The effect of ionizing radiation on the Ca2+, Mg2(+)-ATPase activity of plasma membranes]

A study was made of a change in Ca2+, Mg(2+)-ATPase activity induced by the effect of ionizing radiation (5-10(4) Gy) on a thymocyte plasma membrane suspension. The Michaelis' constant and maximum rate of enzymic reactions were determined. With a dose of 10(3) Gy the structural changes in Ca2+, Mg(2+)-ATPase were shown to reduce the affinity of the substrate to an active enzyme center and to decrease the rate of the enzyme/substrate complex degradation.     

Ca2(+)-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2(+)-ATPase as detected by the use of proteolytic enzyme (V8)

Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.     

Modification of ATP regulatory function in sarcoplasmic reticulum Ca2(+)-ATPase by hydrophobic molecules

The effects of the three hydrophobic molecules triphenylphosphine, trifluoperazine and 3-nitrophenol on Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles was investigated. When ATP was the substrate, triphenylphosphine (3 microM) increased the amount of Ca2+ accumulated by the vesicles. At high concentrations triphenylphosphine inhibited Ca2+ uptake. This effect varied depending on the ATP concentration and the type of nucleotide used. With ITP there was only inhibition and no activation of Ca2+ uptake by triphenylphosphine. On the other hand, trifluoperazine inhibited Ca2+ accumulation regardless of whether ATP or ITP was used as substrate. When 5 mM oxalate was included in the medium in order to avoid binding of Ca2+ to the low-affinity Ca2(+)-binding sites of the enzyme, both stimulation by triphenylphosphine and inhibition by trifluoperazine were reduced. In leaky vesicles at low Ca2+ concentrations, triphenylphosphine and 3-nitrophenol were competitive inhibitors of ATPase activity at the regulatory site of the enzyme (0.1-1 mM ATP). A striking difference was observed when both the high- and low-affinity Ca2(+)-binding sites were saturated. In this condition, triphenylphosphine and 3-nitrophenol promoted a 3-4-fold increase in the apparent affinity for ATP at its regulatory site.     

Abnormal Ca2(+)-ATPase activity in erythrocytes of non-insulin-dependent diabetic rats

Non-insulin-dependent diabetic (NIDD) rats have an increased Ca2(+)-ATPase activity in their kidney basolateral membranes. We find that a similar increased activity occurs in erythrocytes of the NIDD animals. This alteration in membrane ATPase activity appears to be specific for the Ca2(+)-ATPase as (Na(+) + K+) and Mg2(+)-ATPase and Na, K and Mg concentrations in the erythrocyte were not affected by the diabetic condition in these animals. Thus, abnormalities in membrane Ca2(+)-ATPase activity in the NIDD rats are not restricted to one tissue and appear to be a generalized pathology in the NIDD animals.     

Cytochemical localization of high-affinity Ca2(+)-ATPase activity in synaptic terminals

High-affinity Ca2(+)-ATPase activity in the optic tectum of the brain of cichlid fish was cytochemically localized using cerium ions to precipitate phosphate. Activation of the enzyme with micromolar instead of millimolar calcium concentrations (i.e., physiological cytoplasmic instead of extracellular concentrations) resulted in intracellular localization of reaction product attached to the cytoplasmic side of plasma membranes and to synaptic vesicles. The plasmalemmal enzyme activity was concentrated in synaptic regions. Synaptic vesicles in some terminals exhibited high amounts of ATPase activity, whereas others were free of reaction product. By use of electron energy-loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) techniques, even small amounts of cerium-containing precipitates could be analyzed and precisely localized. The cytochemical observations are in good agreement with biochemical findings and therefore indicate that the calcium pump of neuronal plasma membranes can be successfully localized.     

Age-related determination of the basal and calmodulin-stimulated human RBC Ca2(+)-ATPase activity

The basal and calmodulin-stimulated activity of the human erythrocyte Ca2(+)-ATPase was determined in 56 healthy individuals of different ages in order to set control values. The basal activity of all 56 healthy subjects was 0.805 +/- 0.525 mumole hydrolyzed ATP/mg protein/hr, while the calmodulin-stimulated activity gave an average of 2.437 +/- 0.785 mumole hydrolyzed ATP/mg protein/hr. There was no significant difference in basal Ca2(+)-ATPase activity in males and females; however, significantly increased levels of the stimulated red cell calcium pump was seen in females. Based on age, the basal activity for newborns and children up the age of 7 years was higher than that for adults and a distinct increase in activation by calmodulin was observed with a maximum at the age range of 8-13 years. It was demonstrated that for the investigation of Ca2(+)-ATPase activities in children different normal values must be taken into consideration than for those already reported for adults. Our data may serve as controls for Ca2(+)-ATPase activities for comparison purposes and for further studies from different diseases in childhood where increased intraerythrocytic calcium levels are reported.     

The bacterial Kdp K(+)-ATPase and its relation to other transport ATPases, such as the Na+/K(+)- and Ca2(+)-ATPases in higher organisms

The Kdp system is a three-subunit member of the E1-E2 family of transport ATPases. There is sequence homology of the 72 kDa KdpB protein, the largest subunit of Kdp, with the other members of this family. The predicted structure of the 21 kDa KdpC subunit resembles that of the beta subunit of the Na+,K(+)-ATPase, suggesting that these subunits may have a similar function. The 59 kDa KdpA subunit has no known homologue; it is very hydrophobic and is predicted to cross the membrane 10-12 times. Genetic studies implicate this subunit in the binding of K+. As the binding site must be close to the beginning of the transmembrane channel, we suggest that KdpA also forms most or all of the latter. KdpA may have evolved from a K+/H+ antiporter that was recruited by the KdpB precursor to achieve the high affinity and specificity for K+, and the activation of transport by low turgor pressure characteristic of Kdp. Turgor pressure controls the expression of Kdp. This action is dependent on the 70 kDa KdpD and 23 kDa KdpE proteins. We are in the process of sequencing these genes. KdpE is homologous to the smaller protein of other members of a family of pairs of regulatory proteins implicated in control of a variety of bacterial processes such as porin synthesis, phosphate regulon expression, nitrogen metabolism, chemotaxis and nodule formation.     

Cholesterol effect on enzyme activity of the sarcolemmal (Ca2+ + Mg2+)-ATPase from cardiac muscle

The effect of cholesterol incorporation and depletion of the cardiac sarcolemmal sacs on (Ca2+ + Mg2+)-ATPase activity was examined. Cholesterol incorporation to the sarcolemmal sacs was achieved utilizing an in vivo and an in vitro procedure. Cholesterol depleted membranes were obtained in vitro after incubation of the sarcolemmal sacs with inactivated plasma. Arrhenius plots of the (Ca2+ + Mg2+)-ATPase activity showed a triphasic curve when the assays were carried out using a temperature range between 0 and 40 degrees C. The sarcolemmal (Ca2+ + Mg2+)-ATPase activity was shown to be inversely proportional to the cholesterol concentration of the membranes, showing a low ATPase activity with a high cholesterol content and a high ATPase activity when the cholesterol concentration was low. Although the (Ca2+ + Mg2+)-ATPase activity was found to be inhibited in the cholesterol incorporated sarcolemmal sacs, the withdrawal of small amounts of cholesterol from the membranes produced an important stimulatory effect. Changes in (Ca2+ + Mg2+)-ATPase activity due to variation in the membrane cholesterol concentration were shown to be reversible. Our results indicate the possibility of a slow exchange of cholesterol between the tightly bound lipid surrounding the (Ca2+ + Mg2+)-ATPase and the bulk lipid of the sarcolemma.     

Ca2+ transport by the synaptosomal plasma membrane Ca2+-ATPase and the effect of thioridazine

Thioridazine inhibits the activity of the synaptic plasma membrane Ca(2+)-ATPase from pig brain and slightly decreases the rate of Ca(2+) accumulation by synaptic plasma membrane vesicles in the absence of phosphate. However, in the presence of phosphate, thioridazine increases the rate of Ca(2+) accumulation into synaptic plasma membrane vesicles. Phosphate anions diffuse through the membrane and form calcium phosphate crystals, reducing the free Ca(2+) concentration inside the vesicles and the rate of Ca(2+) leak. The higher levels of Ca(2+) accumulation obtained in the presence of thioridazine could be explained by a reduction of the rate of slippage on the plasma membrane ATPase.     

Characterization of a putative Ca2(+)-transporting Ca2(+)-ATPase in the pellicles of Paramecium tetraurelia

In Paramecium, no Ca2(+)-ATPases with the properties of Ca2+ pumps have been identified. Here we report a pellicle associated Ca2(+)-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca2(+)-ATPase activity requires 3 mM Mg for optimal Ca2+ stimulation (KCa = 90 nM) and is specific for ATP as substrate (Km = 75 microM). Vanadate and calmidazolium inhibit Ca2(+)-stimulated activity with an EC50 of about 2 microM and 0.5 microM, respectively. Likewise, 10 microM trifluoperazine inhibits 80% of Ca2(+)-ATPase activity, but bovine calmodulin fails to stimulate. The Ca2(+)-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 micrograms/ml) or ouabain (0.2 mM). Incubation of pellicles with [gamma-32P]ATP specifically labels a 133 kDa protein in a Ca2(+)-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 microM La3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca2(+)-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La3+. Ca2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca2(+)-stimulated ATP hydrolysis (Ca2+ transported/ATP hydrolysed less than 0.2) and is much less sensitive to vanadate inhibition (EC50 approx. 20 microM) compared to the total Ca2(+)-ATPase activity. Therefore, the majority of the Ca2(+)-ATPase activity is likely to be plasma membrane associated.     

The modulation of Ca2+-ATPase activity of sarcoplasmic reticulum by membrane cholesterol. The effect of enzyme coupling

The Ca2+-ATPase activity of sarcoplasmic reticulum is relatively low (less than 2 I.U.) in vesicles where enzyme activity is geared to calcium accumulation. Modulation of membrane fluidity by enriching the membrane with cholesterol has no significant effect on enzyme activity. Collapsing the Ca2+ gradient with the calcium ionophore, A23187, unmasks the inhibitory effect of membrane cholesterol on enzyme activity.     

High efficiency Ca2+ transport by the sarcoplasmic reticulum Ca2(+)-ATPase in the absence of the 53-kilodalton glycoprotein

Although the Ca2(+)-ATPase is the predominant protein species of the skeletal sarcoplasmic reticulum membrane, the functional significance of other minor protein species remains unresolved. The proposition has been tested that the membrane-bound 53-kDa glycoprotein (GP-53) may be required or significantly involved in regulating the coupling of ATP hydrolysis to Ca2+ transport by the Ca2(+)-ATPase. Ca2(+)-ATPases originating from preparations with and without GP-53 were reconstituted into phosphatidylcholine liposomes, and Ca2+ uptake and pumping efficiency were determined. The reconstituted Ca2+ pump from all preparations transported Ca2+ with high efficiency (Ca2+:ATP greater than 1.5). The results demonstrate that GP-53 is not required to couple ATP hydrolysis to Ca2+ transport. Additionally, the observed high coupling efficiency is inconsistent with GP-53 functioning as a substantial positive regulator of coupling.     

Retention of enzyme activity by detergent-solubilized sarcoplasmic Ca2+ -ATPase.

The Ca2+ -activated ATPase of sarcoplasmic reticulum can exist in true solution in the presence of some nonionic detergents, with retention of enzymatic activity for several days. The soluble active particles retain about 30 mol of phospholipid per mol of polypeptide chain even in the presence of a large excess of detergent, indicating the existence of relatively strong attractive forces between protein and lipid, as previous work from other laboratories has already suggested. Deoxycholate is much more effective than nonionic detergents in removing protein-bound lipid and, when used at solubilizing concentrations, completely delipidates and inactivates the ATPase. Preliminary molecular weight measurements indicate that the Ca2+ -ATPase exists as an oligomer in the native membrane: fully active enzyme in Tween 80 has a minimal protein molecular weight of about 400 000, corresponding to a trimer or tetramer of the ATPase polypeptide chain, and even the inactive enzyme in deoxycholate contains a substantial fraction of dimeric protein.     

Monomeric state and Ca2+ transport by sarcoplasmic reticulum Ca2(+)-ATPase, reconstituted with an excess of phospholipid

The structural basis for Ca2+ transport was examined in vesicles reconstituted with an excess of phospholipid by a cholate dialysis procedure. Unincorporated protein and vesicles with a relatively high protein content were removed by sucrose density centrifugation (3-12%), leaving a fraction of lipid-rich vesicles (lipid to protein weight ratio 800-900:1) with a high coupling ratio (1.0) and transport capacity (25 mumol/mg protein, after Ca-phosphate loading). Freeze-fracture analysis showed that the reconstituted vesicles had a remarkably narrow size distribution (diameter 794 +/- 77 A (S.D.], suitable for stereological analysis. Intramembranous particles were dispersed and occurred with a low frequency in the fractured shells, also before sucrose fractionation. It was calculated that the number of intramembranous particles corresponded to the number of Ca2(+)-ATPase polypeptide/vesicle. A ratio of unity between particles and polypeptide chains was also obtained from the density of particle distribution on flat surfaces of fused vesicles, prepared by sucrose fractionation. The size of the particles formed a broad distribution, having a peak value around 60-67 A, both in the reconstituted preparation and sarcoplasmic reticulum vesicles. No evidence for protein-protein interactions was found in chemical cross-linking experiments. It is concluded that the intramembranous particles in the reconstituted preparations are referable to monomeric Ca2(+)-ATPase which is capable of transporting Ca2+ inside the vesicles. The implications of the observations for the associational state of Ca2(+)-ATPase at high protein concentration are considered in relation to previous ultrastructural investigations of membranous Ca2(+)-ATPase in native and two-dimensional-crystalline forms.     

Characteristics of a presynaptic plasma membrane Ca2(+)-ATPase activity from electric organ

Ca2(+)-ATPase activity was measured in electric organ synaptosomal homogenates and their derived presynaptic plasma membranes using a low ionic strength medium, low in Ca2+ and Mg2+, and devoid of K+. The enzyme activity showed a high apparent affinity for Ca2+ (KCa:0.5 microM) and was: (1) 5-fold stimulated by 120 nM calmodulin, (2) highly sensitive to LaCl3 inhibition, and (3) not affected by 20 mM NaN3 or 0.1 mM ouabain. The addition of Mg2+ promoted the disappearance of Ca2(+)-ATPase activity. Incubation of synaptosomal homogenates in the above-mentioned assay medium with [gamma -32P]ATP resulted in the appearance of a 140 kDa band as revealed by SDS-gel electrophoresis. Labeling of this band with 32P was inhibited by 1 mM EGTA or 10 mM NH2OH, indicating that the isotope incorporation required the presence of Ca2+ and the formation of an acyl-phosphate derivative. The results indicate that the Ca2(+)-ATPase activity from synaptosomal homogenates had characteristics corresponding to those of the enzyme that catalyzes an outward transport of Ca2+ in nerve terminals. Preincubation of synaptosomes in Ca2+ plus K+, a depolarizing procedure, induced a large and rapid decrease in the Ca2(+)-ATPase activity, possibly mediated via Ca2+ entry through voltage-gated Ca2+ channels. Furthermore, the muscarinic cholinergic agonist oxotremorine (at 15 microM concentration) did not significantly affect either the enzyme activity or the intensity of the Ca2(+)-dependent 32P incorporation into the 140 kDa band, suggesting that the enzyme is not coupled to muscarinic binding sites.     

Ca2+-induced phase separation in black lipid membranes and its effect on the transport of a hydrophobic ion

Voltage jump-current relaxation studies have been performed with dipicrylamine-doped black membranes of binary lipid mixtures. As in the case of the carrier-mediated ion transport (Schmidt, G., Eibl, H. and Knoll, W. (1982) J. Membrane Biol. 70, 147-155) no evidence was found that the neutral lipid phosphatidylcholine (DPMPC) and the charged phosphatidic acid (DPMPA) are heterogeneously distributed in the membrane over the whole range of composition. However, besides a continuous dilution of the surface charges of DPMPA by the addition of DPMPC molecules, different structural properties of mixed membranes influence the kinetics of the dipicrylamine transport. The addition of Ca2+ to the electrolyte induces a lipid phase separation within the membrane into two fluid phases of distinctly different characteristics of the translocation of hydrophobic ions. Thus, it is possible to determine a preliminary composition phase diagram for the DPMPA/DPMPC mixtures as a function of the Ca2+ concentration.     

Ca2+ transport against its electrochemical gradient in cytochrome oxidase vesicles reconstituted with mitochondrial hydrophobic proteins

Cytochrome oxidase vesicles have recently been shown to accumulate Ca²⁺ in an energy-dependent manner. Energization of these vesicles with internally trapped cytochrome c and externally added ascorbate and phenazine methylsulfate generated an internally positive membrane potential and prevented Ca²⁺ influx (R. N. Rosier and T. E. Gunter, 1980, FEBS Lett.109, 99–103). In contradistinction, when cytochrome oxidase vesicles were reconstituted with complex V, a mitochondrial protein fraction containing the uncoupler binding site (Y. Hatefi, D. L. Stiggall, Y. Galante and W. G. Hanstein, 1974, Biochem. Biophys. Res. Commun.61, 313–321), both Ca²⁺ uptake and generation of an internally positive membrane potential were observed. The uptake was specifically dependent on energization of electron transport. Control experiments verified that the energization conditions used produced appropriately oriented membrane potentials. Other partially purified hydrophobic mitochondrial protein complexes were found to be less effective than complex V. The reconstituted system showed cation selectivity since Ca²⁺, Mn²⁺, and Rb⁺ were transported, while Na⁺ was not. Low levels of uncoupler, which did not affect oxidation rates, were found to partially inhibit Ca²⁺ uptake regardless of the membrane potential polarity. Uncoupling levels of uncoupler markedly inhibited Ca²⁺ uptake in internally negative cytochrome oxidase vesicles; however, inhibition in internally positive cytochrome oxidase vesicles was less relative to that at lower levels of uncoupler. The uncoupling combination of nigericin, valinomycin, and K⁺ was inhibitory to uptake regardless of membrane potential polarity. A reconstituted system of oxidative phosphorylation, which contains a hydrophobic protein fraction, energized with cytochrome oxidase similarly accumulated Ca²⁺ despite formation of an internally positive membrane potential. The results suggest that cytochrome oxidase, when coupled to appropriate hydrophobic mitochondrial proteins, can act as an electrogenic Ca²⁺ pump deriving its energy directly from electron transport.     

Effects of calmodulin on erythrocyte Ca2(+)-ATPase activation and oligomerization

The study was performed on the purified human erythrocyte Ca2(+)-ATPase to test whether or not calmodulin promotes enzyme oligomerization. Two physiologically significant modes of activation of this enzyme were considered, by calmodulin binding to monomeric enzyme and by enzyme oligomerization [Kosk-Kosicka & Bzdega (1988) J. Biol. Chem. 263, 18184]; it was not clear whether the two modes were interdependent or operated independently. Fluorescence resonance energy transfer (FRET) between separately labeled Ca2(+)-ATPase molecules was used to monitor oligomerization. No change in energy transfer efficiency was observed upon subsequent addition of calmodulin at different enzyme concentrations. Lack of decrease in the enzyme concentration at which the half-maximal oligomerization occurred indicated that calmodulin did not facilitate oligomerization. The calmodulin inhibitor compound 48/80 had no effect on either the Ca2(+)-ATPase activity of oligomers or the extent of oligomerization measured by FRET while it drastically decreased the calmodulin-stimulated activity of the monomeric Ca2(+)-ATPase. The findings demonstrate that calmodulin is not involved in the oligomerization-induced activation pathway; it neither promotes oligomerization nor stimulates the Ca2(+)-ATPase activity of oligomers. We have demonstrated that calmodulin added before mixing donor- and acceptor-labeled enzyme populations prevented the occurrence of energy transfer. This inhibition of the formation of mixed donor-acceptor enzyme oligomers by calmodulin was dose dependent. Also, the reversal of the inhibition by compound 48/80 proceeded in a dose-dependent manner. Further, calmodulin prevented the apparent decrease of energy transfer efficiency that resulted from dilution of mixed donor-acceptor enzyme oligomers with unlabeled enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of acylphosphatase on human erythrocyte membrane Ca2(+)-ATPase

We studied the effect of human acylphosphatase on the activity of human erythrocyte membrane Ca2(+)-ATPase. Both the acylphosphatase that is contained in hemolysate and the purified enzyme isolated from red blood cells were able to stimulate Ca2(+)-ATPase activity in erythrocyte membranes. Given the same acylphosphatase activity, however, the hemolysate showed higher stimulatory effect than the purified enzyme. Acylphosphatase stimulation was additive to that induced by calmodulin, thus indicating that acylphosphatase acts in a calmodulin-independent manner. Trifluoperazine, a calmodulin antagonist, did not inhibit acylphosphatase-induced stimulation of Ca2(+)-ATPase activity. Acylphosphatase significantly decreased the rate of Ca2+ influx into inside-out erythrocyte membrane vescicles, thus acting as Ca2+ pump inhibitor. Taken together these findings indicate that acylphosphatase is a soluble, non-calmodulin activator of erythrocyte membrane Ca2(+)-ATPase and might be involved in the control of calcium transport across the plasma membrane.