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1.
8-azido-ATP, when used in the 0.2–5 μM concentration range, fulfills the criteria for a specific photoaffinity label for the (Ca+Mg)ATPase of sarcoplasmic reticulum. It is a substrate for the enzyme. It is a mixed inhibitor of ATPase activity. When photolyzed at 0° it is an inhibitor of ATPase activity. The photoinduced binding of 8-azido-ATP to the (Ca+Mg)ATPase is promoted by Ca2+. The dependence of the labeling of the (Ca+Mg)ATPase on 8-azido-ATP, Ca2+ and Mg2+ concentrations strongly suggests that 2 classes of sites are labeled. When 10–60 μM 8-azido-ATP was used to label sarcoplasmic reticulum, proteins in addition to the (Ca+Mg)ATPase were labeled. 相似文献
2.
(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
3.
Calmodulin copurifies with platelet plasma membranes isolated by glycerol-induced lysis and density gradient centrifugation. These membranes also bind 125I-labeled calmodulin in vitro in the presence of Ca2+. Binding is largely reduced by replacing Ca2+ by Mg2+ or by addition of an excess unlabeled calmodulin. The specific component of binding is saturable, with an apparent Kd of 27 nM and a maximum of 15.9 pmol binding sites per mg of membrane protein. This is equivalent to approx. 4100 binding sites per platelet. Binding was inhibited by addition of phenothiazines, a group of calmodulin antagonists. Half-maximal inhibition was attained with approx. 20 μM trifluoperazine or 50 μM chlorpromazine. In contrast, chlorpromazine-sulfoxide which is inactive towards calmodulin, did not affect the binding. Calmodulin binding polypeptides of the plasma membrane were identified by a gel-overlay technique. A major calmodulin-binding component of molecular weight 149 000 was detected. Binding to this band was Ca2+-dependent and inhibited by chlorpromazine. The molecular weight of this polypeptide is similar to that of glycoprotein I and also that of the red cell (Ca2+ + Mg2+)-stimulated ATPase, which is known to bind calmodulin. The possible role of calmodulin in platelet activation is analysed. 相似文献
4.
Vijai S. Shankar Christopher M. R. Bax Bridget E. Bax A. S. M. Towhidul Alam Baljit S. Moonga Bruce Simon Michael Pazianas Christopher L.-H. Huang Mone Zaidi 《Journal of cellular physiology》1993,155(1):120-129
Earlier studies have demonstrated that a high (mM) extracellular Ca2+ concentration triggers intracellular [Ca2+] signals with a consequent inhibition of bone resorptive activity. We now report that micromolar concentrations of the divalent cation, Ni2+, elicited rapid and concentration-dependent elevations of cytosolic [Ca2+]. The peak change in cytosolic [Ca2+] increased monotonically with the application of [Ni2+] in the 50–5,000 μM range in solutions containing 1.25 mM-[Ca2+] and 0.8 mM-[Mg2+]. The resulting concentration-response function suggested Ni2+-induced activation of a single class of binding site (Hill coefficient = 1). The triggering process also exhibited a concentration-dependent inactivation in which conditioning Ni2+ applications in the range 5–1,500 μM-[Ni2+] inhibited subsequent responses to a maximally effective [Ni2+] of 5,000 μM. Ni2+-induced cytosolic [Ca2+] responses were not dependent on extracellular [Ca2+]. Thus, when 5,000 μM-[Ni2+] was applied to osteoclasts in Ca2+-free, ethylene glycol bis-(aminoethyl ether) tetraacetic acid (EGTA)-containing medium (≤5 nM-[Ca2+] and 0.8 mM-[Mg2+]), cytosolic [Ca2+] responses resembled those obtained in the presence of 1.25 mM-[Ca2+]. Prior depletion of intracellular Ca2+ stores by ionomycin prevented Ni2+-induced cytosolic [Ca2+] responses, suggesting a major role for intracellular Ca2+ redistribution in the response to Ni2+. The effects of Ni2+ were also modulated by the extracellular concentration of the divalent cations, Ca2+ and Mg2+. When these cations were not added to the culture medium (0 μM-[Ca2+] and [Mg2+]), even low [Ni2+] ranging between 5 pM and 50 μM elicited progressively larger cytosolic [Ca2+] transients. However, the response magnitude decreased at higher, 250–5,000 μM-[Ni2+], resulting in a “hooked” concentration-response curve. Furthermore, increasing extracellular [Mg2+] or [Ca2+] (0–1 mM) diminished the response to 50 μM-[Ni2+], a concentration on the rising phase of the “hook.” Similar increases (0–10 mM) in extracellular [Mg2+] or [Ca2+] increased the response to 5,000 μM-[Ni2+], a concentration on the falling phase of the “hook”. These findings are consistent with the existence of a membrane receptor strongly sensitive to Ni2+ as well as the divalent cations, Ca2+ and Mg2+. Receptor occupancy apparently activates intracellular Ca2+ release followed by inactivation. Furthermore, repriming is independent of intracellular Ca2+ stores, suggesting that such inactivation operates at a transduction step between receptor occupancy and intracellular Ca2+ release. © 1993 Wiley-Liss, Inc. 相似文献
5.
P. E. Lindahl 《Experimental cell research》1973,81(2):413-431
Mg2+, Ca2+ and Mn2+ were found to act as activators of the ATP-dependent surface reaction, leading to head-to-head association in bull spermatozoa. Ca2+ was more efficient than Mg2+, while Zn2+, like Na+ + K+ in combination with Mg2+, seemed to have no such effect. High ionic strength induced head-to-head association, as did higher concentrations of Mg2+ and Ca2+ than those necessary for the activation of ATP, Ca2+ acting in a lower conc. than Mg2+. To this effect was added that of the ATP-dependent reaction when ATP was also present. As activators, Mg2+ and Ca2+ did not potentiate each other; their effects were cumulative when the ions acted together.When the ATP concentration within the range 1 × 10−5 to 8 × 10−5 M was increased stepwise in the presence of 2 × 10−5 M Mg2+ or Ca2+, the association resulting from each single concentration step progressively increased. At low cation concentrations, the increase was about the same for the two cations: at higher concentrations it was much steeper in the presence of Ca2+ than in that of Mg2+. In the latter case, it was not statistically significant above 4 × 10−5 M ATP.Increasing the cation concentration in the range 1 × 10−5 to 4 × 10−5 M in the presence of 2 × 10−5 M ATP produced an immediate high increase in association, which was followed by a lower increase. The optimum concentration ratio for Mg2+:ATP was at least 1:1 and for Ca2+: ATP at least 1.5:1.Oubain, containing enone structure, abolishes association. 相似文献
6.
Karl E. O. Åkerman 《Journal of bioenergetics and biomembranes》1977,9(1):65-72
Plots relating the initial rate of mitochondrial Ca2+ transport to the Ca2+ concentration (kinetic plots) have a hyperbolic shape in a Ca2+ concentration range of 2.5–100 µM as measured in sucrose or KCl media. In the presence of Mg2+ or a polyamine spermine, which both are competitive inhibitors of Ca2+ binding to low affinity sites at the membrane surface, the shape of the plots becomes sigmoidal. At higher concentrations of these agents linear kinetic plots are obtained as measured in a sucrose medium. In a KCl medium the sigmoidality of the kinetic plots is enhanced by an increase in the Mg2+ or spermine concentration. It is suggested that Mg2+ and spermine affect the kinetics of Ca2+ transport by interfering with Ca2+ binding to low affinity sites of the membrane surface and that the binding of Ca2+ to these sites is the first step of the mitochondrial Ca2+ transport. 相似文献
7.
Gillian M. Burgess Françoise Giraud Josiane Poggioli Michel Claret 《生物化学与生物物理学报:生物膜》1983,731(3):387-396
Noradrenaline (0.1–5 μM, in the presence of 5 μM propranolol to block β-receptors), ATP (100 μM) and angiotensin II (0.1 μM), which are thought to increase cytosolic Ca2+ concentration by mobilizing Ca2+ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the α-antagonists phenoxybenzamine (50 μM) and phentolamine (1 μM). The response to a maximal dose of noradrenaline (5 μM) and that to ATP (100 μM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 μM) along with the foreign amphiphile Na+-oleate (1–30 μM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg2+, reducing free Ca2+ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca2+ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 μM) and angiotensin II (0.1 μM) also promoted a small release of 45Ca2+ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the α-antagonist phentolamine (5 μM). It is proposed that noradrenaline, via α-adrenergic receptors and other Ca2+-mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca2+ bound to phospholipids, removing thus the mechanical constraints brought about by this ion. 相似文献
8.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,889(2):225-235
Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent Vmax without altering the Km. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl2 also increased uptake. Lowering of the perfusate Ca2+ concentration from 1.27 to 0.1 mM Ca2+, addition of the Ca2+ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca2+ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca2+, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca2+ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca2+ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca2+ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca2+ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca2+-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca2+-dependent and a Ca2+-independent component. Reversal studies suggest an additional role for Ca2+ in maintaining the activated transport state when activated by either epinephrine or insulin. 相似文献
9.
Eugene E. Quist 《Biochemical and biophysical research communications》1980,92(2):631-637
In the presence of 1.0 mM ATP and MgCl2, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca2+ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca2+ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca2+ (1 to 5 μM) also inhibited Mg2+ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca2+ on viscosity and phosphorylation may be due to a membrane bound Ca2+ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg2+ dependent kinase activity. 相似文献
10.
《BBA》1986,850(1):49-56
Mitochondria isolated from corn (Zea mays L.) coleoptiles by an improved procedure which yields functionally intact preparations are much more active in respiration-coupled Ca2+ accumulation than those employed in most earlier studies. Ca2+ uptake by these mitochondria is phosphate-dependent and is accompanied by decrease in Δψ, H+ extrusion and increase in the rate of respiration. A sigmoidal plot with a Hill coefficient of 2.22 was obtained when the rates of Ca2+ uptake were plotted as a function of free Ca2+ concentration. The K0.5 for Ca2+ influx was about 31 μM and a Vmax of 140 nmol Ca2+ per min per mg was attained at a free-Ca2+ concentration of about 120 μM. Ca2+ uptake is sensitive to inhibition by ruthenium red and Mg2+. The external free-Ca2+ concentration maintained at steady state was about 2 μM and was independent of the respiratory substrate and of external Na+, but was increased by exogenous Mg2+. In addition, this preparation of corn mitochondria has shown a much higher ability for Ca2+ retention in the presence of phosphate and NAD(P)H oxidants than liver mitochondria. 相似文献
11.
《Molecular membrane biology》2013,30(3):169-183
The underlying principles of binding equilibria of arsenazo III with Ca2+ and Mg2+ are presented. Ca2+ and Mg2+ can bind arsenazo III in several different protonated forms depending on pH. The binding affinities of these different protonated forms of arsenazo III with Ca2+ increase in the order of H4A4- <H3A5- >H2A6- and with Mg2+, H4A4- > H3A5- > H2A6-. Arsenazo III is not membrane bound. The sensitivity ratio of arsenazo III with Ca2+ to arsenazo III with Mg2+ is close to two orders of magnitude. Arsenazo III and its complexes are extremely sensitive to pH changes. With 5 μM arsenazo III, the minimum detectable amount of Ca2+ can be as low as 0.08 μM. Contrary to current belief, we found that Mg2+ can bind to arsenazo III in a slightly acidic medium. Potential applications of arsenazo III to the study of membrane Ca2+ transport are also discussed. 相似文献
12.
The effects of divalent cations on photoactivation of the latent water-oxidation system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination were investigated by using A23187, an ionophore for divalent cations, and the following results were obtained. (a) Photoactivation in the intact chloroplasts was inhibited by A23187, but was restored on addition of a low concentration of Mn2+ (10 μM). (b) A high concentration of Mn2+ (70 μM) was inhibitory, in contrast, for photoactivation, but the inhibition was restored by the coexistence of a suitable concentration of Ca2+ (5 mM). (c) The Ca2+-dependent restoration was inhibited by a high concentration of Mg2+ or Sr2+, but the inhibition was restored by the coexistence of Ca2+. (d) Kinetic analyses of these competitive effects between divalent cations revealed that: (i) High concentration of Ca2+ inhibits photoactivation in competition with Mn2+. (ii) High concentration of Mn2+ inhibits photoactivation in competition with Ca2+. (iii) High concentration of Mg2+ affects photoactivation by inhibiting Ca2+-dependent restoration in competition with Ca2+. Based on these results, we propose that the latent water-oxidation center has two binding sites, each specific for Mn2+ and Ca2+, and that photoactivation takes place in the center having both Mn2+ and Ca2+ on their respective binding sites. 相似文献
13.
B S Tuana A Dzurba V Panagia N S Dhalla 《Biochemical and biophysical research communications》1981,100(3):1245-1250
The sarcolemmal membranes obtained from rat heart by sucrose-density gradient method were found to exhibit Ca2+ stimulated Mg2+ dependent ATPase and ATP-dependent Ca2+ binding activities. The Ca2+ stimulated ATPase activity was increased by calmodulin; maximal effect was seen at 1 to 5 μg/ml concentrations of calmodulin. The observed activation of the enzyme was associated with an increase in Vmax value from 3.45 to 5.26 μmol Pi/mg protein/hr and a decrease in Ka value from 2.78 to 0.84 μM Ca2+. Calmodulin was also found to increase ATP-dependent Ca2+ binding by 1.6 to 2.2 fold. These results suggest that the activity of Ca2+ pump mechanism in heart sarcolemma is regulated by calmodulin. 相似文献
14.
The effect of polyamines on the kinetics of Ca2+- and Mg2+-mediated membrane fusion was studied by following the intermixing of the contents of vesicles composed of phosphatidate/phosphatidylserine/ phosphatidylethanolamine/cholesterol (1:2:3:2). Addition of polyamines at specific concentration ranging from 40 to 400 μM promoted aggregation of the vesicles. In addition, low levels of spermine (50–100 μM) enhanced both Ca2+ - and Mg2+-mediated fusion. The initial fusion rate of this membrane system increased more than 200-fold when fusion was initiated by Ca2+ after 5 min pre-incubation of vesicles with 50 μM spermine. These results indicate that in addition to their other known effects on cellular metabolism, polyamines may be involved in modulating intracellular membrane fusion. 相似文献
15.
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
16.
17.
《Biochimica et Biophysica Acta (BBA) - Enzymology》1978,522(1):151-160
In the presence of 10 μM Ca2+ and 5 mM Mg2+ (or 0.25 mM Mg2+), the addition of 100 μM Zn2+, Ni2+, Co2+, Fe2+, Cu2+ or 1 mM Mn2+ resulted in varying degrees of stimulation or inhibition of 10−6 M cyclic GMP and cyclic AMP hydrolysis by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart in the absence or presence of phosphodiesterase activator. The substrate specificity of the enzyme was altered under several conditions. The addition of Zn2+ in the presence of 5 mM Mg2+ and the absence of activator resulted in the stimulation of cyclic GMP hydrolysis over a narrow substrate range while reducing the V 65% due to a shift in the kinetics from non-linear with Mg2+ alone to linear in the presence of Zn2+ and Mg2+. Zn2+ inhibited the hydrolysis of cyclic GMP and cyclic AMP in the presence of activator with Ki values of 70 and 100 μM, respectively. Zn2+ inhibition was non-competitive with substrate, activator and Ca2+ but was competitive with Mg2+. In the presence of 10 μM Ca2+ and activator, a Ki of 15 μM for Zn2+ vs. Mg2+ was noted in the hydrolysis of 10−6 M cyclic GMP. Several effects of Zn2+ are discussed which have been noted in other studies and might be due in part to changes in cyclic nucleotide levels following phosphodiesterase inhibition. 相似文献
18.
The effect of phorbol 12-myristate 13-acetate (PMA) on Ca2+-ATPase activity in rat liver nuclei was investigated. Ca2+-ATPase activity was calculated by subtracting Mg2+-ATPase activity from (Ca2+-Mg2+)-ATPase activity. The nuclear Ca2+-ATPase activity was significantly increased by the presence of PMA (2–20 μM) in the enzyme reaction mixture; the maximum effect was seen at 10 μM. The PMA (10 μM)-increased Ca2+-ATPase activity was not blocked by the presence of staurosporine (2 μM) or dibucaine (2 and 10 μM), an inhibitor of protein kinase. Meanwhile, vanadate (20 and 100 μM) caused a significant reduction in the nuclear Ca2+-ATPase activity increased by PMA (10 μM). The present finding suggests that PMA has an activating effect on liver nuclear Ca2+-ATPase independent of protein kinase. © 1994 Wiley-Liss, Inc. 相似文献
19.
Mild proteolysis by trypsin activates the purified (Ca2+ + Mg2+) - ATPase protein from human red cells in a way which is similar to the effect obtained by addition of calmodulin. The trypsin concentration required to reach half maximal effect in 3 minutes at 37°C is 2.5 – 3.5 μg/ml. SDS-poly-acrylamide gel electrophoresis reveals a degradation of the main protein (150'000 Dalton) into a large fragment (95'000 – 100'000 Dalton) and a small fragment (35'000 – 40'000 Dalton). Increasing ATPase activity correlates with the degree of proteolysis.The
Ca of the digested (Ca2+ + Mg2+)-ATPase is 0.85 ± 0.1 μM Ca2+ as compared to 8.0 ± 0.75 μM Ca2+ before digestion and is statistically significantly different from
Ca = 1.66 ± 0.22 μM Ca2+ observed in activation by a saturating calmodulin concentration. Addition of calmodulin to the trypsinized enzyme has neither an effect on the Ca2+-affinity nor achieves any large increase of the maximal rate.High Ca2+ concentrations (above 0.05 – 0.1 mM) after trypsin treatment still inhibit the (Ca2+ + Mg2+)-ATPase activity. Mg2+ activates in the same concentration range (
Mg = 25 μM) as in the undigested preparation (
Mg = 27 μM) and retains its competitive behaviour towards Ca2+ after trypsin treatment.It is concluded that (1) trypsin treatment unmasks high affinity sites for Ca2+ (
Ca 1 μM) and that, therefore, such sites are not added to the system by calmodulin, and (2) that inhibition by high Ca2+-concentrations is not due to Ca - Mg competition at sites located on the calmodulin molecule. 相似文献
20.
Gerhard Meissner 《生物化学与生物物理学报:生物膜》1973,298(4):906-926
The binding of ATP and Ca2+ by the Ca2+ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca2+ pump protein has been found to contain one specific ATP and two specific Ca2+ binding sites per phosphorylation site. ATP binding is dependent on Mg2+ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca2+. In the presence of Mg2+ and the absence of Ca2+, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca2+ binding. In the absence of ATP, Ca2+ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg2+, Sr2+ and La3+, in that order, decrease Ca2+ binding by the Ca2+ pump protein. The affinity of the Ca2+ pump protein for both ATP and Ca2+ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca2+ pump protein-MgATP2? and Ca2+ pump protein-calcium complexes are approx. 0.25 and 0.5 μM?1, respectively. The unphosphorylated Ca2+ pump protein does not contain a Mg2+ binding site with an affinity comparable to those of the ATP and Ca2+ binding sites.The affinity of the Ca2+ pump protein for Ca2+ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca2+ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca2+. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca2+.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca22+-MgATP2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca2+ and probably Mg2+. 相似文献