The interaction of calcium and ryanodine with cardiac sarcoplasmic reticulum

The binding of [3H]ryanodine with cardiac sarcoplasmic reticulum vesicles depends on the calcium concentration. Binding in the absence of calcium appears to be non-specific because it shows no saturation up to 20 microM ryanodine. The apparent Km value for calcium varied between 2 and 0.8 microM when the ryanodine concentration varied between 10 and 265 nM. The Hill coefficient for the calcium dependence of [3H]ryanodine binding was near two. Scatchard analysis of ryanodine binding indicated a high-affinity site with a Bmax of 5.2 +/- 0.4 pmol/mg with a Kd of 6.8 +/- 0.1 nM. Preincubation under conditions in which the high-affinity sites were saturated did not result in stimulation of the calcium uptake rate indicative of closure of the calcium channel. Stimulation of calcium uptake rate occurred only at higher concentrations of ryanodine (apparent Km = 17 microM). This stimulation of the calcium uptake rate also required calcium in the submicromolar range. The data obtained support the hypothesis that ryanodine binding to the low-affinity site (Km about 17 microM) is responsible for closure of the calcium release channel and the subsequent increase in the calcium uptake rate of the sarcoplasmic reticulum. Because the number of ryanodine-binding sites is much less than the number of calcium transport pumps the channel is probably distinct from the pump.     

ATP-dependent calcium accumulation in brain microsomes. Enhancement by phosphate and oxalate.

1. ATP-dependent calcium uptake by a rabbit brain vesicular fraction (microsomes) was studied in the presence of phosphate or oxalate. These anions, which are known to form insoluble calcium salts, increased the rate of calcium uptake and the capacity of the vesicles for calcium accumulation. 2. The degree of activation depended on the concentration of phosphate or oxalate. Under optimal conditions, phosphate promoted a 5-fold increase in the amount of calcium stored at steady state. This level was 200-250 nmol Ca-2+/mg protein. 3. Initial rate of calcium uptake followed Michaelis-Menten kinetics with an apparent Km for calcium of 6.7-10-minus 5 M and a V of 44 nmol/min per mg protein. Optimal pH was 7.0. With 2 mM ATP, optimal Mg-2+ concentration was 2 mM. 4. Dintrophenol and NaN3 inhibited calcium uptake in a mitochondria-enriched fraction but not in the microsomal fraction. 5. Calcium uptake activity was compared in the six subfractions prepared from the whole microsomal fraction by means of a sucrose density gradient fractionation. 6. The Mg-2+-dependent ATPase activity of brain microsomes was activated by calcium. Maximal activation was attained with 100 muM CaCl2. Greater calcium concentrations caused a progressive inhibition. 7. The data suggest that the ATP-dependent calcium uptake in brain microsomes, as in muscle microsomes, is brought about by an active transport process, calcium being accumulated as a free ion inside the vesicles.     

Calcium sequestration activity in rat liver microsomes. Evidence for a cooperation of calcium transport with glucose-6-phosphatase

Mechanisms regulating the energy-dependent calcium sequestering activity of liver microsomes were studied. The possibility for a physiologic mechanism capable of entrapping the transported Ca2+ was investigated. It was found that the addition of glucose 6-phosphate to the incubation system for MgATP-dependent microsomal calcium transport results in a marked stimulation of Ca2+ uptake. The uptake at 30 min is about 50% of that obtained with oxalate when the incubation is carried out at pH 6.8, which is the pH optimum for oxalate-stimulated calcium uptake. However, at physiological pH values (7.2-7.4), the glucose 6-phosphate-stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. The Vmax of the glucose 6-phosphate-stimulated transport is 22.3 nmol of calcium/mg protein per min. The apparent Km for calcium calculated from total calcium concentrations is 31.9 microM. After the incubation of the system for MgATP-dependent microsomal calcium transport in the presence of glucose 6-phosphate, inorganic phosphorus and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. In the system for the glucose 6-phosphate-stimulated calcium uptake, glucose 6-phosphate is actively hydrolyzed by the glucose-6-phosphatase activity of liver microsomes. The latter activity is not influenced by concomitant calcium uptake. Calcium uptake is maximal when the concentration of glucose 6-phosphate in the system is 1-3 mM, which is much lower than that necessary to saturate glucose-6-phosphatase. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose-6-phosphatase multicomponent system. The physiological implications of such a cooperation are discussed.     

Uptake of calcium ions into microsomes isolated from Physarum polycephalum.

Membranous vesicles (microsomes) were isolated from plasmodia of the acellular slime mold, Physarum polycephalum. The microsomes were about 0.2 about 0.2 micronM in diameter, and about 10 nm thick. The main protein component of the vesicles had a molecular weight of 100,000 daltons. Calcium ions were taken up by the microsomes only in the presence of Mg2+- ATP. The maximum amount of Ca2+ ions accumulated in the microsomes was 0.24 micronmole/mg protein. The Ca2+ uptake was not accelerated by oxalate. The ATPase [EC 3.6.1.3] activity required Ca2+ ions for full activation. The concentration of Ca2+ ions required for half-maximum activation was about 1 micronM. The Km and Vm values were 53 micronM and 1.6 micronmole/(mg-min), respectively. About 0.2 mole of Ca2+ ions was taken up by the microsomes, coupled with the hydrolysis of 1 mole of ATP. THE ATPase activity and Ca2+ uptake of the microsomes were not inhibited by sodium azide. Furthermore, electron microscopic examination showed that mitochondrial contamination was slight. These results suggest that a vesicular calcium transport system, analogous to the sacroplasmic reticulum in skeletal muscle, is involved in regulation of the Ca2+ concentration in plasmodia of Physarum.     

ATP-dependent calcium transport in rat parotid microsomes. I. Localization, properties, Ca2+-ATPase activity and phosphoenzyme formation

ATP-dependent Ca2+ transport was studied in rat parotid microsomes; the activity appears to be associated with rough endoplasmic reticulum vesicles, as indicated by marker distribution in subcellular fractions and by electron microscopic observations. Purified rough microsomes exhibit an ATP-dependent Ca2+ accumulation and a Ca2+-dependent ATPase activity; these activities are similarly stimulated by K+ and display an apparent Km for free calcium of 0.6-0.7 microM. A phosphoprotein, with a molecular weight of about 110,000, was detected after short incubation with [gamma 32P] ATP and CaCl2; it is suggested that this compound represents a phosphorylated intermediate form of the Ca2+-ATPase.     

Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump

Mild trypsin treatment of canine cardiac microsomes consisting largely of sarcoplasmic reticulum vesicles produced a severalfold activation of oxalate-facilitated calcium uptake. The increase in calcium uptake was associated with an increase in ATP hydrolysis. Proteases other than trypsin were also effective although to a lesser degree. Trypsin produced a shift of the Ca2+ concentration dependency curve for calcium uptake toward lower Ca2+ concentrations, which was almost identical with that produced by phosphorylation of microsomes by cyclic AMP dependent protein kinase when the trypsin and the protein kinase were present at maximally activating concentrations. The Hill numbers (+/- SD) of the Ca2+ dependency after treatment of microsomes with trypsin (1.5 +/- 0.1) or protein kinase (1.7 +/- 0.1) were similar and were not significantly different from those for untreated control microsomes (1.6 +/- 0.1 and 1.8 +/- 0.1, respectively). Autoradiograms of sodium dodecyl sulfate-polyacrylamide electrophoretic gels indicate that 32P incorporation into phospholamban (Mr 27.3K) or its presumed monomeric subunit (Mr 5.5K) was markedly reduced when trypsin-treated microsomes were incubated in the presence of cyclic AMP dependent protein kinase and [gamma-32P]ATP compared to control microsomes incubated similarly but pretreated with trypsin inhibitor inactivated trypsin. The activation of calcium uptake by increasing concentrations of trypsin was paralleled by the reduction of phosphorylation of phospholamban. Trypsin treatment of microsomes previously thiophosphorylated in the presence of cyclic AMP dependent protein kinase and [gamma-35S]thio-ATP did not result in a loss of 35S label from phospholamban, which suggests that phosphorylation of phospholamban protects against trypsin attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ischemia-Induced Inhibition of Calcium Uptake into Rat Brain Microsomes Mediated by Mg2+/Ca2+ ATPase

Abstract: It is well established that ischemia is associated with prolonged increases in neuronal intracellular free calcium levels. Recent data suggest that regulation of calcium uptake and release from the endoplasmic reticulum is important in maintaining calcium homeostasis. The endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase is the major mechanism for sequestering calcium in this organelle. Inhibition of this enzyme may play a causal role in the loss of calcium homeostasis. In order to investigate the effect of ischemia on calcium sequestration into the endoplasmic reticulum, microsomes were isolated from control and ischemic whole brain homogenates by differential centrifugation. Calcium uptake was measured by radioactive calcium (⁴⁵Ca²⁺) accumulation in the microsomes mediated by Mg²⁺/Ca²⁺ ATPase. Ischemia caused a statistically significant inhibition of presteady-state and steady-state calcium uptake. Duration of ischemia was directly proportional to the degree of inhibition. Decreased calcium uptake was shown not to be the result of increased calcium release from ischemic compared with control microsomes nor the result of selective isolation of ischemic microsomes from the homogenate with a decreased capacity for calcium uptake. The data demonstrate that ischemia inhibits the ability of brain microsomes to sequester calcium and suggest that loss of calcium homeostasis is due, in part, to ischemia-induced inhibition of endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase.     

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats.

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of MG-ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of mitochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg-ATP and 20 muM calcium approximately 38 nmol of calcium per mg of microsomal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 muM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction. Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.     

Ca2+ transport studied with arsenazo III in Tetrahymena microsomes. Effects of calcium ionophore A23187 and trifluoperazine

Transport of Ca2+ in microsomal membrane vesicles of the Tetrahymena has been investigated using arsenazo III as a Ca2+ indicator. The microsomes previously shown to carry a Mg2+-dependent, Ca2+-stimulated ATPase (Muto, Y. and Nozawa, Y. (1984) Biochim. Biophys. Acta 777, 67-74) accumulated calcium upon addition of ATP and Ca2+ sequestered into microsomal vesicles was rapidly discharged by the Ca2+ ionophore A23187. Kinetic studies indicated that the apparent Km for free Ca2+ and ATP are 0.4 and 59 microM, respectively. The Vmax was about 40 nmol/mg protein per min at 37 degrees C. The calcium accumulated during ATP-dependent uptake was released after depletion of ATP in the incubation medium. Furthermore, addition of trifluoperazine which inhibited both (Ca2+ + Mg2+)-ATPase and ATP-dependent Ca2+ uptake rapidly released the calcium accumulated in the microsomal vesicles. These observations suggest that Tetrahymena microsome contains both abilities to take up and to release calcium and may act as a Ca2+-regulating site in this organism.     

Localization of ATP-dependent calcium transport activity in mouse pancreatic microsomes

Electron-dense deposits representing calcium oxalate crystals which result from ATP-dependent calcium uptake have been localized within vesicles of of a heavy microsomal fraction prepared from mouse pancreatic acini. In the absence of either ATP or oxalate, no electron-dense deposits could be observed. By subfractionation of microsomes on discontinuous sucrose gradients, it could be shown that the highest energy-dependent calcium transport activity was associated with the rough endoplasmic reticulum. In rough microsomes, the 45Ca2+-uptake measured was 7 times greater than that of smooth microsomes in the presence of ATP and oxalate and about 3 times greater in he presence of ATP alone. When ribosomes were released from the rough endoplasmic reticulum vesicles by treatment with KCl in the presence of puromycin, the stripped microsomes showed a 40% increase in the specific 45Ca2+-uptake activity measured in he presence of ATP and oxalate and an increase of 80 to 90% in the presence of ATP alone. From these results it can be concluded that the calcium transport activity of microsomes prepared from mouse pancreatic acini is located predominantly in the rough endoplasmic reticulum membrane.     

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of Mg · ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of miltochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg · ATP and 20μM calcium approximately 38 nmol of calcium per mg of microsormal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min. with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 μM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction.Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced calcium uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.Rats were treated with the steroid deoxycorticosterone acetate for ten and thirty days to induce hypertension. After ten days of deoxycorticosterone acetate although hypertension is present, there is no change in calcium uptake activity of aorta microsomes, renal microsomes or renal plasma membranes. After 30 days of deoxycorticosterone acetate treatment calcium uptake activity of renal microsomes is reduced. A variable decrease in calcium uptake activity is observed with aorta microsomes. Renal plasma membrane calcium uptake remains unchanged.     

Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.

The role of ATP-dependent calcium uptake into intracellular storage compartments is an essential feature of hormonally induced calcium signaling. Thapsigargin, a non-phorboid tumor promoter, increasingly is being used to manipulate calcium stores because it induces a hormone-like elevation of cytosolic calcium. It has been suggested that thapsigargin acts through inhibition of the endoplasmic reticulum calcium pump. We have directly tested the specificity of thapsigargin on all of the known intracellular-type calcium pumps (referred to as the sarcoplasmic or endoplasmic reticulum Ca-ATPase family (SERCA]. Full-length cDNA clones encoding SERCA1, SERCA2a, SERCA2b, and SERCA3 enzymes were expressed in COS cells, and both calcium uptake and calcium-dependent ATPase activity were assayed in microsomes isolated from them. Thapsigargin inhibited all of the SERCA isozymes with equal potency. Furthermore, similar doses of thapsigargin abolished the calcium uptake and ATPase activity of sarcoplasmic reticulum isolated from fast twitch and cardiac muscle but had no influence on either the plasma membrane Ca-ATPase or Na,K-ATPase. The interaction of thapsigargin with the SERCA isoforms is rapid, stoichiometric, and essentially irreversible. These properties demonstrate that thapsigargin interacts with a recognition site found in, and only in, all members of the endoplasmic and sarcoplasmic reticulum calcium pump family.     

Energy-dependent calcium transport in endoplasmic reticulum of adipocytes.

The endoplasmic reticulum from isolated rat adipocytes has the ability to actively accumulate calcium. The calcium uptake was characterized using the 20,000 X g supernatant (S1 fraction) of total cellular homogenate. Endoplasmic reticulum vesicles isolated from the S1 fraction as a 160,000 X g microsomal pellet prior to testing demonstrated little ability to accumulate calcium. The calcium uptake in the S1 fraction was localized to the endoplasmic reticulum vesicles by morphologic appearance, by the use of selective inhibitors of calcium uptake, and by high speed sedimentation of the accumulated calcium. The uptake was MgATP- and temperature-dependent and was sustained by the oxalate used as the intravesicular trapping agent. Uptake was linear with time for at least 30 min at all calcium concentrations tested (3 to 100 muM) and exhibited a pH optimum of approximately 7.0. The sulfhydryl inhibitor p-chloromercuribenzene sulfonate produced a dose-dependent inhibition of calcium uptake with total inhibition at 0.07 mumol/mg protein. Ruthenium red and sodium azide inhibited less than 5% of the uptake at concentrations (5 muM and 10 mM, respectively) which completely blocked calcium uptake by mitochondria isolated from the same cells. The Km for calcium uptake was 10 muM total calcium which corresponded to approximately 3.6 muM ionized calcium in the assay system. The maximum velocity of the uptake was 5.0 nmol (mg of microsomal protein)-1 (min)-1 at 24 degrees under the assay conditions used and exhibited a Q10 of 1.8. The uptake activity of the endoplasmic reticulum vesicles in the S1 fraction exhibited a marked time- and temperature-dependent lability which might account in part for the lack of uptake in the isolated microsomal fraction. This energy-dependent calcium uptake system would appear to be of physiologic importance to the regulation of intracellular calcium.     

The mechanism of calcium uptake by liver microsomes: effect of anions and ionophores

The mechanism of calcium uptake by liver microsomes was investigated using various anions and ionophores. Calcium uptake was shown to be specific to microsomes and unlikely to be due to contamination by plasma membranes by correlation of calcium uptake to the marker enzymes specific for these two fractions. Under the conditions employed, phosphates, sulfate, chloride, acetate, nitrate, thiocyanate, maleate, succinate and oxalate all stimulated calcium uptake by microsomes, but to different degrees. The greatest effect (4-6-fold) was observed with phosphate. On the contrary, phosphate is the only anion that stimulates the plasma membrane calcium uptake to any significant degree. Treatment of isolated microsomes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) resulted in inhibition of ATP- and anion-dependent calcium uptake. A lipid-permeable organic acid such as maleate retained its ability to promote calcium uptake in DIDS-treated microsomes. However, a lipophilic anion, such as nitrate, stimulated calcium uptake only in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). In addition, 2 microM valinomycin, when added in the absence or presence of 10 to 100 mM K+, had no stimulatory effect on calcium uptake. These results appear to be consistent with a model in which the active uptake of calcium into microsomes involves electroneutral Ca2+-nH+ exchange.     

Uptake and release of calcium by microsomes of nonvascular smooth muscle.

Microsomes prepared from guinea-pig ileum longitudinal smooth muscle and rat uterus continuously sequester calcium for a one hour period in the presence of Mg-ATP as an energy source and oxalate anion as a trapping agent. Dithiothreitol is essential for maximal calcium uptake activity of the rat uterus microsomes. On sucrose density gradients, calcium uptake of the smooth muscle microsomes appears to be associated with intracellular membrane (sarcoplasmic reticulum). Release of sequestered calcium from the longitudinal muscle microsomes is very slow (20% in 50 minutes). A small labile fraction (20%) is released by EGTA (1 mM) in 10 minutes. Rapid release of sequestered calcium (90% in 10 minutes) occurs in presence of the calcium ionophore A23187 (2 μM) or in the presence of chlorpromazine (1 mM).     

Active transport of calcium in membrane vesicles from Mycobacterium phlei.

Active transport of calcium ions has been demonstrated in inside-out membrane vesicles from Mycobacterium phlei mediated by respiratory linked substrates as well as by ATP hydrolysis. The uptake of calcium exhibited an apparent Km of 80 microM and V of 16.6 nmol calcium uptake x min-1 x mg protein-1. A fortyfold concentration gradient for calcium ions was calculated for both the ATP-induced and the respiration-induced transport of calcium. Removal of coupling-factor-latent ATPase resulted in the complete loss of ATP-driven Ca2+ transport whereas the respiration-driven uptake was reduced by 40-50%. The uptake of calcium was inhibited by the proton conducting ionophores carbonylcyanide m-chlorophenylhydrazone and Gramicidin-D. The accumulated calcium was freely exchangeable with external calcium and was rapidly released by the addition of inhibitors of energy transduction, proton-translocating uncouplers or the ionophore A23187. The uptake of the weak base, methylamine, upon the oxidation of respiratory-linked substrates or the hydrolysis of ATP showed the generation of a protein gradient (inside acidic) which was partially collapsed on the addition of calcium ions. These results suggest that a Ca2+/H+ antiport mechanism may be responsible for the transport of calcium.     

The effect of cytochrome P-450 and reduced glutathione on the ATP-dependent calcium pump of hepatic microsomes from male rats

In the presence of ATP hepatic microsomes sequester calcium. This sequestration is thought to be important in the modulation of free cytosolic calcium concentration. We find that on the addition of NADPH the uptake of calcium by the hepatic microsomes is inhibited 27-85%. This inhibition is reversed by the addition of 1 mM reduced glutathione (85-91% of control), incubation under a nitrogen atmosphere (112% of control), or incubation in a 80% carbon monoxide/20% oxygen atmosphere (75% of control). Superoxide dismutase had no effect on the inhibition, while catalase reversed the inhibition by 35%. The addition of 1 mM reduced glutathione at 2 and 5 min after the addition of NADPH led to uptakes of calcium which paralleled the uptake seen when the reduced glutathione was added at the beginning of the incubation. The effect of reduced glutathione showed saturation kinetics with a Km of 10 microM. Together these data suggest that cytochrome P-450 reduces the activity of the microsomal ATP-dependent calcium pump both by the production of hydrogen peroxide and by the direct oxidation of the protein thiols. The reversal of this effect by reduced glutathione appears to be enzymatically catalyzed.     

Stoichiometric and electrostatic characterization of calcium binding to native and lipid-substituted adenosinetriphosphatase of sarcoplasmic reticulum

The stoichiometry of calcium binding to specific sites (i.e., those producing enzyme activation) was found to be 8-10 nmol/mg protein in native sarcoplasmic reticulum vesicles, and 13.9-15.4 nmol/mg of ATPase purified by non-ionic detergent solubilization and anion exchange chromatography. Parallel measurements of phosphoenzyme yielded levels of 4.0-4.9 and 6.0-7.7 nmol/mg of protein in the two preparations, respectively, demonstrating that each 115 kDa ATPase chain includes one catalytic site and two calcium binding sites. The apparent association constant, K = (6 +/- 2) X 10(5) M-1, and the binding cooperativity, nH = 1.9, were unchanged when measurements were carried out with native sarcoplasmic reticulum vesicles and when the membrane surface charge was altered by lipid substitution with phosphatidylcholine or phosphatidylserine, at neutral pH in the presence of 10 mM MgCl2 and 80 mM KCl. On the other hand, the apparent association constant was increased in the absence of Mg2+ or, to a lesser extent, in the absence of monovalent cations. It was also observed that the cooperative character of the calcium binding isotherms was reduced in low ionic-strength media. Analysis of the electrostatic effects indicates that the calcium-binding domain is shielded from the membrane phospholipid surface charge by virtue of its location within the ATPase protein. The effects of various electrolytes are attributed to monovalent-cation binding in the calcium-binding domain. The apparent loss of cooperativity of the calcium binding isotherms at low ionic strength is attributed to a progressive displacement of the titration curve which is minimal at low degrees of saturation and becomes larger at higher degrees of saturation. This behavior is described quantitatively by the progressive effect of calcium binding on an electrostatic potential generated by localized protein charge densities within, or near, the calcium-binding domain.     

Calcium transport ATPase of canine cardiac sarcoplasmic reticulum. A comparison with that of rabbit fast skeletal muscle sarcoplasmic reticulum.

To define the mechanism responsible for the slow rate of calcium transport by cardiac sarcoplasmic reticulum, the kinetic properties of the Ca2+-dependent ATPase of canine cardiac microsomes were characterized and compared with those of a comparable preparation from rabbit fast skeletal muscle. A phosphoprotein intermediate (E approximately P), which has the stability characteristics of an acyl phosphate, is formed during ATP hydrolysis by cardiac microsomes. Ca2+ is required for the E approximately P formation, and Mg2+ accelerates its decomposition. The Ca2+ concentration required for half-maximal activation of the ATPase is 4.7 +/- 0.2 muM for cardiac microsomes and 1.3 +/- 0.1 muM for skeletal microsomes at pH 6.8 and 0 degrees. The ATPase activities at saturating concentrations of ionized Ca2+ and pH 6.8, expressed as ATP hydrolysis per mg of protein, are 3 to 6 times lower for cardiac microsomes than for skeletal microsomes under a variety of conditions tested. The apparent Km value for MgATP at high concentrations in the presence of saturating concentrations of ionized Ca2+ is 0.18 +/- 0.03 ms at pH 6.8 and 25 degrees. The maximum velocity of ATPase activity under these conditions is 0.45 +/- 0.05 mumol per mg per min for cardiac microsomes and 1.60 +/- 0.05 mumol per mg per min for skeletal microsomes. The maximum steady state level of E approximately P for cardiac microsomes, 1.3 +/- 0.1 nmol per mg, is significantly less than the value of 4.9 +/- 0.2 nmol per mg for skeletal microsomes, so that the turnover number of the Ca2+-dependent ATPase of cardiac microsomes, calculated as the ratio of ATPase activity to the E approximately P level is similar to that of the skeletal ATPase. These findings indicate that the relatively slow rate of calcium transport by cardiac microsomes, whem compared to that of skeletal microsomes, reflects a lower density of calcium pumping sites and lower Ca2+ affinity for these sites, rather than a lower turnover rate.     

Characterization of the hormone-sensitive Ca2+ uptake activity of the hepatic endoplasmic reticulum

The characteristics and kinetics of calcium uptake activity were studied in isolated hepatic microsomes. The sustained accumulation of calcium was ATP- and oxalate-dependent. Glucagon increased microsomal Ca2+ uptake upon either in vivo injection, or in vitro perfusion of the hormone in the liver. In contrast, the effect of insulin depended on the route of administration. Calcium accumulation by subsequently isolated hepatic microsomes increased when insulin was injected intraperitoneally whereas it decreased when the hormone was perfused directly into the liver. These effects of glucagon and insulin were dose dependent. When insulin was added to the perfusate prior to the addition of glucagon, insulin blocked the glucagon-stimulated increase in microsomal Ca2+ uptake. Cyclic AMP mimicked the effect of glucagon on microsomal Ca2+ accumulation when the cyclic nucleotide was perfused into the liver. The effects of glucagon and insulin on the kinetics of hepatic microsomal Ca2+ uptake were investigated. In microsomes isolated from perfused rat livers treated with glucagon the V of the uptake was significantly increased over the control values (12.2 vs. 8.6 nmol Ca2+ per min per mg protein, P less than 0.02). In contrast, the addition of insulin to the perfusate significantly decreased the V of Ca2+ uptake by subsequently isolated microsomes (6.8 vs. 8.3 nmol Ca2+ per min per mg protein, P less than 0.05). However, neither hormone had an effect on the apparent Km for Ca2+ (4.1 +/- 0.5 microM) of the reaction. The effect of these hormones on the activity of Ca2+-stimulated ATPase was also studied. No significant changes in either V or Km for Ca2+ of the enzymatic reaction were detected.