ATP-supported Ca2 Research Articles

Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle

The accumulation of covalently modified proteins is an important hallmark of biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification of specific protein targets. Recently, Narayanan et al. [Narayanan, Jones, Xu and Yu (1996) Am. J. Physiol. 271, C1032-C1040] reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significant age-dependent change in ATP-supported Ca2+-uptake activity for slow-twitch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-muscle sarcoplasmic-reticulum (SR) Ca2+-ATPase isoforms SERCA1 and SERCA2a from fast-twitch and slow-twitch muscle respectively. We find a significant age-dependent loss in the Ca2+-ATPase activity (26% relative to Ca2+-ATPase content) and Ca2+-uptake rate specifically in SR isolated from predominantly slow-twitch, but not from fast-twitch, muscles. Western immunoblotting and amino acid analysis demonstrate that, selectively, the SERCA2a isoform progressively accumulates a significant amount of nitrotyrosine with age (approximately 3.5+/-0. 7 mol/mol of SR Ca2+-ATPase). Both Ca2+-ATPase isoforms suffer an age-dependent loss of reduced cysteine which is, however, functionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO-) (but not NO/O2) results in the selective nitration only of the SERCA2a, suggesting that ONOO- may be the source of the nitrating agent in vivo. A correlation of the SR Ca2+-ATPase activity and covalent protein modifications in vitro and in vivo suggests that tyrosine nitration may affect the Ca2+-ATPase activity. By means of partial and complete proteolytic digestion of purified SERCA2a with trypsin or Staphylococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC-electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dependent tyrosine nitration to the sequence Tyr294-Tyr295 in the M4-M8 transmembrane domain of the SERCA2a, close to sites essential for Ca2+ translocation.

Effects of aging on sarcoplasmic reticulum function and contraction duration in skeletal muscles of the rat.

The impact of aging on the Ca2+ pump function of skeletal muscle sarcoplasmic reticulum (SR) was investigated using SR-enriched membrane vesicles isolated from the slow-twitch soleus muscle (SM) and the relatively fast-twitch gastrocnemius muscle (GM) isolated from adult (6-8 mo old) and aged (26-28 mo old) Fischer 344 rats. In addition, isometric twitch characteristics of SM and GM were determined in situ in adult and aged rats under anesthesia. The rates of ATP-supported Ca2+ uptake by SM SR was markedly lower ( approximately 50%) in the aged compared with adult at varying Ca2+ (0.11-8.24 microM) concentrations. Kinetic analysis of the data revealed age-associated decrease in maximum activity reached (Vmax) and increase in the concentration of Ca2+ giving half of Vmax. In contrast, no significant age-related difference was observed in ATP-supported Ca2+ uptake activity of GM SR. The Ca(2+)-stimulated adenosinetriphosphatase (ATPase) activities and the amount of Ca(2+)-ATPase protein did not vary significantly with aging in SM or GM SR. Also, no significant age-related difference was observed in the content of the ryanodine receptor (Ca(2+)-release channel) or the Ca2+ binding protein, calsequestrin in SM and GM SR. In isometrically contracting SM, the time to peak force, half-relaxation time, and contraction duration were significantly prolonged in the aged compared with adult, whereas there was no age-related difference in maximum developed force. None of these isometric twitch parameters differed significantly with age in the GM. These results demonstrate that the effects of aging on skeletal muscle contractile properties and SR function are muscle specific. Furthermore, the data strongly suggest that impairment in SR Ca2+ pump function, apparently due to uncoupling of ATP hydrolysis from Ca2+ transport, contributes to the age-associated slowing of relaxation in the soleus muscle.

Two Residues That May Ligate Ca2+ in Transmembrane Domain Six of the Plasma Membrane Ca2+-ATPase

In order to identify Ca2+ ligands in the putative transmembrane domain 6 of the plasma membrane Ca2+ pump, amino acids Asn879, Met882, Asp883, and Ser887 were singly altered. Asn879, Met882, and Asp883 were chosen because the corresponding amino acids have been proposed as Ca2+ ligands in the sarcoplasmic reticulum Ca2+ pump (Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca2+ with the pump could be studied without interference from calmodulin binding. The mutants at Asn and Asp did not carry out ATP-supported Ca2+ uptake and formed no acylphosphate from [gamma-32P]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca2+ pump, these two are Ca2+ ligands. However, all the mutants at the position of Met882 showed some activity. Indeed, the Met882--> Ile mutant was fully active at a saturating Ca2+ concentration and only the K1/2 for Ca2+ activation was shifted slightly upward. Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca2+ pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca2+ pumps. The mutant Ser887--> Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca2+ ligand and could not replace the missing Thr at position Met882.

In vitro inhibition of calcium binding by alloxan and of calcium transport by isolated vascular smooth muscle microsomes.

The primary purpose of this study was to investigate the possible direct toxic effect of alloxan on the Ca2+ handling by microsomal membranes isolated from rat mesenteric arteries. It was found that preincubation of the vascular muscle microsomal membranes with alloxan had a suppressive effect on both binding of Ca2+ (in the absence of ATP) and ATP-supported Ca2+ transport. Such an inhibition was time, dose, pH, and temperature dependent. ATP-supported Ca2+ transport was more susceptible to the inhibitory action of alloxan than Ca2+ binding. Unlike alloxan, another commonly used diabetogenic drug, streptozotocin, was not effective in causing such an in vitro inhibition of Ca2+ handling.

Ca2+-activated ATPase in microsomes from human liver.

Human liver microsomal fractions exhibit ATP-supported Ca2+ uptake which is half-maximal at 7 X 10(-7) M free Ca2+ in the presence of oxalate. Ca2+ uptake is coupled to a Ca2+-stimulated ATPase activity, which is half-maximal at 4 X 10(-7) M free Ca2+. Catalysis involves formation of an Mr = 116,000 phosphoprotein with stability characteristics of an acylphosphate compound suggested to represent a phosphoryl protein intermediate of the Ca2+-ATPase. Phosphorylation is half-maximal at about 10(-6) M free Ca2+. The Mr = 116,000 protein is highly susceptible to proteolysis with trypsin. The phosphorylated active site was localized in an Mr = 58,000 primary tryptic fragment and in an Mr = 34,000 subfragment. Analyses on the mechanism of the Ca2+-ATPase suggest the following reaction sequence: formation of an ADP-reactive phosphoenzyme (Mr = 116,000) with bound Ca2+, which can transphosphorylate its Pi to ADP, giving rise to synthesis of ATP; reversible transformation of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, which cannot further react with ADP; hydrolytical cleavage, probably catalyzed by Mg2+, of the ADP-unreactive phosphoenzyme with liberation of Pi. Comparison with the Ca2+-transport ATPase in sarcoplasmic reticulum of skeletal muscle led us to suggest that the Mr = 116,000 Ca2+-ATPase belongs to the class of E1P . E2P-ATPases and might be operative as a Ca2+-transport ATPase at the level of the endoplasmic reticulum in human liver.

Calmodulin stimulation of plasmalemmal Ca2+-pump of canine aortic smooth muscle.

Plasma-membrane-enriched fractions of canine aortic smooth muscle possess an ATP-supported Ca2+ accumulation which has an absolute requirement for Mg2+ and a high affinity for Ca2+ (Km approximately 0.5 microM). The rate of ATP-supported Ca2+ transport is not affected by several calmodulin antagonists, but is stimulated by exogenously added calmodulin. The maximal effect of calmodulin on the rate of ATP-dependent Ca2+ transport (at 5.0 microM Ca2+) occurs at 10 micrograms/ml calmodulin and represents an approximately 3-fold stimulation. This calmodulin stimulation of Ca2+ transport does not require pretreatment of the membranes by EGTA and is an intrinsic property of the plasma membranes. A high-affinity Ca2+-ATPase (Km for Ca2+ approximately 0.5 microM) is also present in the aortic smooth muscle plasma membrane. This high-affinity Ca2+-ATPase does not require Mg2+ for catalytic activity, but is in fact inhibited by increasing Mg2+ concentrations. Calmodulin at concentrations effective for the stimulation of the ATP-dependent Ca2+ transport has no effect on the high-affinity Ca2+-ATPase activity or on the basal ATPase activity stimulated by 5 mM Mg2+ or Ca2+. Our results indicate that isolated plasma membranes of canine aortic smooth muscle contain no endogenous calmodulin. The ability of exogenously added calmodulin to stimulate the rate of ATP-dependent Ca2+ transport by vascular smooth muscle plasma membranes suggests that calmodulin may play a role in lowering the cytoplasmic concentration of ionized calcium during vasodilatation. An Mg2+-independent, but not an Mg2+-dependent high-affinity Ca2+-ATPase, was identified in the plasma membranes. This may be separate from the plasmalemmal Ca2+-pump.

The calcium pump in rat liver endoplasmic reticulum. Demonstration of the phosphorylated intermediate.

Rat liver microsomal fractions enriched with smooth-surfaced vesicles possess an ATP-supported Ca2+ transport activity, which is stimulated by oxalate and half-maximally activated at 3.5 X 10(-7) M free Ca2+. Catalysis of Ca2+ transport involves transient covalent binding of the terminal phosphate from ATP by the vesicles, resulting in the formation of a Mr 118,000 phosphopeptide, which is acid precipitable and unstable in the presence of hydroxylamine, which may be characteristic for an acylphosphate. Phosphorylation of the Mr 118,000 peptide requires the presence of Ca2+, while dephosphorylation is markedly accelerated by Mg2+. In the presence of Ca2+ and Mg2+ phosphorylation proceeds much faster than dephosphorylation, indicating that the latter may be rate limiting for the hydrolysis of ATP. The Mr 118,000 peptide is estimated to represent about 2% of total smooth-surfaced endoplasmic reticulum membrane protein. Comparative studies with sarcoplasmic reticulum from rat skeletal muscle suggest extensive homology of the Ca2+ transport ATPases.

Membrane fractionation of canine aortic smooth muscle: Subcellular distribution of calcium transport activity

Various subcellular membrane fractions were isolated from dog aortic smooth muscle by conventional differential centrifugation followed by isopycnic centrifugation on a sucrose density gradient. These subcellular fractions were characterized by membrane marker enzyme activities, morphological features and the electrophoretic patterns on a sodium dodecyl sulfate polyacrylamide gel. Our results showed that the microsomal membrane fraction isolated by differential centrifugation was very heterogeneous and contained substantial amount of plasma membranes which could be further enriched as a light density fraction on the sucrose density gradient. The subcellular distribution of Ca2+ binding in the absence of ATP and Ca2+ transport in the presence of ATP closely paralleled the distribution of plasma membrane markers. The ATP-supported Ca2+ transport was inhibited by several Ca2+ ionophores, enhanced by inorganic phosphate and oxalate ions and cosedimented toward higher density in a continuous source density gradient with plasma membrane marker enzyme activity in the presence of digitonin. Our present work strongly suggests that plasma membrane is the predominant component of microsomal fraction and responsible for most, if not all, of the azide-insensitive ATP-supported Ca2+ accumulation.

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. II. (Ca2+ + Mg2+)-ATPase-dependent Ca2+ transport activity.

Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.

An analytical approach to the preparation and characterization of subcellular membranes from canine mesenteric arteries.

Subcellular membrane fractions were isolated from dog mesenteric arteries by differential and isopynic sucrose density gradient centrifugations. Isolated membrane fractions were characterized by marker enzyme activities, morphological features and sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns. Our results show that the microsomal fraction isolated by conventional differential centrifugation was highly heterogenous and contained substantial amount of plasma membranes which could be further enriched as a light density membrane fraction on a discontinuous sucrose density gradient. The microsomal fraction and its subfractions were vesicular in appearance under electron microscope and were capable of binding and actively transporting Ca2+. The binding of Ca2+ and ATP-supported Ca2+-transport in the presence or absence of oxalate paralleled the distribution of plasma membrane marker enzyme activities suggesting that plasma membranes in vascular smooth muscle may play a major role in handling Ca2+ and thus the control of contractile function.

Anion effects on in vitro sarcoplasmic reticulum function. The relationship between anions and calcium flux.

Isolated sarcoplasmic reticulum vesicles exhibited different functional characteristics in the presence of zwitterionic as compared to anionic buffers. In the absence of oxalate, dicarboxylic anions (e.g. maleate, succinate) in a dose-dependent manner enhanced ATP-supported Ca2+ accumulation, the ensuing spontaneous Ca2+ release, and Ca2+-dependent ATPase activity compared to zwitterionic buffers (e.g. piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Hepes). This was not attributed to ionic strength and osmotic effects. The additional anion-dependent Ca2+ accumulation was linked to augmented Ca2+-dependent ATPase activity, and both could be induced by the addition of anion at any time during Ca2+ accumulation as long as ATP was present. Since the initial Ca2+ accumulation rates and acyl phosphoenzyme formation were the same between the two buffer classes, and the presence of either oxalate (a Ca2+-precipitating anion) or A23187 (a Ca2+ ionophore) abolished differences in Ca2+-dependent ATPase activity between the two buffer classes, it is likely that conditions favoring high intravesicular Ca2+ concentration allow the expression of the observed effect of the anions. Initial spontaneous Ca2+ release in the presence of maleate was not caused by ATP depletion, and it was virtually absent in Pipes buffer. The rate of spontaneous release was also stimulated in a dose-dependent manner by the dicarboxylic anions, with the time of release being related to the time of anion addition and not ATP addition. A later, more rapid release phase in either maleate or Pipes buffer corresponded to ATP depletion, and could be duplicated at any time in the Ca2+ accumulation/release cycle by the addition of an ATP trap. With an ATP-regenerating system present or with very high ATP concentrations, the maximal peak Ca2+ accumulation in Pipes buffer could approach that in maleate buffer. The data suggest that dicarboxylic anions stimulate the filling of a Ca2+ compartment from which spontaneous Ca2+ release occurs.

Transport of calcium ions by Ehrlich ascites-tumour cells.

Ehrlich ascites-tumour cells accumulate Ca2+ when incubated aerobically with succinate, phosphate and rotenone, as revealed by isotopic and atomic-absorption measurements. Ca2+ does not stimulate oxygen consumption by carefully prepared Ehrlich cells, but des so when the cells are placed in a hypo-osmotic medium. Neither glutamate nor malate support Ca2+ uptake in 'intact' Ehrlich cells, nor does the endogenous NAD-linked respiration. Ca2+ uptake is completely dependent on mitochondrial energy-coupling mechansims. It was an unexpected finding that maximal Ca2+ uptake supported by succinate requires rotenone, which blocks oxidation of enogenous NAD-linked substrates. Phosphate functions as co-anion for entry of Ca2+. Ca2+ uptake is also supported by extra-cellular ATP; no other nucleoside 5'-di- or tri-phosphate was active. The accumulation of Ca2+ apparently takes place in the mitochondria, since oligomycin and atractyloside inhibit ATP-supported Ca2+ uptake. Glycolysis does not support Ca2+ uptake. Neither free mitochondria released from disrupted cells nor permeability-damaged cells capable of absorbing Trypan Blue were responsible for any large fraction of the total observed energy-coupled Ca2+ uptake. The observations reported also indicate that electron flow through energy-conserving site 1 promotes Ca2+ release from Ehrlich cells and that extra-cellular ATP increase permeability of the cell membrane, allowing both ATP and Ca2+ to enter the cells more readily.

Rhodamine 6G: A POTENT INHIBITOR OF MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION

Abstract The lipophilic dye, rhodamine 6G, has been shown to be a potent inhibitor of oxidative phosphorylation, with a Ki of about 3 µm, corresponding to 1.2 nmoles of dye per mg of mitochondrial protein. ATP-supported Ca2+ accumulation was blocked, but not that driven by succinate oxidation. Concentrations of rhodamine 6G above 20 µm slowly uncouple respiration and inhibit respiration-dependent Ca2+ uptake. Neither the dinitrophenol-stimulated ATPase nor uncoupled respiration of intact mitochondria were inhibited by the dye, even at 50 µm. Arsenate-stimulated respiration, on the other hand, was partially blocked by 7.5 µm dye, but energy-linked phosphate accumulation was unaffected. Below 10 µm rhodamine 6G, no mitochondrial swelling, loss of matrix protein, or endogenous K+, Ca2+, or Mg2+, was observed. The dye was bound very tightly to mitochondria, being present at 1.3 and 0.8 nmoles per mg of protein for the inner and outer membranes, respectively. Binding was also monitored by direct measurement of H+ release into the suspending medium in the presence of rhodamine 6G. Some 0.6 to 0.75 mole of H+ was ejected per mole of dye added, of which only about 50% was firmly bound. A red shift occurred on binding, the λmax increasing from 527 to 535 nm. The related compound, rhodamine B, a free acid and uncharged at pH 7, was completely without influence on mitochondrial energy-linked functions. Kinetic studies on the rate of ADP-stimulated H+ uptake in the presence and absence of rhodamine 6G yielded nonlinear, Lineweaver-Burk plots and were of noncompetitive type. The Km for ADP decreased from 56 to 29 µm in the presence of 4 µm rhodamine 6G. When the data is expressed as a Hill plot, straight lines are obtained, with the value of (η), the interaction coefficient, increasing from 1.28 to 1.72 after dye addition. Rhodamine 6G did not inhibit the Mg2+ATPase in sonicated submitochondrial particles, unlike aurovertin. It did, however, block adenine nucleotide binding to intact mitochondria, both for [14C]ATP and [14C]ADP. Inhibition by 10 µm dye for low levels of added nucleotide was identical with that caused by 10 µm atractyloside, but was only very slight at higher nucleotide concentrations (0.2 mm). The results of this study support the conclusion that rhodamine 6G blocks the adenine nucleotide translocase apparently being similar to atractyloside and bongkrekic acid. However, in sharp contrast to these inhibitors, rhodamine 6G did not inhibit the 2,4-dinitrophenol-stimulated ATPase of intact mitochondria. Consequently, uncouplers destroy the dye's ability to interfere with the translocase, and this suggests a lipid binding to be involved in the action of rhodamine 6G.

Uncoupler-stimulated Adenosine Triphosphatase Activity: DEFICIENCY IN INTACT MITOCHONDRIA FROM MORRIS HEPATOMAS AND ASCITES TUMOR CELLS

Abstract 1. Well coupled mitochondria isolated from six hepatomas differing widely in growth rate and degree of differentiation were found to exhibit little or no 2,4-dinitrophenol-stimulated ATPase activity in sharp contrast to normal liver mitochondria. Well coupled mitochondria from L1210 and Ehrlich ascites tumor cells were also found to have markedly reduced uncoupler-stimulated ATPase activity when compared with normal liver mitochondria. 2. When intact mitochondria from one of the hepatomas (7800) were studied in greater detail, it was found that normal uncoupler-dependent ATPase activity could not be demonstrated under a variety of conditions. These included (a) the use of uncouplers more potent than 2,4-dinitrophenol; (b) alteration of osmotic and ionic conditions; (c) variation of temperature between 20 and 40°; (d) variation of pH between 6.9 and 9.5; and (e) inclusion of either defatted bovine albumin or oxidizable substrate in the assay medium. 3. Neither forward nor reverse energy-dependent reactions in hepatoma 7800 mitochondria are altered, nor is their sensitivity to uncoupling agents. In the forward direction, hepatoma mitochondria were found to exhibit normal acceptor control and P:O ratios sensitive to uncouplers, and to exhibit uncoupler-stimulated respiration. In the reverse direction, the ATP-Pi exchange rate was found to be the same in hepatoma 7800 and control liver mitochondria and inhibited almost completely in both cases by 2,4-dinitrophenol. In addition, the capacity for ATP-supported Ca2+ accumulation was similar for hepatoma 7800 and control liver mitochondria. 4. Unlike hepatoma mitochondria, mitochondria isolated from regenerating liver 24, 48, and 72 hours after partial hepatectomy exhibit normal levels of uncoupler-stimulated ATPase activity. 5. These results are interpreted most simply by assuming that hepatoma mitochondria, and perhaps mitochondria from L1210 and Ehrlich ascites tumor cells, contain two principal sites of action for uncoupling agents, one of which may become expressed during the normal to neoplastic transition.

The role of mitochondria in modifying the cellular ionic environment: studies of the kinetic accumulation of calcium by rat liver mitochondria.

1. The affinity of ATP-supported Ca2+ accumulation for both Ca2+ and ATP was determined from initial rate studies employing isolated rat liver mitochondria. TheKm values for “free” Ca2+ and ATP were calculated to be of the order of 2 μM and 100 μM, respectively. TheKm for ATP decreased as the Ca2+ concentration was increased. 2. The curve relating initial rates of Ca2+ accumulation to Ca2+ concentration was singmoidal in shape; values obtained for the Hill coefficient were in the range 1.5–1.9. 3. Concomitant with the ATP-stimulated accumulation of Ca2+, ATP translocation was itselt increased in the presence of Ca2+. This stimulation took place independently of Ca2+ accumulation. 4. Decreasing the pH of the incubation medium decreased the rate of Ca2+ accumulation. This inhibition was competitive in that the affinity of mitochondrial for Ca2+ could be altered. The maximal rate of accumulation did not change with change in pH. 5. The permeant anions inorganic phosphate and acetate stimulated the accumulation of Ca2+ in a non-competitive manner. Both theVmax and Km varied when either of the anions were present. 6. The data are discussed in relation to the role that mitochondria play in controlling the cellular ionic environment.