The distribution and properties of the Mg2(+)-ATPase in the membranes of functionally different muscles in the hen]

From striated (m. pectoralis and myocardium) and smooth (myometrium) muscle tissues of hen, by means of differential centrifugation with Ca-oxalate loading, membrane preparations were obtained with high activity of Mg(2+)-ATPase, i.e. a marker enzyme of tubular membranes of T-system of skeletal muscles. Some properties (pH and temperature optima) of this enzyme were investigated and compared to those of Ca(2+)-ATPase from membranes of the sarcoplasmic reticulum. It was shown that in all the investigated muscles, Mg(2+)-ATPase is associated with membrane fraction which in its density corresponds to tubular membranes of T-system. Activation of this enzyme is characterized by similar optimal levels of pH (7.2) and temperature (25 degrees C). The activity of Ca(2+)-ATPase in the membranes of the sarcoplasmic reticulum, in contrast to that of Mg(2+)-ATPase, is observed in more narrow bands of pH and temperature, exhibiting tissue specificity. The data obtained, indicating a possibility of chromatographic separation of these enzymes, confirm their biochemical individuality.     

Inhibition of calcium-dependent ATPase from sarcoplasmic reticulum by a new class of indolizidine alkaloids, pumiliotoxins A, B, and 251D

Pumiliotoxins (PTX) A, B, and 251D, members of a new class of indolizidine alkaloids isolated from the skin of poison frogs of the family Dendrobatidae, inhibit Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from frog and rat hind-limb muscles. PTX-B and PTX-A appear to be relatively specific inhibitors of Ca2+-ATPase; PTX-A is much less potent than PTX-B. PTX-251D is a potent inhibitor of Ca2+-ATPase, and was also found to inhibit Na+, K+, and Mg2+-ATPases in rat brain synaptosomes. Caffeine and verapamil, two drugs known to affect calcium translocation, are very weak inhibitors of the Ca2+-ATPase. The Ki values for inhibition of the Ca2+-ATPase of rat and frog sarcoplasmic reticulum by PTX-B were comparable and ranged between 22 and 36 microM. Inhibition of calcium-dependent ATPase in sarcoplasmic reticulum by pumiliotoxin-B is noncompetitive with calcium and is not readily reversible. Based on structure-activity profiles, it is concluded that inhibition of Ca2+-ATPase by the indolizidine alkaloids is responsible for the alkaloid-elicited prolongation of twitch in intact muscle.     

Kinetic characteristics of Ca2+, Mg2+-ATPases in cells of the submandibular salivary gland of rats

Kinetic properties of Ca2+, Mg2+-ATPases membranes from acinar cells of rat submandibular salivary glands have been investigated. It was found that kinetics of ATP hydrolysis dependent on Ca2+, Mg2+-ATPases corresponds to the first-order reaction during first 2 min. It was found that the initial velocity of the reaction (V0), maximal amount of the reaction product (Pmax) and characteristic time of the reaction (T) comprised 1.8 +/- 0.4 and 1.6 +/- 0.2 mmole Pi/min per 1 mg protein, 7.5 +/- 1.3 and 1.4 +/- 0.2 mmole Pi/mg protein and 4.1 +/- 0.7 min and 1.1 +/- 0.1 for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. High- and low-affinity sites of ATP and Ca2+-binding in Ca2+-ATPases from plasma and endoplasmic reticulum membranes were identified. Negative cooperation in ATP binding to Ca2+-ATPase from plasma membrane and a positive cooperation for Ca2+-ATPase from endoplasmic reticulum has been found. Ca2+ binding to low-affinity sites of both Ca2+-ATPases showed no cooperation, while Ca2+ binding to high-affinity sites showed the positive cooperation. Using the Hill's coordinates we have found the values of the Mg2+ Michaelis constant (K(Mg)) which yielded 3.89 x 10(-5) and 3.80 x 10(-5) mole/l for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. It is supposed that obtained data are important for further studies of molecular and membrane mechanisms involved in the regulation of intracellular calcium signalling and secretion by salivary acinar cells.     

Effect of SH-reagents on ATPase systems of rabbit skeletal muscle nuclei]

The influence of sulfhydryl reagents on ATPase systems of rabbit sceletal muscles nuclei was studied. It is found that p-ChMB at low concentration similarly inhibits both Mg2+- and Mg2+, Ca2+-ATPases. p-ChMB at higher concentrations inhibits completely Mg2+, Ca2+-ATPase, while Mg2+- ATPase--only by 60%. N-EM is lesser specific inhibitor of SH-groups, than p-ChMB. The degree of nuclear ATPases inhibition by N-EM is practically identical. Using inhibitory analysis, two hypes of skeletal muscles nuclei SH-groups are found: easily reacting with N-EM, and those reacting with N-EM at more high concentrations, which are essential for ATPase ATP-hydrolysing activity. ATP defends Mg2+, Ca2+-ATPase, but not the Mg2+-ATPase from N-EM inhibitory action. Cysteine completely eliminates the inhibitory effect of p-ChMB on Mg2+-ATPase but only 40% on MG2+, Ca2+-ATPase. Mg2+, Ca2+-ATPase of nuclei is more sensitive to the sulfhydryl venoms action than Mg2+-ATPase.     

Mechanism of regulation of phosphorylation-dephosphorylation of Ca2+, Mg2+ and Ca2+ -Atpases by modulator proteins isolated from rat brain cytosol

Two proteins of molecular mass 13 kDa, a specific inhibitor of Na+, K+ -ATPase and another of 12 kDa, which can distinguish between Ca2, Mg2+ and Ca2+ -ATPase activities have been obtained from the pooled fractions isolated from rat brain, using Sephadex G-100 chromatography. In order to determine the key step(s), which is affected by the modulators, we have designed an in vitro experiment of phosphorylation and dephosphorylation of these ATPases in the absence and presence of the modulators. The results suggest that the phosphorylation step of Mg2+ -independent Ca2+ -ATPase is inhibited, while in Mg2+ -dependent Ca2 -ATPase, the dephosphorylation step is stimulated by the modulators. The findings support our earlier observation that the modulators are able to distinguish between Mg2+ -independent and dependent Ca2+ -ATPases activities.     

Na+,K+-ATPase activity of the subcellular fractions of the rat brain in aging

The Na+, K+-ATPase activity in the homogenate and in subcellular fractions of different parts of the brain of adult and old rats was studied in comparison. The content of cholesterol in the above fractions was also determined. In old age the Na+, K+-ATPase activity in the homogenate and microsomal fraction of the cerebral hemispheres' cortex decreases, while the Mg2+-ATPase activity in the cortex microsomal fraction increases. The age-related Na+, K+- and Mg2+-ATPase activity in the myelin of the stem in the synaptic plasma membranes of hemispheres and the brain stem remains unchanged whereas in the myelin fraction of hemispheres it grows. The content of cholesterol in the brain of old rats as compared with adult ones increases in the microsomal fraction and remains unchanged in synaptic membranes. The possible role of age-related modification of lipid component of plasma membranes in the above changes of Na+, K+-ATPase activity is discussed.     

Localization of Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle

Localization of the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in rat papillary muscle was determined by indirect immunofluorescence and immunoferritin labeling of cryostat and ultracryotomy sections, respectively. The Ca2+ + Mg2+-ATPase was found to be rather uniformly distributed in the free sarcoplasmic reticulum membrane but to be absent from both peripheral and interior junctional sarcoplasmic reticulum membrane, transverse tubules, sarcolemma, and mitochondria. This suggests that the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum is antigenically unrelated to the Ca2+ + Mg2+-ATPase of the sarcolemma. These results are in agreement with the idea that the sites of interior and peripheral coupling between sarcoplasmic reticulum membrane and transverse tubules and between sarcoplasmic reticulum and sarcolemmal membranes play the same functional role in the excitation-contraction coupling in cardiac muscle.     

Comparative study of calixarene effect on Mg2+ -dependent ATP-hydrolase enzymatic systems from smooth muscle cells of the uterus

Investigation the influence of calyx[4]arenes C-90, C-91, C-97 and C-99 (codes are indicated) on the enzymatic activity of four functionally different Mg2+ -dependent ATPases from smooth muscle of the uterus: actomyosin ATPase, transporting Ca2+, Mg2+ -ATPase, ouabain-sensible Na+, K+ -ATPase and basal Mg2+ -ATPase. It was shown that calixarenes C-90 and C-91 in concentration 100 microM act multidirectionally on the functionally different Mg2+ -dependent ATP-hydrolase enzymatic systems. These compounds activate effectively the actomyosin ATPase (Ka = 52 +/- 11 microM [Ukrainian character: see text] 8 +/- 2 microM, accordingly), at the same time calixarene C-90 inhibited effectively activity of transporting Ca2+, Mg2+ -ATPase of plasmatic membranes (I(0,5) = 34.6 +/- 6.4 microM), but influence on membrane-bound Na+, K+ -ATPase and basal Mg2+ -ATPase. Calixarene C-91 reduce effectively basal Mg2+ -ATPase activity, insignificantly activating Na+, K+ -ATPase but has no influence on transporting Ca2+, Mg2+ -ATPase activity of plasmatic membranes. Calixarenes C-97 and C-99 (100 microM), which have similar structure, have monodirectional influence on activity of three functionally different Mg2+-dependent ATPases of the myometrium: actomyosin ATPase and two ATPases, that related to the ATP-hydrolases of P-type--Ca2+, Mg2+ -ATPase and Na+, K+ -ATPase of plasmatic membranes. Basal Mg2+ -ATPase is resistant to the action of these two connections. Results of comparative experiments that were obtained by catalytic titration of calixarenes C-97 and C-99 by actomyosin ATPase (I(0,5) = 88 +/- 9 and 86 +/- 8 microM accordingly) and Na+, K+ -ATPase from plasmatic membranes (I(0,5) = 33 +/- 4 and 98 +/- 8 nM accordingly) indicate to the considerably more sensitiveness of Na+, K+ -ATP-ase to these calixarenes than ATPase of contractile proteins. Thus, it is shown that calixarenes have influence on activity of a number of important enzymes, involved in functioning of the smooth muscle of the uterus and related to energy-supplies of the process of the muscle contracting and support of intracellular ionic homeostasis. The obtained results can be useful in further researches, directed at the use of calixarenes as pharmaceutical substance, able to normalize the contractile function of the uterus at some pregnancy pathologies in women's.     

Electron-cytochemical study of the localization and properties of ATPases in the isolated nuclei of rabbit skeletal muscle under normal conditions and in experimental muscular dystrophy]

The properties and localization of ATPase system in nuclei of skeletal muscle of normal rabbit and of those with experimental muscle dystrophy were studied by electron cytochemistry. The product of cytochemical reaction of ATP hydrolysis, which is a marker of ATPase activity localization in nuclear ultrastructures, was detected on the nuclear membrane, in chromatin and in the nucleolus, ATPase activity in the nuclei was detected in the presence of both, Mg2+ and Ca2+. Addition to the incubation medium, originally containing Mg2+, Na+ and K+, resulted in an increased formation of the product reaction in all the nuclear ultrastructures in both in the norm and under experimental muscle dystrophy. However, specific inhibitor of Mg2+, Na+, K+-ATPase--ouabain--suggests the absence in the nuclei of skeletal muscles of rabbit of transport ATPase working in the "Na-pump" system. The results of experiments with a specific complex of Ca2+--EGTA allow to suppose that Mg2+, Ca2+-ATPase of skeletal muscle nuclei of normal rabbits is localized in the nucleoplasm, whereas Mg2+-ATPase is found on the nuclear membrane. Using EGTA we failed to detected the localization of Mg2+, Ca2+-ATPase in nuclear ultrastructures upon experimental muscular dystrophy.     

Effects of denervation on the contents of cholesterol and membrane systems involved in muscle contraction in rabbit fast-twitch sarcotubular system.

Denervated fast-twitch rabbit muscles were progressively losing their fresh weight and the yield of sarcotubular protein was increasing. The activity of Ca(2+)-ATPase was affected but very slightly, the basal Mg(2+)-ATPase and the Mg(2+)-ATPase/Ca(2+)-ATPase ratio however increased together with a simultaneous depression of the membrane-bound acetylcholinesterase activity. We did not observe any differences in density properties of sarcotubular fractions between control and denervated muscle. However, a relative enrichment in SM and H fraction could be seen after denervation with small changes in the content of the Ca(2+)-pump protein, increased levels of calsequestrin and cholesterol, mostly in the heavy and the SM fraction. After denervation the binding sites for 3H-PN-200-110 did not show any changes in receptor affinity, but the number of putative Ca(2+)-channels increased twice along with a depression of 3H-ouabain binding sites. We suggest that the denervation of fast-twitch muscle leads to the hypertrophy of the junctional sarcoplasmic reticulum and the T-system. Changes in the cholesterol content, in the number of putative Ca(2+)-channels and in Na+, K(+)-ATPase can affect the muscle contraction.     

Mg2+- or Ca2+-activated ATPase activities of plasma membranes isolated from vascular smooth muscle

Studies of ATP hydrolysis by various subcellular fractions isolated from rat mesenteric arteries and veins indicate that an apparent ATPase activity, which can be activated by Mg2+ or Ca2+, is primarily associated with the plasma membranes. Although both Mg2+-activated and Ca2+-activated ATPase activities under the optimal condition are substantially lower in venous than in arterial plasma membrane fraction, their dependence on the concentration of Mg2+ and Ca2+ are quite similar in arterial as well as venous plasma membrane fractions. No synergistic effect on ATP hydrolysis was observed in the presence of both Mg2+ and Ca2+. In addition, Mg2+-activated and Ca2+-activated ATPase activities show similar pH dependence, inhibition by deoxycholate, stability toward heat inactivation and substrate specificity. Furthermore, Mg2+-activated and Ca2+-activated ATPase activities were similarly reduced in vascular smooth muscles of spontaneously hypertensive rats. These results suggest that the activation of ATP hydrolysis by Mg2+ or Ca2+ may represent a single enzyme moiety in the plasma membrane of vascular smooth muscle. The possible involvement of such ATPase in the Ca2+ transport function of vascular smooth muscle is discussed.     

H+-adenosine triphosphatases from plasma membranes

New data are presented on the organization of H+-pumps in plasma membranes of cells of bacteria fungi, plants and animals. It is shown that H+-ATPase of bacteria differs in principle from H+-ATPases of plasma membranes of other organisms. The transport H+, K+-ATPase functioning in cells of mucous membrane of the animal stomach as an electroneutral H+-pump is similar by its properties to Na+, K+-ATPase of plasma membranes of animal cells. H+-ATPase of plasma membranes in cells of fungi and higher plants which functions as an electrogenic H+-pump differs essentially from H+-ATPases of F0 X F1-type. Distribution of H+-ATPases in cells of different organisms and their evolution are under discussion.     

Polyunsaturated fatty acids inhibit Mg2+ -ATPase in basolateral membranes from rat enterocytes.

It is known that certain polyunsaturated fatty acids of the n-6 family, for example linoleic and arachidonic acids, can activate both Na+, K+-ATPase and Ca2+-ATPase. These enzymes drive active absorption processes in the duodenal enterocyte. This study presents data which show a 30-50% inhibition of Mg2+-ATPase activity in enterocyte basolateral membrane preparations by linoleic and gamma-linolenic acids (also a member of the n-6 family.) Mg2+-ATPase activity has several possible roles in the enterocyte: involvement in Mg2+ and Ca2+ absorption (as part of Ca2+-ATPase and also myosin I activity) as well as control of phospholipid distribution in the membrane by a class of Mg2+-ATPases called 'flippases'. The action of linoleic and gamma-linolenic acids on basolateral membrane Mg2+-ATPase may thus modulate several cellular transport processes.     

Effect of fluoroaluminate on the ATP-hydrolysing and Ca2+-transporting activity of the purified Ca2+, Mg2+-ATPase of myometrium cell plasma membranes

Fluoroaluminate, known modulator of G-proteins, inhibits ATP-hydrolase activity of purified solubilized Ca2+, Mg(2+)-ATPase from myometrium cell plasma membranes and Ca(2+)-transporting activity of this enzyme reconstituted into azolectin liposomes: 10 mM NaF plus 10 microM AlCl3 inhibited the primary activity by 95% and--by 81%. Inhibition of purified both solubilized and reconstituted Ca2+, Mg(2+)-ATPases by fluoroaluminate evidences for the possibility of direct interaction AlF4- with this enzyme without involvement of G-protein. The sensitivity to fluoroaluminate of sarcolemmal Ca2+, Mg(2+)-ATPase from myometrium is similar to that of Ca2+, Mg(2+)-ATPase from stomach smooth muscle.     

Transverse tubules from frog skeletal muscle. Purification and properties of vesicles sealed with the inside-out orientation

Transverse tubule vesicles were isolated from frog skeletal muscle by a procedure initially described by Rosemblatt et al. (J. Biol. Chem. 256, 8140-8148 (1981)) and later modified by Hidalgo et al. (J. Biol. Chem. 258, 13937-13945 (1983]. A large fraction of the isolated vesicles (80-90%) were sealed, as indicated by the detergent induced increase in (Na+ + K+)-ATPase activity and ATP-dependent ouabain binding. To determine the orientation of the sealed vesicles binding of digoxin, a lipid soluble derivative of ouabain, was measured. The same values of ATP-dependent digoxin binding were found with or without detergents, indicating that all the vesicles that are sealed have the ATP site accessible, and hence are sealed with the cytoplasmic side-out (inside-out orientation). The transverse tubule preparation isolated from frog muscle is highly purified, as indicated by its cholesterol content and its (Na+ + K+)-ATPase activity; negligible contamination with sarcoplasmic reticulum was observed, as indicated by the protein composition and the lack of measurable Ca2+-ATPase activity of the isolated transverse tubules. High initial rates of Mg2+-ATPase activity were found, with the peculiar property of being inhibited during the course of the reaction. Addition of lysophosphatidylcholine or saponin partially prevented the inhibition of Mg2+-ATPase activity during the reaction.     

The animal and human plasma membrane (Ca(2+)+Mg2+)-ATPases--approaches to molecular arrangements of functional parts and oxidative changes.

The molecular structures of animal and human plasma membrane (Ca(2+)+Mg2+)-ATPases are not completely understood in part due to the fact that no suitable single crystal is available. The elucidation of the two-dimensional structure is in progress. The amino acid sequences of human erythrocyte and rat plasma membrane Ca2+ pump isoforms as well as of the pig smooth muscle plasma membrane Ca2+ pump are already known. This article reviews the present state of the knowledge in (Ca(2+)+Mg2+)-ATPase research of animal and human plasma membranes performed in the past few years, concerning in particular arrangements of proteolytically cleaved fragments, and relations between the erythrocyte (Ca(2+)+Mg2+)-ATPase in situ and the purified red cell enzyme, oxidative changes. Results of different experimental approaches concerning the structure of (Ca(2+)+Mg2+)-ATPases rather than the applications of the methods used are emphasized.     

Observations on the (Ca2+ plus Mg2+)-ATPase activator found in various mammalian erythrocytes.

1. A soluble activator of membrane (Ca2+ plus Mg2)-ATPase is present in hemolysates of the newborn calf and cow, the new born and adult pig as well as human erythrocytes. 2. The activator is also found in reticulocytes of the adult pig. 3. The activator obtained from any of the above species is capable of stimulating the membrane (Ca2+ plus Mg2+)-ATPases of the other species, regardless of the age of the animals. 4. The results obtained from density fractionation of human erythrocytes revealed that the soluble factor has little simulatory effect on membranes of young erythrocytes from which it is derived but caused a marked stimulation on (Ca2+ plus Mg2+)-ATPase activity of the intermediate aged and old erythrocyte membranes. 5. The above observations support the following conclusions: (a) the extremely low levels of (Ca2+ plus Mg2+)-ATPase in cow erythrocytes is not due to the lack of a (Ca2+ plus Mg2+)-ATPase activator; (b) the distribution of (Ca2+ plus Mg2+)-atpase activator is not species specific and the differences in the level of membrane (Ca2+ plus Mg2+)-ATPase activity in various species of cells is an inherent property of that particular membrane (c) the (Ca2+ plus Mg2+)-ATPase activator is present at least from the time of reticulocyte formation and remain during tthe life span of the erythrocyte.     

A high-affinity plasma membrane Ca2+-ATPase in Dictyostelium discoideum: its relation to cAMP-induced Ca2+ fluxes

Chemotactic stimulation of Dictyostelium discoideum induces an uptake of Ca2+ by the cells followed by a release of Ca2+. In this study we investigated the mechanism of Ca2+ release and found that it was inhibited by La3+, Cd2+ and azide. Ca2+ release occurred in the absence of external Na+, indicating that an Na+/Ca2+ exchange was not involved. Plasma membranes contained high- and low-affinity ATPase activities. Apparent K0.5 values were 8 microM for the major Mg2+-ATPase and 1.1 microM for the high-affinity Ca2+-ATPase, respectively. The Mg2+-ATPase activity was inhibited by elevated concentrations of Ca2+, whereas both Ca2+-ATPases were active in the absence of added Mg2+. The activities of the Ca2+-ATPases were not modified by calmodulin. The high-affinity Ca2+-ATPase was competitively inhibited by La3+ and Cd2+; we suggest that this high-affinity enzyme mediates the release of Ca2+ from D. discoideum cells.     

Characterization of the Ca2+- or Mg2+-ATPase of transverse tubule membranes isolated from rabbit skeletal muscle

Transverse tubule membranes isolated from rabbit skeletal muscle have high levels of a Ca2+- or Mg2+-ATPase with Km values for Ca-ATP or Mg-ATP in the 0.2 mM range, but do not display detectable levels of ATPase activity activated by micromolar [Ca2+]. The transverse tubule enzyme is less temperature or pH dependent than the Ca2+-ATPase of sarcoplasmic reticulum and hydrolyzes equally well ATP, ITP, UTP, CTP, and GTP. Of several ionic, non-ionic, and zwitterionic detergents tested, only lysolecithin solubilizes the transverse tubule membrane while preserving ATPase activity. After extraction of about 50% of the transverse tubule proteins by solubilization with lysolecithin most of the ATPase activity remains membrane bound, indicating that the Ca2+- or Mg2+-ATPase is an intrinsic membrane enzyme. A second extraction of the remaining transverse tubule proteins with lysolecithin results in solubilization and partial purification of the enzyme. Sedimentation of the Ca2+- or Mg2+-ATPase, partially purified by lysolecithin solubilization, through a continuous sucrose gradient devoid of detergent leads to additional purification, with an overall 3- to 5-fold purification factor. The purified enzyme preparation contains two main protein components of molecular weights 107,000 and 30,000. Cholesterol, which is highly enriched in the transverse tubule membrane, copurifies with the enzyme. Transverse tubule membrane vesicles also display ATP-dependent calcium transport which is not affected by phosphate or oxalate. The possibility that the Ca2+- or Mg2+-ATPase is the enzyme responsible for the Ca2+ transport displayed by isolated transverse tubules is discussed.     

Fusion of proteoliposomes and cells. ATP-dependent Ca2+ uptake into erythrocytes catalyzed by Ca2+-ATPase from skeletal muscle

Purified Ca2+-ATPase from rabbit skeletal muscle has been incorporated into intact erythrocyte membranes by a two-step procedure. The isolated protein was reconstituted into proteoliposomes composed of phosphatidylethanolamine, phosphatidylcholine, and cardiolipin (50:20:30%, respectively). The resulting proteoliposomes were fused with erythrocytes in presence of La3+, Ca2+, or Mg2+. Subsequently, 45Ca uptake into the cells could be demonstrated. It was dependent on externally added ATP, inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate, and enhanced by inactivation of the endogenous Ca2+-ATPase which catalyzes Ca2+ extrusion from the cells. The insertion of the protein did not induce cell lysis, but the cells did become more fragile. Functional insertion of isolated membrane proteins into cell membranes allows a new approach to research of plasma membranes.