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Kinetics of bidirectional active sodium fluxes in the toad bladder

Daniel Wolff Alvin Essig 《生物化学与生物物理学报:生物膜》1977,468(2):271-283

The kinetics of isotopic Na⁺ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal ²²Na⁺ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na⁺ flow. The electrical current

I

and mucosal to serosal ²²Na⁺ influx were then measured with transmembrane potential clamped at

Δψ = 0, 25, 50, 75 or 100 mV

. Subsequent elimination of active Na⁺ transport mucosal amiloride permitted calculation of the rates of active Na⁺ transport

J_{Na}^{a}

and active and passive influx

J^{_{Na}}^{Na}

and

J_{Na}^{a}

and

J_{Na}^{p}

. The results indicate that for Dominican toad bladders mounted in chambers only Na⁺ contributes significantly to transepithelial active ion transport; hence

J_{Na}^{a} = J^{a}

J^{a}

was abolished at

Δψ = E = 96.3 ± 1.9 (S.E.) mV

. As Δψ approached

E

, active efflux

J^{a}

became demonstrable. At

Δ = 100 mV, J^{a}

exceeded

J^{a}

, so that

J^{a}

was negative. Experimental values of

J^{a}

agreed well with theoretical values predicted by a thermodynamic formulation:

J_{exp}^{a} = 0.985 J_{theor}^{a} (r = 0.993)

. The dependence of

J^{a}

on Δψ is curvilinear. 相似文献

Electrokinetic properties of (

Na^{+}

K^{+})- ATPase

vesicles as studied by laser doppler spectroscopy

Peter Schlieper Ram Mohan Raimund Kaufmann 《生物化学与生物物理学报:生物膜》1981,644(1):13-23

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na⁺,⁺)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a

p K = 3.9

; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na⁺ and K⁺; (3) Mg²⁺ binds with an association constant of

K_{a} = 1.1 · 10^{2} M^{?1}

while ATP binds with an apparent

K_{a} = 1.1 · 10^{4} M^{?2}

for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl₂, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg²⁺ concentrations. There is no ATP binding in the absence of Mg²⁺. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is

203.7 ± 15.2 nm

. In the presence of ATP a reduction in size is observed. 相似文献

Characterization of the (

Na^{+} + K^{+})-ATPase

in a membranous preparation from the optic ganglion of the squid (Loligo pealei)

G.E. Breitwieser 《生物化学与生物物理学报:生物膜》1982,689(3):457-463

(1) A membrane fraction enriched in

(Na^{+} + K^{+})-ATPase

(EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an

(Na^{+} + K^{+})-ATPase

specific activity of approx. 2 units/mg and was

>95%

ouabain-sensitive. (2) The

(Na^{+} + K^{+})-ATPase

had a

K_{m}

for ATP of

0.42 ± 0.04 mM

and a pH optimum of 7.0. It was inhibited by ouabain with a

K_{i}

0.32 ± 0.04 μ M

. (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with

NaCl + KCl = 300 mM

. (4) The Mg²⁺ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the

K_{m}

for Mg²⁺ was

0.86 ± 0.10 mM

, and at 6 mM ATP, the

K_{m}

was

1.86 ± 0.44 mM

. High levels of Mg²⁺ caused inhibition of hydrolysis. (5) The interactions of Na⁺ and K⁺ were examined over a range of conditions. K⁺ levels caused modulations in the Na⁺ dependence in the range of 1–150 mM. (6) The

(Na^{+} + K^{+})-ATPase

prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves. 相似文献

Interaction of lymphocytes and macrophage cell line cells (M1 cells). I. Functional maturation and appearance of Fc receptors im M1 cells 总被引：2，自引：0，他引：2

J Yodoi T Masuda M Miyama M Maeda Y Ichikawa 《Cellular immunology》1978,39(1):5-17

M₁ cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells (

M_{1}^{?}

) to mature macrophages (

M_{1}^{+}

) within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While

M_{1}^{?}

cells have no phagocytic activity nor Fc receptor (FcR),

M_{1}^{+}

cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on

M_{1}^{+}

cells is resistant to trypsin and pronase.

M_{1}^{+}

cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens.

M_{1}^{?}

cells lack this macrophage-substituting capacity. Mm₁ cells, mutant cells from M₁ cells, having FcR and higher phagocytic activity than

M_{1}^{+}

cells, are also devoid of this capacity. 相似文献

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts Y.-F. Fu G.A.J. Kuijpers J.J.H.H.M. de Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1984,774(2):277-287

(1) Treatment of

(Na^{+} + K^{+})-ATPase

from rabbit kidney outer medulla with the

γ-^{35} S

labeled thio-analogue of ATP in the presence of

Na^{+} + Mg^{2+}

and the absence of K⁺ leads to thiophosphorylation of the enzyme. The

K_{m}

value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient

n H = 2.6

. (2) The thio-analogue (

K_{m= 35 μM}

) can also support overall

(Na^{+} + K^{+})-ATPase

activity, but

V_{max}

at 37°C is only

1.3 γ mol · (mg protein)^{?} ·

h^?1 or 0.09% of the specific activity for ATP (

K_{m= 0.43 mM}

). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1,

K_{m}

ADP = 48 μM at 0.1 mM ATP

) and is relatively K⁺-insensitve. The

K_{m}

for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation. 相似文献

Effects of oligomycin and quercetin on the hydrolytic activities of the (

Na^{+} + K^{+})- dependent ATPase

Joseph D. Robinson Lisa J. Robinson Nancy J. Martin 《生物化学与生物物理学报:生物膜》1984,772(3):295-306

Quercetin inhibited a dog kidney (

Na^{+} + K^{+})- ATPase

preparation without affecting

K_{m}

for ATP or

K_{0.5}

for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E₂ enzyme conformations, blocked this inhibition, while the K⁺-phosphatase activity was at least as sensitive to quercetin as the (

Na^{+} + K^{+})- ATPase

activity, all consistent with quercetin favoring E₁ conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the

K_{m}

for ATP and the

K_{0.5}

for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K⁺-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E₂ conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E₁ conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on

V_{max}

K_{m}

for substrate, and

K_{0.5}

for K⁺, as well as for stimulation of phosphatase activity by both these reagents at low K⁺ but high Na⁺ concentrations. 相似文献

Energetics of coupled Na⁺ and Cl⁻ entry into epithelial cells of bullfrog small intestine

W.McD. Armstrong W.R. Bixenman K.F. Frey J.F. Garcia-Diaz M.G. O&#x;Regan Jeanie L. Owens 《生物化学与生物物理学报:生物膜》1979,551(1):207-219

Na⁺, K⁺ and Cl^? concentrations (

c_{j}^{i}

) and activities (

a_{j}^{i}

), and mucosal membrane potentials (

E_{m}

) were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na⁺ and Cl^? and 5.4 mM K⁺. Na⁺ and K⁺ concentrations were determined by atomic absorption spectrometry and Cl^? concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO₃. ¹⁴C-labelled inulin was used to determine extracellular volume.

E_{m}

was measured with conventional open tip microelectrodes,

a_{Cl}^{i}

with solid-state Cl^?-selective silver microelectrodes and

a_{Na}^{i}

and

a_{K}^{i}

with Na⁺- and K⁺-selective liquid ion-exchanger microelectrodes. The average

E_{m}

recorded was ?34 mV.

c_{Na}^{i}

c_{K}^{i}

and

c_{Cl}^{i}

were 51, 105 and 52 mM. The corresponding values for

a_{Na}^{i}

a_{K}^{i}

and

a_{Cl}^{i}

were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na⁺ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K⁺ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl^?.

a_{Cl}^{i}

significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na⁺ and Cl^? is implicated in intracellular Cl^? accumulation in this tissue. This study permitted estimation of the steady-state transapical Na⁺ and Cl^? electrochemical potential differences (Δμ̄Na and Δμ̄Cl). Δμ̄Na (?7000 J · mol^?1; cell minus mucosal medium) was energetically more than sufficient to account for Δμ̄Cl (1000–2000 J · mol^?1). 相似文献

Relationship between H+, anion, and monovalent cation movements and Ca2+ transport in sarcoplasmic reticulum: further proof of a cation exchange mechanism for the Ca2+-Mg2+-ATPase pump

D H Haynes 《Archives of biochemistry and biophysics》1982,215(2):444-461

Two spectroscopic probes of free internal Ca²⁺ were used to determine the influence of H⁺ and anion permeation on the active transport of Ca²⁺ by skeletal sarcoplasmic reticulum. The studies were carried out on a well-characterized Ca²⁺-Mg²⁺-ATPase-rich sarcoplasmic reticulum fraction. Studies of D. McKinley and G. Meissner (1977, FEBS Lett., 82, 47–50) show that this fraction consists of two populations of vesicles: type I which has an electrically active monovalent cation (M⁺) permeability and type II which lacks it. The present study distinguishes between electrically active (charge-carrying) and electrically silent (e.g., countertransport) mechanisms of ion permeation in the two vesicles and shows how the active transport of Ca²⁺ is influenced by these permeabilities. The major results are as follows: (1) Both type I and II vesicles have an electrically active H⁺ permeability. (2) Type I vesicles have electrically active anion (A^?) permeabilities; type II vesicles do not. (3) At low concentrations of nonpenetrating buffers, ion imbalances across the membrane can create pH imbalances. This is due to the coupling of M⁺ and A^? movements with H⁺ movements. Following a jump in KCl concentration internal acidification is observed in type I vesicles while internal alkalinization is observed in type II vesicles. These pH gradients are dissipated on a time scale of seconds and tens of minutes for type I and II vesicles, respectively. (4) Tris(hydroxymethyl)aminomethane (Tris) was shown to be effective in dissipating pH gradients in type II vesicles. A model is proposed whereby HCl is equilibrated across the membrane by a Tris-catalyzed transport cycle involving transport of an ion pair between Tris-H⁺ and Cl^? and return of the unprotonated form of the buffer. (5) The permeabilities of several physiological and nonphysiological anions were determined for type I and II vesicles. Electrically active permeability was demonstrated for Cl^? and phosphate in type I vesicles. Type II vesicles lacked electrically active mechanisms for these two anions. Evidence is given for slow

Cl^{?} OH^{?}

exchange and for rapid

Cl^{?} HCO_{3}^{?}

exchange in type II vesicles. Electrically silent phosphate influx probably occurs by

H_{2} PO_{4}^{?} OH^{?}

exchange. (6) Under normal conditions the Ca²⁺ uptake of type II vesicles is masked. It can be unmasked by addition of nigericin in the presence of Tris. The combination of ionophore and penetrating buffer render the type II vesicles KCl permeable, allowing the replenishment of internal K⁺ during active transport. The results are analyzed and shown to be in agreement with the Ca²⁺-Mg²⁺-ATPase pump acting as a

Ca^{2+} K^{+}

exchanger. The results are shown to be in disagreement with electrogenic models of pump function. 相似文献

Relationship between calcium ion transport and (Ca²⁺ + Mg²⁺)-ATPase activity in adipocyte endoplasmic reticulum

Betty L. Black Leonard Jarett Jay M. Mcdonald 《生物化学与生物物理学报:生物膜》1980,596(3):359-371

Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca²⁺ transport in this preparation were characterized and compared to those of (Ca²⁺ + Mg²⁺)-ATPase activity. The time course of Ca²⁺ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8–11.5 nmol Ca²⁺/mg protein after 3–4 min of incubation. The rate of Ca²⁺ transport was increased by higher oxalate concentrations with a near linear rate of uptake at 20 mM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca²⁺/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent

K_{m}

values of 100 μM and 7 μM for ATP concentration ranges above and below 50 μM, respectively. The apparent

K m

values for Mg²⁺ and Ca²⁺ were 132 μM and 0.36–0.67 μM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal (

Ca^{2+} + Mg^{2+}

)-ATPase. The presence of potassium was required for maximum Ca²⁺ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca²⁺ transport and (

Ca^{2+} + Mg^{2+} - ATPase

activity was

K^{+} > Na^{+} = NH_{4}^{+} > Li^{+} . Ca^{2+}

transport and (

Ca^{2+} + Mg^{2+})- ATPase

activity were both inhibited 10–20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 μM

p- chloromercuribenzene

sulfonate. The results indicate that Ca²⁺ transport in adipocyte endoplasmic reticulum is mediated by a

(Ca^{2+} + Mg^{2+})-ATPase

and suggest an important role for endoplasmic reticulum in control of intracellular Ca²⁺ distribution. 相似文献

10.

Nanosecond reduction kinetics of photooxidized chlorophyll-aII (P-680) in single flashes as a probe for the electron pathway,H+-release and charge accumulation in the O2-evolving complex

K. Brettel E. Schlodder H.T. Witt 《BBA》1984,766(2):403-415

相似文献

11.

Effects of 5-hydroxylysine on acetylene reduction and NH4+-assimilation in the cyanobacterium Anabaena cylindrica.

J K Ladha P Rowell W D Stewart 《Biochemical and biophysical research communications》1978,83(2):688-696

5-hydroxylysine, an analogue of glutamate and lysine, causes

NH_{4}^{+}

production by N₂-fixing A. cylindrica; it also reversibly inhibits GS activity in vitro but has no effect on alanine dehydrogenase or GOGAT. On adding 5-hydroxylysine intracellular pools of glutamine, glutamate and aspartate decrease; those of alanine and serine increase. 5-hydroxylysine alleviates the inhibitory effect of

NH_{4}^{+}

on heterocyst production and C₂H₂ reduction and in

NH_{4}^{+} -grown

cultures results in heterocyst synthesis and in C₂H₂ reduction. The data suggest that the GS-GOGAT pathway is the sole route of importance in primary

NH_{4}^{+}

assimilation in A. cylindrica, that

NH_{4}^{+}

alone does not inhibit nitrogenase and heterocyst production, and that GS and/or a product is involved in regulating the production of both. 相似文献

12.

Studies on (

K^{+} + H^{+}

)-ATPase IV. Effects of phospholipase C treatment

J.J. Schrijen A. Omachi W.A.H.M. Van Groningen-Luyben J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1981,649(1):1-12

(1) The total phospholipid content of a gradient purified (

K^{+} + H^{+}

)-ATPase preparation from pig gastric mucosa is 105 μmol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 μmol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The (

K^{+} + H^{+}

)-ATPase and K⁺ stimulated

p- nitrophenylphosphatase

activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH,

K_{m}

value for ATP and temperature dependence of the gastric (

K^{+} + H^{+}

)-ATPase, but slightly decreases the

K_{a}

value for K⁺. (5) Phospholipase C treatment lowers the

Ado PP[NH]P

binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules. 相似文献

13.

The effect of formate on cytochrome

aa_{3}

and on electron transport in the intact respiratory chain

Peter Nicholls 《BBA》1976,430(1):13-29

1. Formate inhibits cytochrome

c

oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome

aa_{3}

. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome

aa_{3} (a^{3+} a_{3}^{3+}

) and in the half-reduced species (

a^{2+} a_{3}^{3+}

). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the

aa_{3}

spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of

a_{3}^{2+}

minus

a_{3}^{3+}

, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome

a_{3}^{3+}

) and azide (which induces peak shifts of cytochrome

a^{2+}

towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome

a^{2+} a_{3}^{3+} - HCOOH

is faster than its rate of dissociation from

a^{3+} a_{3}^{3+} - HCOOH

, especially in the presence of cytochrome

c

. The

K_{i}

for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome

c

reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus

N,N,N′,N′- tetramethyl -p- phenyl-enediamine

oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome

c

reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome

aa_{3}

level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion. 相似文献

14.

Crystallization patterns of membrane-bound (Na+ + K+)-ATPase

Hans Hebert Peter L. Jørgensen Elisabeth Skriver Arvid B. Maunsbach 《生物化学与生物物理学报:生物膜》1982,689(3):571-574

Extensive formation of two-dimensional crystals of the proteins of the pure membrane-bound

(Na^{+} + K^{+})-ATPase

is induced during prolonged incubation with vanadate and magnesium. Some membrane crystals are formed in medium containing magnesium and phosphate. Computer-averaged images of the two-dimensional crystals show that the unit cell in vanadate-induced crystals contains a protomeric αβ-unit of the enzyme protein. In phosphate-induced crystals an

(αβ)_{2} - unit

occupies one unit cell suggesting that interactions between αβ-units can be of importance in the function of the Na⁺, K⁺ pump. 相似文献

15.

The presence of two (

Na^{+} + K^{+})-ATPase

inhibitors in equine muscle ATP: Vanadate and a dithioerythritol-dependent inhibitor

Eugene E. Quist Lowell E. Hokin 《生物化学与生物物理学报:生物膜》1978,511(2):202-212

A potent inhibitor of

(Na^{+} + K^{+})-ATPase

activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V₂O₅ inhibit sarcolemma

(Na^{+} + K^{+})-ATPase

with an

I_{50}

of 1 μM in the presence of 1 mM

ethyleneglycol-bis-(β-aminoethylether) -N,N′- tetraacetic

acid (EGTA), 145 mM NaCl, 6mM MgCl₂, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate in increased by free Mg²⁺. The inhibition is half maximally reversed by 250 μM epinephrine. Equine muscle ATP was also found to contain a second

(Na^{+} + K^{+})-ATPase

inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg²⁺ and is half maximally reversed by 2 μM epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the

(Na^{+} + K^{+})-ATPase

to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified

(Na^{+} + K^{+})-ATPase

from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg²⁺-ATPase, Ca²⁺-ATPase and

(Ca^{2+} + Mg^{2+})-ATPase

. 相似文献

16.

Rat enterocyte Na⁺ transport in vitro action of parathyroid hormone and calcitonin

Bernard Lacour Tilman Drüeke Evelyne Pierandreï Bernadette Nabarra Jean-Louis Funck-Brentano 《生物化学与生物物理学报:生物膜》1981,648(2):151-161

Parathyroid hormone (PTH) and calcitonin exert well known effects on the renal tubule which are thought to involve specific hormone receptors and adenyl cyclase. In the intestine, it is not clear whether the action of PTH and calcitonin is only indirect or also direct, and their mechanisms of action are much less well established. In the present study, possibly direct effects of PTH and calcitonin on Na⁺ transport in isolated intestinal epithelial cells of rats were investigated. In the presence of bovine PTH (1.2 I.U./ml) in the incubation medium, the Na⁺ efflux rate constant (

^{o} K_{Na}

) of isolated enterocytes was significantly reduced when compared to that in control experiments with the hormone vehicle only. The mean depression of

^{o} K_{Na}

induced by bovine PTH was 26% as compared to the control (100%) and to that induced by ouabain (4.0mM) which was 44%. No depressant effect of bovine PTH on

^{o} K_{Na}

was observed when the isolated enterocytes were incubated with ouabain (4.0 mM). Thus, bovine PTH appeared to inhibit the ouabain-sensitive Na⁺ pump. When incubating the isolated epithelial cells in an EGTA-containing Ca²⁺-free medium, bovine PTH lost its capacity to inhibit (

^{o} K_{Na}

). Thus, the presence of extracellular Ca²⁺ appeared necessary for the inhibitory effect of bovine PTH. In contrast to its effect on

^{o} K_{Na}

, bovine PTH induced no change in net Na⁺ uptake by isolated enterocytes. Moreover, no significant effect on enterocyte Na⁺ transport could be demostrated for salmon or porcine calcitonin at two different concentrations in the incubation medium. Neither bovine PTH nor salmon calcitonin induced significant changes in enterocyte cyclic AMP or cyclic GMP concentrations. It was concluded that bovine PTH, but not calcitonin, exerted a direct inhibitory effect on the ouabain-sensitive

^{o} K_{Na}

of isolated rat enterocytes. The effect of bovine PTH occured without measurable activation of the cyclic nucleotide system but needed the presence of Ca²⁺ in the incubation medium to be operative. 相似文献

17.

A model for the reaction pathways of the K⁺-dependent phosphatase activity of the (

Na^{+} + K^{+}

)-dependent ATPase

Joseph D. Robinson Grace M. Levine Lisa J. Robinson 《生物化学与生物物理学报:生物膜》1983,731(3):406-414

(Na^{+} + K^{+})-dependent

ATPase preparations from rat brain, dog kidney, and human red blood cells also catalyze a K⁺-dependent phosphatase reaction. K⁺ activation and Na⁺ inhibition of this reaction are described quantitatively by a model featuring isomerization between E₁ and E₂ enzyme conformations with activity proportional to E₂K concentration:

Differences between the three preparations in

K_{0.5}

for K⁺ activation can then be accounted for by differences in equilibria between E₁K and E₂K with dissociation constants identical. Similarly, reductions in

K_{0.5}

produced by dimethyl sulfoxide are attributable to shifts in equilibria toward E₂ conformations. Na⁺ stimulation of K⁺-dependent phosphatase activity of brain and red blood cell preparations, demonstrable with KCl under 1 mM, can be accounted for by including a supplementary pathway proportional to E₁Na but dependent also on K⁺ activation through high-affinity sites. With inside-out red blood cell vesicles, K⁺ activation in the absence of Na⁺ is mediated through sites oriented toward the cytoplasm, while in the presence of Na⁺ high-affinity K⁺-sites are oriented extracellularly, as are those of the

(Na^{+} + K^{+})-dependent

ATPase reaction. Dimethyl sulfoxide accentuated Na⁺-stimulated K⁺-dependent phosphatase activity in all three preparations, attributable to shifts from the E₁P to E₂P conformation, with the latter bearing the high-affinity, extracellularly oriented K⁺-sites of the Na⁺-stimulated pathway. 相似文献

18.

Ouabain receptor interactions in

(Na^{+} + K^{+})-ATPase

preparations. II. Effect of cations and nucleotides on rate constants and dissociation constants

Erland Erdmann Wilhelm Schoner 《生物化学与生物物理学报:生物膜》1973,330(3):302-315

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant (

K_{D}

) of 13 nM was found in the presence of (

Mg^{2+} + P_{i}

) and (

Na^{+} + Mg^{2+} + ATP

). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of (

Mg^{2+} + P_{i}

), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of

(Na^{+} + K^{+})-ATPase

. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot). 相似文献

19.

The insect brain (

Na^{+} + K^{+})- ATPase

Binding of ouabain in the hawk moth, Manduca sexta

Andrew L. Rubin Arthur F. Clark William L. Stahl 《生物化学与生物物理学报:生物膜》1981,649(2):202-210

(1) A quantitative study has been made of the binding of ouabain to the (

Na^{+} + K^{+})- ATPase

in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain (

Na^{+} + K^{+})- ATPase

will bind ouabain either in the presence of Mg²⁺ and P_i, (‘Mg²⁺, P_i’ conditions) or in the presence of Na⁺, Mg²⁺, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (

Na^{+} + K^{+})- ATPase

. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the

K_{D}

) of ouabain to the M. sexta brain (

Na^{+} + K^{+})- ATPase

under both binding conditions. However, ouabain binding is more sensitive to K⁺ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K⁺ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta (

Na^{+} + K^{+})- ATPase

has a higher

K_{D}

for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta (

Na^{+} + K^{+})- ATPase

can bind ouabain. 相似文献

20.

The effect of ammonium ions upon isolated reactions of the glycolytic scheme

MUNTZ JA HURWITZ J 《Archives of biochemistry and biophysics》1951,32(1):137-149

In the glycolytic system derived from rat brain acetone powder, ammonium ion has been found to stimulate three different reactions: (a) the transphosphorylase reaction from phosphoenolpyruvate, (b) the phosphohexokinase reaction, and (c) the hexokinase reaction. The transphosphorylases are affected differently depending upon whether adenosine diphosphate or adenylic acid is the phosphate acceptor; in the case of the latter, the dependency upon

NH_{4}^{+}

is particularly marked. A highly active myokinase is present in these extracts and its activity influences the transphosphorylase reaction to a considerable extent. The phosphohexokinase reaction is stimulated to a greater extent by

NH_{4}^{+}

than is the hexokinase reaction. In contrast to these reactions which require the participation of the adenylic system, triose phosphate oxidase activity is uninfluenced by the presence of

NH_{4}^{+}

. 相似文献