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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.
《Comparative biochemistry and physiology. B, Comparative biochemistry》1990,95(2):343-354
- 1.1. The (Na+ + K+)- and Na+-ATPases, both present in kidney microsomes of Sparus auratus L., have different activities and optimal assay conditions as, in the first of the two stocks of fish used (A), the spec. act. of the former is 51.7 μmol Pi mg prot−1 hr−1 at pH 7.5, 100 mM Na+, 10 mM K+, 17.5 mM Mg2+, 7.5 mM ATP and that of the latter is 6.5 μmol Pi mg prot−1 hr−1 at pH 6.5, 40 mM Na+, 4.0 mM Mg2+, 2.5 mM ATP.
- 2.2. Ouabain and vanadate specifically inhibit the (Na+ + K+)-ATPase but not the Na+-ATPase that is preferentially inhibited by ethacrynic acid.
- 3.3. While the (Na+ + K+)-ATPase is strictly specific for ATP and Na+, Na+-ATPase can be activated by various monovalent cations and, apart from ATP, hydrolyses CTP, though less efficiently.
- 4.4. The second stock B, subjected to higher salinity than A, shows an acidic shifted Na+-ATPase optimal pH, opposed to the stability of that of the (Na+ + K+)-ATPase, a decreased (Na+ + K+)-ATPase and a strikingly depressed Na+-ATPase.
- 5.5. The results are compared with literature data and discussed on the basis of the presumptive different roles as well as functional prevalence in various salinities of the two ATPases.
3.
H.K. Kimelberg 《生物化学与生物物理学报:生物膜》1975,413(1):143-156
The (Na++K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na++K+)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg2+: ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (Ea) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the Ea below this was very high (> 100 kcal/mol).Incresed Mg2+ concentrations at Mg2+: ATP ratios in excess of 1:1 inhibited the (Na++K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na++K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and Ea values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km for ATP.Since both of cholesterol and Mg2+ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context. 相似文献
4.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1983,736(1):73-78
(1) Contrary to what has usually been assumed, (Na+ + K+)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na+ + Mg2+ to ADP-NH2 and Pi. The activity is ouabain-sensitive and is not detected in the absence of either Mg2+ or Na2+. The specific activity of the Na+ + Mg2+ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K+. The activity is not stimulated by K+, nor can K+ replace Na+ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na+ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The Km value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH2]P are not detectably hydrolysed by (Na+ + K+)-ATPase in the presence of Na+ + Mg2+. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg2+ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na+ + Mg2+ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na+ + K+)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C. 相似文献
5.
6.
A Ca2+-ATPase and a Mg2+/H+-antiporter are present on tonoplast membranes from roots of Zea mays L. 总被引:1,自引:0,他引:1
The primary or secondary energized transport of Ca2+, Mg2+ and H+ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca2+ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca2+ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca2+-pumping and tonoplast H+-pyrophosphatase (PPiase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca2+-pumping activity could be detected. The ATP-dependent Ca2+ uptake into tonoplast vesicles (using Ca2+ concentrations from 0.8–1 μM) was completely inhibited by the Ca2+ ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H+ accumulation had no effect. Vanadate (IC50 = 43 μM) and diethylstilbesterol (IC50 = 5.2 μM) were potent inhibitors of this type of Ca2+ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%–50% of the ATP-dependent activity (K m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca2+ transport mechanism is the only functioning Ca2+ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca2+ concentration. In contrast, the secondary energized Ca2+-transport mechanism of the tonoplast, the low-affinity Ca2+/H+-antiporter, which was reported to allow the uptake of Ca2+ in exchange for H+, functions chiefly as an Mg2+ transporter under physiological conditions because cytosolic Mg2+ is several orders of magnitude higher than the Ca2+ concentration. This conclusion was deduced from experiments showing that Mg2+ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H+ from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H+-ATPase and H+-PPiase was found to be influenced by Ca2+ differently from and independently of the Mg2+ concentration. Calcium was a strong inhibitor for the H+-PPiase (IC50 = 18 μM, Hill coefficient nH = 1.7) but a weak one for the H+-ATPase (IC50 = 330 μM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca2+-and Mg2+-regulated H+-PPiase, (ii) the newly described high-affinity Ca2+-AT-Pase, (iii) the low-affinity Mg2+(Ca2+)/H+-antiporter and (iv) the H2+-ATPase. 相似文献
7.
Several plasma-membrane proteins from beet root (Beta vulgaris L.) have been functionally incorporated into reconstituted proteoliposomes. These showed H+-ATPase activity, measured both as ATP hydrolysis and H+ transport. The proton-transport specific activity was 10 times higher than in plasma membranes, and was greatly stimulated
by potassium and valinomycin. These proteoliposomes also showed calcium-regulated protein kinase activity. This kinase activity
is probably due to a calmodulin-like domain protein kinase (CDPK), since two protein bands were recognized by antibodies against
soybean and Arabidopsis CDPK. This kinase phosphorylated histone and syntide-2 in a Ca2+-dependent manner. Among the plasma-membrane proteins phosphorylated by this kinase, was the H+-ATPase. When the H+-ATPase was either prephosphorylated or assayed in the presence of Ca2+, both the ATP-hydrolysis and the proton-transport activities were slower. This inhibition was reversed by an alkaline-phosphatase
treatment. A trypsin treatment (that has been reported to remove the C-terminal autoinhibitory domain from the H+-ATPase) also reversed the inhibition caused by phosphorylation. These results indicate that a Ca2+-dependent phosphorylation, probably caused by a CDPK, inhibits the H+-ATPase activities. The substrate of this regulatory phosphorylation could be the H+-ATPase itself, or a different protein influencing the ATPase activities.
Received: 1 May 1997 / Accepted: 25 June 1997 相似文献
8.
Jinling Zhai Haixia Xu Xinli Cong Yongchuan Deng Zhihui Xia Xi Huang Gangping Hao Xingyu Jiang 《Acta Physiologiae Plantarum》2013,35(1):161-173
A large number of plant Ca2+/H+ exchangers have been identified in endomembranes, but far fewer have been studied for Ca2+/H+ exchange in plasma membrane so far. To investigate the Ca2+/H+ exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca2+/H+ exchange in plasma membrane vesicles was measured by Ca2+-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca2+/H+ exchange was optimal at pH 7.0, and displayed transport specificity for Ca2+ with saturation kinetics at K m = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca2+-dependent transport of H+ out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca2+/H+ exchange increased comparing to control without valinomycin effect, suggesting that the Ca2+/H+ exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H+:Ca2+. Eosin Y, a Ca2+-ATPase inhibitor, drastically inhibited Ca2+/H+ exchange in plasma membrane as it does for the purified Ca2+-ATPase in proteoliposomes, indicating that measured Ca2+/H+ exchange activity is mainly due to a plasma membrane Ca2+ pump. These suggest that calcium (Ca2+) is transported out of Arabidopsis cells mainly through a Ca2+-ATPase-mediated Ca2+/H+ exchange system that is driven by the proton-motive force from the plasma membrane H+-ATPase. 相似文献
9.
Selective chemical modification was used to examine amino acid residues that might be critical for the operation of the gastric K+-stimulated ATPase. Modification of amino groups with the fluorigenic reagent 2-methoxy-2,4-diphenyl-3-dihydrofuranone resulted in selective inhibition of the K+-stimulated ATPase and H+-transporting activities of the gastric microsomes, while the Mg2+-ATPase was not affected. Half-maximal inhibition occurred at about 3 μg 2-methoxy-2,4-diphenyl-3-dihydrofuranone/ml at pH 8.5. ATP provided complete protection against inhibition; the apparent Km for ATP protection was about 50 μM. Nucleotide selectivity for protection was ATP > ADP > ITP > GTP > CTP > AMP. Sodium dodecyl sulfate gel electrophoresis of the reacted microsomes showed that virtually all the fluorescent label was on the Mr 100 000 peptide band, a very small peptide, and aminolipids. In the presence of ATP there was about 75% reduction in the fluorescent label on the Mr 100 000 peptide, but no change in the labeling of the other components. The arginine specific reagent, butanedione, inhibited Mg2+-ATPase and K+-ATPase activities, with the former being much less reactive. Similar to 2-methoxy-2,4-diphenyl-3-dihydrofuranone, ATP provided complete protection from butanedione treatment. It is concluded that amino and guanidino groups are critical to the function of the K+-ATPase and may be actually at the ATP binding site. 相似文献
10.
31P NMR chemical shifts of salts of adenosine 5′-triphosphate and diphosphate: ATPH2?22(Me4N+) · H2O, ATPH2?22 Na+ · 3.5 H2O, ATPH2?2Mg2+ · 4 H2O, ATPH2?2Ca2+ · 2 H2O, ADPH2?2(Me4N+) · H2O and ADPH2?Mg2+ · 4 H2O have been measured in 0.02 M 2H2O solutions at 145.7 MHz (22° C) at constant p2H values (8.20 and 6.20). The results are compared with those obtained from salts of adenosine 5′-monophosphate and other simpler phosphomonoesters, e.g. AMP2?2(Me4N+), AMP2?Mg2+, AMPH?Me4N+ and (AMPH?)2Mg2+. It is concluded that the effects exerted by Mg2+ and Ca2+ on the 31P NMR shifts of dipoly- and tripolyphosphates relative to monovalent cations are due mainly to changes in conformation of the polyphosphate chain rather than to purely electronic factors associated with the binding of divalent cations to the phospho-oxyanions. The data are consistent with the existence of the following complexes at p2H 8.20: (MgPαPβ)ADP? and (MgPαPγ)ATP2?af (MgPαPβ)ATP2?af (MgPβPγ)ATP2? with the latter equilibrium relatively fast in the NMR time scale. Monoprotonation of the terminal phosphate appears to weaken the Mg2+-polyphosphate binding, particularly at Pβ of MgADPH and at Pβ and Pγ of MgATPH?. The Mg2+-polyphosphate binding weakens further at p2H 3.70, i.e. in MgATPH2. Possible implications of the results in the mechanism of actomyosin Mg2+-ATPase in muscle contraction are discussed. 相似文献
11.
Summary Characteristics of the native and reconstituted H+-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H+-ATPase retained characteristics similar to those of the native plasma membrane H+-ATPase following reconstitution into proteoliposomes. ATPase activity and H+ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO
3
–
was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H+ transport showed a pH optimum of 6.5, required a divalent cation (Co2+>Mg2+>Mn2+>Zn2+>Ca2+), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aK
m
of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H+-ATPases were stimulated by monovalent ions. Native plasma membrane H+-ATPase showed an order of cation preference of K+>NH
4
+
>Rb+>Na+>Cs+>Li+>choline+. This basic order was unchanged following reconstitution, with K+, NH
4
+
, Rb+ and Cs+ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H+ transport, both enzymes required a cation such as K+ for maximal transport activity, but were stimulated preferentially by Cl– even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H+-ATPase. 相似文献
12.
Magnesium-dependent adenosine triphosphatase has been purified from sheep kidney medulla plasma membranes. The purification, which is based on treatment of a kidney plasma membrane fraction with 0.5% digitonin in 3 mm MgCl2, effectively separates the Mg2+-ATPase from (Na+ + K+)-ATPase present in the same tissue and yields the Mg2+-ATPase in soluble form. The purified enzyme is activated by a variety of divalent cations and trivalent cations, including Mg2+, Mn2+, Ca2+, Co2+, Fe2+, Zn2+, Eu3+, Gd3+, and VO2+. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme shows two bands with Rf values corresponding to molecular weights of 150,000 and 77,000. The larger peptide is phosphorylated by [γ-32P]ATP, suggesting that this peptide may contain the active site of the Mg2+-ATPase. The Mg2+-ATPase activity is unaffected by the specific (Na+ + K+)-ATPase inhibitor ouabain. 相似文献
13.
The specific activity of (Na+ + Mg2+)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na+ + K+ + Mg2+)-dependent ATPase in both cases.(Na+ + Mg2+)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI2, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na+ + Mg2+)-dependent ATPase activity of gills arises from the presence of a (Na+ + K+ + Mg2+)-dependent ATPase. 相似文献
14.
Chick brain microsomal ATPase was strongly inhibited by Cu2+. (Na+ + K+)-ATPase was more susceptible to low levels of Cu2+ than Mg2+-ATPase. The inhibition of (Na+ + K+)-ATPase could be partially protected from Cu2+ in the presence of ATP in the preincubation period. When Cu2+ (6 μM) was preincubated with the enzyme in the absence of ATP, only sulfhydryl-containing amino acids (d-penicillamine and l-cysteine) could reverse the inhibition. At lower concentrations of Cu2+ (< 1.4 μM), in the absence of ATP during preincubation, the inhibition could be completely reversed by the addition of 5 mM l-phenylalanine and l-histidine as well as d-penicillamine and l-cysteine.Kinetic analysis of action of Cu2+ (1.0 μM) on (Na+ + K+)-ATPase revealed that the inhibition was uncompetitive with respect to ATP. At a low concentration of K+ (5 mM), V with Na+ was markedly decreased in the presence of Cu2+ and Km was about twice that of the control. However, at high K+ concentration (20 mM), the Km for Na+ was not affected. At both low (25 mM) and high (100 mM) Na+, Cu2+ displayed non-competitive inhibition of the enzyme with respect to K+.On the basis of these data, we suggest that Cu2+ at higher concentrations (> 6 μM) inactivates the enzyme irreversibly, but that at lower concentrations (< 1.4 μM), Cu2+ interacts reversibly with the enzyme. 相似文献
15.
《Process Biochemistry》2010,45(8):1236-1244
Keratins are important bioresources for apparels and feedstuffs, but recalcitrant to common enzymes. Now, it is popular and essential to develop keratinolytic enzymes for environmental prevention and improvement of keratin product quality. In the study, the medium optimization, purification, characterization and application of the keratinase from a newly isolated Chryseobacterium L99 sp. nov. were conducted. Exogenous sucrose, malt sugar, glucose, starch, tryptone, Mg2+, Zn2+, Ca2+ and Cu2+ could promote the keratinase production, while exogenous urea, NH4Cl and yeast extract exhibited strong inhibition effects. Response surface methodology predicted a maximum keratinase yield of 213.8 U mL−1, at (g L−1) sucrose 16.8, MgCl2·6H2O 1.9, feather keratin 40.0, NaH2PO4·2H2O 6.0 and K2HPO4·6H2O 1.0, where dry cell weight nearly had a minimum 8.58 g L−1. Then, a serine keratinase about 33 kDa was purified, and its optimal activity was acquired at 40 °C and pH 8.0 with K+, Zn2+or Co2+. Compared with Savinase 16 L and transglutaminase, the L99 keratinase could efficient prevent shrinkage and eliminate directional frictional effect of wool, indicating it as a promising prospect in the biotreatment of wool fibres. 相似文献
16.
《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1999,122(1):93-99
Halenaquinol, a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata, was found to be a powerful inhibitor of the rat brainstem and of the rat brain cortex Na+, K+-ATPases and the rabbit muscle sarcoplasmic reticulum Ca2+-ATPase with I50 values of 7.0×10−7, 1.3×10−6 and 2.5×10−6 M, respectively. Halenaquinol also inhibited K+-phosphatase activity of the rat brain cortex Na+, K+-ATPase with an I50 value of 3×10−6 M. Ouabain-insensitive Mg2+-ATPase activity of the microsomal fraction of the rat brain cortex was weakly inhibited by halenaquinol. Inhibition was irreversible, dose- and time-dependent. Naphthohydroquinone fragment in structures of halenaquinol, related natural and model compounds was very important for an inhibiting effect. 相似文献
17.
(1) The effects of calmodulin binding on the rates of Ca2+-dependent phosphorylation and dephosphorylation of the red-cell Ca2+ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg2+-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg2+-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca2+ + Mg2+)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca2+ affinity for sustaining ATP hydrolysis is raised at least 20-fold, Km(Ca) ? 10 μM (—calmodulin) and Km(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca2+ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (Km(Ca) ? 3–4 · 10-8 M). (Ca2+ + Mg2+)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca2+ + Mg2+)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca2+ + Mg2+)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (Km(MgATP) ? 360 μM), and CaATP is an inhibitor (Ki(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca2+- pump active site, producing a high (Ca2+ + Mg2+)-ATPase activity, high Ca2+ affinity and regulation of activity by MgATP. 相似文献
18.
LOCALIZATION OF Na+, K+-ATPASE AND OTHER ENZYMES IN TELEOST PSEUDOBRANCH : I. Biochemical Characterization of Subcellular Fractions 总被引:2,自引:1,他引:1
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Leslie A. Dendy Russell L. Deter Charles W. Philpott 《The Journal of cell biology》1973,57(3):675-688
In an effort to determine the subcellular localization of sodium- and potassium-activated adenosine triphosphatase (Na+, K+-ATPase) in the pseudobranch of the pinfish Lagodon rhomboides, this tissue was fractionated by differential centrifugation and the activities of several marker enzymes in the fractions were measured. Cytochrome c oxidase was found primarily in the mitochondrial-light mitochondrial (M+L) fraction. Phosphoglucomutase appeared almost exclusively in the soluble (S) fraction. Monoamine oxidase was concentrated in the nuclear (N) fraction, with a significant amount also in the microsomal (P) fraction but little in M+L or S. Na+, K+-ATPase and ouabain insensitive Mg2+-ATPase were distributed in N, M+L, and P, the former having its highest specific activity in P and the latter in M+L. Rate sedimentation analysis of the M+L fraction indicated that cytochrome c oxidase and Mg2+-ATPase were associated with a rapidly sedimenting particle population (presumably mitochondria), while Na+, K+-ATPase was found primarily in a slowly sedimenting component. At least 75% of the Na+, K+-ATPase in M+L appeared to be associated with structures containing no Mg2+-ATPase. Kinetic properties of the two ATPases were studied in the P fraction and were typical of these enzymes in other tissues. Na+, K+-ATPase activity was highly dependent on the ratio of Na+ and K+ concentrations but independent of absolute concentrations over at least a fourfold range. 相似文献
19.
N.Charle Morcos 《生物化学与生物物理学报:生物膜》1982,688(3):747-756
N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1·10?4 M. The sarcolemmal markers, ouabain-sensitive (Na+ + K+)-ATPase and Na+/Ca2+-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na+ pump (K+-p-nitrophosphatase) were not affected. Almost 20% of the (Ca2+ + Mg2+)-ATPase and Ca2+ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27–39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca2+ + Mg2+-ATPase and Ca2+ pump compared to control hearts. (Ca2+ + Mg2+)-ATPase and Ca2+ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class ( for inhibition approx. 1.5 μM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg2+-ATPase and Ca2+-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles. 相似文献
20.
《Comparative biochemistry and physiology. A, Comparative physiology》1995,110(3):235-241
Cl− absorption across isolated, perfused gills of freshwater adapted Chinese crabs (Eriocheir sinensis) was analysed by measuring transepithelial potential differences (PDte) and radioactive tracer fluxes across isolated, perfused posterior gills. Applying hemolymph-like NaCl salines on both sides of the epithelium PDte amounted to −30±1 mV (n=14). Undirectional Cl− influxes of 470±38 and effluxes of 245±27 μmol·hr−1·g−1 wet weight (ww) (n=14) resulted in a Cl− net influx of 226±31 μmol·hr−1·g−1 ww. Symmetrical substitution of Na+ by choline resulted in a substantial hyperpolarisation of the gill. Cl− influx was unchanged under these conditions. However, net influx of Cl− decreased by 40%, due to an increase of the Cl− efflux.Nevertheless, a significant Cl− net influx remained which was independent of the presence of Na+. When 2 mmol/l ouabain were added to the internal perfusion medium, PDte increased, although the fluxes remained unchanged. Following external application of 1μmol/l of the V-type H+-ATPase inhibitor bafilomycin, Al PDte and Cl− effluxes were not significantly affected. However, Cl− influxes decreased. These findings suggest that Cl− can be taken up independently of Na+ and that active Na+ independent Cl− uptake across the posterior gill of Eriocheir sinensis is probably driven by a V-type H+-ATPase localized in the apical membrane. 相似文献