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.

     

Further Characterization of the Red Beet Plasma Membrane Ca-ATPase Using GTP as an Alternative Substrate

The GTP-driven component of Ca²⁺ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca²⁺-translocating ATPase and assess its utility as a probe for this transport system. Uptake of ⁴⁵Ca²⁺ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum, sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca²⁺ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca²⁺-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of ⁴⁵Ca²⁺-uptake by exogenous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven ⁴⁵Ca²⁺ uptake represents the capacity of the plasma membrane Ca²⁺-translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ-³²P]-GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca²⁺-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca²⁺-ATPases present in animal cells.     

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of ¹²⁵I-labeled calmodulin to bovine cerebellar membranes have been determined and correlted with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca²⁺, calmodulin binds to a finite number of specific membrane sites with a dissociation constant (K_d) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca²⁺-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be infered from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca²⁺ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca²⁺ atoms. The concentration of the active calmodulin ·Ca²⁺ species required for half-maximal activation of adenylate cyclase is very similar to the K_d of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca²⁺-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.     

Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons

Evidence for the involvement of Ca²⁺ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-¹⁴C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-¹⁴C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca²⁺, whereas lipolytic acyl hydrolase proved to be insensitive to Ca²⁺. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca²⁺. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC₅₀ values ranging from 10 to 15 micromolar. Thus the Ca²⁺-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca²⁺ on phospholipase D is independent of calmodulin. The role of Ca²⁺ as a second messenger in the initiation of membrane lipid degradation is discussed.     

The spinach plasma membrane Ca2+ pump is a 120‐kDa polypeptide regulated by calmodulin‐binding to a terminal region

The spinach (Spinacia oleracea L.) leaf plasma membrane Ca²⁺-ATPase is regulated by calmodulin (3-fold stimulation) and limited proteolysis (trypsin; 4-fold stimulation). The plasma membrane Ca²⁺-ATPase was identified as a 120-kDa polypeptide on western immunoblots using two different antibodies. During trypsin treatment the 120-kDa band diminished and a new band appeared at 109 kDa. The appearance of the 109-kDa band correlated with the increase in enzyme activity following trypsin treatment. The stimulations by calmodulin and trypsin were not additive, suggesting that the 109-kDa polypeptide represents a Ca²⁺-ATPase lackin a terminal fragment involved in calmodulin regulation. This was confirmed by ¹²⁵I-calmodulin overlay studies where calmodulin labeled the 120-kDa band in the presence of Ca²⁺, while the 109-kDa band did not bind calmodulin. The effects of calmodulin and limited proteolysis on ATP-dependent accumulation of ⁴⁵Ca²⁺ in isolated inside-out plasma membrane vesicles were studied, and kinetical analyses performed with respect to Ca²⁺ and ATP. Calmodulin increased the V_max. for Ca²⁺ pumping 3-fold, and reduced K_m for Ca²⁺ from 1.6 to 0.9 µM. The K_m for ATP (11 µM) was not affected by calmodulin. The effects of limited proteolysis on the affinities for Ca²⁺ and ATP were similar to those obtained with calmodulin. Notably, however, limited proteolysis increased the V_max. for Ca²⁺ pumping to a higher extent than calmodulin, indicating incomplete calmodulin activation, or removal of an additional inhibitory site by trypsin.     

Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.     

A high affinity,calmodulin-responsive (Ca2+ + Mg2+)-ATPase in isolated bone cells

Although acute alterations in Ca²⁺ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca²⁺-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca²⁺-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca²⁺ of 280 nM, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg²⁺ could replace Ca²⁺ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg²⁺ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca²⁺-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca²⁺-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca²⁺ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.     

Volume regulation in poterioochromonas: involvement of calmodulin in the ca-stimulated activation of isofloridoside-phosphate synthase

In Poterioochromonas malhamensis Peterfi (syn. Ochromonas malhamensis Pringsheim) osmotically induced shrinkage is reversed by an accumulation of isofloridoside. Addition of Ca²⁺ ions to homogenates from standard volume cells initiates an enzyme system for the activation of isofloridoside-phosphate synthase. This process is stimulated in the presence of Ca²⁺ by calmodulin, isolated from the same alga or from bovine brain, and requires the presence of membranes. The stimulation observed when Ca²⁺ is added without exogenous calmodulin is inhibited by the calmodulin-binding substance R 24571. These results show that the effect of Ca²⁺ is mediated by calmodulin. The Ca²⁺/calmodulin-dependent activation is enhanced when fluoride or molybdate ions are present in the homogenization buffer. This might indicate the involvement of a phosphorylated compound in the activation mechanism.     

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca²⁺ upon addition of MgATP. MgATP-dependent Ca²⁺ uptake co-migrates with the plasma membrane H⁺-ATPase on a sucrose gradient. Ca²⁺ uptake is insensitive to oligomycin, inhibited by vanadate (IC₅₀ 40 micromolar) and erythrosin B (IC₅₀ 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca²⁺ uptake is insensitive to protonophores. These results indicate that Ca²⁺ transport in these microsomal vesicles is catalyzed by a Mg²⁺-dependent ATPase localized on the plasma membrane. Ca²⁺ strongly reduces ΔpH generation by the plasma membrane H⁺-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca²⁺ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca²⁺ uptake into plasma membrane vesicles. The Ca²⁺-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca²⁺-induced decrease of ΔpH generation by the plasma membrane H⁺-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H⁺-ATPase, is accelerated by MgATP-dependent Ca²⁺ uptake, indicating that the decrease of ΔpH generation induced by Ca²⁺ reflects the efflux of H⁺ coupled to Ca²⁺ uptake into plasma membrane vesicles. It is therefore proposed that Ca²⁺ transport at the plasma membrane is mediated by a Mg²⁺-dependent ATPase which catalyzes a nH⁺/Ca²⁺ exchange.     

Stimulation by calmodulin of Ca2+ uptake and (Ca2+-Mg2+) ATPase activity in membrane fractions from ox neurohypophyses

Calmodulin stimulated ⁴⁵Ca²⁺ uptake into a plasma membrane enriched fraction from ox neurohypophysial nerve endings and into a microsome fraction. The ⁴⁵Ca²⁺ uptake and the (Ca²⁺-Mg²⁺) ATPase activity in the plasma membrane fraction exhibited similar pCa and calmodulin sensitivities, suggesting that the enzyme activity is the biochemical expression of a high affinity Ca²⁺ pump. Calmodulin thus seems to play a role in regulation of the intracellular free Ca²⁺ concentration in the neurohypophysis.     

Regulation of endogenously catalyzed ADP-ribosylation in adipocyte plasma membranes by Ca and calmodulin

The effects of Ca²⁺ and calmodulin on endogenously catalyzed ADP-ribosylation were investigated in adipocyte plasma membranes. Four specific proteins of 70, 65, 61 and 52 kDa were labeled with [³²P]ADP-ribose and ADP-ribosylation of the proteins was highly dependent upon the conditions employed. ADP-ribosylation of the 70 kDa protein was observed only in membranes supplemented with Ca²⁺. Maximal incorporation of [³²P] into the protein was achieved with free Ca²⁺ concentrations of 90 μM. Calcium-stimulated ADP-ribosylation of the 70 kDa protein was inhibited by calmodulin. Half-maximal inhibition was observed in membranes incubated with 1.2 μM calmodulin. The effect of calmodulin was characterized by an inhibition of the incorporation of [³²P]ADP-ribose as opposed to a stimulation of its removal. ADP-ribosylation of the 61 kDa protein was not altered by added Ca²⁺ and/or calmodulin whereas ADP-ribosylation of the 65 kDa protein was partially (50%) inhibited by free Ca²⁺ concentrations between 10⁻⁶ – 10⁻⁵ M. These results provide evidence that the adipocyte plasma membrane contains ADP-ribosyltransferase activities and demonstrate that ADP-ribosylation of a 70 kDa protein is regulated by Ca²⁺ and calmodulin.     

Biochemical characteristics of the Ca2+ pumping ATPase in the peribacteroid membrane from broad bean root nodules

Ca²⁺-ATPase in the peribacteroid membrane (PBM) of symbiosomes isolated from Vicia faba root nodules was characterized in terms of its hydrolytic and transport activities. Both activities were found to be pH-dependent and exhibit pH optimum at pH 7.0. Translocation of Ca²⁺ through the PBM by the Ca²⁺-ATPase was shown to be fueled by ATP and other nucleotide triphosphates in the following order: ATP?>?ITP???GTP???UTP???CTP, the K _m of the enzyme for MgATP being about 100 μM. Ca-dependent ITP-hydrolytic activity of symbiosomes was investigated in the presence of the Ca-EGTA buffer system and showed the affinity of PBM Ca²⁺-ATPase for Ca²⁺ of about 0.1 μM. The transport activity of Ca²⁺-ATPase was inhibited by erythrosin B as well as orthovanadate, but markedly stimulated by calmodulin from bovine brain. These results allowed us to conclude that this enzyme belongs to IIB-type Ca²⁺-ATPases which are present in other plant membranes.     

A high-affinity calcium-stimulated ATPase activity in the hen oviduct shell gland

The hen oviduct shell gland is a highly active calcium-transporting epithelial tissue which is responsible for the mineralization of the egg shell. We have identified a calcium-stimulated ATPase present at high specific activity in membrane preparations from shell gland mucosal shavings. In the presence of optimal MgCl₂ (5 mm) and a Ca²⁺ buffer, ATP hydrolysis was stimulated by addition of low concentrations of free Ca²⁺ (K_0.5 ~0.4 μm); but not by similar concentrations of Mn²⁺, Zn²⁺, Co²⁺, or La²⁺. This stimulation was specific for ATP; there was little or no effect of Ca²⁺ on hydrolysis of ADP, AMP, GTP, ITP, or p-nitrophenyl phosphate. Calcium-stimulated ATPase activity was inhibited by chlorpromazine, trifluoperazine, and quercetin, as well as by sulfhydryl-blocking agents, but not by oligomycin or ouabain. No significant effect of calmodulin was observed. Finally, low concentrations of free Ca²⁺ (10 to 100 μm) in the presence or absence of Mg²⁺ stimulated transfer of ³²P from [γ-³²P]ATP to a 105,000 molecular weight shell gland membrane protein. This phosphoprotein was sensitive to hydrolysis by heating or by hydroxylamine treatment at acidic pH, and its formation was not inhibited by addition of K⁺. The specific activity of Ca²⁺-ATPase in total membrane preparations from laying hen shell gland ranged from 80 to 150 nmol/min/ mg protein, similar to or greater than levels found in purified plasma membrane fractions from a variety of tissues. No significant activity was found in membrane preparations from the magnum or isthmus regions of the oviduct, which are not involved in egg shell calcification. The characteristics of the Ca²⁺-ATPase, its high specific activity, and its preferential localization in the shell gland region of the oviduct suggest a role for an ATP-dependent calcium transport system in egg shell mineralization.     

Controlled Proteolysis Activates the Plasma Membrane Ca2+ Pump of Higher Plants (A Comparison with the Effect of Calmodulin in Plasma Membrane from Radish Seedlings)

The effects of calmodulin and of controlled trypsin treatments on the activity of the Ca2+ pump were investigated in plasma membrane purified from radish (Raphanus sativus L.) seedlings. Treatment of the plasma membrane with ethylenediaminetetra-acetate (EDTA), which removed about two-thirds of the plasma membrane-associated calmodulin, markedly increased the stimulation of the Ca2+ pump by calmodulin. In EDTA-treated plasma membrane, stimulation by calmodulin of the Ca2+ pump activity was maximal at low free Ca2+ (2-5 [mu]M) and decreased with the increase of free Ca2+ concentration. The Ca2+ pump activity was stimulated also by a controlled treatment of the plasma membrane with trypsin: the effect of trypsin treatment depended on the concentration of both trypsin and plasma membrane proteins and on the duration of incubation. Stimulation of the Ca2+ pump activity by trypsin treatment of the plasma membrane was similar to that induced by calmodulin both in extent and in dependence on the free Ca2+ concentration in the assay medium. Moreover, the Ca2+ pump of trypsin-treated plasma membrane was insensitive to further stimulation by calmodulin, suggesting that limited proteolysis preferentially cleaves a regulatory domain of the enzyme that is involved in its activation by calmodulin.     

Signal tranduction: regulation of cAMP concentration in cardiac muscle by calmodulin-dependent cyclic nucleotide phosphodiesterase

The bovine heart calmodulin-dependent phosphodiesterase can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme's affinity for calmodulin. The phosphorylation of calmodulin-dependent phosphodiesterase is blocked by Ca²⁺ and calmodulin and reversed by the calmodulin-dependent phosphatase. The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for calmodulin. The CaM-dependent phosphodiesterase isozymes of heart and brain are regulated by calmodulin, but the affinity for calmodulin are different. Furthermore, the bovine heart CaM-dependent phosphodiesterase isozyme in stimulated at much lower Ca²⁺ concentration than the bovine brain isozymes. Results from this study suggest that the activity of this phosphodiesterase is precisely regulated by cross-talk between Ca²⁺ and cAMP signalling pathways.     

Relationship between plasma membrane Ca2+-ATPase activity and acrosome reaction in guinea pig sperm

The results obtained by biochemical measurement demonstrated for the first time that significant decrease of the plasma membrane Ca²⁺-ATPase activity occurred during capacitation and acrosome reaction of guinea pig sperm. Ethaorynic acid, one kind of Ca²⁺-ATPase antagonists, inhibited the plasma membrane Ca²⁺-ATPase activity, but calmodulin (50μg/mL) and trifluoperazine (200- 500μmol/L) did not, suggesting that calmodulin is not involved in ATP-driven Ca²⁺ efflux from sperm. However, calmodulin is involved in the control of Ca²⁺ influx. TFP, one kind of calmodulin antagonists, accelerated the acrosome reaction and Ca²⁺ uptake into sperm cells significantly. Ca²⁺-ATPase antagonists, quercetin, sodium orthovandate, furosemide and ethacrynic acid promoted the acrosome reaction, but inhibited Ca2+ uptake, which cannot be explained by their inhibitory effects on the plasma membrane Ca²⁺-ATPase activity. It is speculated that this phenomenon might be caused by simultaneous inhibitions of the activities of Ca²⁺-ATPase present in the plasma membrane, the outer acrosome membrane and the outer mitochondrion membrane resulting in Ca²⁺ accumulation in the cytoplasm, which in turn blocks further Ca2+ entry through some negative feedback mechanism(s). The inhibitory effect of Ca²⁺-ATPase antagonist on glycolytic activity may also be the reason for Ca²⁺ accumulation in cytoplasm and inhibition of Ca²⁺ uptake.     

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.     

Ca2+-independent stimulation of cyclic GMP-dependent protein kinase by calmodulin

Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca²⁺ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10^?8 M and 5.0 × 10^?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. $\text{251}$, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca²⁺-dependently and independently.     

Identification and Characterization of the Ca-ATPase which Drives Active Transport of Ca at the Plasma Membrane of Radish Seedlings

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca²⁺ uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MI De Michelis [1987] Plant Physiol 83: 994-1000). A Ca²⁺-dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca²⁺-dependent ATPase is stimulated by the Ca²⁺ ionophore A₂₃₁₈₇ and is localized at the plasma membrane. Ca²⁺-dependent ATPase activity and ATP-dependent Ca²⁺ uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca²⁺ (half-maximal rate at about 70 nanomolar), and MgATP (K_m 15-20 micromolar). Ca²⁺ uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca²⁺ uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca²⁺-ATPase described in this paper is the enzyme which drives active transport of Ca²⁺ at the plasma membrane of higher plants.     

Decreases in plasma membrane Ca2+‐ATPase in brain synaptic membrane rafts from aged rats

Precise regulation of free intracellular Ca²⁺ concentrations [Ca²⁺]_i is critical for normal neuronal function, and alterations in Ca²⁺ homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca²⁺]_i is the plasma membrane Ca²⁺‐ATPase (PMCA), the high‐affinity transporter that fine tunes the cytosolic nanomolar levels of Ca²⁺. We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In this study, we isolated raft and non‐raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess calmodulin to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age‐related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.