共查询到20条相似文献,搜索用时 31 毫秒
1.
Elizabeth Zubrzycka-Gaarn Glen MacDonald Laurie Phillips Annelise O. Jorgensen David H. MacLennan 《Journal of bioenergetics and biomembranes》1984,16(5-6):441-464
In order to determine whether polymorphic forms of the Ca2+ + Mg2+-dependent ATPase exist, we have examined the cross-reactivity of five monoclonal antibodies prepared against the rabbit skeletal muscle sarcoplasmic reticulum enzyme with proteins from microsomal fractions isolated from a variety of muscle and nonmuscle tissues. All of the monoclonal antibodies cross-reacted in immunoblots against rat skeletal muscle Ca2+ + Mg2+-dependent ATPase but they cross-reacted differentially with the enzyme from chicken skeletal muscle. No cross-reactivity was observed with the Ca2+ + Mg2+-dependent ATPase of lobster skeletal muscle. The pattern of antibody cross-reactivity with a 100,000 dalton protein from sarcoplasmic reticulum and microsomes isolated from various muscle and nonmuscle tissues of rabbit demonstrated the presence of common epitopes in multiple polymorphic forms of the Ca2+ + Mg2+-dependent ATPase. One of the monoclonal antibodies prepared against the purified Ca2+ + Mg2+-dependent ATPase of rabbit skeletal muscle sarcoplasmic reticulum was found to cross-react with calsequestrin and with a series of other Ca2+-binding proteins and their proteolytic fragments. Its cross-reactivity was enhanced in the presence of EGTA and diminished in the presence of Ca2+. Its lack of cross-reactivity with proteins that do not bind Ca2+ suggests that it has specificity for antigenic determinants that make up the Ca2+-binding sites in several Ca2+-binding proteins including the Ca2+ + Mg2+-dependent ATPase.This paper is dedicated to the memory of Dr. David E. Green. 相似文献
2.
The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two-phase partitioning. Its purity was examined with marker enzymes, Mg2+-dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH-cytochrome c reductase, as well as the sensitivity of Mg2+-dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid-chromic acid. Mg2+- or Ca2+-dependent ATPases were associated with the plasma membrane. Ca2+-dependent ATPase was activated at physiological cytoplasmic concentrations of Ca2+ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg2+-dependent ATPase. The isolated plasma membrane vesicles were mostly right side-out. To test for H+-translocation, right side-out vesicles were inverted; 27% of vesicles were inside-out after treatment with Triton X-100. The inside-out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca2+. The reduced fluorescence was recovered with the addition of 10 mmol/L NH4Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca2+-dependent ATPase might pump H+ out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification. 相似文献
3.
Oligomycin effects on ATPase and photophosphorylation of pea chloroplast thylakoid membranes
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Oligomycin inhibited the membrane-bound, Ca2+-dependent ATPase of pea (Pisum sativum var. Progress No. 9) chloroplasts up to 50%, but only after treating the membranes with trypsin, whether or not the trypsin step was needed for full activity. The energy-linked Mg2+-dependent (light- and dithiothreitol (DTT)-activated) ATPase of pea thylakoids could be inhibited up to 100% under specified conditions. The data indicate that oligomycin does not interfere with activation processes, and it failed to inhibit the ATPase of solubilized chloroplast coupling factor 1 under any circumstances. Photophosphorylation, previously thought insensitive to oligomycin, was inhibited 30% in the case of pea chloroplasts, and this increased to 50% inhibition after pretreating the chloroplasts with either trypsin or DTT. The nature of inhibition of phosphorylation was complex, with apparent small components of electron transport inhibition and uncoupling, as well as energy transfer inhibition. 相似文献
4.
Two distinct adhesion mechanisms in embryonic neural retina cells. II. An immunological analysis 总被引:5,自引:0,他引:5
Antibodies were raised against neural retina cells prepared by dissociation in EGTA alone (E cells, Ca2+-independent aggregation), in trypsin + Ca2+ (TC cells, Ca2+-dependent aggregation), or in trypsin + EGTA (TE cells, nonadhesive). Anti-E-cell Fab selectively inhibited Ca2+-independent aggregation, anti-TC-cell Fab selectively inhibited Ca2+-dependent aggregation, and anti-TE-cell Fab inhibited neither. Fab from a fourth preparation, also raised against E cells, inhibited both Ca2+-independent and Ca2+-dependent aggregation but was separated by immunoadsorption into two fractions, one specific for each mode of aggregation. In cells which utilize both modes simultaneously (LTC cells), each was inhibited exclusively by the appropriate Fab. The immunological data presented here demonstrate the existence in the same cells of two distinct and functionally independent adhesion mechanisms, each responsible for one of the two modes of aggregation. The differing adhesive properties of retinal cells prepared by different procedures are explained by the presence, absence, or degree of activity of these two mechanisms, qualities regulated by the concentrations of trypsin and Ca2+ used in the tissue dissociation. 相似文献
5.
Gethyn J. Allen Francisco Morais Shelagh Muir R. Gareth Wyn Jones Roger A. Leigh 《Physiologia plantarum》1995,94(3):506-514
Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca2+ than Mg2+. Here we characterise the Ca2+-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca2+-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg2+. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca2+-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca2+ on the plasma membrane K+, Mg2+-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca2+-dependent activity is stimulated by free Ca2+ with a Km of 8.1 μM in the absence of Mg2+ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca2+-dependent than Mg2+-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca2+ and Mg2+ The Ca2+-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg2+-dependent activity functioned as a bafilomycin sensitive H+-pump in vacuolar membrane vesicles. Ca2+-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K+. Possible functions of Ca2+-dependent hydrolysis as a H+-pump or a Ca2+-pump was investigated using vacuolar membrane vesicles. No H+ or Ca2+ translocating activity was observed under conditions when the Ca2+-dependent ATP hydrolysis was active. 相似文献
6.
《生物化学与生物物理学报:生物膜》1987,899(1):83-92
(1) Calmodulin-depleted red cell membranes catalyse a Ca2+, Mg2+-dependent ATP-[3H]ADP exchange at 37° C. The Ca2+, Mg2+-dependent exchange, measured at 20 μM CaCl2, 1.5 mM MgCl2, 1.5 mM ADP and 1.5 mM ATP, is comparable to the (Ca2+ + Mg2+)-ATPase activity, between 0.3 and 0.8 mmol/litre original cells per h. (2) EDTA-washed membranes present a Ca2+-dependent ATP-ADP exchange whose rate is not more than 7% of that found in a Mg2+-containing medium, while their Ca2+-dependent ATPase is essentially zero. Addition of 1.5 mM MgCl2 to the medium restores both activities to the levels found with membranes not treated with EDTA. (3) Calmodulin (16 μg/ml) produces an eight-fold stimulation of the Ca2+-dependent ATP-ADP exchange, slightly less than it stimulates the Ca2+-dependent ATP hydrolysis. The effect of 1.5 mM MgCl2 on the exchange is greater in the presence than in the absence of calmodulin. (4) It is proposed that the reversal of the initial phosphorylation of the Ca2+ pump, occurring at a fast rate at 37° C, involves a conformational change in the phosphoenzyme. Thus, it would be an ADP-liganded phosphoenzyme of the form EP(ADP) that would experience the fast conformational transition at 37° C. The great difficulty in producing an overall reversal of the Ca2+ pump should then be due to one or more reaction steps later than and including Ca2+ release and dephosphorylation. 相似文献
7.
Melchiorre Cervello Daniela Di Ferro Lucia D'Amelio Francesca Zito Valeria Matranga 《Biology of the cell / under the auspices of the European Cell Biology Organization》1992,74(2):231-234
Summary— Sea urchin embryos can be easily dissociated into single cells by exposure to Ca2+- and Mg2+-free seawater. When transferred back to normal seawater, isolated cells spontaneously form aggregates capable of development. Here, the Ca2+-dependent self-aggregation of toposome, a 22S glycoprotein complex which mediates cell-cell adhesion in sea urchin embryos, has been investigated using the purified molecule. Results show that the 22S complex is completely converted to 15S particles by sedimentation on sucrose isokinetic gradients in the presence of EDTA. Reconstitution of the 22S complex is achieved by readdition of Ca2+. We propose that the 15S particle constitutes the toposome functional unit on the cell surface. 相似文献
8.
Pseudomonas marina (ATCC 27 129) rapidly aggregates when suspended in buffered artificial seawater (ASW). Light microscopic observations of stained preparations, showed that flagella-flagella contact was responsible for this phenomenon. Aggregation did not occur if flagella were sheared off, or if motility was inhibited with NaN3. Aggregates were not observed when Mg2+ was omitted from ASW, even though the bacteria remained motile. Other divalent cations, including Ca2+, Mn2+, and Ba2+ could replace Mg2+. However, there is no absolute requirement for divalent cations, since aggregation occurred in ASW containing Cs+ or Li+ instead of Mg2+. P. marina aggregates developed from pH 5.8–8.4, but not below pH 5.8 even though motility continued unimpaired to pH 4.5.Abbreviation ASW
artificial seawater 相似文献
9.
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
10.
Reaggregation of mechanically dissociated mouse cerebellar cells (M cells) was compared with cells that received an additional trypsinization either before (T cells) or after (MT cells) the dissociation step. Reaggregation behaviour was followed by measuring the number and size distribution of particles with a Coulter counter. Aggregation rates which were calculated as percentage of decrease of particles could be measured reproducibly. Since the percentage of very large particles (> 100 cells) formed during aggregation varied considerably from one experiment to the next, size distribution curves of particles were used more to distinguish qualitative differences in a less quantitative way.Whereas aggregation rates and size distribution of particles with M cells were almost identical when aggregation occurred in medium of high (1.1 mM) or low (0.1 mM) Ca2+ concentrations, T and MT cells aggregated better at high Ca2+ concentration. Their aggregation rates were reduced by approx. 50% at low Ca2+ concentrations and larger aggregates were hardly formed under these conditions. The aggregation rates of T and MT cells showed a clear dependence on Ca2+ concentration, being half maximal at approx. 0.1 mM Ca2+.The ability of M cells to aggregate at low or high Ca2+ concentrations was influenced by subsequent trypsinization to produce MT cells. When the trypsin concentration was changed from 0.001 to 0.1% during this procedure the aggregation rates at high Ca2+ concentration were reduced to approx. 80% of the maximal value, whereas those at low Ca2+ concentrations were reduced to 35%. Variation of the Ca2+ concentration between 1.1 and 0.1 mM during the trypsinization step (0.015% trypsin) revealed no difference on the aggregation rates.We propose that M cells aggregate mainly or exclusively by a Ca2+-independent binding mechanism, whereas T or MT cells aggregate using a Ca2+-dependent one which may be functionally silent in M cells. 相似文献
11.
The ATPase activity of the chloroplast coupling factor 1 (CF1) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca2+ -dependent activity. The Ca2+ dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca2+ to Mg2+. Both the Ca2+ -dependent and Mg2+ -dependent ATPase activities of heat-treated Dunaliella CF1 are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF1. However, when assayed under identical conditions, the Ca2+ -dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg2+ -dependent activity. These data are interpreted as indicating that soluble Dunaliella CF1 can exist in a variety of conformations, at least one of which catalyzes a Ca2+ -dependent ATPase and two or more of which catalyze an Mg2+ -dependent ATPase. 相似文献
12.
Immunological detection of cell surface components related with aggregation of Chinese hamster and chick embryonic cells. 总被引:6,自引:0,他引:6
Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca2+-dependent and Ca2+-independent ones. We could prepare cells with only the Ca2+-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca2+. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca2+ retain only the Ca2+-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca2+-independent mechanism, but did not inhibit the aggregation of these cells by the Ca2+-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Ca2+-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types. 相似文献
13.
Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca2+,Mg2+-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca2+ + Mg2+) > Mn2+ = (Ca2+ + Mn2+) > (Mg2+ + EGTA) > Ca2+. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn2+ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca2+,Mn2+-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca2+ + Mn2+) > (Ca2+ + Mg2+) > Mn2+ > (Mg2+ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed. 相似文献
14.
We examined similarities in adhesive properties of mouse cleaving embryos at one- to eight-cell stages and of teratocarcinoma cells by aggregation studies. Teratocarcinoma cells and fibroblastic cells have a Ca2+-dependent cell-cell adhesion site (CDS), which is resistant to trypsin in the presence of Ca2+ but sensitive in the absence of Ca2+. When several embryos treated with trypsin in the presence of Ca2+ (TC) were kept in contact with each other, they fused into a single aggregate in the medium with Ca2+ but not without Ca2+. Embryos treated with trypsin in the absence of Ca2+ (TE) did not show such Ca2+-dependent aggregation. Aggregation of TC-treated embryos was inhibited by Fab fragments of antibody raised against TC-treated teratocarcinoma F9 cells. The aggregation-inhibitory effect of the Fab was removed by absorption with TC-treated teratocarcinoma cells, but not with TE-treated teratocarcinoma cells. This effect was not removed by absorption with fibroblasts and some other tissue cells. TC-treated embryos adhered to TC-treated teratocarcinoma cells, but not to TC-treated fibroblastic cells. These results suggest that early mouse embryos share a common CDS molecule with teratocarcinoma cells but not with fibroblastic cells. 相似文献
15.
Summary By using an in vitro functional assay, we have shown that Drosophila embryonic cells possess Ca2+-dependent adhesive sites, which resemble in many respects those described for vertebrate cells and tissues. The cells, obtained by mechanical disruption of gastrulastage embryos, form aggregates within 30 min when maintained under constant rolling. The aggregation is completely dependent on the presence of Ca2+ in the medium. In its absence, the cells remain dispersed but the process is reversible by readdition of Ca2+. In addition the aggregation is temperature-dependent. No aggregation occurs at 4° C but it can be restored by raising the temperature to 25° C. These properties are characteristic of these cells: established cell lines do not aggregate under the same conditions and mixing of cell lines and embryonic cells does not result in chimeric aggregates, thus pointing towards cell-type selectivity with respect to aggregability. Observations in electron microscopy have shown that the embryonic cells in the aggregates tightly adhere to one another and form, as early as after 30 min, maculae adherens junctions. Drosophila embryonic cells have adhesion sites that are protected from trypsin proteolysis in the presence of Ca2+ and sensitive in its absence. The cells' aggregation can be inhibited by a mouse antiserum directed against cell-surface components and a good correlation exists between neutralization of the inhibitory activity of the antiserum and the presence of trypsin-sensitive sites on the cells. These data are in favour of cell-cell adhesion mediated by specific adhesion proteins. 相似文献
16.
E E Quist 《Archives of biochemistry and biophysics》1980,203(1):123-133
In the presence of MgCl2 and ATP, the specific viscosity of suspensions of unsealed freezethawed erythrocyte membranes decreased slowly with time at 37 °C. The decrease in viscosity was found to be an index of Mg-ATP-specific induced folding of these membranes. Mg-ATP-dependent shape or viscosity changes were found to be highly temperature dependent and the viscosity of these membranes did not decrease in the presence of 2 mm 5′-adenyl imidodiphosphate and MgCl2. Cyclic AMP, NaCl, or KCl did not have any effect on the rate of Mg-ATP-induced viscosity decreases. The Mg-ATP-dependent viscosity decreases were inhibited 100% by 1 mm chlorpromazine or 1 mmN-ethylmaleimide. Mg-ATP-dependent viscosity decreases were half-maximally inhibited by 1 μm Ca2+ and completely inhibited by 3–5 μm Ca2+. Ca2+ (5 μm) also inhibited Mg2+-dependent phosphorylation 25 to 30% in these membranes. However, if these membranes were preincubated in the absence of Ca2+ for greater than 10 min at 37 °C, 5 μm Ca2+ no longer inhibited Mg-ATP-dependent viscosity decreases and only inhibited Mg2+-dependent phosphorylation 5% in these preincubated membranes. Preincubation of these membranes at 37 °C for 10 min in the absence of Ca2+ also resulted in the loss of approximately 40 to 50% of the high-Ca2+ affinity Ca + Mg-ATPase activity. The presence of 5 μm Ca2+ in the preincubation medium protected against the loss of the inhibitory effect of Ca2+ on Mg2+-dependent phosphorylation and Mg-ATP-dependent viscosity decreases. The presence of Ca2+ in the preincubation medium also protected against the loss of Ca + Mg-ATPase activity in these membranes. It is hypothesized that freeze-thawed erythrocyte membranes contain a Ca2+ phosphatase activity which is temperature labile in the absence of Ca2+ and that this Ca2+ phosphatase activity may be involved in the regulation of shape of these membranes. Also discussed is the possible relationship of this Ca2+ phosphatase with Ca + Mg-ATPase activity and the problems inherent in studying Ca2+-regulated functions in freeze-thawed erythrocyte membranes. 相似文献
17.
Mikhail V. Dubinin Victor N. Samartsev Anastasia E. Stepanova Ekaterina I. Khoroshavina Nikita V. Penkov Valery A. Yashin Vlada S. Starinets Irina B. Mikheeva Sergey V. Gudkov Konstantin N. Belosludtsev 《Journal of bioenergetics and biomembranes》2018,50(5):391-401
The paper examines membranotropic Ca2+-dependent effects of ω-hydroxypalmitic acid (HPA), a product of ω-oxidation of fatty acids, on the isolated rat liver mitochondria and artificial membrane systems (liposomes). It was established that in the presence of Ca2+, HPA induced aggregation of liver mitochondria, which was accompanied by the release of cytochrome c from the organelles. It was further demonstrated that the addition of Ca2+ to HPA-containing liposomes induced their aggregation and/or fusion. Ca2+ also caused the release of the fluorescent dye sulforhodamine B from liposomes, indicating their permeabilization. HPA was shown to induce a high-amplitude swelling of Ca2+-loaded mitochondria, to decrease their membrane potential, to induce the release of Ca2+ from the organelles and to result in the oxidation of the mitochondrial NAD(P)H pool. Those effects of HPA were not blocked by the MPT pore inhibitor CsA, but were suppressed by the mitochondrial calcium uniporter inhibitor ruthenium red. The effects of HPA were also observed when Ca2+ was replaced with Sr2+ (but not with Ba2+ or Mg2+). A supposition is made that HPA can induce a Ca2+-dependent aggregation of mitochondria, as well as Ca2+dependent CsA-insensitive permeabilization of the inner mitochondrial membrane – with the subsequent lysis of the organelles. 相似文献
18.
Experiments on the effects of varying concentrations of Ca2+ on the Mg2+ + Na+-dependent ATPase activity of a highly purified preparation of dog kidney (Na+ + K+)-ATPase showed that Ca2+ was a partial inhibitor of this activity. When Ca2+ was added to the reaction mixture instead of Mg2+, there was a ouabain-sensitive Ca2+ + Na+-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg2+ + Na+-dependent activity. The apparent affinities of the enzyme for Ca2+ and CaATP seemed to be higher than those for Mg2+ and MgATP. Addition of K+, along with Ca2+ and Na+, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca2+ + Na+ + K+-dependent ATPase was about two orders of magnitude smaller than that of Mg2+ + Na+ + K+-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca2+, Na+, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K+. The enzyme also exhibited a Ca2+ + Na+-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca2+ on Mg2+-dependent activities, nor the Ca2+-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg2+ in all reaction mixtures, it could not be determined whether the apparent Ca2+-dependent activities involved enzyme-substrate complexes containing Ca2+ as the divalent cation or both Ca2+ and Mg2+. 相似文献
19.
《Journal of liposome research》2013,23(3):571-580
AbstractPhospholipid vesicle aggregation is usually mediated by phospholipid-binding proteins such as the annexins in a Ca2+-dependent manner. Here, we describe aggregation of unilamellar liposomes by trypsin and papain in the absence of cations. Cations including Ca2+ inhibited the aggregation. While both trypsin and papain promoted aggregation of liposomes made of phosphatidylcholine and phosphatidylglycerol, only papain elicited aggregation of liposomes made of exclusively phosphatidylcholine. Incubation of trypsin for 30 min at 37°C destroyed its liposome aggregating activity, similar treatment had no effect on papain's. Chymotrypsin and pepsin had no liposome aggregating activity. 相似文献
20.
Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na+ were found to take up Ca2+ when incubated in K+ media or in sucrose media containing micromolar concentrations of free Ca2+. Li+- or choline+loaded ghosts did not take up Ca2+. The Ca2+ accumulated by Na+-loaded ghosts could be released by the Ca2+ ionophore A23187, but not by EGTA. Ca2+ uptake was inhibited by external Sr2+, Na +, Li +, or choline +. All the 45Ca2+ accumulated by Na+-dependent Ca2+ uptake could be released by external Na +, indicating that both Ca2+ influx and efflux occur in a Na+-dependent manner. Na + -dependent Ca2+ uptake and release were only slightly inhibited by Mg2+. In the presence of the Na+ ionophore Monensin the Ca2+ uptake by Na +-loaded ghosts was reduced. Ca2+ sequestered by the Na+-dependent mechanism could also be released by external Ca2+ or Sr2+ but not by Mg2+, indicating the presence of a Ca2+/Ca2+ exchange activity in secretory membrane vesicles. This Ca2+/Ca2+ exchange system is inhibited by Mg2+, but not by Sr2+. The Na + -dependent Ca2+ uptake system in the presence of Mg2+ is a saturable process with an apparent Km of 0.28 μM and a Vmax= 14.5 nmol min?1 mg protein?1. Ruthenium red inhibited neither the Na+/Ca2+ nor the Ca2+/Ca2+ exchange, even at high concentrations. 相似文献