Characterization of Ca Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots

The characteristics of Ca²⁺ transport into endoplasmic reticulum vesicles isolated from roots of Lepidium sativum L. cv Krause have been investigated. The concentration of free Ca²⁺ and ATP needed for half-maximal activity were 2.5 and 73 micromolar, respectively, and the enzyme obeyed Michaelis-Menten-like kinetics. The pH maximum occurred at 7.5 and the activity was greatly reduced at either pH 7.0 or 8.0.

The Ca²⁺-dependent modulation protein, calmodulin, was tested for its effect on Ca²⁺ transport into endoplasmic reticulum vesicles. Although the phenothiazine inhibitors chlorpromazine, fluphenazine, and trifluoperazine all inhibited Ca²⁺ transport activity with a half-maximal effect at approximately 35 micromolar, authentic bovine brain calmodulin did not alter the activity at concentrations of 0.5 to 8 micrograms per milliliter. Calmodulin also showed no influence on the time-dependent accumulation of Ca²⁺ into vesicles. The membranes did not contain endogenously bound calmodulin since washing with (ethylenebis[oxyethylenenitrile])tetraacetic acid or fluphenazine, treatments which disrupt calmodulin binding, did not alter Ca²⁺ transport activity. The inhibition of Ca²⁺ transport by phenothiazine drugs was likely related to their nonspecific interaction with the membrane. Thus, there was no indication that calmodulin regulated Ca²⁺ uptake into root endoplasmic reticulum.

     

Reconstitution and Characterization of a Calmodulin-Stimulated Ca-Pumping ATPase Purified from Brassica oleracea L

Purification and functional reconstitution of a calmodulin-stimulated Ca²⁺-ATPase from cauliflower (Brassica oleracea L.) is described. Activity was purified about 120-fold from a microsomal fraction using calmodulin-affinity chromatography. The purified fraction showed a polypeptide at 115 kD, which formed a phosphorylated intermediate in the presence of Ca²⁺, together with a few polypeptides with lower molecular masses that were not phosphorylated. The ATPase was reconstituted into liposomes by 3-([cholamidopropyl]-dimethylammonio-)1-propanesulfonate (CHAPS) dialysis. The proteoliposomes showed ATP-dependent Ca²⁺ uptake and ATPase activity, both of which were stimulated about 4-fold by calmodulin. Specific ATPase activity was about 5 μmol min⁻¹ (mg protein)⁻¹, and the Ca²⁺/ATP ratio was 0.1 to 0.5 when the ATPase was reconstituted with entrapped oxalate. The purified, reconstituted Ca²⁺-ATPase was inhibited by vanadate and erythrosin B, but not by cyclopiazonic acid and thapsigargin. Activity was supported by ATP (100%) and GTP (50%) and had a pH optimum of about 7.0. The effect of monovalent and divalent cations (including Ca²⁺) on activity is described. Assay of membranes purified by two-phase partitioning indicated that approximately 95% of the activity was associated with intracellular membranes, but only about 5% with plasma membranes. Sucrose gradient centrifugation suggests that the endoplasmic reticulum is the major cellular location of calmodulin-stimulated Ca²⁺-pumping ATPase in Brassica oleracea inflorescences.     

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.     

Calcium-pumping ATPases in vesicles from carrot cells : stimulation by calmodulin or phosphatidylserine, and formation of a 120 kilodalton phosphoenzyme

Ca²⁺-ATPases keep cytoplasmic [Ca²⁺] low by pumping Ca²⁺ into intracellular compartments or out of the cell. The transport properties of Ca²⁺-pumping ATPases from carrot (Daucus carota cv Danvers) tissue culture cells were studied. ATP-dependent Ca²⁺ transport in vesicles that comigrated with an endoplasmic reticulum marker, was stimulated three- to fourfold by calmodulin. Cyclopiazonic acid (a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase) partially inhibited oxalate-stimulated Ca²⁺ transport activity; however, it had no effect on calmodulin-stimulated Ca²⁺ uptake driven by ATP or GTP. The results would suggest the presence of two types of Ca²⁺-ATPases, an endoplasmic reticulum- and a plasma membrane-type. Interestingly, incubation of membranes with [gamma³²P]ATP resulted in the formation of a single acyl [³²P]phosphoprotein of 120 kilodaltons. Formation of this phosphoprotein was dependent on Ca²⁺, but independent of Mg²⁺. Its enhancement by La³⁺ is characteristic of a phosphorylated enzyme intermediate of a plasma membrane-type Ca-ATPase. Calmodulin stimulated Ca²⁺ transport was decreased by W-7 (a calmodulin antagonist), ML-7 (myosin light chain kinase inhibitor) or thyroxine. Acidic phospholipids, like phosphatidylserine, stimulated Ca²⁺ transport, similar to their effect on the erythrocyte plasma membrane Ca²⁺-ATPase. These results would indicate that the calmodulin-stimulated Ca²⁺ transport originated in large part from a plasma membrane-type Ca²⁺ pump of 120 kilodaltons. The possibility of calmodulin-stimulated Ca²⁺-ATPases on endomembranes, such as the endoplasmic reticulum and secretory vesicles, as well as the plasma membrane is suggested.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Inhibition by Orthovanadate of ATP-Dependent Ca2+ Transport in Microsomes Isolated from Rat Liver

A technique employing sucrose-density centrifugation for the enrichment of rat liver microsomes and rat liver plasma membranes in separate subcellular fractions is described. The fractions are enriched in glucose 6-phosphatase and 5′-nucleotidase, respectively, and are free of cytochrome oxidase activity. Vanadate-sensitive Ca²⁺ transport activity (half-maximal inhibition at ～10 μM vanadate, corresponding to ～12 nmol/mg of protein) was detected in only that fraction enriched in microsomal membranes. Inhibition by vanadate of ATP-dependent Ca²⁺ transport is noncompetitive with respect to added Ca²⁺ but competitive with respect to added ATP. Because it inhibits ATP-dependent Ca²⁺ transport in rat liver microsomes but not in rat liver plasma membranes, vanadate becomes a useful tool to distinguish in vitro between these two transport systems.     

Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome $\text{c}$ reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Differentiation of Ca pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome c reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

In vitro and in vivo phosphorylation of polypeptides in plasma membrane and tonoplast-enriched fractions from barley roots

Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with γ-[³²P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg²⁺ was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46, and 28 kilodaltons required millimolar Mg²⁺ concentrations and was greatly enhanced by Ca²⁺. When roots of intact plants were labeled with [³²P]orthophosphate, polypeptides at approximately 135, 116, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mm NaCl had no effect.     

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein: Is PAM involved in the capacitative calcium entry?

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca²⁺ signalling and maintenance of Ca²⁺ homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca²⁺-ATPase, Na⁺, K⁺-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca²⁺ ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca²⁺ entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca²⁺ entry, and their formation and rebuilding have an important regulatory role in cellular Ca²⁺ homeostasis.     

Association of Phytochrome with Rough-surfaced Endoplasmic Reticulum Fractions from Soybean Hypocotyls

Distribution of phytochrome (as Pfr) among membranes from soybean hypocotyls (Glycine max L. cv. Wayne) was determined by the combined techniques of cell fractionation, difference spectrometry, and electron microscopic morphometry. More than 90% of the phytochrome was found in the soluble fraction. With homogenates prepared in the presence or absence of Mg²⁺, the portion associated with membrane was only 6.5% and 1%, respectively. In the presence of Mg²⁺, the content of particulate phytochrome correlated with the amount of endoplasmic reticulum with attached ribosomes in the fractions but not with mitochondria or other membranes (including endoplasmic reticulum membranes from which the ribosomes may have been lost during cell fractionation). In the absence of Mg²⁺, phytochrome was associated with a “heavy” plasma membrane fraction. The phytochrome content was sufficiently low to be accounted for by a contamination of less than 10% by rough-surfaced fragments of endoplasmic reticulum. The findings show association of phytochrome with a particulate fraction enriched in rough-surfaced fragments of endoplasmic reticulum but do not rule out cosedimentation of some unknown or unspecific phytochrome aggregate with this fraction.     

Darier disease : A disease model of impaired calcium homeostasis in the skin

The importance of extracellular calcium in epidermal differentiation and intra-epidermal cohesion has been recognized for many years. Darier disease (DD) was the first genetic skin disease caused by abnormal epidermal calcium homeostasis to be identified. DD is characterized by loss of cell-to-cell adhesion and abnormal keratinization. DD is caused by genetic defects in ATP2A2 encoding the sarco/endoplasmic reticulum Ca²⁺-ATPase isoform 2 (SERCA2). SERCA2 is a calcium pump of the endoplasmic reticulum (ER) transporting Ca²⁺ from the cytosol to the lumen of ER. ATP2A2 mutations lead to loss of Ca²⁺ transport by SERCA2 resulting in decreased ER Ca²⁺ concentration in Darier keratinocytes. Here, we review the role of SERCA2 pumps and calcium in normal epidermis, and we discuss the consequences of ATP2A2 mutations on Ca²⁺ signaling in DD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Senescence-Related Changes in ATP-Dependent Uptake of Calcium into Microsomal Vesicles from Carnation Petals

Microsomal membrane vesicles isolated from the petals of young carnation (Dianthus caryophyllus L. cv White Sim) flowers accumulate Ca²⁺ in the presence of ATP. The specific activity of ATP-dependent uptake is ~20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca²⁺ stimulation of ATP hydrolysis was not discernible above the high background of Ca²⁺-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca²⁺ concentration, suggesting that Ca²⁺ serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca²⁺ at half maximal velocity of uptake (S_0.5) was 12.5 micromolar and the Hill coefficient (n_H) was 2.5. The addition of calmodulin to membrane preparations that had been isolated in the presence of chelators did not promote ATP-dependent accumulation of Ca²⁺, although this may reflect the fact that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca²⁺ into membrane vesicles was unaffected by 50 micromolar ruthenium red and 5 micromolar sodium azide, indicating that uptake is primarily into vesicles of non-mitochondrial origin. By subfractionating the microsomes on a linear sucrose gradient, it was established that the ATP-dependent Ca²⁺ transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca²⁺ into microsomal vesicles declined by ~70%. This occurred before the appearance of petal-inrolling and the climacteric-like rise in ethylene production, parameters that denote the onset of senescence. There were no significant changes during this period in S_0.5 or n_H, but V_max for ATP-dependent Ca²⁺ uptake decreased by ~40%. A similar decline in ATP-dependent uptake of Ca²⁺ into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene.     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin     

Ca-transport in sea urchin unfertilized eggs: Regulation by endogenous sulfated polysaccharides and K

Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca²⁺ by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K⁺ leading to an enhancement of the Ca²⁺ transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca²⁺ at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K⁺ but not by Li⁺ and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K⁺. These data suggest that the regulation of the SERCA pump by K⁺ and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.     

Calcium Transport in the Green Alga Mesotaenium caldariorum: Preliminary Characterization and Subcellular Distribution

The subcellular localization and biochemical characterization of calcium transport were studied in the unicellular green alga Mesotaenium caldariorum. Membrane fractions prepared by osmotic lysis of Mesotaenium protoplasts exhibit high rates of ATP-dependent calcium uptake. Sucrose gradient centrifugation separates two pools of activity, which display specific activities for calcium transport as high as 15 nanomoles Ca²⁺ per minute per milligram of protein. Marker enzyme analysis shows that this dual distribution of calcium transport activity is similar to that of vanadate-insensitive ATPase and pyrophosphatase, activities considered to be associated with the tonoplast. Plasma membranes, endoplasmic reticulum vesicles, mitochondrial membranes, and thylakoids band at higher densities than either calcium transport fraction. Both pools of ATP-dependent calcium uptake contain two components which are not separable on sucrose gradients but can be distinguished on the basis of inhibitor sensitivity. One component is inhibited by nigericin or trimethyltin chloride (I₅₀ values of 3 nanomolar and 4 micromolar, respectively), while the other component is vanadate sensitive (I₅₀ of 25 micromolar). These results suggest that direct Ca²⁺ transport and Ca²⁺/H⁺ antiport activities are present in both sucrose gradient fractions.     

Ion-translocating ATPases in tendrils ofBryonia dioica Jacq.

Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils ofBryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as Mg²⁺ATP-driven transport of⁴⁵Ca²⁺. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca²⁺-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca²⁺-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K⁺, Mg²⁺-ATPase and the Ca²⁺-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling.Abbreviations CAM calmodulin - CCCP carbonylcyanidem-chlorophenylhydrazone - IC₅₀ concentration giving 50% inhibition - PM plasma membrane - rER rough endoplasmic reticulum - sER smooth endoplasmic reticulum - FC fusicoccin - U₃+U₃ the two PM-rich upper phases obtained after phase partitioning of microsomal membranes The authors wish to thank the Deutsche Forschungsgemeinschaft, Bonn, Germany, and the Fonds der Chemischen Industrie, Frankfurt, Germany (literature provision) for financial support.     

A Ca/H Antiport System Driven by the Proton Electrochemical Gradient of a Tonoplast H-ATPase from Oat Roots

Two types of ATP-dependent calcium (Ca²⁺) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca²⁺ uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO₃⁻. The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg²⁺.

Like the tonoplast H⁺-ATPase activity, vanadate-insensitive Ca²⁺ accumulation was stimulated by 20 millimolar Cl⁻ and inhibited by 10 micromolar 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid or 50 micromolar N,N′-dicyclohexylcarbodiimide. This Ca²⁺ transport system had an apparent K_m for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca²⁺ transport was abolished by compounds that eliminated a pH gradient and Ca²⁺ dissipated a pH gradient (acid inside) generated by the tonoplast-type H⁺-ATPase. These results provide compelling evidence that a pH gradient generated by the H⁺-ATPase drives Ca²⁺ accumulation into right-side-out tonoplast vesicles via a Ca²⁺/H⁺ antiport. This transport system was saturable with respect to Ca²⁺ (K_m apparent = 14 micromolar). The Ca²⁺/H⁺ antiport operated independently of the H⁺-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca²⁺ accumulation. Ca²⁺ transport by this system may be one major way in which vacuoles function in Ca²⁺ homeostasis in the cytoplasm of plant cells.

     

Environmental cadmium is not sequestered by metallothionein in rainbow trout

Smooth endoplasmic reticulum vesicles from rat liver display an ATP-supported Ca²⁺ transport which is mediated by a (Ca²⁺ + Mg²⁺)-ATPase. During the catalytic cycle the terminal phosphate from ATP is incorporated to form an acid-precipitable reaction product(118 000-M_r in SDS-gel electrophoresis) with stability characteristics of an acylphosphate. Comparative studies with sarcoplasmic reticulum vesicles from fast-twitch skeletal muscle suggest that the 118 000-M_r phosphopeptide may be identified with the phosphorylated reaction intermediate of a Ca²⁺ transport ATPase in endoplasmic reticulum, similar to that in sarcoplasmic reticulum of muscle.     

Thermogenic activity of Ca-ATPase from skeletal muscle heavy sarcoplasmic reticulum: The role of ryanodine Ca channel

The sarcoplasmic reticulum Ca²⁺ ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca²⁺ transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca²⁺ channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (ΔH^cal) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg²⁺ or ruthenium red, conditions that close the ryanodine Ca²⁺ channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the ΔH^cal values of ATP hydrolysis increased significantly. Neither Mg²⁺ nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the ΔH^cal of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca²⁺ channel is opened or closed.