Separation of adenosine diphosphate-adenosine triphosphate–exchange activity from the cerebral microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase

1. A microsomal fraction from ox cerebral cortex catalysed [(14)C]ADP-ATP exchange at a speed similar to that at which it liberated P(i) from ATP in the presence of Na(+), K(+) and Mg(2+). 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na(+), K(+) or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide-cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na(+)-plus-K(+)-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP-ATP-exchange activity does not take part in the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP-ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na(+)-plus-K(+)-stimulated adenosine triphosphatase is indicated.     

Effect of Ni2+ on Ca2+-stimulated adenosine triphosphatase in the microsomal fraction of the rat parotid gland in vitro

Ni2+ inhibited Ca2+-stimulated adenosine triphosphatase activity in the microsomal fraction of the rat parotid gland in vitro. The Ni2+ concentration required for 50% inhibition was 0.45 mM. Inhibition mechanisms of Ni2+ for Ca2+ and ATP were of the competitive type and the noncompetitive type, respectively. The Ki values of Ni2+ for Ca2+ and ATP were 0.52 and 0.59 mM, respectively. The inhibitory effect of Ni2+ was reversible.     

Calcium transport in endoplasmic reticulum of the rat liver during lipid peroxidation

Some parameters of calcium transport in rat liver microsomes under conditions of lipoperoxidation activation modelled by antioxidant deficiency (AOD) were studied. This process was shown to be associated with a sharp stimulation of NADPH- and ascorbate-dependent lipid peroxidation in hepatocyte endoplasmic reticulum. The activation of lipid peroxidation was accompanied by disturbances in the kinetic properties of Ca2(+)-ATPase. This was paralleled with a considerable decrease of the ATP-dependent 45Ca-accumulation, increase in the passive permeability of microsomal vesicles for Ca2+ and Ca2+ elevation in the microsomal fraction. The AOD-induced diminution of the Ca2(+)-pump efficiency was slightly prevented by injections of rats with the antioxidants, alpha-tocopherol acetate and ionol which enable Ca2+ compartmentation correction in liver cytosol and membrane fractions.     

Calcium ion as a probe of the monovalent cation center of sodium, potassium ATPase

Sodium and potassium ion-transport adenosine triphosphatase from dog kidney was incubated with 0.4-2 mM Ca2+ at 23 degrees C for more than 2 min in the absence of monovalent inorganic cations, cooled to 0 degrees C, and phosphorylated from 1 mM Pi with 2.4 mM MgCl2. The resultant phosphoenzyme resembled that obtained by incubating the enzyme with K+ in place of Ca2+ in six respects. It was concluded that Ca2+ can occupy the monovalent cation-binding center for K+. The rate constant for release of Ca2+ from the dephosphoenzyme at 0 degrees C was 0.17 s-1. The rate of release from the phosphoenzyme was at least 7-fold slower. Phosphorylation stabilized the binding of Ca2+ to the enzyme in contrast to its destabilization of the corresponding K X enzyme complex. K-sensitive phosphoenzyme did not respond to free Ca2+. Thus Ca2+ was not easily accepted by nor released from the phosphoenzyme and would not be an effective substrate for transport. A selective barrier against Ca2+ between the monovalent cation binding center and the extracellular solution is proposed. Release of calcium from the dephosphoenzyme yielded a conformation that was not phosphorylated from Pi. The enzyme changed the conformation of its center for phosphorylation before or at the same time that it changed the conformation of its center for ion transport.     

Characterization of the mutant-unc D-gene product in a strain of Escherichia coli K12. An altered beta-subunit of the magnesium ion-stimulated adenosine triphosphatase.

Membranes from a mutant strain of Escherichia coli K12 carrying the uncD409 allele were washed in low-ionic-strength buffers in the presence or absence of the proteinase inhibitor p-aminobenzamidine. Unlike membranes from a normal strain, those from strain AN463 (uncD409) did not become proton-permeable, as judged by NADH-induced atebrinfluorescence quenching, when the membranes were washed in the absence of p-aminobenzamide. Furthermore, ATP-dependent atebrin-fluorscence quenching in such washed membranes could not be reconstituted by the addition of solubilized Mg2+-stimulated adenosine triphosphatase preparations. The examination by two-dimensional polyacrylamide-gel electrophoresis of the polypeptide composition of the washed membranes from strain AN463 (uncD409) indicated the presence of a polypeptide of similar molecular weight to the normal beta-subunit of the Mg2+-stimulated adenosine triphosphatase, but with an altered isoelectric point. Both the normal and abnormal beta-subunits were identified in membranes prepared from a partial diploid strain carrying both the unc+ and uncD409 alleles. It is concluded that the uncD gene codes for the beta-subunit of the Mg2+-stimulated adenosine triphosphatase.     

Behaviour of cardiac microsomal Ca2+ pump under conditions that may simulate pathological situations

The behaviour of Ca2+ ATPase activity in relation to Ca2+ transport process was studied under different experimental conditions in canine cardiac microsomal fraction predominantly containing sarcoplasmic reticulum. The total Ca2+ concentration required for half maximal activation (Ka) of microsomal Ca2+ ATPase and Ca2+ uptake did not differ significantly, unless 0.1 mmol/l EGTA was present in the incubation media. Pretreatment of cardiac microsomes with membrane disruptive agents like phospholipase A, trypsin as well as deoxycholate strongly increased (2-3 fold) Ca2+ ATPase activity but uptake rate of Ca2+ declined. Only in phospholipase C and beta-glucuronidase pretreatment, a parallel decrease of Ca2+ ATPase and uptake was observed. In presence of excess (free)Ca2+ (greater than 10 mumol/l) both Ca2+ ATPase as well as Ca2+ uptake were inhibited, however, Ca2+ binding process remained unaltered. Likewise, low pH completely altered the relation between Ca2+ binding and ATPase activity; whereas Ca2+ ATPase was inhibited, Ca2+ binding did not change. Our present data provide evidence for some cellular factors that may be involved in producing uncoupling of microsomal Ca2+ ATPase from Ca2+ accumulation process that was previously observed in various pathological situations.     

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.     

Effect of phospholipase A2 on Ca2+-stimulated adenosine triphosphatase activity in microsomal fraction of rat submandibular gland

The effect of phospholipase A2 on the Ca2+-ATPase (EC 3.6.1.3) activity in the microsomal fraction of rat submandibular gland was kinetically studied in vitro. The Ca2+-ATPase activity was significantly increased by the treatment with phospholipase A2 in the presence of bovine serum albumin as a scavenger for hydrolyzed products. When the microsomal fraction was incubated with phospholipase A2 in the absence of bovine serum albumin, the Ca2+-ATPase activity was not altered. The Vmax and Km values for both ATP and Ca2+ were increased by the phospholipase A2 treatment, respectively. These results indicated that the activation of Ca2+-ATPase by the phospholipase A2 treatment is due to the increase of Vmax.     

Separation and characterization of the aldehydic products of lipid peroxidation stimulated by ADP-Fe2+ in rat liver microsomes.

1. Methods using t.l.c. and high-pressure liquid chromatography (h.p.l.c.) have been used to separate the complex variety of substances possessing a carbonyl function that are produced during lipid peroxidation. 2. The major type of lipid peroxidation studied was the ADP-Fe2+-stimulated peroxidation of rat liver microsomal phospholipids. Preliminary separation of the polar and non-polar products was achieved by t.l.c.: further separation and identification of individual components was performed by h.p.l.c. Estimations were performed on microsomal pellets and the supernatant mixture after incubation of microsomes for 30 min at 37 degrees C. 3. The polar fraction was larger than the non-polar fraction when expressed as nmol of carbonyl groups/g of liver. In the non-polar supernatant fraction the major contributors were n-alkanals (31% of the total), alpha-dicarbonyl compounds (22%) and 4-hydroxyalkenals (37%) with the extraction method used. 4. Major individual contributors to the non-polar fraction were found to be propanal, 4-hydroxynonenal, hexanal and oct-2-enal. Other components identified include butanal, pent-2-enal, hex-2-enal, hept-2-enal, 4-hydroxyoctenal and 4-hydroxyundecenal. The polar carbonyl fraction was less complex than the non-polar fraction, although the identities of the individual components have not yet been established. 5. Since these carbonyl compounds do not react significantly in the thiobarbituric acid reaction, which largely demonstrates the presence of malonaldehyde, it is concluded that considerable amounts of biologically reactive carbonyl derivatives are released in lipid peroxidation and yet may not be picked up by the thiobarbituric acid reaction.     

Calcium ion-dependent adenosine triphosphatase activity and plasma-membrane phosphorylation in the human neutrophil.

A plasma-membrane fraction was isolated from a post-nuclear extract of human neutrophils by centrifugation through a linear sucrose density gradient. This fraction exhibited a Ca2+-dependent adenosine triphosphatase (ATPase) activity that could be differentiated from mitochondrial or myosin ATPase and from plasma-membrane Mg2+-dependent ATPase. When assayed in the presence of [gamma-32P]ATP, the Ca2+-dependent ATPase reaction resulted in the formation of an acid-resistant hydroxylamine-sensitive bond between the gamma-[32P] phosphate group and a membrane protein subunit with an apparent mol.wt. of 135000. Half-maximal activating effect of Ca2+ was found at 82nM and 0.18 microM for the ATPase and the formation of the 32P-membrane complex respectively. Generation of the phosphorylated product attained the steady state at 0 degrees C by about 30s, and was rapidly reversed by ADP. These results suggest that the Ca2+-activated ATPase reaction occurs through the formation of a phosphoprotein intermediate, similar to that described for some Ca2+-dependent ATPase enzymes associated with Ca2+ transport. The possibility thus exists that the neutrophil Ca2+-dependent ATPase catalyses a process of Ca2+ extrusion from the cell, thereby participating in the regulation of several Ca2+-dependent neutrophil functions.     

Microsomal glutathione-dependent protection against lipid peroxidation acts through a factor other than glutathione peroxidase and glutathione S-transferase in rat liver

Ascorbate-Fe3+-induced and NADPH-induced lipid peroxidation of rat liver microsomes were inhibited by glutathione (GSH). This inhibition was due to microsomal GSH-dependent factor. This factor was heat labile, and storage of microsomes at 4 degrees C for 1 week diminished the activity. GSH could not be substituted by other sulfhydryl compounds tested. Deoxycholate (1 mM) and bromosulfophthalein (0.1 mM) inhibited GSH-dependent protection but did not inhibit microsomal GSH peroxidase activity. Iodoacetate (10 mM) inhibited GSH-dependent protection but did not inhibit microsomal GSH S-transferase. N-Ethylmaleimide (0.1 mM) and oxidized glutathione (10 mM) inhibited GSH-dependent protection but activated microsomal GSH S-transferase activity. These results indicate the existence of a heat-labile, microsomal GSH-dependent protective factor against lipid peroxidation that acts through a factor other than GSH-peroxidase and GSH S-transferase.     

Liver cytosolic non-dialysable factor(s) can counteract GTP-dependent Ca2+ release in rat liver microsomal fractions

Readdition to rat liver microsomes of dialysed liver post-microsomal supernatant resulted in an almost complete inhibition of the Ca2+-releasing effect of GTP. Such inhibition was heat-labile, and was associated with non-ultrafiltrable supernatant components with a molecular weight higher than 30,000 D. A preliminary fractionation of liver supernatant showed that the inhibitory effect is recovered in the 40-50% ammonium sulfate-precipitated proteins, with an approx. 10-fold enrichment. The active ammonium sulfate fraction did not modify the GTP-induced Ca2+ increase of passive Ca2+ efflux from microsomes, nor did it affect microsomal GTP hydrolysis, which is likely required for its Ca2+ releasing effect. The active ammonium sulfate fraction appears to markedly favour the translocation of GTP-released Ca2+ into a microsomal GTP-insensitive pool. Separation of liver microsomes in smooth and rough fractions revealed that such GTP-insensitive Ca2+ pool is almost completely associated with smooth microsomes.     

Phospholipid-protein interactions in the Ca2+-adenosine triphosphatase of sarcoplasmic reticulum.

Ca2+-adenosine triphosphatase from sarcoplasmic reticulum has been delipidated by gel filtration through a Sephadex G-200 column equilibrated with buffer containing cholate. The delipidated Ca2+-adenosine triphosphatase had negligible adenosine triphosphatase activity, but up to 50% of the ATPase activity was restored when the delipidated enzyme was recombined with phosphilipids. It was shown with the delipidated preparation that the phosphorylation of the enzyme by either ATP or Pi was entirely dependent on phospholipids. Among the purified phospholipids, phosphatidylcholine reactivated the adenosine triphosphatase activity better than phosphatidylethanolamine. Vesicles capable of translocating Ca2+ were reconstituted from delipidated Ca2+-adenosine triphosphatase and phosphatidylethanolamine, but not with phosphatidylcholine alone. We conclude that the firmly bound phospholipids which are purified together with the adenosine triphosphatase protein are not essential for the pump since they can be substituted by phosphatidylethanolamine isolated from soybeans.     

Effects of lipid peroxidation on membrane-bound enzymes of the endoplasmic reticulum

1. Induction of the formation of lipid peroxide in suspensions of liver microsomal preparations by incubation with ascorbate or NADPH, or by treatment with ionizing radiation, leads to a marked decrease of the activity of glucose 6-phosphatase. 2. The effect of peroxidation can be imitated by treating microsomal suspensions with detergents such as deoxycholate or with phospholipases. 3. The substrate, glucose 6-phosphate, protects the glucose 6-phosphatase activity of microsomal preparations against peroxidation or detergents. 4. The loss of glucose 6-phosphatase activity is not due to the formation of hydroperoxide or formation of malonaldehyde or other breakdown products of peroxidation, all of which are not toxic to the enzyme. 5. All experiments lead to the conclusion that the loss of activity of glucose 6-phosphatase resulting from peroxidation is a consequence of loss of membrane structure essential for the activity of the enzyme. 6. In addition to glucose 6-phosphatase, oxidative demethylation of aminopyrine or p-chloro-N-methylaniline, hydroxylation of aniline, NADPH oxidation and menadione-dependent NADPH oxidation are also strongly inhibited by peroxidation. However, another group of enzymes separated with the microsomal fraction, including NAD⁺/NADP⁺ glycohydrolase, adenosine triphosphatase, esterase and NADH–cytochrome c reductase are not inactivated by peroxidation. This group is not readily inactivated by treatment with detergents. 7. Lipid peroxidation, by controlling membrane integrity, may exert a regulating effect on the oxidative metabolism and carbohydrate metabolism of the endoplasmic reticulum in vivo.     

Characterization of calcium-ion-activated adenosine triphosphatase in the plasma membrane of rat mast cells.

The properties of a Ca2+ activated adenosine triphosphatase shown to be present in homogenates of purified rat peritoneal mast cells were investigated. The enzyme was activated by Ca2+, Mg2+, and to a lesser extent by Mn2+ and Co2+. Ca2+ alone was necessary for full activity and the further addition of Mg2+ did not have any effect. The chelating agents EGTA (ethanedioxybis(ethylamine)tetra-acetate) and EDTA completely inhibited the reaction. The pH optimum was 7.8. Reduced glutathione, cysteine, dithiothreitol, N-ethylmaleimide, urea, ADP, NaF, increasing ionic strength and Triton X-100 all inhibited the reaction. On subcellular fractionation of mast-cell homogenates by density-gradient centrifugation, the distribution of Ca2+ activated adenosine triphosphatase resembled that of 5'-nucleotidase, but differed from that of the other markers used, suggesting localization in the plasma membrane. Further experiments indicated that the enzyme is present on the external surface of the plasma membrane.     

Abnormal membrane properties of the sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia: modes of action of halothane, caffeine, dantrolene, and two other drugs

The role of sarcoplasmic reticulum (SR) in malignant hyperthermia (MH) was studied using the heavy microsomal fraction prepared from semitendinosus muscles of both normal and genetically MH-susceptible pigs. In the presence of ATP, SR was loaded with 70 nmol Ca2+/mg SR protein. Under these conditions, MH-SR demonstrated Ca2+-induced Ca2+ release (Ca-ICaR) and halothane-induced Ca2+ release (halothane-ICaR; halothane concentrations as low as 10 microM). Normal SR did not demonstrate these release phenomena. Dantrolene inhibited the halothane-ICaR, but did not inhibit the Ca-ICaR. Ruthenium red and tetracaine inhibited both types of Ca2+ release. From the measurement of passive Ca2+ efflux, it was shown that dantrolene did not affect the Ca2+ permeability of the SR itself, but suppressed only the halothane-induced increment of the permeability. The membrane order parameter of the SR, as measured by the spin-probe EPR technique, indicated that halothane disordered the lipid bilayer of MH-SR to a greater extent than it did of normal SR. This halothane disordering effect on MH-SR was antagonized by dantrolene. Ruthenium red and tetracaine did not antagonize the halothane disordering effect. These results raise the possibility that halothane could disturb the structure of the lipoprotein complex in MH-SR in such a way that it could open the Ca2+-release channels. The Ca2+ thus released further opens the channel through the Ca-ICaR mechanism in a positive feedback fashion, thus triggering the MH syndrome. The efficacy of dantrolene in ameliorating the MH syndrome might be related to the inhibition of this halothane effect.     

Possible regulation of thiamine diphosphatase activity in rat brain microsomes by lipids.

The effects of various treatments, which affect membrane structure, on microsomal thiamine diphosphatase and thiamine triphosphatase activities of rat brain, were examined. The treatment of micorosomes at alkaline pH caused a 2-fold activation of the thiamine diphosphatase, this being related to a change in membrane structure which was evidenced by a decrease of the turbidity of the microsomal suspension. Repeated freezing and thawing after hypo-osmotic treatment also increased the activity of microsomal thiamine diphosphatase. In addition, the thiamine diphosphatase activity was enhanced by treatment of the microsomes with phospholipase C or acetone. This lipid depletion resulted in a marked reduction in the apparent Km value of the thiamine diphosphatase with a corresponding loss in heat stability of the enzyme. We found further that brain thiamine diphosphatase was solubilized by Triton X-100. This decreased the phospholipid content in the preparation, but did not affect the apparent Km value and heat stability of the enzyme. In contrast with thiamine diphosphatase, thiamine triphosphatase was inactivated by treatment at alkaline pH or with acetone. However, treatment with phospholipase C did not affect the activity of thiamine triphosphatase.     

Inhibition of microsomal lipid peroxidation by glutathione and glutathione transferases B and AA. Role of endogenous phospholipase A2.

Lipid peroxidation in vitro in rat liver microsomes (microsomal fractions) initiated by ADP-Fe3+ and NADPH was inhibited by the rat liver soluble supernatant fraction. When this fraction was subjected to frontal-elution chromatography, most, if not all, of its inhibitory activity could be accounted for by the combined effects of two fractions, one containing Se-dependent glutathione (GSH) peroxidase activity and the other the GSH transferases. In the latter fraction, GSH transferases B and AA, but not GSH transferases A and C, possessed inhibitory activity. GSH transferase B replaced the soluble supernatant fraction as an effective inhibitor of lipid peroxidation in vitro. If the microsomes were pretreated with the phospholipase A2 inhibitor p-bromophenacyl bromide, neither the soluble supernatant fraction nor GSH transferase B inhibited lipid peroxidation in vitro. Similarly, if all microsomal enzymes were heat-inactivated and lipid peroxidation was initiated with FeCl3/sodium ascorbate neither the soluble supernatant fraction nor GSH transferase B caused inhibition, but in both cases inhibition could be restored by the addition of porcine pancreatic phospholipase A2 to the incubation. It is concluded that the inhibition of microsomal lipid peroxidation in vitro requires the consecutive action of phospholipase A2, which releases fatty acyl hydroperoxides from peroxidized phospholipids, and GSH peroxidases, which reduce them. The GSH peroxidases involved are the Se-dependent GSH peroxidase and the Se-independent GSH peroxidases GSH transferases B and AA.     

Cryoresistance of rat liver endoplasmic reticulum membranes]

The effects of freezing of microsomes in liquid nitrogen and those of storage of microsomal suspensions at 2-4 degrees C and -3 - -5 degrees C for 24 hrs, on the enzymatic activities and hydrophobicity of membranes were studied. The hydrophobicity was determined by fluorescence of bound 1,8-anilino-naphthalene sulfonate. Rapid freezing of the microsomal suspension in liquid nitrogen followed by rapid warming did not change the hydrophobicity of the membranes, the rate of enzymatic lipid peroxidation, the level of cytochrome P-450 and the activity of NADH- and NADPH-cytochrome c reductase. A considerable decrease in the rate of enzymatic lipid peroxidation and membrane hydrophobicity was observed in the microsomes stored for 24 hrs at 2-4 degrees C. The 24-hr storage at -3 - -5 degrees C with subsequent thawing resulted in a rapid aggregation of the microsomes.     

Effect of temperature on [3H]ryanodine binding to sarcoplasmic reticulum from bullfrog skeletal muscle

It has been clarified that ryanodine binds to Ca2(+)-induced Ca release channels in the open state in sarcoplasmic reticulum. While the pharmacological action of ryanodine is known to be retarded at a low temperature, the Ca-releasing action of caffeine is potentiated at a low temperature. In order to obtain deeper insight into the molecular mechanism underlying Ca-release, the effect of temperature on ryanodine binding to the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle was examined. Although Ca2+ is indispensable for ryanodine binding, Ca2+ alone cannot cause ryanodine binding in a reaction medium of a salt concentration similar to that of the sarcoplasm. In addition to Ca2+, caffeine and/or beta,gamma-methylene adenosine triphosphate (AMPOPCP) are necessary. [3H]Ryanodine binding at 25 degrees C closely paralleled the Ca release activity in respect of the Ca2(+)-dependence in the presence of caffeine and/or AMPOPCP, and the effects of inhibitors. A Scatchard plot for ryanodine binding gave a straight linear line, indicating a single class of homogeneous binding sites. At 0 degrees C, the rate of ryanodine binding decreased. Q10 being about 3 on average. The affinity for ryanodine was reduced to about half that at 25 degrees C, with no change in the maximum number of binding sites. The temperature-dependent change in apparent affinity for Ca2+ on ryanodine binding is not always consistent with that in the case of Ca-release activity. The bound ryanodine may be in an occluded state because it did not dissociate for up to 90 h at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)