Contact-mediated changes in ATPase activity at the surface of primary cultured hepatoma cells.

Hepatoma cells grown in monolayer culture display certain alterations in their Mg-ATPase activity present on the cell surface as a function of time during a exponential growth. Levels of enzyme estimated biochemically and expressed as activity per cell increase as the cell population density increases. Histochemical investigation shows that Mg-ATPase activity is located intensively on the surface of cell contact and the activity is not encountered on the cell surface facing the free space. No enzyme activity is detected histochemically on the cell surface of sparse culture. Deposits of acidic polysaccharide are also seen on the surface of cell contact.     

Membrane phosphatase activities in isolated and cultured adult rat hepatocytes

Membrane bound phosphohydrolysing enzymes, such as Na-K-ATPase, Mg-ATPase, ALPase and G-6-Pase were assayed in intact liver, in freshly isolated cells and in cultured hepatocytes to evaluate the effects of the isolation procedure and culture on these enzyme activities. Na-K-ATPase and Mg-ATPase are significantly reduced following cell dispersion while ALPase and G-6-Pase are nearly unaffected. During culture, Na-K-ATPase is restored to the "in vivo" level within the first two days, but rapidly declines in the following days. The Na dependent, energy requiring AIB uptake shows a similar pattern; Mg-ATPase is practically unmodified. A significant increase in ALPase activity and a net decrease of G-6-Pase activity, as a function of the culture time has been observed.     

Different effects of inhibitors on two mutants of Escherichia coli K12 affected in the Fo portion of the adenosine triphosphatase complex.

The effects of the inhibitors dicyclohexyl-carbodiimide (DCCD), bathophenanthroline and tertiary octylcatechol, on some enzyme activities in membranes from strains of Escherichia coli carrying mutations in the uncB or uncC genes have been studied. Membranes prepared from uncC mutants retain a normal DCCD-sensitive Mg2+-stimulated adenosine triphosphatase (Mg-ATPase) activity whereas in uncB mutants this enzyme activity is insensitive to DCCD. The membrane-bound Mg-ATPase activity from the uncC mutant strain, as compared with that from the normal strain, is only partially sensitive to the inhibitors bathophenanthroline or tertiary-octylcatechol. Both of these inhibitors stimulate the membrane-bound Mg-ATPase from uncB mutant strains. A DCCD-insensitive Mg-ATPase activity is found in the cytoplasmic fraction following cell disruption of either the uncB or the uncC mutants. The lipophilic chelators bathophenanthroline and tertiary-octylcatechol stimulate the activity of the 'soluble' Mg-ATPase in the uncB mutant but partially inhibit the activity in the uncC mutant. The NADH oxidase activities in membranes from both mutant and normal strains are strongly inhibited by tertiary-octylcatechol and bathophenanthroline but not by DCCD.     

Post-mortem changes in cytochemical localization and enzymological measurement of marker enzymes of the mitochondria, SDH and Mg-ATPase, of porcine muscle stored at 4 degrees C, -18 degrees C, or -80 degrees C

As a series of studies on postmortem changes in the fine structure of porcine muscle, activity of two mitochondrial marker enzymes, succinate dehydrogenase (SDH) and magnesium dependent adenosine triphosphatase (Mg-ATPase), was measured and localized in cardiac, red and white muscles stored at 4 degrees C, -18 degrees C or -80 degrees C. The postmortem loss of SDH activity was most remarkable in cardiac muscle. The variation of SDH activity was proportional to the amount of absolute activity. The postmortem change of Mg-ATPase was more variable than SFH, though the activity was well preserved up to 15 weeks in all three types of porcine muscle stored at -80 degrees C. The loss of Mg-ATPase was most remarkable in red muscle stored at -18 degrees C or -80 degrees C. Cytochemical localization of SDH was between the outer and the inner mitochondrial membranes while that of Mg-ATPase was on the inner surface or matrix side of the inner membrane. Those localization was not altered by the difference in temperature and the duration of storage.     

THE LOCALIZATION OF Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE ON RED CELL GHOST MEMBRANES

The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO₃)₂ resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb²⁺ and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of P_i on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis P_i is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.     

The effect of anions on Mg-ATPase in red blood cells.

Anions exert an influence on the passive permeability of Na+ and K+ in erythrocytes. THE EFFECT ON Mg-ATPase activity has been studied in human erythrocytes. 40 mM bicarbonate increased the activity as compared to the effect of 40 mM chloride; 20 mM sulphate inhibited it. Salicylate acted first as an activator then as an inhibitor of Mg-ATPase; maximum activity was reached at 60 mM CONCENTRATION. Thiocyanate inhibited saponin-stimulated Mg-ATPase, Ki = 1.85 X 10(-2)M. The probable mechanisms of action of the above anions on Mg-ATPase and possible relation to passive permeability of Na+ and K+ ions are discussed.     

Common structural domains in the sarcoplasmic reticulum Ca-ATPase and the transverse tubule Mg-ATPase

Transverse tubule (TT) membranes isolated from chicken skeletal muscle possess a very active magnesium-stimulated ATPase (Mg-ATPase) activity. The Mg-ATPase has been tentatively identified as a 102-kD concanavalin A (Con A)-binding glycoprotein comprising 80% of the integral membrane protein (Okamoto, V.R., 1985, Arch. Biochem. Biophys., 237:43-54). To firmly identify the Mg-ATPase as the 102-kD TT component and to characterize the structural relationship between this protein and the closely related sarcoplasmic reticulum (SR) Ca-ATPase, polyclonal antibodies were raised against the purified SR Ca-ATPase and the TT 102-kD glycoprotein, and the immunological relationship between the two ATPases was studied by means of Western immunoblots and enzyme-linked immunosorbent assays (ELISA). Anti-chicken and anti-rabbit SR Ca-ATPase antibodies were not able to distinguish between the TT 102-kD glycoprotein and the SR Ca-ATPase. The SR Ca-ATPase and the putative 102-kD TT Mg-ATPase also possess common structural elements, as indicated by amino acid compositional and peptide mapping analyses. The two 102-kD proteins exhibit similar amino acid compositions, especially with regard to the population of charged amino acid residues. Furthermore, one-dimensional peptide maps of the two proteins, and immunoblots thereof, show striking similarities indicating that the two proteins share many common epitopes and peptide domains. Polyclonal antibodies raised against the purified TT 102-kD glycoprotein were localized by indirect immunofluorescence exclusively in the TT-rich I bands of the muscle cell. The antibodies substantially inhibit the Mg-ATPase activity of isolated TT vesicles, and Con A pretreatment could prevent antibody inhibition of TT Mg-ATPase activity. Further, the binding of antibodies to intact TT vesicles could be reduced by prior treatment with Con A. We conclude that the TT 102-kD glycoprotein is the TT Mg-ATPase and that a high degree of structural homology exists between this protein and the SR Ca-ATPase.     

Inhibition, by a protease inhibitor, of the solubilization of the F1-portion of the Mg2+-stimulated adenosine triphosphatase of Escherichia coli.

The effects of two protease inhibitors on the solubilization of the membrane-bound Mg2+-adenosine triphosphatase (Mg-ATPase) of Escherichia coli were investigated. p-Aminobenzamidine prevented the solubilization of the Mg-ATPase during treatment of membranes with low-ionic-strength buffers containing ethylenediaminetetraacetic acid. p-Aminobenzamidine did not prevent subsequent solubilization of the Mg-ATPase by treatment of the membranes with chloroform. This method of solubilization yielded a preparation of similar apparent molecular weight but with a 10-fold-increased specific activity as compared with the Mg-ATPase solubilized by washing with low-ionic-strength buffer. However, in contrast to the latter preparation, the chloroform-solubilized Mg-ATPase did not reconstitute ATP-dependent energization of stripped membranes, which were prepared by low-ionic-strength washing in the absence of p-aminobenzamidine. Another protease inhibitor, epsilon-amino-n-caproic acid, did not effect the solubilization of the Mg-ATPase, but did inhibit the loss of activity occurring during concentration, by ultrafiltration, of the Mg-ATPase solublized by the low-ionic-strength treatment.     

Tributyltin inhibits the oligomycin-sensitive Mg-ATPase activity in Mytilus galloprovincialis digestive gland mitochondria

Tributyltin (TBT), widely employed in the past in antifouling paints, is one of the most toxic organic pollutants. Although recently banned, it still threatens coastal water ecosystems and accumulates in filter-feeding molluscs. TBT is known to act as a membrane-active toxicant; however data on mussels are scanty and exposure effects on mitochondrial ATPase activities remain hitherto unexplored. TBT effects on the mitochondrial Mg-ATPase activities in the digestive gland of Mytilus galloprovincialis were investigated both in vitro and in TBT-exposed mussels. Both an oligomycin-sensitive Mg-ATPase (OS Mg-ATPase) (70% of total Mg-ATPase activity) and an oligomycin-insensitive ATPase (OI Mg-ATPase) (30%) were found. The OS-Mg-ATPase was as much as 70% in vitro inhibited by 0.7 μM (203 μg/L) TBT, while higher concentrations promoted a partial inhibition release up to 5.0 μM TBT; higher than 10.0 μM TBT concentrations yielded nearly complete enzyme inhibition. Concentrations higher than 1 μM TBT enhanced the OI Mg-ATPase. Mussels exposed to 0.5 and 1.0 μg/L TBT in aquaria showed a 30% depressed OS Mg-ATPase activity, irrespective of TBT dose and exposure time (24 and 120 h). The OI Mg-ATPase activity was apparently refractory to TBT exposure and halved both in control and TBT-exposed mussels after 120 h exposure.     

Ultrastructural localization of the membrane-bound Mg-adenosine triphosphatase activity in rat meninges

The distribution of the membrane-bound magnesium ions-dependent adenosine triphosphatase (Mg-ATPase) activity has been studied ultracytochemically in rat meninges by the method of Wachstein and Meisel (1957). A device specially constructed to avoid preparation artefacts has been used to obtain sections from the parietal region of the head. The meninges display an intense though irregularly distributed ATPase activity marked by depositions of electron-dense reaction product (RP) which is almost absent in the outer and middle dural layers. In the borderline zone between dura mater and the arachnoid the RP deposits are found at the outer surface of the inner dural cells and at the contact sites between these cells and the dural neurothelium. The intercellular cleft(s) between the neurothelium and the outer arachnoidal layer, occupied by an "electron-dense band", remains free of RP. The strongest accumulations of reactions granules are observed on the surface of the leptomeningeal cells of the arachnoidal space. In the contact region between the inner arachnoidal and the outer pial layers the distribution of the RP is similar to the one observed in the interface zone dura mater/arachnoid, while the pial cells themselves are definitely reaction-positive. In all meningeal vessels RP is found at the lumenal and abluminal aspects of the endothelium as well as at the cell membranes of the perivascular cells. These results emphasize the importance of the dural neurothelium for the functions of the blood-cerebrospinal fluid (CSF)-barrier between the dural blood vessels and the CSF.     

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.     

Cytochemical and biochemical demonstration of an ATPase in membranes of human peroxisomes.

We demonstrated a neutral Mg-ATPase activity in human peroxisomal membranes. To establish the precise experimental conditions for detection of this ATPase, both cytochemical and biochemical characterizations were first carried out in liver peroxisomes from control and cipofibrate-treated rats. The results demonstrated an Mg-ATPase reaction in both normal and proliferated peroxisomes. The nucleotidase activity, with marked preference for ATP, was sensitive to the inhibitors N-ethylmaleimide and 7-chloro-4-nitro-benzo-2-oxadiazole (NBDCl). An ultrastructural cytochemical analysis was developed to evaluate the peroxisomal localization, which localized the reaction product to the peroxisomal membrane. These characteristics can help to differentiate the peroxisomal ATPase from the activity found in mitochondria and endoplasmic reticulum. The conditions established for detecting the rat peroxisomal ATPase were then applied to human peroxisomes isolated from liver and skin fibroblasts in culture. A similar Mg-ATPase activity was readily shown, both cytochemically and biochemically, in the membranes of human peroxisomes. These results, together with previous evidence, strongly support the presence of a specific ATPase in the human peroxisomal membrane. This ATPase may play a crucial role in peroxisome biogenesis.     

Does the conformation of Mg-ATPase (spectrin-dependent ATPase) influence the passive permeability to K+?

The processing of human erythrocytes disclosed changes in Mg-ATPase activity following action of Pb2+ and Nile blue, and changes of permeability of K+ after treatment with Nile blue. The obtained results and those from previous papers can be summarized as follows : Substances decreasing the activity of stimulated membrane Mg-ATPase (spectrin-dependent ATPase) in red blood cells increase the passive permeability to K+, and substances increasing the stimulated Mg-ATPase activity decrease the passive permeability to K+. A hypothesis is proposed that the conformation of Mg-ATPase is secondarily reflected in the state of the proper path for K+ transport through the membrane; thus the rate of passive permeability to K+ is influenced.     

The distribution of ATPase activities in purified transverse tubular membranes

Vesiculated fragments of transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes were purified from heterogeneous microsomal membrane fractions of chicken breast muscle by a modification of an iterative calcium-oxalate loading technique. The distribution of ATPase activities were determined for the TT and SR and were compared to enriched fractions of sarcolemma (SL) membranes. The TT membranes were characterized by high rates of magnesium-stimulated ATPase (Mg-ATPase) and 5′-nucleotidase activities but were virtually devoid of calcium-stimulated, magnesium-dependent ATPase (Ca,Mg-ATPase) activity. Moderate levels of a latent sodium and potassium-stimulated ATPase (Na,K-ATPase) were observed for TT membranes when unmasked with valinomycin and monensin. In contrast to the behavior of TT membranes, highly purified SR membranes displayed an active Ca,Mg-ATPase but negligible Na,K-ATPase, Mg-ATPase, and 5′-nucleotidase activities. High levels of Na,K-ATPase and 5′-nucleotidase activities were observed for SL membranes; however, the SL displayed no appreciable Ca,Mg-ATPase and Mg-ATPase activities. The lack of significant Mg-ATPase activity in the SR and SL fractions suggested that the Mg-ATPase was uniquely associated with the TT membranes. The TT Mg-ATPase was further characterized by its pH and temperature dependences, and its sensitivity to pharmacologic agents. The Mg-ATPase of the TT was insensitive to inhibition by sodium azide and oligomycin in concentrations shown to exert maximum inhibition on the F₁ ATPase of submitochondrial particles. The Mg-ATPase was also resistant to the effects of ouabain and orthovanadate in concentrations which abolished the Na,K-ATPase and Ca,Mg-ATPase activities of the SL and SR, respectively. The Mg-ATPase displayed temperature and pH optima (25 °C, pH 7.3) which were distinguishable from the Ca,Mg-ATPase (45 °, pH 7.0) of highly purified SR fractions but which were very similar to the temperature and pH dependencies of the mixed microsomal fractions (MMF) from which the TT membranes were derived. Similarities in the pH and temperature dependencies of the TT and MMF Mg-ATPases plus the absence of appreciable Mg-ATPase activity in highly purified SR membranes suggests that the “basic” Mg-ATPase often seen in crude SR fractions may originate from TT membrane contamination. The resistance of the TT Mg-ATPase to inhibition by the pharmacologic agents tested plus its unique temperature and pH dependences indicate that this ATPase is distinguishable from other ATPases and may, therefore, be of value as a specific biochemical marker for TT membranes.     

Demonstration of a Mg-ATPase and a Na,K-ATPase from the arteria carotis communis of the sheep.

Besides the Mg-ATPase, a Na,K-ATPase can be demonstrated in different fractions of smooth muscles of the A. carotis communis of the sheep. The highest activity of Mg-ATPase is observed in the heavy microsomal fraction. The Ca-ion may act as a complete substitute for the Mg-ion in the Mg-ATPase. The proportion of Na,K-ATPase is between 10 and 40%, depending on the preparative conditions used in the individual fractions. Fractionated salt treatment (LiBr, KC1, KBr) improved the assay of Na,K-ATPase but increased strength of the Tris-HC1-buffer considerably reduced its activity.     

Specific in vitro activation of Ca,Mg-ATPase by vitamin D-dependent rat renal calcium binding protein (calbindin D28K)

The vitamin D-dependent, calcium-binding protein from rat kidney, calbindin D28k (renal CaBP) specifically stimulates Ca,Mg-ATPase activity of human erythrocyte plasma membranes in a dose-dependent, calcium-sensitive manner. This stimulation was about two-fold compared to a three-fold stimulation by calmodulin. The effect was specific since other calcium-binding proteins and low molecular weight proteins did not stimulate Ca,Mg-ATPase activity. Renal CaBP did not stimulate cyclic nucleotide phosphodiesterase at concentrations greater than those which stimulated Ca,Mg-ATPase activity. This is the first report of a specific in vitro effect of renal CaBP on an enzyme system.     

Calmodulin-dependent regulation of Ca,Mg-ATPase activity in plasma membranes of the swine myometrium

Highly purified plasma membrane (PM) preparations of pig myometrium were found to contain 0.91 +/- 0.22 microgram calmodulin per mg of PM protein. Treatment of membranes with 1 mM EGTA in the presence of 0.2 M NaCl causes the diminution of the calmodulin content down to 3% of the original level. The activity of Ca, Mg-ATPase is thereby decreased by 40%. Exogenous calmodulin restores the enzyme activity up to 1.94 +/- +/- 0.30 mumol Pi/mg protein/hour. The maximal activation of Ca, Mg-ATPase is observed with 10(-7) M calmodulin. Calmodulin increases the total ATPase activity of myometrium PM without affecting the Mg-ATPase activity. Trifluoroperazine (20 microM) diminishes the activating effect of exogenous calmodulin on Ca, Mg-ATPase. Calmodulin stimulates Ca, Mg-ATPase at low concentrations of Ca2+(10(-8)-10(-6) M) by decreasing Km for Ca2+ from 0.4.10(-6) M to 2.10(-8) M as well as by increasing Vmax--from 0,8 to 1.42 mumol Pl/mg protein/hour. It is supposed that the activating effect of calmodulin on Ca, Mg-ATPase is based on electrostatic interactions of Ca2+-free calmodulin with the enzyme.     

Conditioned media from cultured cystic fibrosis fibroblasts inhibits Na/K ATPase activity

Conditioned culture media taken from fibroblast cell lines derived from skin biopsies of control or of patients with Cystic Fibrosis (CF) were incubated with membranes of rat submandibular glands. The Na/K - ATPase activity of these membranes was inhibited when treated with CF-media, including both ouabain sensitive and insensitive activities. However, the membrane associated Mg-ATPase, Ca-ATPase, and both basal and hormone-stimulated adenylate cyclase activities were relatively unaffected. Thus, a factor or factors produced by CF-fibroblasts was shown to be active in a cell-free system derived from an exocrine gland.     

Some properties of hog thyroidal membrane-bound adenosine tri-phosphatase: proteolytic activation of Mg-dependent activity.

The ATPase preparations from the hog thyroid was preincubated with various amounts of trypsin. The activity of Mg-ATPase was consistently elevated. On the contrary, the Na, K-ATPase activity decreased with increasing amounts of trypsin. The effects were similar to those which were observed in the enzyme preparations treated with basis polyamino acids as previously reported. This phenomenon seemed to be specific in the preparations from the thyroid. The Mg-dependent activity was increased after pretreatment with trypsin or poly-L-lysine (PLL) when CTP, ITP and UTP were used as substrate. Thus the substrate specificity of Mg-ATPase was low. The enzyme-kinetics using ATP as substrate showed that the increase in activity was due to an increase in Vmax and not to a change in Km. The activity of Mg-ATPase was increased even after 30 min of preincubation with trypsin, while the Na, K-ATPase activity was almost diminished. These results suggest that the activity of Mg-ATPase in the preparation from the thyroid is specifically changed by the modification of the molecular environment of the enzyme with trypsin or basic polyamino acids.     

Activation of Mg-ATPase (spectrin-dependent ATPase) by Ca2+.

The dependence of saponin-stimulated Mg-ATPase activity in the erythrocyte membrane on Ca2+ concentration was studied. In the membrane of freshly sampled human erythrocytes we found for this enzyme and Ca2+ an apparent dissociation constant of 0.611 mumol/l (SE +/- 0.106 mumol/l) and Hill coefficient of 0.93 (SE +/- 0.05). The enzyme is in most probability identical with Ca,Mg-ATPase of high affinity to Ca2+ described also as spectrin-dependent Ca,Mg-ATPase.