ADENOSINE TRIPHOSPHATE-LINKED CONCENTRATION OF CALCIUM IONS IN A PARTICULATE FRACTION OF RABBIT MUSCLE

ATPase and ATP-dependent calcium ion concentration was studied with a membrane fraction isolated from homogenized rabbit skeletal muscle by differential centrifugation. Electron micrographs of the fraction indicate that it consists mainly of resealed tubules and vesicles of the endoplasmic reticulum. The up-to-1400-fold concentration of calcium in this fraction might be explained by proposing the existence of an energy-requiring system for the transport of calcium ions into the tubules or vesicles.     

Effects of Mg2+ on calcium accumulation by two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium accumulation by two fractions of sarcoplasmic reticulum presumably derived from longitudinal tubules (light vesicles) and terminal cisternae (heavy vesicles) was examined radiochemically in the presence of various free Mg2+ concentrations. Both fractions of sarcoplasmic reticulum exhibited a Mg2+-dependent increase in phosphate-supported calcium uptake velocity, though half-maximal velocity in heavy vesicles occurred at a much higher free Mg2+ concentration than that in light vesicles (i.e., approx. 0.90 mM vs. approx. 0.02 mM Mg2+). Calcium uptake velocity in light vesicles correlated with Ca2+-dependent ATPase activity, suggesting that Mg2+ stimulated the calcium pump. Calcium uptake velocity in heavy vesicles did not correlate with Ca2+-dependent ATPase activity, although a Mg2+-dependent increase in calcium influx was observed. Thus, Mg2+ may increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles. Analyses of calcium sequestration (in the absence of phosphate) showed a similar trend in that elevation of Mg2+ from 0.07 to 5 mM stimulated calcium sequestration in heavy vesicles much more than in light vesicles. This difference between the two fractions of sarcoplasmic reticulum was not explained by phosphoenzyme (EP) level or distribution. Analyses of calcium uptake, Ca2+-dependent ATPase activity, and unidirectional calcium flux in the presence of approx. 0.4 mM Mg2+ suggested that ruthenium red (0.5 microM) can also increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles, with no effect in light vesicles. These functional differences between light and heavy vesicles suggest that calcium transport in terminal cisternae is regulated differently from that in longitudinal tubules.     

A comparison of vesicles derived from terminal cisternae and longitudinal tubules of sarcoplasmic reticulum isolated from rabbit skeletal muscle

Sarcoplasmic reticulum vesicles were separated into heavy (derived from terminal cisternae) and light (derived from longitudinal tubules) fractions, according to Meissner [Biochim. Biophys. Acta, 389, 51-68 (1975)]. The similar Ca2+ sensitivities of phosphoprotein formation, ATPase activity and calcium uptake, and the similar phosphoprotein turnover rates (ATPase/phosphoprotein formation) of both fractions indicate that the same ATPase enzyme is present in the terminal cisternae and longitudinal sarcoplaxmic reticulum. The higher V for Ca2+-activated ATPase activity and calcium uptake in the light fraction correlated with the higher concentration of ATPase enzyme per mg of membrane protein in this fraction. In both the presence and absence of calcium-precipitating anions, the light fraction stored more calcium than the heavy. The Ca2+ dependence of calcium release after addition of EGTA appeared similar in both fractions, but the rate of calcium release was more rapid in the light fraction. These findings suggest that calcium release may occur more rapidly from longitudinal than terminal cisternae portions of the sarcoplasmic reticulum and that calcium release, like calcium uptake, may be mediated by the ATPase enzyme in the sarcoplasmic reticulum membrane. Although the activation energies for Ca2+-activated ATPase activity above and below the transition temperature were significantly different for the heavy and light fractions, their transition temperatures were similar. Partial purification of the ATpase enzyme by deoxycholate treatment modified the activation energies of the light but not the heavy fraction and caused the activation energies to become similar. The phosphoprotein levels of heavy and light vesicles did not become similar after deoxycholate treatment, although gel electrophoretograms indicated both samples contained > 90% ATPase protein. These results indicate the protein-lipid associations in these two fractions may be different.     

Calcium transport in transverse tubules isolated from rabbit skeletal muscle

Isolated transverse tubule vesicles free of sarcoplasmic reticulum transport calcium with high affinity in the presence of ATP. The calcium transport by transverse tubules differs from calcium transport by sarcoplasmic reticulum. It is not increased by oxalate or phosphate, it has a different temperature dependence, it is inhibited by sub-micromolar concentrations of orthovanadate, it is stimulated by calmodulin, and is inhibited by quercetin without causing calcium release. The rates of calcium transport by transverse tubules are two orders of magnitude lower than those of sarcoplasmic reticulum, suggesting that the calcium pump protein of transverse tubules is a minor component of the membrane. Addition of calmodulin to transverse tubule vesicles--treated with high salt in the presence of EGTA to remove endogenous calmodulin--caused a marked stimulation of transport rates at low concentrations of calcium, and decreased from 1.0 to 0.3 microM the calcium concentration at which half-maximal rates of transport were obtained. A role for the transverse tubule calcium pump in maintaining low sarcoplasmic calcium concentrations is proposed.     

Effects of Mojave toxin on rat skeletal muscle sarcoplasmic reticulum.

Effects of the lethal fraction (MD-9) from the venom of the Mojave rattlesnake, Crotalus scutulatus, on sarcoplasmic reticulum were investigated. The calcium sequestering activity of the vesicles was reduced by the lethal fraction and subsequent release of calcium was enhanced. These effects were observed to be dependent upon MD-9 concentration and the length of preincubation time with the vesicles. An enhanced ATPase activity that was affected by concentration and MD-9 preincubation time was also observed. Both calcium uptake and ATPase activity effects may be due to a phospholipase activity associated with the fraction.     

The Calcium Sensitivity of Individual Secretory Vesicles Is Invariant with the Rate of Calcium Delivery

Differences in the calcium sensitivity of individual secretory vesicles can explain a defining feature of calcium-regulated exocytosis, a graded response to calcium. The role of the time dependence of calcium delivery in defining the observed differences in the calcium sensitivity of sea urchin egg secretory vesicles in vitro was examined. The calcium sensitivity of individual secretory vesicles (i.e., the distribution of calcium thresholds) is invariant over a range of calcium delivery rates from faster than micromolar per millisecond to slower than micromolar per second. Any specific calcium concentration above threshold triggers subpopulations of vesicles to fuse, and the size of these subpopulations is independent of the time course required to reach that calcium concentration. All evidence supports the hypothesis that the magnitude of the free calcium is the single controlling variable that determines the fraction of vesicles that fuse, and that this fraction is established before the application of calcium. Submaximal responses to calcium cannot be attributed to alterations in the calcium sensitivity of individual secretory vesicles arising from the temporal properties of the calcium delivery. Models that attempt to explain the cessation of fusion using changes in the distribution of calcium thresholds arising from the rate of calcium delivery and/or adaptation are not applicable to this system, and thus cannot be general.     

The mechanism of calcium control of smooth muscle tonic contraction

It has been shown in the experiments carried out on a fraction of inverted vesicles of myometrium sarcolemma that ATP-dependent Ca2+ transport system prevents dissipation of the calcium gradient directed from the intervesicular space outward with subsequent establishment of the stationary level of cation content inside the membrane vesicles (a blocker of electro-controlled calcium channels diltiasems was present in the incubation medium). Ortovanadatean inhibitor of the sarcolemma calcium pump suppressed Ca2+ stationary exchange in the vesicles fraction. The value of calcium stationary content in the vesicle membrane was regulated both by a change of the calcium pump activity (by varying Mg2+ concentration in the ATP-containing incubation medium), and by modification of calcium permeability of the vesicles (by varying concentration of ionophore A-23187 in this medium). In the presence of diltiasem and ortovanadate the Ca2+ basal current entering the myocytes from hyperpotassium washing solution activated the smooth muscle tonic contraction. In the absence of ortovanadate no contractile response was observed. On the basis of the evidence obtained a mechanism of calcium control of myometrium tonic contraction is proposed. According to this mechanism the Ca2+ current entering the unexcited myocytes under physiological conditions is efficiently compensated by the calcium pump of the sarcolemma. The inhibition of the latter (or an increase of the sarcolemma basal calcium permeability) provides further slow transition of the stationary value of Ca2+ concentration in the myoplasm to a new higher level and activation of the smooth muscle contraction accordingly.     

Regulation of actin polymerization by membrane fraction of platelets

We studied the interaction between the purified membrane fraction of human platelets and the polymerization of skeletal actin. The viscosity of actin was measured by the falling ball method. The fraction suppressed the polymerization of actin in the presence of 20 mM KCl and 0.4 mM EGTA. The addition of calcium ion or thrombin to the fraction did not cause suppression. A DNase I affinity column bound the membrane fraction in the presence of calcium ion. The frozen membrane fraction and the vesicles reconstituted with lipids from the platelet membrane enhanced the polymerization of actin. Trypsinized membrane fraction and the membrane fraction treated with phospolipase A2 enhanced the polymerization of actin, but membrane fraction treated with phospholipase C had no effect. The reconstituted membrane vesicles mentioned above lowered the critical concentration for actin polymerization. These findings suggested that the polymerization of intracellular actin is enhanced not only by the mobilization of calcium ion, but also by biochemical changes in the membrane lipids.     

Submaximal Responses in Calcium-triggered Exocytosis Are Explained by Differences in the Calcium Sensitivity of Individual Secretory Vesicles

A graded response to calcium is the defining feature of calcium-regulated exocytosis. That is, there exist calcium concentrations that elicit submaximal exocytotic responses in which only a fraction of the available population of secretory vesicles fuse. The role of calcium-dependent inactivation in defining the calcium sensitivity of sea urchin egg secretory vesicle exocytosis in vitro was examined. The cessation of fusion in the continued presence of calcium was not due to calcium-dependent inactivation. Rather, the calcium sensitivity of individual vesicles within a population of exocytotic vesicles is heterogeneous. Any specific calcium concentration above threshold triggered subpopulations of vesicles to fuse and the size of the subpopulations was dependent upon the magnitude of the calcium stimulus. The existence of multiple, stable subpopulations of vesicles is consistent with a fusion process that requires the action of an even greater number of calcium ions than the numbers suggested by models based on the assumption of a homogeneous vesicle population.     

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.     

Static and dynamic pH-dependent components of calcium exchange in the fraction of smooth muscle sarcolemma vesicles

It is proved that in the fraction of inverted vesicles of the myometrium sarcolemma there are two components of calcium metabolism which depend on the proton concentration in the incubation medium. The first component, a static one, identified under alkalization of the incubation medium from pH 6.0 up to pH 8.0 under equilibrium conditions (Ca2+ concentration inside and outside vesicles is the same) is manifested as an increase of the calcium capacity of vesicles at the expense of Ca2+-binding centres of the inner surface of membrane vesicles. The second component, a dynamic one, is represented as a passive transmembrane flow of Ca2+ outflowing from the vesicles induced by alkalization of the extravesicle space. Alkalization-stimulated Ca2+ release from vesicles is analyzed kinetically. Possible functional role of two components of pH-dependent metabolism of Ca2+ in providing the electrical and pharmacological-mechanical conjugation in the smooth-muscular tissue is under discussion.     

ELECTROPHORETIC AND IMMUNOLOGICAL STUDIES OF THE MALE ACCESSORY REPRODUCTIVE GLAND PROTEINS OF POECILOCERA PICTA FABR. (ACRIDIDAE: ORTHOPTERA)

Electrophoretic separation of the protein extracts of the whole accessory reproductive gland (ARG) reveals a progressive increase in number of fractions from day 0 to day 12 of adult life. The number of fractions secreted by the different types of tubules, viz., white tubules, hyaline tubules and seminal vesicles, varies. The protein concentration of the ARG of 12-day-old insects is four times higher than that of newly emerged adult insects. Three protein fractions of the ARG extract are immunologically precipitatale by antiserum to whole accessory gland complex. Immunoelectrophoresis of the oocyte extract from mated females and total protein extract of empty spermatophores revealed that one fraction of the oocyte protein and two of the spermatophores have their immunological counterparts in the whole ARG extract.     

Calcium release from two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium release from sarcoplasmic reticulum vesicles presumably derived from longitudinal tubules (LSR) and terminal cisternae (HSR) of rabbit skeletal muscle was investigated by dual wavelength spectrophotometry using the calcium-indicator antipyrylazo III. In 120 mM KCl, 5 mM MgCl2, 30 microM, CaCl2, 50 microM MgATP, 100 microM antipyrylazo III, 40 mM histidine (pH 6.8, 25 degrees C), LSR and HSR sequestered approx. 115 nmol calcium/mg, and then spontaneously released calcium. Analysis of ATP hydrolysis and phosphoenzyme level during LSR and HSR calcium sequestration indicated that this calcium release process was passive, occurring in the virtual absence of ATP and phosphoenzyme. Moreover, subsequent addition of ATP reinitiated the calcium sequestration-release sequence. Calcium release by HSR was more than 4-times faster than that by LSR. Analysis of the calcium release phase demonstrated a biexponential decay for both LSR (0.10 and 0.63 min-1) and HSR (0.26 and 1.65 min-1), suggestive of heterogeneity within each fraction. Replacement of 120 mM KCl with either 120 mM choline chloride, 240 mM sucrose, or H2O reduced maximal calcium sequestration by LSR, but had less effect on LSR calcium release rate constants. In the case of HSR, these changes in the ionic composition of the medium drastically reduced calcium release rate constants with little effect on calcium content. These marked differences between LSR and HSR are consistent with the hypothesis that the calcium permeability of the terminal cisternae is greater and more sensitive to the ionic environment than is that of the longitudinal tubules of sarcoplasmic reticulum.     

Effects of the fraction (1-10 kDa) of the brain of a Yakut horse on kinetic parameters of Ca2+ transport system in sarcolemma vesicles of cardiomyocytes]

The effect of the fraction (1-10 kDa) obtained from the brain of cold-adapted animal (Yakut horse) on Ca2+ transport in sarcolemma vesicles of cardiomyocytes was investigated. It was shown that during insertion of Yakut horse brain fraction into incubation medium at the concentration from 10(-9) M to 3.10(-5) M at Ca2+ transport substrate concentration from 0.1 mM to 1.0 mM, the rate of Ca2+ passive penetration into vesicles slightly increased and at Ca2+ transport substrate concentration 3 mM, which is physiologic, a decrease of rate values was established for all concentrations of the fraction (1-10 kDa) of Yakut horse. While studying the kinetics of an active Ca2+ transport for all investigated concentrations of the fraction (1-10 kDa) of Yakut horse brain from 10(-9) M to 3.10(-5) M at Ca2+ concentration in incubation medium from 10(-7) to 3.10(-6) M, calcium accumulation rates by vesicles exceeded control values. So we can suppose that application of brain fraction (1-10 kDa) of genotypically cold-adapted animal, results in a decrease of intracellular Ca2+ concentration.     

Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder. Studies using a fast-reaction apparatus

Regulation of the sodium permeability of the luminal membrane is the major mechanism by which the net rate of sodium transport across tight epithelia is varied. Previous evidence has suggested that the permeability of the luminal membrane might be regulated by changes in intracellular sodium or calcium activities. To test this directly, we isolated a fraction of the plasma membrane from the toad urinary bladder, which contains a fast, amiloride-sensitive sodium flux with characteristics similar to those of the native luminal membrane. Using a flow-quench apparatus to measure the initial rate of sodium efflux from these vesicles in the millisecond time range, we have demonstrated that the isotope exchange permeability of these vesicles is very sensitive to calcium. Calcium reduces the sodium permeability, and the half-maximal inhibitory concentration is 0.5 microM, well within the range of calcium activity found in cells. Also, the permeability of the luminal membrane vesicles is little affected by the ambient sodium concentration. These results, when taken together with studies on whole tissue, suggest that cell calcium may be an important regulator of transepithelial sodium transport by its effect on luminal sodium permeability. The effect of cell sodium on permeability may be mediated by calcium rather than by sodium itself.     

The synthesis of 15N- and deuterium-substituted, spin-labeled analogues of NAD+ and their use in EPR studies of dehydrogenases

Calcium efflux and EGTA-induced calcium release from an internal platelet membrane fraction have been studied after the oxalate-supported calcium uptake had reached steady state. Increasing external calcium concentrations stimulate the calcium efflux velocity, with an apparent half-maximal stimulation at about 5 microM outside calcium concentration and a maximal velocity of calcium efflux of 4.66 +/- 2.32 nmol X min-1 X mg-1. Moreover, the ratio of the liberated calcium on the loaded calcium seems to be independent of the increasing external calcium concentration. Increasing the calculated internal calcium concentration by varying the oxalate potassium concentration from 10 mM to 1 mM results in an increase of the liberated calcium from the membrane vesicles from 7.4% to 63%, respectively, without changing the calcium efflux velocity. Similar conclusions can be drawn from the observation of results from the calcium efflux and EGTA-induced calcium release methods. Moreover, calcium pump reversal does not seem to be responsible for the calcium efflux or calcium release. All these different points added to the previously described regulation of calcium efflux by the catalytic subunit of cAMP protein kinase suggest us that the mechanism of calcium liberation by the platelet membranes is different from the calcium uptake.     

Preparation and characterization of longitudinal tubules of sarcoplasmic reticulum from fast skeletal muscle

Sarcoplasmic reticulum (SR) serves a central role in calcium uptake and release, thereby regulating muscle relaxation and contraction, respectively. Recently, we have isolated fractions referable to longitudinal tubules (R2) and terminal cisternae (R4), the two major types of sarcoplasmic reticulum (A. Saito et al. (1984) J. Cell Biol. 99, 875-885). The terminal cisternae contain two types of membranes, the calcium pump membrane and the junctional face membrane. The terminal cisternae are filled with electron-opaque contents which serve as a Ca2+ reservoir. The longitudinal tubules consist mainly of the calcium pump membrane. In this study, we describe a new longitudinal tubule fraction (F2) and characterize it together with the R2 and R4 SR fractions. The calcium pump membrane of the longitudinal tubules is a highly specialized membrane consisting of about 90% calcium pump protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive changes in morphology can be observed in the SR fractions referable to osmotic differences during the fixation conditions using either glutaraldehyde-tannic acid or osmium tetroxide fixatives. The changes include swelling or shrinkage and aggregation of the compartmental contents when the fixative contains calcium ions. The two types of SR have different osmotic permeability to the same medium, as indicated by differential swelling or shrinkage. Both longitudinal tubule and terminal cisternae vesicles of SR appear larger and are spherical vesicles when the glutaraldehyde-tannic acid fixative is isotonic as compared with the "standard" fixation method. We have previously reported that the ruthenium red-sensitive calcium release channels are localized to the terminal cisternae. The terminal cisternae as isolated are leaky to Ca2+ since these channels are in the "open state" (S. Fleischer et al. (1985) Proc. Natl. Acad. Sci USA 82, 7256-7259). Thus, the Ca2+, Mg2+-dependent ATPase (Ca2+ ATPase) rate is only slightly enhanced in the presence of a Ca2+ ionophore, which dissipates the Ca2+ gradient across the SR membrane. We now find that preincubation with ruthenium red restores the tight coupling of the Ca2+ ATPase activity to Ca2+ transport. That is to say, ATPase activity is reduced and the addition of ionophore stimulates the Ca2+ ATPase activity 4- to 7-fold. The Ca2+ ATPase activity in longitudinal tubules is already tightly coupled. It is minimal after a Ca2+ gradient has been generated, but can be stimulated 9- to 20-fold when the Ca2+ gradient is dissipated with ionophore. This finding suggests that the Ca2+ ATPase activity in SR is tightly coupled to Ca2+ transport in situ.     

Electron cytochemistry of oxalate-stimulated calcium uptake in microsomes from the smooth muscle of the pig stomach

Summary A microsomal fraction was isolated from the smooth muscle of the antrum of the pig stomach by differential centrifugation. Electron microscopy of the negatively stained material showed that this fraction is heterogeneous in composition. The microsomes accumulated calcium in the presence of ATP, magnesium and oxalate. The amount of calcium taken up per mg protein was in the same range as observed for other smooth muscle microsomal preparations. Although this amount is much smaller than that in the microsomal fraction of skeletal muscle, calcium oxalate crystals were formed in some vesicles, as occurs in the skeletal muscle fragmented sarcoplasmic reticulum. Through the presence of the calcium oxalate crystals, many of these vesicles acquired sufficient mass and density to allow them to be isolated by centrifugation. A purification of about 40 fold in terms of calcium content was reached.     

Calcium homeostasis in larval and adult Drosophila melanogaster

Calcium homeostasis in Drosophila melanogaster was examined in response to the challenges imposed by growth, reproduction and variations in dietary calcium content. Turnover time for calcium, calculated as the time for (45)Ca(2+)to accumulate to half the steady state value of 3.46 nmol/fly, was 3.3 days. Although larvae weighed 2x as much as adults, they contained 3-4x as much calcium. Anterior Malpighian tubules (Mts) contain much more calcium than posterior Mts, accounting for 25-30% of the calcium content of the whole fly. In response to a 6.2-fold increase in dietary calcium level, calcium content of whole flies increased only 10%. Hemolymph calcium concentration ( approximately 0.5 mM) was similar in males and females and in animals raised on diets differing in calcium content. Fluid secretion rate, secreted fluid calcium concentration, and transepithelial calcium flux in tubules isolated from flies raised on high and low calcium diets did not differ significantly. Malpighian tubules secrete calcium at rates sufficient to eliminate whole body calcium content in 0.5 and 3 days for tubules secreting fluid at basal and maximal rates, respectively. It is suggested that flies absorb high quantities of calcium from the diet and maintain homeostasis through the combined effects of elimination of calcium in fluid secreted by the Malpighian tubules and the sequestration of calcium in granules, especially within the distal segment of the anterior pair of Malpighian tubules.     

Role of matrix vesicles in calcification.

The role of matrix vesicles in the calcification process was investigated in vitro. Isolated vesicles were unable to transport calcium actively. The ATPase activity was not stimulated by calcium in the presence of an optimal magnesium concentration. At a physiological substrate concentration of pyrophosphate, the pyrophosphatase had a pH optimum around 7.0. The vesicles nucleated calcium phosphate precipitation independently of the presence of hydrolyzable phosphate compounds. It is suggested that vesicles induce calcification by nucleating calcium phosphate precipitation and through the local destruction of pyrophosphate, a crystallization inhibitor.