Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca+++Mg++)-ATPase of sarcoplasmic reticulum

Summary We have shown that a Ca⁺⁺-ionophore activity is present in the (Ca⁺⁺+Mg⁺⁺)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A.E. Shamoo & D.H. MacLennan, 1974.Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca⁺⁺+Mg⁺⁺)-ATPase and Ca⁺⁺ transport, but had no effect on the activity of the Ca⁺⁺ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca⁺⁺ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca⁺⁺ transport by blocking essential-SH groups. However, it appears that there are no essential-SH groups in the Ca⁺⁺ ionophore and that mercuric chloride inhibited the Ca⁺⁺ ionophore activity by competition with Ca⁺⁺ for the ionophoric site. Blockage of Ca⁺⁺ transport by mercuric chloride probably occurs both at sites of essential-SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca⁺⁺ ionophoric activity.     

Active Uptake of Ca++ and Ca++-Activated Mg++ ATPase in Red Cell Membrane Fragments

Isolated human red blood cell membrane fragments (RBCMF) were found to take up Ca⁺⁺ in the presence of ATP.¹ This ATP-dependent Ca⁺⁺ uptake by RBCMF appears to be the manifestation of an active Ca⁺⁺ transport mechanism in the red cell membrane reported previously (Schatzmann, 1966; Lee and Shin, 1969). The influences of altering experimental conditions on Ca⁺⁺-stimulated Mg⁺⁺ ATPase (Ca⁺⁺ ATPase) and Ca⁺⁺ uptake of RBCMF were studied. It was found that pretreatment of RBCMF at 50°C abolished both Ca⁺⁺ ATPase and Ca⁺⁺ uptake. Pretreatment of RBCMF with phospholipases A and C decreased both Ca⁺⁺ ATPase and Ca⁺⁺ uptake, whereas pretreatment with phospholipase D did not significantly alter either Ca⁺⁺ ATPase or Ca⁺⁺ uptake. Both Ca⁺⁺ ATPase and Ca⁺⁺ uptake had ATP specificity, similar optimum pH's, and optimum incubation temperatures. From these results, it was concluded that Ca⁺⁺ uptake is intimately linked to Ca⁺⁺ ATPase.     

R 24571: a new powerful inhibitor of red blood cell Ca++-transport ATPase and of calmodulin-regulated functions

Compound R 24571 (1-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-β-(2,4-dichlorobenzyloxy)phenethyl]imidazoliniumchloride) is found to be a powerful inhibitor of red blood cell Ca⁺⁺-ATPase as well as Ca⁺⁺ transport into inside-out red blood cell vesicles with an IC₅₀-value of 0.5 and 2 μM, respectively. The inhibitory action of R 24571 is more specific on the calmodulin-dependent fraction of Ca⁺⁺-transport ATPase as compared to the basal Ca⁺⁺-transport ATPase (determined in the absence of calmodulin) and can be antagonized by increasing concentrations of calmodulin in an apparently competitive manner. With respect to other ATPases the action of R 24571 is relatively specific for red blood cell Ca⁺⁺-transport ATPase. Mg⁺⁺-ATPase requires a 40 times higher concentration for halfmaximal inhibition (IC₅₀ = 20 μM) whereas (Na⁺ + K⁺)-transport ATPase is only slightly affected in the investigated concentration range (≤20 μM).     

Effects of morphine tolerance and dependence on Mg++ dependent ATPase activity of synaptic vesicles.

The effect of morphine on ATPase of synaptic plasma membranes (SPM) and synaptic vesicles isolated from the mouse brain was studied. The activity of synaptic vesicle Mg⁺⁺-dependent ATPase from mice rendered morphine tolerant and dependent by pellet implantation was 40% higher than that from placebo implanted mice. However, the activities of Mg⁺⁺-dependent ATPase and Na⁺, K⁺ activated ATPase of SPM of tolerant and nontolerant mice were not significantly different. The activity of synaptic vesicular Mg⁺⁺-dependet ATPase was dependent on the concentration of Mg⁺⁺ but not of Ca⁺⁺; maximum activity was obtained with 2 mM MgCl₂. On the other hand, Mg⁺⁺-dependent ATPase activity of SPM was dependent on both Mg⁺⁺ and Ca⁺⁺, activity being maximum using 2 mM MgCl₂ and 10^?5 M CaCl₂. It is suggested that this stimulation of ATPase activity may alter synaptic transmission and may thus be involved in some aspects of morphine tolerance and dependence.     

Effect of chemical modification of cytoplasmic activator protein on human red cell membrane Ca++ + Mg+ ATPase.

A highly purified cytoplasmic activator protein of human red cell membrane Ca⁺⁺ + Mg⁺⁺ ATPase was prepared by two step purification scheme utilizing Diethylaminoethyl cellulose (DE-52) and sephadex (G-100) column chromatography. This purified protein can elicit a maximum activation of membrane Ca⁺⁺ + Mg⁺⁺ ATPase at low calcium concentrations. The stimulatory effect of this protein can be rendered totally ineffective by chemical modification with N-bromosuccinimide. The results suggest a possible role of methionine oxidation in the regulation of the Ca⁺⁺ + Mg⁺⁺ ATPase activator activity.     

Calmodulin in the membrane transport of Ca++

The involvement of calmodulin in a Ca⁺⁺-transporting process was demonstrated for the first time by Jarrett and Penniston and by Gopinath and Vincenzi in 1977. They observed that the Ca⁺⁺-pumping ATPase of the erythrocyte membrane was specifically activated by a soluble protein factor which shared the properties of calmodulin, and was indeed found to be identical with it. This fundamental observation has triggered a considerable amount of activity in the various areas of membrane transport of Ca⁺⁺. As a result, it is now known that calmodulin is involved in many processes of Ca⁺⁺ transport located in various membranes, and it has also become known that it plays different roles in different transport systems. The fine details of the interaction(s) between calmodulin and the various transport systems are at the moment obscure. It has become obvious, however, that some Ca⁺⁺ transporting enzymes interact with calmodulin directly, whereas some do not, and are influenced by it via intermediate (modulating) proteins. At the present initial state of knowledge, this difference offers a convenient means to classify the type of calmodulin involvment in the various Ca⁺⁺ transporting processes. It will thus be used in the synthetic survey presented in this article.     

Noradrenergic β-Adrenoceptor-Mediated Intracellular Molecular Mechanism of Na–K ATPase Subunit Expression in C6 Cells

Rapid eye movement sleep deprivation-associated elevated noradrenaline increases and decreases neuronal and glial Na–K ATPase activity, respectively. In this study, using C6 cell-line as a model, we investigated the possible intracellular molecular mechanism of noradrenaline-induced decreased glial Na–K ATPase activity. The cells were treated with noradrenaline in the presence or absence of adrenoceptor antagonists, modulators of extra- and intracellular Ca⁺⁺ and modulators of intracellular signalling pathways. We observed that noradrenaline acting on β-adrenoceptor decreased Na–K ATPase activity and mRNA expression of the catalytic α2-Na–K ATPase subunit in the C6 cells. Further, cAMP and protein kinase-A mediated release of intracellular Ca⁺⁺ played a critical role in such decreased α2-Na–K ATPase expression. In contrast, noradrenaline acting on β-adrenoceptor up-regulated the expression of regulatory β2-Na–K ATPase subunit, which although was cAMP and Ca⁺⁺ dependent, was independent of protein kinase-A and protein kinase-C. Combining these with previous findings (including ours) we have proposed a working model for noradrenaline-induced suppression of glial Na–K ATPase activity and alteration in its subunit expression. The findings help understanding noradrenaline-associated maintenance of brain excitability during health and altered states, particularly in relation to rapid eye movement sleep and its deprivation when the noradrenaline level is naturally altered.     

Studies on the Active Transport of Calcium in Human Red Cells

The Ca⁺⁺ transport mechanism in the red cell membrane was studied in resealed ghost cells. It was found that the red cell membrane can transport Ca⁺⁺ from inside the cell into the medium against great concentration gradient ratios. Tracing the movement of ⁴⁵Ca infused inside red cells indicated that over 95% of all Ca⁺⁺ in the cells was transported into media in 20 min incubation under the optimum experimental conditions. The influence of temperature on the rate constant of transport indicated an activation energy of 13,500 cal per mole. The optimum pH range of media for the transport was between 7.5 and 8.5. As energy sources, ATP¹, CTP, and UTP were about equally effective, GTP somewhat less effective, and ITP least effective among the nucleotides tested. The Ca⁺⁺ transport does not appear to involve exchange of Ca⁺⁺ with any monovalent or divalent cations. Also, it is not influenced by oligomycin, sodium azide, or ouabain in high concentrations, which inhibit the Ca⁺⁺ transport in mitochondria or in sarcoplasmic reticulum. In these respects, the Ca⁺⁺ transport mechanism in the red cell membrane is different from those of mitochondria and the sarcoplasmic reticulum.     

ATPase Activity of Myosin Correlated with Speed of Muscle Shortening

Myosin was isolated from 14 different muscles (mammals, lower vertebrates, and invertebrates) of known maximal speed of shortening. These myosin preparations were homogeneous in the analytical ultracentrifuge or, in a few cases, showed, in addition to the main myosin peak, part of the myosin in aggregated form. Actin- and Ca⁺⁺-activated ATPase activities of the myosins were generally proportional to the speed of shortening of their respective muscles; i.e. the greater the intrinsic speed, the higher the ATPase activity. This relation was found when the speed of shortening ranged from 0.1 to 24 muscle lengths/sec. The temperature coefficient of the Ca⁺⁺-activated myosin ATPase was the same as that of the speed of shortening, Q₁₀ about 2. Higher Q₁₀ values were found for the actin-activated myosin ATPase, especially below 10°C. By using myofibrils instead of reconstituted actomyosin, Q₁₀ values close to 2 could be obtained for the Mg⁺⁺-activated myofibrillar ATPase at ionic strength of 0.014. In another series of experiments, myosin was isolated from 11 different muscles of known isometric twitch contraction time. The ATPase activity of these myosins was inversely proportional to the contraction time of the muscles. These results suggest a role for the ATPase activity of myosin in determining the speed of muscle contraction. In contrast to the ATPase activity of myosin, which varied according to the speed of contraction, the F-actin-binding ability of myosin from various muscles was rather constant.     

Technical considerations for assessing alterations in skeletal muscle sarcoplasmic reticulum Ca++-sequestration functionin vitro

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca⁺⁺-ATPase activity and Ca⁺⁺-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca⁺⁺-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca⁺⁺-activated Ca⁺⁺-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca⁺⁺-uptake was measured at a range of physiological free [Ca⁺⁺] using the Ca⁺⁺ fluorescent dye Indo-1. Maximum Ca⁺⁺-uptake rates when corrected for initial [Ca⁺⁺]_f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca⁺⁺ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca⁺⁺-uptake and Ca⁺⁺-ATPase activity measured in the presence of the Ca⁺⁺ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca⁺⁺-uptake (r=+0.44, P<0.05) and between HOM and CM Ca⁺⁺-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca⁺⁺-uptake function and maximal Ca⁺⁺-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994)     

Coordinated, coenzyme Q reversible, 2,5-dibromothymoquionine inhibition of electron transport and ATPase in Escherichia coli.

2,6-dibromothymoquinone (DBMIB) and other coenzyme Q analogs partially inhibit electron transport and the membrane-bound Mg⁺⁺ stimulated ATPase of $\text{E. coli}$ membranes. The inhibitions by DBMIB are fully reversed by coenzyme Q₆, and other analogs show partial reversal by coenzyme Q₆. Electron transport reactions inhibited are NADH and lactate oxidase, NADH menadione reductase, lactate phenazinemethosulfate reductase and duroquinol oxidase. The concentrations of DBMIB required are similar for electron transport and ATPase inhibition and inhibitions are all increased by uncouplers. Electron transport and ATPase are not inhibited in a DBMIB insensitive mutant. Soluble ATPase extracted from the membranes does not show DBMIB inhibition under either high or low Mg⁺⁺ conditions. Lipophilic chelators show additional inhibition over DBMIB. It appears that coenzyme Q functions at three sites in $\text{E. coli}$ electron transport where ATPase activity is controlled. Coenzyme Q deficient mutants also show decreased electron transport and ATPase activity which is restored by coenzyme Q.     

Membrane transport properties differ following return of serum-deprived versus Ca++-deprived human fibroblasts to a proliferative state

Human lung fibroblasts (W138) can be brought to a quiescent state by removal of serum from the medium or by lowering of the extracellular Ca⁺⁺. Upon return of Ca⁺⁺ or serum, the cells enter the G1 phase and progress to S within 15–18 hours. Since multiple G1 phase blocks have been demonstrated, we wished to determine whether the Ca⁺⁺ and serum block were equivalent since previous data suggested that these two medium components may act at a common point in the initiation of proliferation. We have evaluated the membrane transport of ⁸⁶Rb, 3-O-methylglucose, AIB, and cycloleucine following stimulation of quiescent cells by Ca⁺⁺ or serum. Serum stimulation results in large increases in the influx of all the substances tested. These increases are prevented if Ca⁺⁺ is absent upon serum stimulation or they are rapidly diminished following Ca⁺⁺ removal. In contrast, Ca⁺⁺ stimulation of Ca⁺⁺-deprived cells causes little or no enhancement of any of the transport systems, yet the cells progress to S phase in a manner similar to serum-stimulated cells. These results indicate that the Ca⁺⁺ and serum G0 or G1 block are not equivalent and that the serum-induced change in transport of these components does not appear necessary for successful G1 phase progression. Furthermore, the data suggest that the sequence in which Ca⁺⁺ or serum are presented to the cells alters the ability of Ca⁺⁺ to modulate the transport systems. Quiescent cells which are exposed to Ca⁺⁺ prior to serum possess a Ca⁺⁺ modulation of several transport systems. Cells which are exposed to Ca⁺⁺ subsequent to serum do not appear to possess this Ca⁺⁺ regulation.     

Effect of lipid on the access of ATP and calcium to the delipidated Ca++-ATPase of intestinal brush border membrane.

The delipidated Ca⁺⁺-ATPase prepared from intestinal brush border membranes showed a higher activity of Ca⁺⁺-independent ATPase, a lower Km value for ATP and a higher Km value for Ca⁺⁺ than its original membrane Ca⁺⁺-ATPase. The addition of phosphatidylcholine re-activated the delipidated Ca⁺⁺-ATPase to approximately 89 % of its original membrane Ca⁺⁺-ATPase activity but did not restore the affinity for Ca⁺⁺. This phospholipid raised the Km value for ATP but had little effect on the Km value for Ca⁺⁺. Palmitic acid elevated the Km value for Ca⁺⁺ but did not change the Km value for ATP. Kinetic analyses of these data suggest that the hydrocarbon chain of phosphatidylcholine is an important rate-limiting factor for the access of Ca⁺⁺ to the enzyme and the polar head groups of phosphorylcholine and ester bond may be the factor for the access of ATP.     

Inhibition of Na+-coupled solute transport by calcium in brush border membrane vesicles

Intracellular Ca⁺⁺ is known to influence Na⁺ flux in luminal membranes. Abnormally elevated Ca⁺⁺ levels in some cells is believed to be the primary pathophysiologic defect in cystic fibrosis (CF). This in turn is thought to alter Na⁺ transport which accounts for certain clinical manifestations of this disease. Two Na⁺-dependent intestinal transport mechanisms have been reported to be suppressed or missing in CF. To examine whether alterations in cell Ca⁺⁺ may account for these findings, studies were performed to examine the influence of Ca⁺⁺ on Na⁺-solute co-transport across intestinal luminal membranes. Purified brush border membrane vesicles prepared from rat small bowel were preincubated in either Ca⁺⁺-free buffer or buffer containing 2.5 mM CaCl₂. Ca⁺⁺ loaded vesicles showed marked inhibition of Na⁺ co-transport of taurocholic acid, taurochenodeoxycholic acid, glucose and valine when compared to controls. The uptake of Na⁺ was also significantly reduced by intravesicular Ca⁺⁺. These data demonstrate that intravesicular Ca⁺⁺ inhibits Na⁺-coupled solute transport as well as Na⁺ influx across intestinal brush border membranes. These data suggest that intracellular Ca⁺⁺ may suppress Na⁺-dependent solute absorption in the intestine. Results presented here further support the theory that elevated intracellular Ca⁺⁺ may account for intestinal malabsorption and other altered transport phenomena reported in CF.     

Evidence for solvent-induced conformational changes of the soluble Dunaliella chloroplast coupling factor 1 (CF1)

The ATPase activity of the chloroplast coupling factor 1 (CF₁) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca²⁺ -dependent activity. The Ca²⁺ dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca²⁺ to Mg²⁺. Both the Ca²⁺ -dependent and Mg²⁺ -dependent ATPase activities of heat-treated Dunaliella CF₁ are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF₁. However, when assayed under identical conditions, the Ca²⁺ -dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg²⁺ -dependent activity. These data are interpreted as indicating that soluble Dunaliella CF₁ can exist in a variety of conformations, at least one of which catalyzes a Ca²⁺ -dependent ATPase and two or more of which catalyze an Mg²⁺ -dependent ATPase.     

Correlations of Length and Volume Measurements in Myofibril Suspensions

Instrumentation has been developed for the rapid electronic sizing of large numbers of myofibrils. The response of myofibrils in the presence of ATP to changes in Ca⁺⁺ concentration was examined. Shortening of myofibrils upon addition of Ca⁺⁺ was accompanied by an increased protein effective volume of approximately 10-40%. Whereas ATPase activation and increased turbidity of myofibrils upon addition of Ca⁺⁺ were reversible upon subsequent addition of EGTA, the shortening and swelling were irreversible. It is proposed that the swelling may result from the breaking of hydrophobic bonds within myosin. The ATPase activity and turbidity are measures of the input, while the shortening and swelling are measures of the output of a coupled nonequilibrium process; failure of reversal of the output indicates an uncoupling under the experimental conditions.     

Comparison of the reaction of N-ethylmaleimide with myosin and heavy meromyosin subfragment 1

Myosin reacted at low ionic strength with NEM forms an actomyosin which is Ca⁺⁺ insensitive. With HMM S-1 the reaction with NEM causes a marked loss of the actin activated ATPase activity and the Ca⁺⁺ sensitivity is reduced but not eliminated. The presence of actin during the sulfhydryl reaction does not significantly alter this result. HMM S-1 prepared from myosin previously desensitized by NEM regains Ca⁺⁺ sensitivity. These results indicate that the conformations of myosin and HMM S-1 are different and could reflect a difference between insoluble (filamentous) myosin and myosin, or its fragments, in solution.     

Effects of beta-bungarotoxin on calcium uptake by sarcoplasmic reticulum from rabbit skeletal muscle

A fluorescent chelate probe and a Millipore filtration technique have been used to study the effects of β-bungarotoxin (β-toxin) on passive and active Ca⁺⁺ uptake and ATPase in fragmented sarcoplasmic reticulum (SR) of rabbit skeletal muscle. β-Toxin at 3 × 10^?6 M did not affect ATPase activity. In the absence of ATP, β-Toxin increased the passive uptake of Ca⁺⁺; in the presence of ATP, active Ca⁺⁺ uptake was inhibited. The effect of β-toxin in SR can be detected at concentrations as low as 10^?9 M. The results suggest that β-toxin induces Ca⁺⁺ leakage in SR membranes.     

Calcium-dependent phosphatidylinositol phosphorylation in lamellibranch gill lateral cilia

Summary Pure lateral (L) cilia may be separated from the remaining (R) cilia types ofMytilus edulis gill by serotonin activation after hypertonic shock. The two classes of cilia were permeabilized with 0.012% Triton X-100 and incubated with³²P-labeled ATP at low Ca⁺⁺ (10^–7 M), where L cilia beat, or in high Ca⁺⁺ (2–20 M), where L cilia arrest but R cilia are active. The labeled cilia were separated into axoneme and membrane-matrix fractions by detergent extraction, subjected to SDS-PAGE on 5–15% gels, and autoradio-graphed. Neither cilia type undergoes Ca⁺⁺-dependent phosphorylation of specific proteins, suggesting that neither Ca⁺⁺-induced arrest in L cilia nor the Ca⁺⁺ activation of other cilia is phosphorylation-dependent. However, lipid phosphorylation in L cilia is highly Ca⁺⁺-dependent. Identified by thin-layer chromatography, the phospholipid that is phosphorylated in a Ca⁺⁺-dependent manner is phosphatidylinositol 4-phosphate (PIP), yielding the 4,5-bisphosphate (PIP₂). PIP₂ increases at least 3-fold under Ca⁺⁺-arrest conditions.Aequipecten gill lateral cilia, which require higher Ca⁺⁺ levels for arrest, show even more striking changes. In both cases, the effect is maximal at micromolar Ca⁺⁺ levels. Phosphorylation of other lipids is Ca⁺⁺-independent. In the Ca⁺⁺-insensitive or activated R cilia, PIP₂ levels are intermediate, increasing only marginally with increased [Ca⁺⁺]. The formation of PIP₂ in response to Ca⁺⁺, as opposed to its breakdown to form inositol 1,4,5-trisphosphate and diacylglycerol, may be characteristic of a Ca⁺⁺ transport system. Mechanically sensitive, the L cilia arrest as a consequence of an inward flux of Ca⁺⁺ ions, acting directly on the axoneme. After Ca⁺⁺-induced arrest, the formation of PIP₂ may be involved in sequestering Ca⁺⁺ or in augmenting Ca⁺⁺ pump activity, thus reducing Ca⁺⁺ levels so that motility may resume quickly.     

Phosphatidate releases calcium from cardiac sarcoplasmic reticulum

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