Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.     

Effect of tris(hydroxymethyl)aminomethane on isolated sarcoplasmic reticulum vesicles.

The preincubation of isolated sarcoplasmic reticulum vesicles in Tris-Cl (pH 7.3) increases their (Ca²⁺ + Mg²⁺)-dependent adenosine triphosphatase activity and decreases their ATP-dependent Ca uptake capacity. These effects of Tris are dependent on the preincubation time and the Tris concentration; they are maximal below 10 μm Ca and decrease upon the increase of Ca concentration in the preincubation media, and they increase upon the increase of the preincubation pH. Differences in ATPase activity between preincubated and control vesicles are abolished by A23187 but not by carbonyl cyanide p-trifluoromethoxy phenyl hydrazone. The results suggest that: (i) Preincubation of the vesicles in Tris causes an increase of their permeability for Ca, or a membrane damage. (ii) Tris must diffuse within the vesicles to promote these effects. (iii) Ca prevents these effects by decreasing the membrane permeability for Tris. The basic findings were reproduced replacing Tris by imidazole.     

The mechanism of increase in the ATPase activity of sarcoplasmic reticulum vesicles treated with n-alcohols.

Treatment of sarcoplasmic reticulum vesicles with aqueous n-alcohols caused inhibition of calcium uptake and enhancement of ATPase activity. With increasing alcohol concentration, the ATPase activity reached a maximum (in the case of n-butanol, at about 350 mM) and then decreased. The effect of n-butanol was extensively studied. The purified ATPase enzyme and leaky vesicles treated with Triton X-100 or phospholipase A showed high ATPase activity in the absence of n-butanol. With increasing n-butanol concentration, their atpase activities began to decrease above about 250 mM n-butanol, without any enhancement. In the presence of ATP, the turnover rate of calcium after calcium accumulation had reached a steady level was the same as that at the initial uptake. n-Butanol did not affect these rates. Kinetic analyses of these experiments were carried out. The mechanisms of calcium transport and of increase of ATPase activity in the presence of alcohol were interpreted as follows. After calcium accumulation had reached a steady level, fast influx and efflux continued; the influx was coupled with phosphorylated enzyme (E-P) formation and most of the efflux was coupled with rephosphorylation of ATP from ADP and E-P. The observed ATPase activity is the difference between these two reactions. If alcohol molecules make the vesicles leaky, calcium ions will flow out without ATP synthesis and the apparent ATPase activity will increase. The effect of alcohols on sarcoplasmic reticulum vesicles was separated into two actions. The enhancement of ATPase activity was attributed to a leakage of calcium ions from the vesicles, while the decrease of ATPase activity at higher concentrations of alcohols was attributed to denaturation of the ATPase enzyme itself. The two effects were interpreted in terms of equilibrium binding of alcohol molecules to two different sites of the vesicles; leakage and denaturation sites. Similar analysis was carried out for various n-alcohols from methanol to n-heptanol. The apparent free energies of binding of the methylene groups of n-alcohols were evaluated to be -863 cal/mol for the leakage site, and -732 cal/mol for the denaturation site.     

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.     

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.     

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.     

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.     

Mechanism of action of ryanodine on cardiac sarcoplasmic reticulum

Ryanodine was found to initially inhibit calcium uptake by cardiac sarcoplasmic reticulum. This initial depression was followed by a later marked stimulation of calcium uptake. These effects were noted when calcium uptake was measured in the presence or absence of oxalate. The requirement for preincubation with ryanodine was highly dependent on ryanodine concentration and temperature. The mechanism of action of ryanodine clearly was not an effect on oxalate entry or calcium oxalate precipitation because the effects were also observed in the absence of oxalate. Ryanodine also had no effect on passive calcium efflux from actively loaded vesicles. Because ryanodine had no effect on Ca2+-ATPase activity under defined conditions of an ATP-regenerating system and no calcium gradient, we suggest ryanodine does not change the stoichiometry of the pump. Our results are consistent with the hypothesis that ryanodine closes a calcium channel in a subpopulation of the vesicles.     

Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

A platelet subcellular fraction, sedimenting between 14,000 and 40,000 g and consisting primarily of membrane vesicles, accumulates up to 200–400 nmoles calcium/mg protein in the presence of ATP and oxalate. Steady-state levels of calcium accumulation are attained in 40–60 min. Calcium uptake requires adenosine triphosphate (ATP), is enhanced by oxalate, and is accompanied by the release of inorganic phosphate. Calcium accumulation and phosphate release require magnesium and are inhibited by Salyrgan (10 µM) and adenosine diphosphate (ADP) (1 mM), but not by ouabain (0.1 mM). The ATPase activity is stimulated by low concentrations of calcium (5–10 µM) and is inhibited by 2 mM EGTA. Electron microscopic histochemistry using lead nitrate to precipitate released phosphate results in lead precipitates localized primarily at the inner surface of membrane vesicles. These results provide evidence for a membrane ATPase that is stimulated by low concentrations of calcium and may be involved in the transport of calcium across the membrane. It is postulated that the observed calcium uptake activity is an in vitro manifestation of a calcium extrusion pump in the intact platelet.     

Activation of phosphorylating vesicles by net transfer of phosphatidyl choline by phospholipid transfer protein

Vesicles formed with phosphatidyl ethanolamine, phosphatidyl choline, cardiolipin, coupling factors and hydrophobic proteins from bovine heart mitochondria catalyzed a rapid³²P_i-ATP exchange. When phosphatidyl choline was deleted during the assembly of the vesicles, little³²P_i-ATP exchange was observed. Exchange activity was induced by incubating such deficient vesicles with phosphatidyl choline liposomes in the presence of a phosphatidyl choline transfer protein isolated from bovine heart. Transfer of [³²P] phosphatidyl choline was demonstrated by isolation of the activated vesicles by sucrose density centrifugation.     

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : I. Characterization of a Ca-Pumping ATPase Associated with the Endoplasmic Reticulum

Calcium transport was examined in microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue using chlorotetracycline as a fluorescent probe. This probe demonstrates an increase in fluorescence corresponding to calcium accumulation within the vesicles which can be collapsed by the addition of the calcium ionophore A23187. Calcium uptake in the microsomal vesicles was ATP dependent and completely inhibited by orthovanadate. Centrifugation of the microsomal membrane fraction on a linear 15 to 45% (w/w) sucrose density gradient revealed the presence of a single peak of calcium uptake which comigrated with the marker for endoplasmic reticulum. The calcium transport system associated with endoplasmic reticulum vesicles was then further characterized in fractions produced by centrifugation on discontinous sucrose density gradients. Calcium transport was insensitive to carbonylcyanide m-chlorophenylhydrazone indicating the presence of a primary transport system directly linked to ATP utilization. The endoplasmic reticulum vesicles contained an ATPase activity that was calcium dependent and further stimulated by A23187 (Ca(2+), A23187 stimulated-ATPase). Both calcium uptake and Ca(2+), A23187 stimulated ATPase demonstrated similar properties with respect to pH optimum, inhibitor sensitivity, substrate specificity, and substrate kinetics. Treatment of the red beet endoplasmic reticulum vesicles with [gamma-(32)P]-ATP over short time intervals revealed the presence of a rapidly turning over 96 kilodalton radioactive peptide possibly representing a phosphorylated intermediate of this endoplasmic reticulum associated ATPase. It is proposed that this ATPase activity may represent the enzymic machinery responsible for mediating primary calcium transport in the endoplasmic reticulum linked to ATP utilization.     

SeO_3~（2－）及SeO_3~（2－）和半胱氨酸或还原型谷胱甘肽联用对牛脑V

Ｓｅ_３~（２－）对牛脑Ｖ型质子转运ＡＴＰ酶复合体活力呈现明显的抑制作用。这种抑制作用具有对剂量和预保温时间的依赖关系,且不被稀释效应所恢复,但过量的ＤＴＴ可去除这种抑制作用。半胱氨酸对这种抑制作用呈双功能效应。低浓度时促进抑制作用,高浓度时部分恢复抑制作用。还原型谷胱甘肽与半胱氨酸有类似效应。但促进抑制作用没有半胱氨酸强,而恢复抑制作用却较半胱氨酸强。ＳＤＳ聚丙烯酰胺凝胶电泳和放射自显影图谱表明,ＳｅＯ_３~（２－）和半脱氨酸优先结合在７０ｋＤ亚基上。并且验证了ＡＴＰ酶复合体的－ＳＨ基和ＳｅＯ_３~（２－）及半胱氨酸的－ＳＨ基以－Ｓ－Ｓｅ－Ｓ－键相连接。根据上述结果,讨论了７０ｋＤ亚基的－ＳＨ基及其相关的结构域涉及到ＡＴＰ酶的活性中心,７０ｋＤ亚基可能是ＡＴＰ酶复合体的水解活性亚基。     

Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.     

Phosphatidate releases calcium from cardiac sarcoplasmic reticulum

Phosphatidate (PA) inhibits calcium accumulation by cardiac sarcoplasmic reticulum (SR) and enhances its Ca⁺⁺ ATPase activity. These effects seem to be related to a phosphatidate-induced increase in the calcium permeability of the SR membrane with resultant calcium release. The amount of calcium released by phosphatidate is dependent both on the calcium concentration outside the SR vesicles and the internal calcium concentration. The ionophoric effects of phosphatidate on the sarcoplasmic membrane provide a novel pathway for controlling Ca⁺⁺ transport in the cardiac cell.     

Tegumental Ca-stimulated adenosine triphosphatase activity in adult Schistosoma mansoni worms

A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6) was histochemically visualized within the tegument of fixed worms on the cytoplasmic leaflet of both the double surface membrane and the basement membrane; this reaction was inhibited by 1 microM chlorpromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosomes are discussed.     

Detergent-solubilized sarcoplasmic reticulum ATPase. Hydrodynamic and catalytic properties

Solubilization of the ATPase of sarcoplasmic reticulum vesicles with the nonionic detergent dodecyl non-aoxyethylene alcohol (C12E9) resulted in a large (about 5-fold) increase in its Ca2+ ATPase activity. Measurements using a calcium ionophore suggest this activation was the result of rendering the vesicles permeable to calcium. Complete activity is preserved at a detergent concentration range in which the detergent is complexed with the monomeric form of the ATPase, as measured by Sepharose 6B chromatography. Using a calibrated column, we found the C12E9 complex to have a Stokes radius of 55 A. As measured by time-resolved fluorescence anisotropy decay experiments, it had a rotational correlation coefficient of 214 ns, which is equivalent to a Stokes radius of 59 A. The axial ratio of the corresponding ellipsoid of revolution is calculated to be 5 to 6, indicating the complex is quite asymmetric. Like the vesicular form of the ATPase, the detergent-solubilized monomeric form bound with high affinity about 9 nmol of Ca2+/mg of protein. Also, like the vesicular enzyme, the solubilized form displayed a Ca2+ dependence of the activation of ATP hydrolysis which was cooperative (Hill coefficient 1.8). These results suggest that the calcium sites interact intramolecularly.     

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.     

Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles

Abstract Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.     

Characterization of a proton translocating ATPase and sucrose uptake in a tonoplast-enriched vesicle fraction from sugarcane

A sucrose gradient fraction was used to characterize the tonoplast ATPase from storage tissue of the sugarcane plant ( Saccharum sp. var. H57–5175). Marker enzyme analyses and characterization of low-density vesicles isolated on a sucrose gradient were consistent with a highly enriched tonoplast fraction. ATPase and proton transport activities were both substantially inhibited by nitrate (80%), but very little by vanadate (10%), indicating a high titer of tonoplast compared to plasma-membrane vesicles in the fraction. Sensitivity toward other inhibitors, as well as ion effects, correlated closely among ATPase and proton translocation activities. Although the vesicles in this fraction showed good proton translocating activity there was no indication that ATP stimulated sucrose uptake in this tonoplast population.     

Plasma membrane vesicles prepared from unadhered monocytes: Characterization of calcium transport and the calcium ATPase

We have purified unadhered human monocytes in sufficient quantities to prepare monocyte plasma membrane vesicles and study vesicular calcium transport. Monocytes were isolated from plateletpheresis residues by counterflow centrifugal elutriation. By combining this source and procedure, 7 x 10(8) monocytes of over 90% purity were obtained. The membranes, isolated on a sucrose step gradient, had an 18-fold enrichment in Na,K-ATPase, a 29-fold diminution of succinate dehydrogenase activity and were vesicular on transmission electron micrographs. The membrane vesicles loaded with oxalate accumulated calcium only in the presence of Mg and ATP. Calcium uptake did not occur if ATP was replaced by any of five nucleotide phosphates or if Mg was omitted. Calcium transport had a maximal velocity of 4 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.53 microM. The ionophore A23187 completely inhibited calcium accumulation while 5 mM sodium cyanide and 10 microM ouabain had no effect. A calcium-activated ATPase was present in the same plasma membrane vesicles. The calcium ATPase had a maximal velocity of 18.0 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.60 microM. Calcium-activated ATPase activity was absent if Mg was omitted or if (gamma - 32P) GTP replaced (gamma - 32P) ATP. Monocyte plasma membranes that were stripped of endogenous calmodulin by EGTA treatment showed a reduced level of calcium uptake and calcium ATPase activity. The addition of exogenous calmodulin restored the transport activity to that of unstripped monocyte plasma membranes. Thus, monocyte plasma membrane vesicles contain a highly specific, ATP-dependent calcium transport system and a calcium-ATPase with similar high calcium affinities.