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1.
Electrode measurements of the average number of H+ ejected and K+ taken up (in the presence of valinomycin) per pair of electrons passing the energy-conserving sites of the respiratory chain of rat liver and rat heart mitochondria have given identical values of the H+/site and 5+/site ratios very close to 4 in the presence of N-ethylmaleimide, an inhibitor of interfering respiration-coupled uptake of H+ + H2PO4-. The K+/site uptake ratio of 4 not only shows that inward movement of K+ provides quantitative charge-compensation for the 4 H+ ejected, but also confirms that 4 charges are separated per pair of electrons per site. When N-ethylmaleimide is omitted, the H+/site ejection ratio is depressed, because of the interfering secondary uptake of H/+ with H2PO4- on the phosphate carrier, but the K+/site uptake ratio remains at 4.0. Addition of phosphate or acetate, which can carry H+ into respiring mitochondria, further depresses the H+/site ratio, but does not affect the K+/site ratio, which remains at 4.0. These and other considerations thus confirm our earlier stoichiometric measurements that the average H+/site ratio is 4.0 and also show that the K+/site uptake ratio can be used as a measure of the intrinsic H+/site ratio, regardless of the presence of phosphate in the medium and without the necessity of adding N-ethylmaleimide or other inhibitors of H+ + H2PO4- transport. 相似文献
2.
Ca 2+ transport activity in mitochondria from some plant tissues 总被引:8,自引:0,他引:8
Mitochondria isolated from some 14 different higher plants and fungi were examined for their capacity to carry out respiration-dependent accumulation of Ca2+. Additions of Ca2+ give little or no stimulation of state 4 respiration of plant mitochondria, although the added Ca2+ was largely accumulated. Accumulation of Ca2+ required phosphate and, in most cases, was stimulated by Mg2+ and ADP or ATP. Ca2+ uptake was abolished by respiratory inhibitors and uncoupling agents. The ratio of Ca2+ ions taken up per pair of electrons per energy-conserving site was normal at about 2.0 for mitochondria from sweet potato and white potato; mitochondria from other plants showed somewhat lower ratios. Accumulated Ca2+ was only very slowly released from previously loaded plant mitochondria. Respiration-inhibited sweet potato mitochondria show both high-affinity and low-affinity Ca2+ binding sites sensitive to uncouplers, La3+, and ruthenium red and thus resemble animal mitochondria. Most other plant mitochondria lack high affinity sites. In general, mitochondria from sweet potato and white potato tubers resemble those from animal tissues, but mitochondria from carrots, beets, turnips, onions, cabbage, artichokes, cauliflower, avocados, mung bean and corn seedlings, and mushrooms show rather low affinity and activity in accumulation of Ca2+, probably due to lack of a specific Ca2+ carrier. 相似文献
3.
The number of H+ ejected during passage of 2e- through each energy-conserving site of the mitochondrial respiratory chain (the H+/site ratio) was measured in three ways. In each case transmembrane movements of endogenous phosphate were minimized. (1) Measurement of the uptake of weak acids during loading of mitochondria with Ca2+ demonstrated that 2.0 weak acid anions were accumulated per Ca2+ ion. Since 1.7 to 2.0 Ca2+ ions were were taken up per site, these data correspond to an H+/site ratio of 3.5 to 4.0. (2) More direct measurement of H+ ejection using the oxygen pulse technique demonstrated that the H+/site ratio was 3.0. In these experiments phosphate movements were prevented by addition of N-ethylmaleimide to inhibit phosphate-hydroxide antiport, by washing the mitochondria to remove endogenous phosphate, or by working at 5 degrees C to reduce the rate of phosphate transport. When phosphate movements were allowed, H+/site ratios of 2.0 were observed. (3) Measurement of the initial steady rates of oxygen consumption and H+ ejection following addition of substrate to aerobic, substrate-limited mitochondria yielded H+/site ratios of 2.0, which were elevated to 4.0 when phosphate transport was prevented as described above. Previous determinations of the H+/site ratio were thus underestimates due to the unrecognized movements of endogenous phosphate; our results show that the H+/site ratio is at least 3.0 andmay be as high as 4.0. 相似文献
4.
The mitochondrial H+/site ratio (i.e. the number of protons ejected per pair of electrons traversing each of the energy-conserving sites of the respiratory chain) has been evaluated employing a new experimental approach. In this method the rates of oxygen uptake and H+ ejection were measured simultaneously during the initial period of respiration evoked by addition of succinate to aerobic, rotenone-inhibited, de-energized mitochondria. Either K+, in the presence of valinomycin, or Ca2+, was used as mobile cation to dissipate the membrane potential and allow quantitative H+ ejection into the medium. The H+/site ratio observed with this method in the absence of precautions to inhibit the uptake of phosphate was close to 2.0, in agreement with values obtained using the oxygen pulse technique (Mitchell, P. and Moyle, J. (1967) Biochem. J. 105, 1147-1162). However, when phosphate movements were eliminated either by inhibition of the phosphate-hydroxide antiporter with N-ethylamaleimide or by depleting the mitochondria of their endogenous phosphate content, H+/site ratios close to 4.0 were consistently observed. This ratio was independent of the concentration of succinate, of mitochondrial protein, of pH between 6 and 8, and of ionic composition of the medium, provided that sufficient K+ (plus valinomycin) or Ca2+ were present. Specific inhibitors of the hydrolysis of endogenous ATP or transport of other ions (adenine nucleotides, tricarboxylates, HCO3-, etc.) were shown not to affect the observed H+/site ratio. Furthermore, the replacement of succinate by alpha-glycerol phosphate, a substrate which is oxidized on the outer surface of the inner membrane and thus does not need to enter the matrix, gave the same H+/site ratios as did succinate. It is concluded that the H+/site ratio of mitochondrial electron transport, when phosphate movements are eliminated, may be close to 4.0. 相似文献
5.
M Hirata M Kukita T Sasaguri E Suematsu T Hashimoto T Koga 《Journal of biochemistry》1985,97(6):1575-1582
Inositol 1,4,5-trisphosphate (InsP3) releases Ca2+ from the non-mitochondrial Ca2+ store site of various types of cells. To study the mechanisms of the Ca2+ release from the store site, the effect of InsP3 on the passive Ca2+ release and influx, and the active Ca2+ uptake in the presence of oxalate, was examined using saponin-treated guinea pig peritoneal macrophages. InsP3 stimulated the passive Ca2+ release and influx. Although InsP3 slightly inhibited the active Ca2+ uptake in the presence of oxalate, it seems unlikely that the Ca2+ release by this agent is caused by the inhibition of the Ca2+ uptake, because the addition of apyrase or hexokinase (which removes ATP within 30 s, so that no more Ca2+ can be accumulated) or vanadate (which inhibits the Ca2+ uptake) resulted in very slow release of Ca2+. These results suggest that the Ca2+ permeability of the Ca2+ store membrane is increased by InsP3. InsP3 did not cause an increase in the Ca2+ permeability of phospholipid vesicles (liposomes), indicating that this agent may bring about Ca2+ release by a specific effect on the physiologically relevant Ca2+ channels or carriers in the non-mitochondrial Ca2+ store site. The passive Ca2+ release by InsP3 was enhanced by ATP and an unhydrolyzable ATP analogue, 5'-adenylyimidodiphosphate, but not by ADP or AMP. The passive Ca2+ release by InsP3 was observed even at 0 degree C. 相似文献
6.
Initial rate measurements of the stoichiometric relationships between H+ ejection, K+ and Ca2+ uptake, and electron transport were carried out on mitochondria from Ehrlich ascites tumor cells grown in mice. With succinate as substrate and N-ethylmaleimide to prevent interfering H+ reuptake via the phosphate carrier, close to 8 H+ were ejected per oxygen atom reduced (H+/O ejection ratio = 8.0); with the NAD-linked substrates pyruvate or pyruvate + malate, the H+/O ejection ratio was close to 12. The average H+/site ratio (H+ ejected/2e-/energy-conserving site) was thus close to 4. The simultaneous uptake of charge-compensating cations, either K+ (in the presence of valinomycin) or Ca2+, was also measured, yielding average K+/site uptake ratios of very close to 4 and Ca2+/site ratios close to 2. It was also demonstrated that each calcium ion enters the respiring tumor mitochondria carrying two positive electric charges. These stoichiometric data observed in mitochondria from Ehrlich ascites tumor cells thus are in complete agreement with similar data on normal rat liver and rat heart mitochondria and suggest that the H+/site ratio of mitochondrial electron transport may be 4 generally. It was also observed that the rate of deltaH+ back-decay in anaerobic tumor mitochondria following oxygen pulses is some 6- to 8-fold greater than in rat liver mitochondria tested at equal amounts of mitochondrial protein. 相似文献
7.
Ca2+ transport and Ca2+-dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands. 总被引:5,自引:0,他引:5
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Ca2+ transport across mammary-gland Golgi membranes was measured after centrifugation of the membrane vesicles through silicone oil. In the presence of 2.3 microM free Ca2+ the vesicles accumulated 5.8 nmol of Ca2+/mg of protein without added ATP, and this uptake was complete within 0.5 min. In the presence of 1 mM-ATP, Ca2+ was accumulated at a linear rate for 10 min after the precipitation of intravesicular Ca2+ with 10 mM-potassium oxalate. ATP-dependent Ca2+ uptake exhibited a Km of 0.14 microM for Ca2+ and a Vmax. of 3.1 nmol of Ca2+/min per mg of protein. Ca2+-dependent ATP hydrolysis exhibited a Km of 0.16 microM for Ca2+ and a Vmax. of 10.1 nmol of Pi/min per mg of protein. The stoichiometry between ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase varied between 0.3 and 0.7 over the range 0.03-8.6 microM-Ca2+. Both Ca2+ uptake and Ca2+-stimulated ATPase were strongly inhibited by orthovanadate, which suggests that the major mechanism by which Golgi vesicles accumulate Ca2+ is through the action of the Ca2+-stimulated ATPase. However, Ca2+ uptake was also decreased by the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone), indicating that it may occur by other mechanisms too. The effect of CCCP may be related to the existence of transmembrane pH gradients (delta pH) in these vesicles: the addition of 30 microM-CCCP reduced delta pH from a control value of 1.06 to 0.73 pH unit. Golgi vesicles also possess a Ca2+-efflux pathway which operated at an initial rate of 0.5-0.57 nmol/min per mg of protein. 相似文献
8.
Right-side-out membrane vesicles of Bacillus subtilis W23 grown on tryptone-citrate medium accumulated Ca2+ under aerobic conditions in the presence of a suitable electron donor. Ca2+ uptake was an electrogenic process which was completely inhibited by carbonyl cyanide m-chlorophenylhydrazone or valinomycin and not by nigericin. This electrogenic uptake of calcium was strongly dependent on the presence of phosphate and magnesium ions. The system had a low affinity for Ca2+. The kinetic constants in membrane vesicles were Km = 310 microM Ca2+ and Vmax = 16 nmol/mg of protein per min. B. subtilis also possesses a Ca2+ extrusion system. Right-side-out-oriented membrane vesicles accumulated Ca2+ upon the artificial imposition of a pH-gradient, inside acid. This system had a high affinity for Ca2+; Km = 17 microM Ca2+ and Vmax = 3.3 nmol/mg of protein per min. Also, a membrane potential, inside positive, drove Ca2+ transport via this Ca2+ extrusion system. Evidence for a Ca2+ extrusion system was also supplied by studies of inside-out-oriented membrane vesicles in which Ca2+ uptake was energized by respiratory chain-linked oxidation of NADH or ascorbate-phenazine methosulfate. Both components of the proton motive force, the pH gradient and the membrane potential, drove Ca2+ transport via the Ca2+ extrusion system, indicating a proton-calcium antiport system with a H+ to Ca2+ stoichiometry larger than 2. The kinetic parameters of this Ca2+ extrusion system in inside-out-oriented membranes were Km = 25 microM and Vmax = 0.7 nmol/mg of protein per min. 相似文献
9.
Rat brain mitochondrial Ca2+ uptake and release were examined in the presence of amiloride (3,5-diamino-6-chloro-N-(diaminomethylene)-pyrazinecarboxamide) and nineteen amiloride analogues. Amiloride, an inhibitor of Na+-Ca2+ exchange in plasmalemma membranes, did not affect energy-dependent Ca2+ uptake, whereas several other analogues were inhibitors. Similarly, amiloride did not alter Ca2+ release in the presence or absence of Na+. However, some analogues were found that stimulated and others that inhibited Ca2+ release. While many of these analogues reduced mitochondrial respiratory control ratios, two analogues were identified which inhibited Ca2+ uptake but did not alter mitochondrial respiratory control. Similarly two analogues were identified which inhibited Ca2+ efflux without affecting respiratory control. 相似文献
10.
The number of protons ejected per pair of electrons passing each energy-conserving site in the electron transport chain (the H+/site ratio) has been investigated in rat liver mitochondria by means of the oxygen pulse technique introduced by Mitchell and Moyle (1967) (Biochem. J. 105, 1147-1162). The usual H+/site values of 2.0 observed by this method were found to be substantially underestimated as a result of the influx of phosphate into the mitochondria. This was shown by three different kinds of experiments. 1. Addition of N-ethylmaleimide or mersalyl, inhibitors of mitochondrial phosphate transport, increased the H+/site ratio from 2.0 to 3.0. The dependence of this effect on the concentration of either inhibitor was identical with that for inhibition of phosphate transport. Added phosphate diminished the H+/site ratio to values below 2.0 in the absence of N-ethylmaleimide. N-Ethylmaleimide protected the elevated H+/site ratio of 3.0 against the deleterious effect of added phosphate, but did not prevent a lowering effect of weak acid anions such as 3-hydroxybutyrate. 2. Prior washing of mitochondria to remove the endogenous phosphate that leaks out during the anaerobic preincubation led to H+/site ratios near 3.0, which were not increased by N-ethylmaleimide. Addition of low concentrations of phosphate to such phosphate-depleted mitochondria decreased the H+/site ratio to 2.0; addition of N-ethylmaleimide returned the ratio to 3.0. 3. Lowering the temperature to 5 degrees, which slows down phosphate transport, led to H+/site values of 3.0 even in the absence of N-ethylmaleimide. The H+/site ratio of 3.0 observed in the absence of phosphate movements was not dependent on any narrowly limited set of experimental conditions. It occurred with either Ca2+ or K+ (in the presence of valinomycin) as mobile permeant cation. It was independent of the concentration of succinate, oxygen, mitochondria, or rotenone, additions of Ca2+, Li+, or Na+ and was independent of medium pH between 6.5 and 7.5. Inhibitors of the transport of ions or acids other than phosphate did not affect the H+/site ratio. These results indicate that re-uptake of endogenous phosphate, lost from mitochondria during anaerobic preincubation, reduces the observed H+ ejection and leads to underestimated H+/site ratios of 2.0 in the oxygen pulse method. When phosphate movements are eliminated by the procedures described above, the observed H+/site ratio is about 3.0. This value appears to be closer to the true H+/site ratio for the primary H+ ejection process during electron transport. 相似文献
11.
Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM. 相似文献
12.
Lanthanides (La3+, Pr3+ and Tb3+) inhibit Na+-gradient-dependent Ca2+ influx into synaptic plasma membrane vesicles. 50% inhibition is obtained by 7 microM lanthanide concentration. The inhibition of the Na+-gradient-dependent Ca2+ uptake exhibits competitive kinetic behaviour. The apparent Km of the Ca2+ influx is increased from 50 microM in the absence of lanthanides to 118 microM in the presence of La3+, 170 microM in the presence of Pr3+ and 130 microM in the presence of Tb3+. The maximal reaction velocity is not altered (8.35 nmol Ca2+ transported per mg protein per min in the absence of lanthanides and 8.16 nmol/mg per min in the presence of lanthanides). Lanthanides also inhibited Na+-gradient-dependent Ca2+ efflux from synaptic plasma membrane vesicles that were preloaded with Ca2+ in a Na+-gradient-dependent manner. Introduction of La3+ into the interior of the synaptic plasma membrane vesicles by rapid freezing of the vesicles in liquid N2 and slow thawing had no effect on either Na+-gradient-dependent Ca2+ influx or efflux. Synaptic plasma membrane vesicles can be preloaded with Ca2+ also in an ATP-dependent manner. This form of Ca2+ uptake is also inhibited by La3+ though at higher concentrations than the Na+-gradient-dependent Ca2+ uptake. Na+-gradient-dependent efflux from synaptic plasma membrane vesicles preloaded in an ATP-dependent fashion ('inside-out' vesicles) unlike efflux from synaptic plasma membrane vesicles preloaded in a Na+-gradient-dependent manner was not inhibited by La3+. These findings suggest that the inhibition by La3+ is manifested asymmetrically on both sides of the synaptic plasma membrane. Lanthanides are probably not transported via the Na+-Ca2+ exchanger since Tb3+ entry measured by fluorescence of Tb3+-dipicolinic acid complex formation occurred at high Tb3+ concentrations only (1.5 mM or above) and was not Na+-gradient dependent. 相似文献
13.
The effects of the three hydrophobic molecules triphenylphosphine, trifluoperazine and 3-nitrophenol on Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles was investigated. When ATP was the substrate, triphenylphosphine (3 microM) increased the amount of Ca2+ accumulated by the vesicles. At high concentrations triphenylphosphine inhibited Ca2+ uptake. This effect varied depending on the ATP concentration and the type of nucleotide used. With ITP there was only inhibition and no activation of Ca2+ uptake by triphenylphosphine. On the other hand, trifluoperazine inhibited Ca2+ accumulation regardless of whether ATP or ITP was used as substrate. When 5 mM oxalate was included in the medium in order to avoid binding of Ca2+ to the low-affinity Ca2(+)-binding sites of the enzyme, both stimulation by triphenylphosphine and inhibition by trifluoperazine were reduced. In leaky vesicles at low Ca2+ concentrations, triphenylphosphine and 3-nitrophenol were competitive inhibitors of ATPase activity at the regulatory site of the enzyme (0.1-1 mM ATP). A striking difference was observed when both the high- and low-affinity Ca2(+)-binding sites were saturated. In this condition, triphenylphosphine and 3-nitrophenol promoted a 3-4-fold increase in the apparent affinity for ATP at its regulatory site. 相似文献
14.
The uptake of Ca2+ by energized liver mitochondria was compared in normal fed as well as in protein-energy malnourished rats. In the presence of phosphate, mitochondria obtained from both groups were able to accumulate Ca2+ from the suspending medium and eject H+ during oxidation of common substrates which activate different segments of the respiratory chain. The rate of Ca2+ uptake was significantly lower in mitochondria from protein-energy malnourished rats. The rates of oxygen consumption and H+ ejection were decreased by 20-30% during oxidation of substrates at the three coupling sites. Similarly, mitochondria from protein-energy malnourished rats exhibit a 34% decrease in the maximal rate of Ca2+ uptake and a 25% lower capacity for Ca2+ load. The stoichiometric relationship of Ca2+/2e- remained unaffected. In steady state, with succinate as a substrate in the presence of rotenone and N-ethylmaleimide, mitochondria from normal fed and protein-energy malnourished rats showed a similar rate of Ca2+ uptake. Furthermore in both groups the stoichiometry of the H+/O ratio was close to 8.0 (H+/site ratio close to 4.0), and of Ca2+/site was close to 2.0. The diminished rate of Ca2+ uptake observed in mitochondria from protein-energy malnourished rats could be explained on the basis of a depressed rate of electron transport in the respiratory chain rather than by an effect at the level of the Ca2+ or H+ transport mechanism per se. 相似文献
15.
Na+-Ca2+ exchange activity in rabbit lymphocyte plasma membranes 总被引:2,自引:0,他引:2
T Ueda 《Biochimica et biophysica acta》1983,734(2):342-346
Plasma membranes of rabbit thymus lymphocytes accumulated Ca2+ when a Na+ gradient (intravesicular greater than extravesicular) was formed across the membranes. Dissipation of the Na+ gradient by the addition of Na+ to the external medium decreased Ca2+ uptake. Ca2+ preloaded into the lymphocytes was extruded when Na+ was added to the external medium. The Ca2+ uptake decreased at acidic pH but increased at alkaline pH (above 8) and the activity was saturable for Ca2+ (apparent Km for Ca2+ was 61 microM and apparent Vmax was 11.5 nmol/mg protein per min). Na+-dependent uptake of Ca2+ was inhibited by tetracaine and verapamil, and partially inhibited by La3+. The uptake was not influenced by orthovanadate. 相似文献
16.
Inhibition of Na+/Ca2+ exchanger activity in cardiac and skeletal muscle sarcolemmal vesicles by monoclonal antibody 44D7 总被引:3,自引:0,他引:3
Monoclonal antibodies 44D7 and 4F2 inhibited specifically the Na+-dependent Ca2+ fluxes characteristic of the Na+/Ca2+ exchanger in cardiac and skeletal muscle sarcolemmal vesicles. Preincubation of membrane vesicles with monoclonal antibody 44D7 inhibited 90% of the Na+-dependent Ca2+ uptake measured in the first 10 s of the reaction and 50% of that measured after 60 s. Ca2+/calmodulin-dependent ATPase activity and ATP-dependent Ca2+ uptake by sarcolemmal vesicles were not affected by monoclonal antibody 44D7 whereas the Na+-dependent release of accumulated Ca2+ was inhibited. In the presence of the 44D7 antigen isolated from human kidney, monoclonal antibody 44D7 could no longer inhibit Na+-dependent Ca2+ fluxes. The distribution of 4F2 antigenic activity in the isolated muscle membrane fractions correlated with that of Na+/Ca2+ exchanger activity; cardiac and skeletal muscle sarcolemmal vesicles expressed higher levels of the antigen than skeletal muscle transverse tubule membrane, while no antigen could be detected in sarcoplasmic reticulum membranes. Our results suggest that monoclonal antibodies 44D7 and 4F2 interact either directly with the Na+/Ca2+ exchanger molecules or with some other protein(s) responsible for the regulation of this activity in the heart and skeletal muscle. 相似文献
17.
MgATP-dependent glucose 6-phosphate-stimulated Ca2+ accumulation in liver microsomal fractions. Effects of inositol 1,4,5-trisphosphate and GTP 总被引:3,自引:0,他引:3
A Benedetti R Fulceri A Romani M Comporti 《The Journal of biological chemistry》1988,263(7):3466-3473
Ca2+ release triggered by inositol 1,4,5-trisphosphate (IP3) and/or GTP has been studied with rough and smooth microsomes isolated from rat liver. Microsomes were loaded with Ca2+ in the presence of MgATP and in the presence or in the absence of glucose 6-phosphate (glucose-6-P) which markedly stimulated the MgATP-dependent Ca2+ accumulation in rough and smooth microsomes (5- and 10-fold, respectively). Upon addition of IP3 (5 microM), rough and smooth microsomes rapidly release a part (not exceeding 20%) of the Ca2+ previously accumulated both in the absence and in the presence of glucose-6-P. Under the same experimental conditions, inositol 1,3,4,5-tetrakisphosphate was ineffective in triggering any Ca2+ release. Upon addition of GTP (10 microM) both the microsomal fractions progressively release the Ca2+ previously accumulated in the presence of glucose-6-P, when 3% polyethylene glycol was also present. In the absence of polyethylene glycol, GTP released Ca2+ from rough microsomes only, and GTP plus IP3 caused a Ca2+ release which was the sum of the Ca2+ releases caused by GTP and IP3 independently. Both IP3 and GTP, added to microsomes at the beginning of the glucose-6-P-stimulated Ca2+ uptake, reduced the Ca2+ accumulation into rough and smooth microsomes without modifying the initial rate (3 min) of Ca2+ uptake. Also in these conditions, the effects of GTP and IP3 were merely additive. These results indicate that both rough and smooth liver microsomes are responsive to IP3 and GTP with respect to Ca2+ release and that IP3 and GTP likely act independently. 相似文献
18.
Ca2+ accumulation and endogenous respiration of sporulating Bacillus megaterium are inhibited to the same extent by electron-transport of inhibitors and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, suggesting that Ca2+ is accumulated by an active transport process. Forespores isolated in stage V of sporulation demonstrated Ca2+-specific carrier-mediated Ca2+ uptake, consistent with downhill transfer [Hogarth & Ellar (1978) Biochem. J. 176, 197-203]. In the present studies forespore Ca2+ uptake was unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and by concentrations of respiratory inhibitor that inhibited forespore endogenous respiration by 85%. These data suggest that Ca2+ enters the isolated forespore by facilitated diffusion. Ca2+ uptake into sporulating protoplasts was completely inhibited by concentrations of respiratory inhibitors that had no effect on either Ca2+ uptake or respiration of stage-V forespores, but which resulted in inhibition of mother-cell membrane NADH oxidase. These results indicate that the mother-cell membrane is a site for active transport of Ca2+ into the sporulating cell. The effects of the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide on mother-cell membrane adenosine triphosphatase, NADH oxidase and protoplast Ca2+ uptake were examined. 相似文献
19.
Release of Ca2+ from a non-mitochondrial store site in peritoneal macrophages treated with saponin by inositol 1,4,5-trisphosphate. 总被引:20,自引:11,他引:9
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The effects of inositol 1,4,5-trisphosphate, prepared from human erythrocyte ghosts, on Ca2+ release from intracellular store sites were studied in saponin-treated guinea pig peritoneal macrophages. Micromolar concentrations of inositol 1,4,5-trisphosphate released Ca2+ within 1 min from store sites which had accumulated Ca2+ in the presence of 10 mM-NaN3. In the presence of 10 mM-NaN3, the Ca2+ accumulated in the presence of oxalate was seen in the endoplasmic reticulum of saponin-treated macrophages by electron microscopy, indicating that the site of Ca2+ released by inositol 1,4,5-trisphosphate may be endoplasmic reticulum-like membranes. When the concentrations of free Ca2+ were over 3.5 X 10(-6) M, the release of Ca2+ by this agent was inhibited. This inhibition may be due to either the higher concentration of extra-vesicular free Ca2+ or the larger accumulation of Ca2+ into the store site or perhaps both effects. MgCl2 also had an inhibitory effect on the Ca2+ release. Inositol 1,4,5-trisphosphate also released Ca2+ from cardiac sarcoplasmic reticulum, but not from erythrocyte inside-out vesicles. 相似文献
20.
On addition of ATP to vesicles derived from the sarcoplasmic reticulum (SR) of skeletal muscle, Ca2+ is accumulated from the external medium. Following uptake, spontaneous release of Ca2+ occurs in the presence or in the absence of ATP. These processes of Ca2+ uptake and release were simulated by using the models derived for ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227; Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730] and for Ca2+ release from passively loaded vesicles [McWhirter, Gould, East & Lee (1987) Biochem. J. 245, 713-722]. The simulations are consistent with measurements of the effects of pH, K+, Ca2+ and Mg2+ on uptake and release of Ca2+. The increase in maximal Ca2+ accumulation observed in the presence of maleate is explained in terms of complexing of Ca2+ and maleate within the SR. The calculated concentration of ADP generated by hydrolysis of ATP has a large effect on the simulations. The effects of an ATP-regenerating system on the measured Ca2+ uptake is explained in terms of both removal of ADP and precipitation of Ca3(PO4)2 within the vesicles. It is concluded that both the process of Ca2+ uptake and the process of Ca2+ release seen with SR vesicles can be interpreted quantitatively in terms solely of the properties of the Ca2+ + Mg2+-activated ATPase. 相似文献