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1.
Activating effect of regucalcin on (Ca2+-Mg2+)-ATPase in rat liver plasma membranes: relation to sulfhydryl group 总被引:1,自引:0,他引:1
The activating mechanism of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca2+–Mg2+)-ATPase in the plasma membranes of rat liver was investigated. (Ca2+–Mg2+)-ATPase activity was markedly increased by a sulfhydryl (SH) group protecting reagent dithiothreitol (DTT; 2.5 and 5 mM as a final concentration), while the enzyme activity was significantly decreased by a SH group modifying reagent N-ethylmaleimide (NEM; 0.5–5 mM). The effect of DTT (5 mM) to increase the enzyme activity was clearly blocked by NEM (5 mM). Regucalcin (0.25–1.0 M) significantly increased (Ca2+-Mg2+)-ATPase activity. This increase was completely blocked by NEM (5 mM). Meanwhile, digitonin (0.04%), which can solubilize the membranous lipids, significantly decreased (Ca2+–Mg2+)-ATPase activity. Digitonin did not have an effect on the DTT (5 mM)-increased enzyme activity. However, the effect of regucalcin (0.25 M) increasing (Ca2+–Mg2+)-ATPase activity was entirely blocked by the presence of digitonin. The present results suggest that regucalcin activates (Ca2+–Mg2+)-ATPase by the binding to liver plasma membrane lipids, and that the activation is involved in the SH groups which are an active site of the enzyme. 相似文献
2.
Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox 总被引:3,自引:0,他引:3
This study examined the status of sarcolemmal Na+/K+-ATPase activity in rat heart under conditions of Ca2+-paradox to explore the existence of a relationship between changes in Na+/K+-pump function and myocardial Na+ as well as K+ content. One min of reperfusion with Ca2+ after 5 min of Ca2+-free perfusion reduced Na+/K+-ATPase activity in the isolated heart by 53% while Mg2+-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca2+ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na+/K+-ATPase activity was consistently more depressed than Mg2+-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in Vmax without any changes in Km values. Low Na+ perfusion and hypothermia, which protect the isolated heart from the Ca2+-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na+ and K+ content to Na+/K+-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na+/K+-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na+ and K+ content, respectively. These results suggest that changes in myocardial Na+ and K+ content during Ca2+-paradox are related to activity of the Na+/K+-pump; the impaired Na+/K+-ATPase activity may lead to augmentation of Ca2+-overload via an enhancement of the Na+/Ca2+-exchange system. 相似文献
3.
Georgina Rodríguez de Lores Arnaiz Amanda Pellegrino de Iraldi 《Neurochemical research》1997,22(3):293-296
Neuronal ATPases comprise a wide variety of enzymes which are not uniformly distributed in different membrane preparations. Since purified vesicle fractions have Mg2+/Ca2+-ATPase, the purpose of the present study was to know whether such enzyme activities have a preferential concentration in a synaptic vesicle fraction in order to be used as markers for these organelles. Resorting to a procedure developed in this Institute, we fractionated the rat cerebral cortex by differential centrifugation following osmotic shock of a crude mitochondrial fraction and separated a purified synaptic vesicle fraction over discontinuous sucrose gradients. Mg2+/Ca2+-ATPase activities and ultrastructural studies of isolated fractions were carried out. It was observed that similar specific activities for Mg2+/Ca2+-ATPases were found in all fractions studied which contain synaptic vesicles and/or membranes. Although the present results confirm the presence of Mg2+ and Ca2+-ATPase activities in synaptic vesicles preparations, they do not favor the contention that Mg2+/Ca2+-ATPase is a good marker for synaptic vesicles. 相似文献
4.
We reported previously that a Ca2+-ATPase in rat testes and goat spermatozoa could be activated by Ca2+ alone without Mg2+, though it has a lot of similarities with the well known Ca2+, Mg2+-ATPase. Recently, we were successful in isolating the phosphorylated intermediate of the former enzyme under control conditions
i.e., in the presence of low concentration of Ca2+ and at low temperature. Increase of the concentration of Ca2+ and/or temperature lead to dephosphorylation. Based on our observations, we proposed a reaction scheme comparable to that
of Ca2+, Mg2+-ATPase. The findings strengthened our previous report that Mg2+-independent Ca2+-ATPase is involved in Ca2+ transport and Ca2+ uptake like Ca2+, Mg2+-ATPase. 相似文献
5.
Characterization of K+-dependent and K+-independentp-nitrophenylphosphatase activity of synaptosomes
M. Guerra Marichal A. Rodríguez Del Castillo P. Martín Vasallo E. Battaner Arias 《Neurochemical research》1993,18(7):751-758
These experiments examined effects of several ligands on the K+
p-nitrophenylphosphatase activity of the (Na+,K+)-ATPase in membranes of a rat brain cortex synaptosomal preparation. K+-independent hydrolysis of this substrate by the synaptosomal preparation was studied in parallel; the rate of hydrolysis in the absence of K+ was approximately 75% less than that observed when K+ was included in the incubation medium. The response to the H+ concentrations was different: K+-independent activity showed a pH optimum around 6.5–7.0, while the K+-dependent activity was relatively low at this pH range. Ouabain (0.1 mM) inhibited K+-dependent activity 50%; a concentration 10 times higher did not produce any appreciable effect on the K+-independent activity. Na+ did not affect K+-independent activity at all, while the same ligand concentration inhibited sharply the K+-dependent activity; this inhibition was not competitive with the substrate,p-nitrophenyl phosphate. K+-dependent activity was stimulated by Mg2+ with low affinity (millimolar range), and 3 mM Mg2+ produced a slight stimulation of the activity in absence of K+, which could be interpreted as Mg2+ occupying the K+ sites. Ca2+ had no appreciable effect on the activity in the absence of K+. However, in the presence of K+ a sharp inhibition was found with all Ca2+ concentrations studied. ATP (0.5 mM) did not affect the K+-independent activity, but this nucleotide behaved as a competitive inhibitor top-nitrophenylphosphate. Pi inhibited activity in the presence of K+, competively to the substrate, so it could be considered as the second product of the reaction sequence.Abbreviations used
p-NPP
p-nitrophenylphosphate
-
p-NPPase
rho-nitrophenylphosphatase activity 相似文献
6.
Pontes ZL Oliveira LS Franzon R Wajner M Wannmacher CM Wyse AT 《Neurochemical research》2001,26(12):1321-1326
Na+,K+-ATPase and Mg2+-ATPase activities were determined in the synaptic plasma membranes from hippocampus of rats subjected to chronic and acute proline administration. Na+,K+-ATPase activity was significantly reduced in chronic and acute treatment by 33% and 40%, respectively. Mg2+-ATPase activity was not altered by any treatment. In another set of experiments, synaptic plasma membranes were prepared from hippocampus and incubated with proline or glutamate at final concentrations ranging from 0.2 to 2.0 mM. Na+,K+-ATPase, but not Mg2+-ATPase was inhibited (30%) by the two amino acids. In addition, competition between proline and glutamate for the enzyme activity was observed, suggesting a common binding site for these amino acids. Considering that Na+,K+-ATPase activity is critical for normal brain function, the results of the present study showing a marked inhibition of this enzyme by proline may be associated with the neurological dysfunction found in patients affected by type II hyperprolinemia. 相似文献
7.
Rita Sikdar Uma Ganguly Pratima Pal Barsanjit Mazumder Parimal C. Sen 《Molecular and cellular biochemistry》1991,103(2):121-130
The goat spermatozoa membranes isolated after treatment with octa (ethylene glycol) mono n-dodecyl ether (C12E8) followed by discontinuous sucrose density gradient centrifugation have been found to contain an ATPase that is stimulated by externally added Ca2+ only. The membrane fraction has also found to contain Mg2+-dependent Ca2+-ATPase activity, however the former activity is about 2 fold higher than the latter. The molecular weight of the enzyme is found to be about 97,000 on SDS-polyacrylamide gel. The optimum concentration of Ca2+ required for maximum activity is 3 mM for both Mg2+-dependent and Mg2+-independent Ca2+-ATPase. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities respectively. ATP with an optimum concentration of 4 mM is observed to be the best substrate than any other nucleotides. The inhibitors like trifluoperazine and vanadate and group specific probes e.g. DTNB and TNBS inhibit these two enzymes but at different rates. Ca2+-uptake study shows that the uptake in the presence of Ca2+ and ATP is higher than in the presence of Mg2+, Ca2+ and ATP. The findings lead us to believe that the Mg2+-independent Ca2+-ATPase has some role in Ca2+ transport like Mg2+-dependent enzyme.Abbreviations Tris
Tris (hydroxymethyl) amino ethane
- Hepes-N
2-hydroxy ethyl piperizine-N1-2-ethane sulfonic acid
- Pipes-Piperizine-N
N1-bis(2-ethane sulfonic acid)
- EGTA
Ethylene Glycol-bis (-amino ethyl ether)
- N, N, N1, N1
Tetraacetic Acid, sodium salt
- TFP
Trifluoperazine
- DTNB
5,51 Dithiobis (2 nitrobenzoic acid)
- TNBS
2, 4, 6-Trinitrobenzene Sulfonate
- C12E8
Octa (ethylene glycol) mono n-dodecyl ether
- PMSF
Phenylmethyl Sulfonyl Fluoride
- PAGE
Polyacrylamide Gel Electrophoresis
- PME
-Mercapto Ethanol 相似文献
8.
The Ca2+/Mg2+ ATPase of the rat heart sarcolemmal particles was solublized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12 – 0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca2+ (Ka = 0.4 mM) and Mg2+ (Ka = 0.2 mM) as well as by other cations in the order Ca2– > Mg2+ > Mn2+ > Sr2+ > Ba2+ > Ni2+ > Cu2+. The ATPase activity in the presence of Ca2+ was markedly inhibited by Mg2+, Mn2+, Ni2+ and Cu2+ whereas the monovalent cations such as Na+ and K+ were without effect. The enzyme did not exhibit Ca2+ stimulated Mg2+ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca2+ or Mg2+ ATPase activity was 8.5 – 9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca2+ metabolism in the myocardium is discussed. 相似文献
9.
B.S. Jena B.K. Patnaik 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1995,111(4):545-552
Hepatic Na+-K+-ATPase and Mg2+-ATPase activities of male green lizards declined during the maturation phase (juvenile to 1-year-old) and stabilized thereafter. On the other hand, the Ca2+-ATPase activity of the liver declined during the later half of the life span (1-year-old to 2–4-year-old). Starvation stress induced a decline in hepatic Na+-K+-ATPase and Mg2+-ATPase activities of juvenile lizards and caused an increase in 1-year-old and 2–4-year-old counterparts. The Ca2+-ATPase activity declined only in starved 1-year-old lizards. Following cold stress, the hepatic Na+-K+-ATPase activity of juvenile lizards showed a higher degree of decline than 2–4-year-old counterparts. The Mg2+-ATPase activity declined in cold-stressed juvenile lizards, but the parameter was elevated in similarly treated 1-year-old lizards. On the other hand, the increase in Ca2+-ATPase activity in response to cold stress was confined only to 2–4-year-old lizards. 相似文献
10.
《Molecular membrane biology》2013,30(4):191-201
The uncoupling of Ca2+ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu3+ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Co2+ and at 25°C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca2+, 3 mM ATP, and at 25°C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu3+. Digest past TD2 in the presence of 5 mM Ca2+ and 3 mM AMP-PNP. (a non-hydrolyzable ATP analog) at 25°C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca2+ transport from ATPase activity is possible by tryptic digestion of the (Ca2+ + Mg2+)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin. 相似文献
11.
K.Damodar Reddy A Chaudhuri K Thangavelu 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1997,116(4):459-466
Tissue-specific age-dependent changes were observed in Na+K+-, Ca2+-, and Mg2+-ATPase activities in tropical tasar silkworm, Antheraea mylitta Drury. Maximum enzyme activity was recorded in all the tissues on day 12 (before spinning) in control group of animals. In testis, Na+K+-, Ca2+-, and Mg2+-ATPase activities gradually increased from day 2 to day 12 during fifth larval age and level was maintained up to adult eclosion while, in ovary, a marked decline was noted up to day of adult emergence. Further, a significant and sharp rise was found in ATPase activity in silk gland tissue up to day 12 and afterwards a drastic fall was noted on day 15 (end of spinning) during fifth larval age.Administration of T4 to fifth stage larvae (1 hr old) at doses 0.5–2.0 μg/g significantly elevated the Na+K+-, Ca2+-, and Mg2+-ATPase activities in larval and pupal gonads in a dose-dependent fashion. But, in moths, the enhancement was very much confined to Na+K+- and Ca2+-ATPase in testes and only Ca2+-ATPase in ovaries. Again, in silk glands thyroxine (0.5–2.0 μg/g) caused a significant rise in the all ion-dependent ATPase activities only during the fifth larval stage. Interestingly, higher doses of T4 (4.0 μg/g) caused a significant reduction in Na+K+-, Ca2+- and Mg2+-ATPase in all the tissues almost all the days studied so far. However, lower doses of T4 (0.1 and 0.25 μg/g) remained ineffective in altering the different ion-specific ATPase activities. This study suggests, that mammalian thyroxine has a metabolic influence showing biphasic nature of action in tasar silkworm ATPase system. 相似文献
12.
Streck EL Zugno AI Tagliari B Franzon R Wannmacher CM Wajner M Wyse AT 《Neurochemical research》2001,26(11):1195-1200
The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na+, K+-ATPase and Mg2+-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na+, K+-ATPase activity, whereas methionine had no effect. Mg2+-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na+, K+-ATPase activity. Tested compounds did not alter Na+, K+-ATPase and Mg2+-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na+, K+-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients. 相似文献
13.
Danijela Krstić Katarina Krinulović Vesna Vasić 《Journal of enzyme inhibition and medicinal chemistry》2013,28(5):469-476
Kinetics and inhibition of Na+/K+-ATPase and Mg2+-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu2+, Zn2+, Fe2+ and.Co2+) and heavy metals (Hg2+and Pb2+) ions were studied. All investigated metals produced a larger maximum inhibition of Na+/K+-ATPase than Mg2+-ATPase activity. The free concentrations of the key species (inhibitor, MgATP2 ? , MeATP2 ? ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu2+/Fe2+ or Hg2+/Pb2+caused additive inhibition, while Cu2+/Zn2+ or Fe2+/Zn2+ inhibited Na+/K+-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu2+/Fe2+ or Cu2+/Zn2+ inhibited Mg2+-ATPase activity synergistically, while Hg2+/Pb2+ or Fe2+/Zn2+ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na+/K+-ATPase activity by reducing the maximum velocities (Vmax) rather than the apparent affinity (Km) for substrate MgATP2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg2+-ATPase activity by Zn2+, Fe2+ and Co2+ as well as kinetic analysis indicated two distinct Mg2+-ATPase subtypes activated in the presence of low and high MgATP2 ? concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na+/K+-ATPase with various potencies. Furthermore, these ligands also reversed Na+/K+-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg2+-induced inhibition was not obtained. 相似文献
14.
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》1981,649(3):533-540
ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na+ + K+)-ATPase in the absence of Mg2+ (EDTA present) with a homogeneous but 15-fold different affinity, the Kd values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na+ + K+)-ATPase, respectively. The Kd value for ATP is equal to the Km for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na+ + K+)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg2+, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The Kd and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg2+ concentration. The Kd increases from 0.06 mM at 0.5 mM Mg2+ to a maximum of 0.26 mM at 2 mM Mg2+ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg2+ to 3.3 nmol/mg protein at 4 mM Mg2+. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg2+ concentration yields a maximal binding capacity at infinite Mg2+ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na+ + K+)-ATPase. The Kd for Mg2+ at the sites, where it exerts this effect, is 0.8 mM. The Kd for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg2+ to a maximum of 4.2 μM at 2 mM Mg2+ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg2+ concentration. Hence, Mg2+ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na+ + K+)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites. 相似文献
15.
Barsanjit Mazumder Shyamali Mukherjee Parimal C. Sen 《Molecular and cellular biochemistry》1990,95(1):13-20
Chlorpromazine, an antipsychotic drug, is found to inhibit Na+,K+-ATPase activity in rat brain microsomal membranes in vitro in concentration and time dependent manner but some inconsistency is observed when the effect was studied with respect to different temperatures. Various ligands and/or substrate affect the inhibition by chlorpromazine in different ways. The in vivo study with this drug shows that the activities of Na+,K+-ATPase, Ca–2-ATPase and acetylcholinesterase in the microsomal membranes of different organs are inhibit with increases in concentration or lengths of time of treatment and then levels off. 相似文献
16.
Heart sarcolemma has been shown to contain an ATPase hydrolizing system which is activated by millimolar concentrations of divalent cations such as Ca2+ or Mg2+. Although Ca2+-dependent ATPase is released upon treating sarcolemma with trypsin, a considerable amount of the divalent cation dependent ATPase activity was retained in the membrane. This divalent cation dependent ATPase was solubilized by sonication of the trypsin-treated dog heart sarcolemma with 1% Triton X-100. The solubilized enzyme was subjected to column chromatography on a Sepharose-6B column, followed by ion-exchange chromatography on a DEAE cellulose column. The enzyme preparation was found to be rather labile and thus the purity of the sample could not be accurately assessed. The solubilized ATPase preparations did not show any cross-reactivity with dog heart myosin antiserum or with Na+ + K+ ATPase antiserum. The enzyme was found to be insensitive to inhibitors such as ouabain, verapamil, oligomycin and vanadate. The enzyme preparation did not exhibit any Ca2+-stimulated Mg2+ dependent ATPase activity. Furthermore, the low affinity of the enzyme for Ca2– (Ka = 0.3 mM) rules out the possibility of its involvement in the Ca2+ pump mechanism located in the plasma membrane of the cardiac cell. 相似文献
17.
We have already described the separation of two brain soluble fractions by Sephadex G-50, one of which stimulates (peak I) and the other inhibits (peak II) Na+, K+-ATPase and K+-p-nitrophenylphosphatase (K+-p-NPPase) activities. Here we examine the features of synaptosomal membrane p-NPPase activity in the presence and absence of brain peak I. It was observed that stimulation of Mg2+, K+-p-NPPase activity by peak I was concentration dependent, The ability of peak I to stimulate p-NPPase activity was lost by heat treatment followed by brief centrifugation. Pure serum albumin also stimulated enzyme activity. K+-p-NPPase stimulation by peak I proved dependent on K+ concentration but independent of Mg2+ and substrate p-nitrophenylphosphate concentrations. Since our determinations were performed in a non-phosphorylating condition reflecting the Na+, K+-ATPase Na+ site, it is suggested that peak I may stimulate the Na+-dependent enzyme phosphorylation known to take place from the internal cytoplasmic side. 相似文献
18.
Anil K. Verma John T. Penniston Schmuel Muallem Virgilio Lew 《Journal of bioenergetics and biomembranes》1984,16(5-6):365-378
Antibodies raised in rabbits against the purified erythrocyte membrane Ca2+ pumping ATPase were affinity-purified using an ATPase-Sepharose column. Addition of a few molecules of the purified antibody per molecule of ATPase was sufficient to inhibit the ATPase activity. Extensively washed ghosts or preincubated pure ATPase sometimes develop an appreciable Mg2+-ATPase activity. In such cases, the antibodies inhibited the Mg2+-ATPase as well as the Ca2+-ATPase. This is consistent with the hypothesis that a portion of the Mg2+-ATPase activity of ghosts is derived from the Ca2+-ATPase. When nitrophenylphosphatase activity was observed, both Mg2+ - and Ca2+-stimulated activities were observed. Only the Ca2+ activity was inhibited by the antibodies, confirming that this activity is due to the Ca2+ pump, and suggesting that the Mg2+-nitrophenylphosphatase is due to a separate enzyme. Amounts of antibody comparable to those which inhibited the Ca2+-ATPases had no effect on the Na+-K+-ATPase; 4-fold higher amounts of antibody significantly stimulated the Na+-K+-ATPase, but this effect of the antibody was not specific: Immunoglobulins from the nonimmune serum also significantly stimulated the Na+-K+-ATPase.In resealed erythrocyte membranes, antibodies incorporated into the ghosts inactivated the Ca2+-ATPase, while antibodies added to the outside had no significant effect. 相似文献
19.
Vijayan Elimban Dayuan Zhao Naranjan S. Dhalla 《Molecular and cellular biochemistry》1987,77(2):143-152
In order to gain some information regarding Ca2+-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca2+-ATPase activity of myosin purified from rat heart. Both Ca2+-dependent ATPase and myosin ATPase were stimulated by Ca2+ but the maximal activation of Ca2+-dependent ATPase required 4 mM Ca2+ whereas that of myosin ATPase required 10 mM Ca2+. These ATPases were also activated by other divalent cations in the order of Ca2+ > Mn2+ > Sr2+ > Br2+ > Mg2+; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca2+-dependent ATPase was not activated by actin. The pH optima of Ca2+-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na+ markedly inhibited Ca2+-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca2+-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca2+-dependent ATPase. Both Ca2+-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH4-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca2+-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca2+ and 3 to 4 mM ATP like sarcolemmal Ca2+-dependent ATPase. K+ stimulated both ATPase activities in the absence of Ca2+ and inhibited in the presence of Ca2+. Both enzymes were inhibited by Na+, Mg2+, La3+, and azide similarly. However, Ca2+ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca2+ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca2+-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca2+-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca2+-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca2+-ATPase. These observations suggest that Ca2+-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils. 相似文献
20.
Enn K. Seppet Frantisek Kolar Ian M. C. Dixon Tomoji Hata Naranjan S. Dhalla 《Molecular and cellular biochemistry》1993,129(2):145-159
In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca2+-channels, Na+–Ca2+ exchange and Ca2+-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca2+-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca2+ and Ca2+ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca2+ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [3H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (Bmax) without any change in the dissociation constant for receptor-ligand complex (Kd) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca2+-stimulated ATPase, ATP-dependent Ca2+ uptake and ouabain-sensitive Na+–K+ ATPase were decreased whereas the Mg2+-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na+–K+ ATPase activity, whereas Ca2+-stimulated ATPase, ATP-dependent Ca2+ uptake, and Mg2+-ATPase activities were unchanged. The Vmax and Ka for Ca2+ of cardiac sarcolemmal Na+–Ca2+ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca2+-transport processes is regulated by thyroid hormones and the modification of Ca2+-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart. 相似文献