondence to: T. Nakayama 相似文献
20.
Summary The initial mechanisms of injury to the proximal tubule following exposure to nephrotoxic heavy metals are not well established. We studied the immediate effects of silver (Ag+) on K+ transport and respiration with extracellular K+ and O2 electrodes in suspensions of renal cortical tubules. Addition of silver nitrate (AgNO3) to tubules suspended in bicarbonate Ringer's solution caused a rapid, dose-dependent net K+ efflux (K
m
=10–4
m,V
max=379 nmol K+/min/mg protein) which was not inhibited by furosemide, barium chloride, quinine, tetraethylammonium, or tolbutamide. An increase in the ouabain-sensitive oxygen consumption rate (QO2) (13.9±1.1 to 25.7±4.4 nmol O2/min/mg,P<0.001), was observed 19 sec after the K+ efflux induced by AgNO3 (10–4
m), suggesting a delayed increase in Na+ entry into the cell. Ouabain-insensitive QO2, nystatin-stimulated QO2, and CCCP-uncoupled QO2 were not significantly affected, indicating preserved function of the Na+, K+-ATPase and mitochondria. External addition of the thiol reagents dithiothreitol (1mm) and reduced glutathione (1mm) prevented and/or immediately reversed the effects on K+ transport and QO2. We conclude that Ag+ causes early changes in the permeability of the cell membrane to K+ and then to Na+ at concentrations that do not limit Na+, K+-ATPase activity or mitochondrial function. These alterations are likely the result of a reversible interaction of Ag+ with sulfhydryl groups of cell membrane proteins and may represent initial cytotoxic effects common to other sulfhydryl-reactive heavy metals on the proximal tubule. 相似文献