Ultrastructural localization of calcium ions in ram spermatozoa before and after cold shock as demonstrated by a pyroantimonate technique

Ram spermatozoa were subjected to cold shock before fixation in pyroantimonate-osmium. Ultrathin sections revealed an electron-dense particulate precipitate in association with the cells. The precipitate was shown to be related to the presence of calcium by exposure of the material to EGTA which reduced or completely eliminated the deposits. In the acrosome region, very little precipitate was evident when the plasma membrane was intact. Cold shock resulted in the disruption of the plasma membrane. When the acrosome remained intact, precipitate was concentrated just anterior to the equatorial segment, but many cells also had acrosomal disruption and then a more even distribution of precipitate was seen on the outer acrosomal membrane. Precipitate was rarely visible within or beneath the acrosome. Post-acrosomally, calcium pyroantimonate deposits were frequently present in the dense lamina beneath the plasma membrane and these became more intense after cold shock. Midpiece sections revealed a few large granules beneath the plasma membrane and a fine particulate precipitate within mitochondria. Similarly, the fine precipitate was also associated with the outer dense fibres in midpieces and tails. Cold shock did not apparently increase the extent or intensity of precipitates in these sites.     

Isolation and assay of corn root membrane vesicles with reduced proton permeability

Conditions promoting the formation of sealed membrane vesicles from corn roots with reduced proton permeability were examined using the probe 9-aminoacridine as a rapid indicator of pH gradient formation and dissipation. Plasma membrane vesicles isolated by differential and density gradient centrifugation were leaky to protons and rapidly equilibrated when exposed to artificially imposed pH gradients. The leaky plasma membrane vesicles showed reduced proton permeability when incubated with calcium or with excess phospholipids. However, these vesicles were unable to form ATP-induced pH gradients. Sealed vesicles isolated by discontinuous Ficoll gradient centrifugation of a microsomal fraction displayed reduced proton permeability and were osmotically active. In contrast to purified plasma membrane vesicles, the microsomal-derived vesicles were more suitable for studies of active proton transport.     

Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum

A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart.     

Subcellular distribution of calcium in zona fasciculata cells of the rat adrenal cortex

Zona fasciculata cells from the adrenal cortex of female Sprague-Dawley rats were fixed by immersion in potassium pyroantimonate-osmium tetroxide and potassium pyroantimonate-glutaraldehyde to study the distribution of calcium. Potassium pyroantimonate-osmium tetroxide treatment gave reproducible patterns of electron-opaque precipitate, whereas inconsistent deposits of reaction product were seen after potassium pyroantimonate-glutaraldehyde fixation. Nuclei showed sparse precipitate over heterochromatin and dense aggregates over areas of nucleoli surrounded by portions of the nucleolar-dense component. Two major cytoplasmic sites of precipitate were identified: mitochondria and vesicles of smooth endoplasmic reticulum. Most of the intramitochondrial precipitate was localized to the intracristal space. Precipitate was also seen in vesicles of Golgi apparatus. The extracellular space was filled with closely packed electron-opaque particles. Observation of tissues treated with control fixative saturated with EGTA showed little if any reaction, confirming that calcium was the primary cation precipitated by potassium pyroantimonate. Our results provide a method suitable for accurate localization of calcium in adrenocortical cells.     

ATP-driven calcium transport in membrane vesicles of Streptococcus sanguis.

Calcium transport was investigated in membrane vesicles prepared from the oral bacterium Streptococcus sanguis. Procedures were devised for the preparation of membrane vesicles capable of accumulating 45Ca2+. Uptake was ATP dependent and did not require a proton motive force. Calcium transport in these vesicles was compared with 45Ca2+ accumulation in membrane vesicles from Streptococcus faecalis and Escherichia coli. The data support the existence of an ATP-driven calcium pump in S. sanguis similar to that in S. faecalis. This pump, which catalyzes uptake into membrane vesicles, would be responsible for extrusion of calcium from intact cells.     

Comparison of the Ca-Mg ATPase and calcium transport in rat and human erythrocytes: Evidence for an electrogenic mechanism

Inside out vesicles prepared from rat erythrocytes transport calcium by an electrogenic mechanism. Calmodulin stimulates the activity of a Ca-Mg ATPase, and also stimulates calcium transport. Permeant anions stimulate calcium transport relative to that observed when an impermeant anion (gluconate) is employed. The inside out vesicles transport phosphate anion in a calmodulin-stimulated, calcium- and ATP-dependent manner. Membrane potential sensitive fluorescent dyes demonstrate a positive interior membrane potential during calcium transport in the absence of permeant anions. The properties of the rat erythrocyte calcium transport system corresponds closely to those of the human erythrocyte; the activity of the Ca-Mg ATPase, rates and stoichiometries of calcium and phosphate transport and the degree of calmodulin stimulation are directly comparable between the two species.     

New Procedure for the Isolation of Membrane Vesicles of Bacillus subtilis and an Electron Microscopy Study of Their Ultrastructure

A rapid procedure for the isolation of membrane vesicles of Bacillus subtilis is described that minimizes the action of proteolytic enzymes, excreted by this organism, on the membrane proteins. The membrane vesicles obtained have, in addition to a low endogenous respiration rate, a low endogenous activity for transport of amino acids and carboxylic acids. In the presence of the electron donor, ascorbate-phenazine methosulfate, the transport activities for these compounds were comparable to the activities of intact cells. In addition, these activities were retained for a prolonged period of time. Electron microscopy examination of thin sections of the vesicles showed that the preparation consisted almost exclusively of membrane vesicles which were not contaminated with other cell components. The membrane vesicles, which are six to seven times smaller in diameter than protoplasts, often enclosed smaller vesicles. Freeze-etching of intact cells, protoplasts, and membrane vesicles showed that the orientation of the membrane of the vesicles was identical to the orientation of the plasma membrane in intact cells and protoplasts. This also held for the majority of the membranes of the enclosed vesicles, only 15% having the opposite orientation.     

Electron-dense precipitates in glomus cells of rat carotid body after fixation in glutaraldehyde and pyroantimonate-osmium tetroxide mixture as possible indicators of calcium localization

Summary An attempt was made to study the subcellular localization of calcium in carotid body glomus cells of adult rats using fixation with glutaraldehyde followed by treatment with a mixture of pyroantimonate and osmium tetroxide. Precipitates were seen as electron-dense particles (EDP) in the glomus cells, mostly within membrane-bound organelles, such as dense-cored vesicles, mitochondria, small clear vesicles, multivesicular bodies, and especially in lysosomes. However, EDP were also seen in the nuclei and in the free cytoplasm of the glomus cells and even outside them.Preincubation of carotid bodies in media containing calcium and either high potassium or calcium-ionophore A 23187 resulted in a marked increase in the general precipitation pattern, there being an increased amount of EDP both in the glomus cell nuclei and in the cytoplasm. Dense-cored vesicles more often showed precipitates than those in the controls. Some dense-cored vesicles contained multiple precipitates, typically located in the electron-lucent area between core and vesicle membrane.Extensive diffusion of ions probably occurred during fixation before precipitation, making the localization of calcium and other precipitating cations unreliable. However, it is possible that precipitates, which were regularly seen in the dense-cored vesicles, may reflect the content of bound calcium. The possible significance of calcium in glomus cell function is discussed, and the need for more adequate methods is emphasized.The present study has been supported by grants from the Finska Läkaresällskapet and the Sigrid Jusélius Foundation, Helsinki, FinlandWe wish to express our gratitude to Dr. Robert Hamill of Eli Lilly Co. for kindly providing us with the ionophore A 23187. Technical assistance by Mrs. S. Huhtaniitty and Mrs. T. Stjernvall is gratefully acknowledged     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

Is vacuole the richest store of IP3-mobilizable calcium in plant cells?

Inositol trisphosphate is known to mobilize calcium from internal stores in plant cells. However, with the exception of the vacuole, the largest plant cell compartment, organelles responsive to inositol trisphosphate have not been extensively identified. In this way, we have separated membrane vesicles from the same carrot microsomal fraction and identified them, both by marker enzyme activities and electron microscopy. These correspond to pure plasma membrane, pure tonoplast and mixed mitochondria, endoplasmic reticulum, Golgi membrane fractions. All the fractions accumulated calcium in a ATP-dependent manner and were tightly sealed. Inositol trisphosphate-dependent calcium releases were accurately measured only in fractions corresponding functionally and structurally to tonoplast, the vacuolar membrane. The process was dose-dependent and fairly specific for inositol trisphosphate. While highly significant, approximately 40% of the mobile calcium only may be released from tonoplast vesicles by inositol trisphosphate which remained basically intact during the release experiments. From these results it is concluded that the vacuole is the richest store of calcium directly mobilizable by inositol trisphosphate in plant cells, but inositol trisphosphate is not able to release the overall mobile vacuolar calcium.     

Intermembrane electron transport in the absence of added water-soluble carriers.

Electron transport from untreated to mersalyzed microsomal vesicles at the level of NADH-cytochrome b5 reductase or cytochrome b5 has been demonstrated in the absence of added water-soluble electron carriers. A similar effect was shown in the systems "intact mitochondria - mersalyzed microsomes" and "mersalyzed mtiochondria - untreated microsomes". No measurable electron transport between intact and mersalyzed particles of inner mitochondrial membrane was found. The obtained data suggest that the capability to carry out intermembrane electron transfer is specific for NADH-cytochrome b5 reductase and/or cytochrome b5, localized in microsomal and outer mitochondrial membranes.     

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.     

Effects of Cations on Hormone Transport in Primary Roots of Zea mays

We examined the influence of aluminum and calcium (and certain other cations) on hormone transport in corn roots. When aluminum was applied unilaterally to the caps of 15 mm apical root sections the roots curved strongly away from the aluminum. When aluminum was applied unilaterally to the cap and ³H-indole-3-acetic acid was applied to the basal cut surface twice as much radioactivity (assumed to be IAA) accumulated on the concave side of the curved root as on the convex side. Auxin transport in the apical region of intact roots was preferentially basipetal, with a polarity (basipetal transport divided by acropetal transport) of 6.3. In decapped 5 mm apical root segments, auxin transport was acropetally polar (polarity = 0.63). Application of aluminum to the root cap strongly promoted acropetal transport of auxin reducing polarity from 6.3 to 2.1. Application of calcium to the root cap enhanced basipetal movement of auxin, increasing polarity from 6.3 to 7.6. Application of the calcium chelator, ethylene-glycol-bis-(β-aminoethylether)-N,N,N′, N′-tetraacetic acid, greatly decreased basipetal auxin movement, reducing polarity from 6.3 to 3.7. Transport of label after application of tritiated abscisic acid showed no polarity and was not affected by calcium or aluminum. The results indicate that the root cap is particularly important in maintaining basipetal polarity of auxin transport in primary roots of corn. The induction of root curvature by unilateral application of aluminum or calcium to root caps is likely to result from localized effects of these ions on auxin transport. The findings are discussed relative to the possible role of calcium redistribution in the gravitropic curvature of roots and the possibility of calmodulin involvement in the action of calcium and aluminum on auxin transport.     

Permeability and channel-mediated transport of boric acid across membrane vesicles isolated from squash roots

Boron is an essential micronutrient for plant growth and the boron content of plants differs greatly, but the mechanism(s) of its uptake into cells is not known. Boron is present in the soil solution as boric acid and it is in this form that it enters the roots. We determined the boron permeability coefficient of purified plasma membrane vesicles obtained from squash (Cucurbita pepo) roots and found it to be 3 x 10(-7) +/-1.4 x 10(-8) cm s(-1), six times higher than the permeability of microsomal vesicles. Boric acid permeation of the plasma membrane vesicles was partially inhibited (30%-39%) by mercuric chloride and phloretin, a non-specific channel blocker. The inhibition by mercuric chloride was readily reversible by 2-mercaptoethanol. The energy of activation for boron transport into the plasma membrane vesicles was 10.2 kcal mol(-1). Together these data indicate that boron enters plant cells in part by passive diffusion through the lipid bilayer of the plasma membrane and in part through proteinaceous channels. Expression of the major intrinsic protein (MIP) PIP1 in Xenopus laevis oocytes resulted in a 30% increase in the boron permeability of the oocytes. Other MIPs tested (PIP3, MLM1, and GlpF) did not have this effect. We postulate that certain MIPs, like those that have recently been shown to transport small neutral solutes, may also be the channels through which boron enters plant cells.     

The effect of calmodulin and far-red light on the kinetic properties of the mitochondrial and microsomal calcium-ion transport system from corn

The kinetic properties of active Ca²⁺ transport into mitochondria and microsomal membrane vesicles prepared from coleoptiles of dark-and light-grown corn seedlings have been studied. The apparent values for K _m and V _max for Ca²⁺ of the mitochondrial transport system from dark-grown plants are about one order of magnitude higher than those from the microsomal transport system. Calmodulin has no effect on the Ca²⁺ accumulation into mitochondria whereas the apparent maximum transport velocity and affinity for Ca²⁺ of the microsomal Ca²⁺-transport system are both increased by calmodulin. When intact corn seedlings are irradiated with far-red light, the calmodulin-induced increase of the apparent maximum transport velocity and affinity for Ca²⁺ can no longer be observed. From these data it can be concluded that the low cytoplasmic Ca²⁺ concentration in the cytoplasm of coleoptile cells from dark-grown corn is maintained by a calmodulin-regulated Ca²⁺ pump. Irradiation with photomorphogenically active far-red light lowers the Ca²⁺-transport activity and thus causes an increase of the cytoplasmic, free-Ca²⁺ concentration. The physiological implications will be discussed.     

Electron-dense endoplasmic reticulum-like profiles closely associated with mitochondria in glomus cells of the carotid body after fixation with oxalate

Fixation in the presence of oxalate was used to demonstrate the electron-dense Ca2+ precipitates in the endoplasmic reticulum in glomus cells of the carotid body. Glomus cells in intact carotid bodies or cells dissociated from the organ by treatment with collagenase were studied electron microscopically. In the intact organ as well as in dissociated glomus cells, electron-dense endoplasmic reticulum-like profiles were seen closely associated with mitochondria, while these lacked reaction product. The interspace between mitochondria was occupied by electron-dense, slightly distended ER, which appeared to contact the outer membrane of the mitochondria. Occasionally, a mitochondrion was in contact with several ER profiles or the ER formed an electron-dense 'cap' on the mitochondrion. The electron-dense precipitates could be removed from ultrathin sections with the calcium chelator ethyleneglycol-2(2-aminoethyl tetra-acetic acid) (EGTA). It is tentatively suggested that the endoplasmic reticulum could be involved in intracellular buffering of Ca2+ in the glomus cell, as has been previously suggested for neurons.     

Characterization of tonoplast polypeptides isolated from corn seedling roots

Tonoplast vesicles were isolated by discontinuous sucrose gradient centrifugation in the presence of Mg²⁺ from 5 day old corn (Zea mays L., Golden Cross Bantam) seedling roots. Marker enzyme assays indicated only a low degree of cross-contamination of tonoplast vesicles at the 10/23% (weight/weight) interface by other membrane components. Severalfold enrichment of tonoplast ATPase and pyrophosphatase was indicated in tonoplast fractions by dot blot studies with antibodies against an oat tonoplast ATPase and a mung bean tonoplast pyrophosphatase. Comparison of two-dimensional electrophoretic gels of tonoplast and microsomal membrane polypeptides revealed approximately 68 polypeptides to be specific to tonoplast by silver staining. Immunoblot analysis with antibodies against a tonoplast holoenzyme ATPase from oat roots revealed the presence of the 72, 60, and 41 kilodalton polypeptides in isolated tonoplast vesicles from corn roots. Affinity blotting with concanavalin A and secondary antibodies indicated the degree of glycosylation of tonoplast polypeptides, where 21 of 68 tonoplast-specific polypeptides contained detectable carbohydrate moieties. Salt and NaOH washes removed 38 of the tonoplast-specific polypeptides, indicating a peripheral association with the membrane. Thirteen of the peripheral polypeptides and eight of the integral polypeptides were identified as glycoproteins. This information on the polypeptide composition of the tonoplast of root cells will aid in gaining insight into the role of this membrane in controlling vacuolar functions.     

Orientation of the protonmotive force in membrane vesicles of escherichia coli

Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D-lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside. The latter type of vesicle, on the other hand, exhibits D-lactate-dependent proline transport but does not accumulate calcium with D-lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D-lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy-linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).     

In vitro biosynthesis, core glycosylation and membrane integration of opsin

A membrane-integrated , core-glycosylated form of bovine opsin was synthesized in vitro when bovine retina mRNA was translated in a wheat germ cell-free system supplemented with dog pancreas microsomal vesicles; glycosylation and integration of opsin into membranes were coupled to translation. Proteolysis with themolysin was used to probe the orientation of opsin within the dog pancreas microsomal membrane, and to compare it with that of opsin in rod cell disk membranes isolated from bovine retina. Intact microsomal or disk vesicles were required for production of discrete, membrane-associated thermolysin fragments of opsin; no discrete opsin fragments were detected when membranes were incubated with thermolysin in the presence of the nonionic detergent, Triton X-100. The major opsin fragments produced by themosylin treatment of intact microsomal vesicles resembled those from disk vesicles in their size, oligosaccharide content, and order of appearance. In each case, the first cleavage of opsin took place at the COOH-terminus, generating a glycosylated fragment, O’, which was only slightly smaller than intact opsin. Both the microsomal and disk membrane forms of O’ were next cleaved internally; glycosylated fragments of similar sizes in both cases were detected which were derived from the NH(2)-terminal portion of O’. Several smaller NH(2)-terminal fragments of opsin were detected only in thermolysin-treated microsomal membranes, and not in disk membranes. The data suggest that the topology of opsin integrated into dog pancreas microsomal vesicles is similar to that in rod cell disk vesicles, although not identical. In each case, the glycosylated NH(2)-terminal region of opsin is located within the lumen of the vesicle, while discrete COOH-terminal and internal segments of opsin apparently emerge at the outer, cytoplasmic face of the membrane. Thus, opsin in the heterologous microsomal membrane, like its counterpart in the native disk membrane, may cross the bilayer at least three times. The internal domain of the polypeptide that emerges at the outer membrane surface is apparently more highly exposed in the case of opsin in microsomal membranes, evidenced by the additional internal thermolysin cleavage sites detected.     

A calmodulin-stimulated Ca2+ pump in rat aorta plasma membranes

An ATP-driven Ca2+-transport system has been characterized in a microsomal fraction from rat aorta. Calmodulin enhanced 2.5-fold 45Ca accumulation by EGTA-treated microsomes incubated with 10 microM Ca2+ (in the absence of oxalate) by increasing markedly the apparent affinity of the transport system for Ca2+. The ionophore A23187 induced a rapid release of the sequestered 45Ca. The vesicles that took up 45Ca were distributed like plasmalemmal marker enzymes when the microsomal fraction was subfractionated by density gradient centrifugation. In particular, these vesicles were markedly shifted towards higher equilibrium densities after addition to the microsomes of 0.2 mg digitonin/mg protein before isopycnic centrifugation. We conclude that the calmodulin-stimulated Ca2+ pump associated with the microsomal fraction is located in plasmalemmal elements.