Case history of a patient with multiple sclerosis and scleroderma.
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A Askari
Publications and source records attributed to A Askari.
Restoration of phosphorylation capacity to the dormant half of the alpha-subunits of Na+, K(+)-ATPase.
Purified kidney Na+, K(+)-ATPase whose alpha-subunit is cleaved by chymotrypsin at Leu266-Ala267, loses ATPase activity but forms the phosphoenzyme intermediate (EP) from ATP. When EP formation was correlated with extent of alpha-cleavage in the course of proteolysis, total EP increased with time before it declined. The magnitude of this rise indicated doubling of the number of phosphorylation sites after cleavage. Together with previous findings, these data establish that half of the alpha-subunits of oligomeric membrane-bound enzyme are dormant and that interaction of the N-terminal domain of alpha-subunit with its phosphorylation domain causes this half-site reactivity. Evidently, disruption of this interaction by proteolysis abolishes overall activity while it opens access to phosphorylation sites of all alpha-subunits.
Adenosine Triphosphate↗
Similarities and differences between the properties of native and recombinant Na+/K+-ATPases.
Progress of mutagenesis studies on the relation of the structure of Na+/K+-ATPase to its reaction mechanism has been impeded by the paucity of information on the properties of small amounts of impure recombinant enzyme obtained in the currently available expression systems, and the uncertainty of whether expression in a new environment alters the various catalytic activities of this membrane enzyme. Hence, our aim was to make a detailed comparison of the properties of the extensively studied canine kidney Na+/K+-ATPase with those of its alpha1,beta1 subunits expressed in the baculovirus-infected Sf-9 cells. The active fraction of the recombinant enzyme, containing 10-20% of the expressed a subunits, was found to have normal molar activity, all the partial reactions, and the ability to catalyze ATP-dependent Na+/K+ exchange after reconstitution into proteoliposomes. Comparison of steady-state kinetics of the hydrolytic activities of recombinant and native enzymes showed that (a) ATP and Na+ plots of Na+-ATPase were the same in the two preparations; (b) apparent K+ affinity of K+-phosphatase of recombinant enzyme was lower than that of kidney enzyme; and (c) for Na+/K+- ATPase activity, apparent K+ affinity of recombinant enzyme was lower, and its apparent Na+ and ATP affinities were higher than those of kidney enzyme. The two enzymes had similar ADP- and K+-sensitive phosphointermediates, identical affinities for ouabain, and similar ligand sensitivities of dissociation rates of ouabain-enzyme complexes. Evidently, the recombinant enzyme has reduced affinity at cytoplasmic K+ sites, but no changes at multiple Na+, ATP, and ouabain binding sites. Likely causes of this selective change include altered glycosylation state of beta and interactions among active and inactive recombinant enzymes. The present results provide the necessary database for the appropriate use of an expression system in structure-function studies on canine alpha1,beta1 isoform of Na+/K+-ATPase, and indicate the need for similar studies on recombinant Na+/K+-ATPases obtained in other expression systems.
Animals↗
Partial inhibition of Na+/K+-ATPase by ouabain induces the Ca2+-dependent expressions of early-response genes in cardiac myocytes.
Exposure of neonatal rat cardiac myocytes to ouabain concentrations that caused partial inhibition of Na+/K+-ATPase but no loss of viability, increased c-fos and c-jun mRNAs and the transcription factor AP-1. The increased mRNAs were proportional to the extent of inhibition of Na+/K+-ATPase and the resulting rise in steady state intracellular Ca2+ concentration. The rapid and sustained increase of c-fos mRNA was shown to be due to increased transcriptional rate. Induction of c-fos by ouabain was prevented when either extracellular or intracellular Ca2+ was lowered and was attenuated by pretreatment of myocytes with a phorbol ester under conditions known to down-regulate protein kinase C. Exposure to ouabain for 24-48 h also increased total transcriptional activity and protein content of myocytes. The findings suggest that the same signal responsible for the positive inotropic action of ouabain, i.e. net influx of Ca2+ caused by partial inhibition of Na+/K+-ATPase, also initiates the rapid protein kinase C-dependent inductions of the early-response genes, the subsequent regulations of other cardiac genes by the resulting transcription factors, and stimulation of myocyte growth. Whether these hitherto unrecognized effects of cardiac glycosides are obtained in the intact heart and their relevance to the therapeutic uses of these drugs remain to be determined.
Animals↗
Activation of Na+/K(+)-ATPase by fatty acids, acylglycerols, and related amphiphiles: structure-activity relationship.
A number of fatty acids and derivatives have been shown to activate Na+/K(+)-ATPase when ATP is suboptimal. To explore the relation of the structures of these amphiphiles to enzyme activation, the effects of varying amphiphile concentrations on the activity of the highly purified kidney Na+/K(+)-ATPase at 50 microM ATP were determined. Among fatty acids, efficacy (maximal level of activation) and potency were found to be dependent, in different ways, on chain length and unsaturation. Compared to fatty acids, the corresponding alcohols had lower efficacies. Methyl esters of fatty acids inhibited, but CoA esters and monoacyl esters of glycerol activated the enzyme. Relation between chain length and potency among CoA esters and monoacylglycerols was the same as that observed with acids. Diacylglycerols did not activate, but they antagonized the effects of the activator amphiphiles. The substantial specificities of the amphiphile effects support the hypothesis that these ligands bind to a distinct amphipathic peptide segment of the intracellular central loop of the alpha-subunit to regulate ATP binding to the enzyme. The findings also suggest that direct effects of the changing intracellular levels of fatty acids and derivatives on Na+/K(+)-ATPase should be considered as a possible mechanism for the regulation of its function in the intact cell.
Animals↗
Intersubunit and intrasubunit contact regions of Na+/K(+)-ATPase revealed by controlled proteolysis and chemical cross-linking.
To identify interfaces of alpha- and beta-subunits of Na+/K(+)-ATPase, and contact points between different regions of the same alpha-subunit, purified kidney enzyme preparations whose alpha-subunits were subjected to controlled proteolysis in different ways were solubilized with digitonin to disrupt intersubunit alpha,alpha-interactions, and oxidatively cross-linked. The following disulfide cross-linked products were identified by gel electrophoresis, staining with specific antibodies, and N-terminal analysis. 1) In the enzyme that was partially cleaved at Arg438-Ala439, the cross-linked products were an alpha,beta-dimer, a dimer of N-terminal and C-terminal alpha fragments, and a trimer of beta and the two alpha fragments. 2) From an extensively digested enzyme that contained the 22-kDa C-terminal and several smaller fragments of alpha, two cross-linked products were obtained. One was a dimer of the 22-kDa C-terminal peptide and an 11-kDa N-terminal peptide containing the first two intramembrane helices of alpha (H1-H2). The other was a trimer of beta, the 11-kDa, and the 22-kDa peptides. 3) The cross-linked products of a preparation partially cleaved at Leu266-Ala267 were an alpha,beta-dimer and a dimer of beta and the 83-kDa C-terminal fragment. Assuming the most likely 10-span model of alpha, these findings indicate that (a) the single intramembrane helix of beta is in contact with portions of H8-H10 intramembrane helices of alpha; and (b) there is close contact between N-terminal H1-H2 and C-terminal H8-H10 segments of alpha; with the most probable interacting helices being the H1,H10-pair and the H2,H8-pair.
Amino Acid Sequence↗
A comparative study of Na+/K(+)-ATPases of duck salt gland and canine kidney: implications for the enzyme's reaction mechanism.
Highly purified preparations of duck salt gland and canine kidney Na+/K(+)-ATPases with comparable specific activities were used to clarify the causes of previously reported differences between the substrate-velocity curves of these enzymes. When assays were done under identical conditions (pH 7.4; 37 degrees C), and a wide range of closely spaced ATP concentrations were used, the curves of both enzymes exhibited intermediary plateaus, as noted before for the salt gland enzyme. The two enzymes also had the same numbers of phosphorylation and ouabain binding sites, and their catalytic subunits were of the alpha 1 isoform type as revealed by immunostaining with specific antibodies. The findings suggest that the substrate-velocity curves of all widely used Na+/K(+)-ATPases may contain an intermediary plateau which is diagnostic of reaction mechanisms that generate rate equations containing powers of substrate concentration greater than two, e.g., a mechanism involving an oligomer with more than two protomers.
Animals↗
Interactions of phosphorylation and dimerizing domains of the alpha-subunits of Na+/K(+)-ATPase.
Chemical cross-linking studies are among a number of experimental approaches that have suggested the functional significance of higher association states of alpha,beta-protomers of Na+/K(+)-ATPase. Formation of the phosphointermediate of the enzyme on Asp369 of the alpha-subunit is known to induce oxidative cross-linking of the alpha-subunits catalyzed by Cu(2+)-phenanthroline. To localize the phosphorylation-induced alpha,alpha-interface, we cleaved alpha at Arg438-Ala439 by controlled proteolysis and exposed the partially cleaved enzyme to the cross-linking reagent. In addition to the alpha,alpha-dimer, two other phosphorylation-induced cross-linked products were obtained. Using gel electrophoretic resolution of the cross-linked 32P-labeled enzyme, N-terminal analyses of the products, and their reactivities with sequence-specific antibodies, the two products were identified as a homodimer of the C-terminal 64-kDa fragment of alpha and a heterodimer of alpha and the 64-kDa peptide. The latter dimer was also obtained when the cross-linked alpha,alpha-dimer was formed first and then subjected to proteolysis. The findings localize the dimerizing domain to the C-terminal side of Ala439 and indicate that intersubunit proximities of dimerizing domains are regulated by phosphorylation-dephosphorylation of Asp369 during the reaction cycle of the enzyme.
Animals↗
Functional coupling of phosphorylation and nucleotide binding sites in the proteolytic fragments of Na+/K(+)-ATPase.
Cleavage of the alpha-subunit of Na+/K(+)-ATPase by trypsin at Arg438-Ala439 causes enzyme inhibition which has been suggested to be due to altered alignment of phosphorylation site on the 48-kDa N-terminal fragment with nucleotide binding site on the 64-kDa C-terminal fragment. Our aims were to test this hypothesis and to assess the effect of the cleavage on the enzyme's two ATP sites. Na(+)-dependent phosphorylation of the partially cleaved enzyme by ATP showed that K0.5 values of ATP for phosphorylations of intact alpha and 48-kDa peptide were the same (0.4 microM). Unchanged interactions among the residues across the cleavage site were also indicated by data showing that reaction of fluorescein isothiocyanate with the 64-kDa peptide blocked phosphorylation of the 48-kDa peptide by ATP. ATP is known to block the reaction of fluorescein isothiocyanate with the enzyme. Experiments on the partially cleaved enzyme showed that K0.5 of ATP for protection of alpha was 30-60 microM, and the value for the protection of interacting 48-kDa and 64-kDa peptides was 1-3 mM. Evidently, while the cleavage does not affect the high affinity catalytic site, it disrupts the allosteric low affinity ATP site. Experiments on reconstituted preparations showed that the cleavage abolished ATP-dependent Na+/K+ exchange, Pi+ATP-dependent Rb+/Rb+ exchange, ATP-dependent Na+/Na+ exchange, and ADP+ATP-dependent Na+/Na+ exchange activities. Selective disruption of the low affinity ATP site accounts for the inhibitions of all functions involving K+(Rb+), based on the established role of this site in the control of K+ access channels. Cleavage-induced inhibitions of other activities, however, suggest additional roles of the low affinity ATP site in the reaction cycle.
Adenosine Diphosphate↗
Different oxidant sensitivities of the alpha 1 and alpha 2 isoforms of Na+/K(+)-ATPase expressed in baculovirus-infected insect cells.
Inhibition of Na+/K(+)-ATPase by partially reduced oxygen metabolites is an early event in the course of cell injury caused by oxidative stress. We showed before that isoforms of the enzyme obtained from different sources have different oxidant sensitivities. To evaluate the role of tissue-specific impurities in this difference, cDNAs of alpha 1 and alpha 2 isoforms were expressed in Sf-9 insect cells, and the effects of H2O2 on the resulting isolated enzymes were studied. The expressed alpha 2 was significantly more sensitive than alpha 1 to H2O2. These findings, together with our previous data showing different oxidant sensitivities of alpha 1 and alpha 3 in a cardiac enzyme preparation, indicate that differential oxidant sensitivities of Na+/K(+)-ATPase isoforms of various tissues are dictated by the primary sequences of alpha 1, alpha 2, and alpha 3 subunits.
Animals↗
Effects of doxorubicin on cardiomyocytes with reduced level of superoxide dismutase.
Our previous studies have shown that isolated adult rat cardiomyocytes with normal and reduced Cu/Zn SOD activities are equally susceptible to extracellularly generated oxidants (hydrogen peroxide, glucose oxidase/glucose and xanthine oxidase/xanthine systems). In the present study we exposed myocytes with reduced SOD activity to doxorubicin (adriamycin). Cardiotoxicity of doxorubicin has been attributed to the production of superoxide anion inside the cell. Cardiomyocytes with reduced SOD activity, but normal ATP content and viability, were obtained by the treatment of isolated cells with diethyldithiocarbamate (DDC). DDC-treated myocytes were significantly less resistant to doxorubicin than controls. Doxorubicin-stimulated superoxide anion formation, measured by the rate of SOD-inhibitable acetylated cytochrome C reduction, was significantly higher in the cytosolic fraction of DDC-treated cells compared to controls. These results indicate that for isolated cardiac myocytes an essential part of cytotoxicity of doxorubicin can be explained by the formation of superoxide anion and that the level of intracellular SOD activity should be considered as a significant factor for cell protection.
Animals↗
Role of intracellular SOD in oxidant-induced injury to normal and copper-deficient cardiac myocytes.
Previous studies have shown that susceptibilities of hepatocytes and endothelial cells to H2O(2)-induced injury are altered by changes in the intracellular activity of Cu,Zn-containing superoxide dismutase (CuZn-SOD). To evaluate the role of intracellular CuZn-SOD in oxidant-induced injury to rat cardiac myocytes, cells with reduced CuZn-SOD activity but normal ATP content were either isolated from the hearts of adult copper-deficient rats or obtained by treatment of normal isolated adult myocytes with diethyldithiocarbamate. These myocytes and controls with normal CuZn-SOD activity were exposed to either reagent H2O2 or oxidants generated by extracellular glucose oxidase plus glucose or xanthine oxidase plus xanthine. It was shown that myocytes with CuZn-SOD activities reduced by 70-90% were equally susceptible to H2O2 and the two oxidant-generating systems as the control myocytes. The findings suggest that in adult cardiac myocytes, in contrast to the situation in some other cells, intracellular CuZn-SOD may not have a significant defensive role against acute H2O(2)-induced injury. The possibility remains, however, that changes in the activity of this enzyme, e.g., in copper deficiency, may be relevant to the ability of myocytes to cope with chronic oxidative stress resulting from imbalance between intracellular oxygen radical-generating and -scavenging systems.
Adenosine Triphosphate↗
Altered expressions of cardiac Na/K-ATPase isoforms in copper deficient rats.
OBJECTIVE: The aim was to determine if copper deficiency affects the expression of Na/K-ATPase alpha isoforms in the rat heart. METHODS: Copper deficiency was induced by placing weanling rats on a copper deficient diet for 4-5 weeks. Adult ventricular tissue, isolated ventricular myocytes, and brain stems of the control and deficient rats were compared for Cu, Zn-superoxide dismutase (CuZn-SOD) activity and for protein and mRNA contents of Na/K-ATPase alpha isoforms. RESULTS: In brain stem, where copper deficiency did not alter CuZn-SOD activity, mRNA and protein levels of alpha isoforms also remained unchanged. In ventricular tissue and ventricular myocytes, copper deficiency reduced CuZn-SOD activity, mRNAs of alpha 1 and alpha 2 isoforms, and the alpha 2 isoform protein. The alpha 1 isoform protein of ventricular tissue and its myocytes was marginally reduced by copper deficiency. CONCLUSIONS: In the rat ventricular tissue, oxidative stress resulting from copper deficiency (1) enhances the turnover of the more oxidant sensitive alpha 2 isoform to a greater extent than the turnover of the alpha 1 isoform; (2) regulates mRNA levels of alpha 1 and alpha 2 isoforms; and (3) contributes to the cardiomyopathy of copper deficiency.
Animals↗
Oxidant-induced activations of nuclear factor-kappa B and activator protein-1 in cardiac myocytes.
Activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappa B), two transcription factors that respond to a wide range of signals, have been shown to be activated by H2O2 in several cell lines. Since H2O2 and related oxidants are implicated in reperfusion injury to the heart, we wished to know if NF-kappa B is present in the myocardium and if cardiac AP-1 and NF-kappa B also respond to oxidants. Rat neonatal cardiac myocytes were exposed to H2O2, and changes in c-fos and c-jun mRNAs, immunoreactive c-Fos and c-Jun proteins (components of AP-1), and immunoreactive p50 subunit of NF-kappa B were determined. Changes in nuclear activities of AP-1 and NF-kappa B were also measured by electrophoretic mobility shift assays. When myocytes were exposed to nonlethal concentrations of H2O2, c-fos and c-jun mRNAs were rapidly induced, reaching peak values at 30-60 min. The levels of c-Fos and c-Jun proteins increased in nuclei as revealed by immunostaining, and DNA binding activity of nuclear AP-1 increased. The presence of p50 subunit of NF-kappa B and its H2O2-induced shift from cytoplasm to nucleus were shown by immunostaining. H2O2-induced myocyte nuclear proteins capable of binding to a DNA probe containing the NF-kappa B element were also demonstrated. The findings suggest that altered expressions of cardiac genes regulated by AP-1 and NF-kappa B may be components of oxidant-induced injury to the heart or a part of the heart's adaptive response to oxidative stress.
Animals↗
Reconstitution and partial purification of cardiac sarcolemmal Na+/H+ (K+/H+) antiporter.
Most of the plasma membrane Na+/H+ antiporters that have been well characterized do not transport K+. Our previous studies showed that cardiac plasma membrane contains a nonselective alkali cation/H+ antiporter that transports either Na+ or K+. The purpose of this work was to develop a convenient assay for the cardiac antiporter to be used in the course of its purification. Sarcolemmal membranes prepared from bovine heart were solubilized with Triton X-100 in the presence of a mixture of phospholipids, and the solubilized proteins were passed through Bio-Beads to form proteoliposomes. Exchange activities were assayed either by measuring H+ gradient-dependent uptakes of 22Na+ and 86Rb+ by proteoliposomes or by recording H+ release from proteoliposomes as reported by the fluorescence of a pH probe that was trapped in the proteoliposomes during reconstitution. Properties of the reconstituted nonselective antiporter were found to be similar to those of the native sarcolemmal antiporter. Using a DEAE-cellulose column, 20-fold purification of the antiporter was achieved, demonstrating the suitability of the reconstitution assay procedures for further purification of the functional antiporter.
Amiloride↗
Different sensitivities of native and oxidized forms of Na+/K(+)-ATPase to intracellular proteinases.
Inactivation of Na+/K(+)-ATPase by partially reduced oxygen metabolites has been implicated in ischemia-reperfusion injury to heart and other organs. Because oxidation of many proteins makes them more susceptible to degradation by intracellular proteinases, we studied the effects of several such proteinases on native and H2O2-oxidized preparations of Na+/K(+)-ATPase from canine kidney (containing alpha 1 isoform of the catalytic subunit) and rat axolemma (containing alpha 2 and alpha 3 isoforms). Lysosomal cathepsin D degraded the native and the oxidized preparations at acid pH, but it was significantly more effective against the oxidized forms. m-Calpain had little or no effect on the native Na+/K(+)-ATPase preparations, but it digested the oxidized alpha-subunits of the axolemma and the kidney enzymes. mu-Calpain's effects were similar to those of m-calpain. Multi-catalytic proteinase which is known to degrade a large number of oxidized proteins, did not affect the native or the oxidized forms of Na+/K(+)-ATPase. The findings suggest that (a) during oxidative stress there may be accelerated degradation of the oxidatively damaged Na+/K(+)-ATPase, either through internalization and transport to lysosomes, or by the action of calpains at the membrane; and (b) those isoforms of the enzyme that are more sensitive to oxidants are more susceptible to degradation by the above processes.
Animals↗
Different sensitivities of the Na+/K(+)-ATPase isoforms to oxidants.
Inhibition of Na+/K(+)-ATPase by partially reduced oxygen metabolites has been suggested to be involved in ischemia-reperfusion injury to heart and other organs. Since various isoforms of the enzyme have different sensitivities to ouabain and several other inhibitors, we studied the effects of H2O2 and the hydroxyl radical on enzyme activity and phosphoenzyme formation in Na+/K(+)-ATPase preparations with known alpha-subunit isoform composition in order to assess the oxidant sensitivities of the isoforms. Rat axolemma enzyme (alpha 2 and alpha 3) which has higher sensitivity than the rat kidney enzyme (alpha 1) to ouabain also showed higher oxidant sensitivity than the kidney enzyme. No significant difference between the oxidant sensitivities of the alpha 2 and alpha 3 of the axolemma was noted. In the ferret heart enzyme (alpha 1 and alpha 3), we confirmed that alpha 3 has higher ouabain sensitivity than alpha 1, and we established that alpha 3 also has higher oxidant sensitivity than alpha 1. The rat kidney enzyme (alpha 1) and the canine kidney enzyme (a variant of alpha 1 with much higher ouabain sensitivity than the rat kidney enzyme) exhibited similar oxidant sensitivities. The findings suggest that (a) oxidant sensitivity is related to structural features that distinguish alpha 1 from alpha 2 and alpha 3, rather than to features that control ouabain sensitivity; and (b) different isoform compositions of the various tissues may contribute to their relative susceptibilities to oxidant stress.
Amino Acid Sequence↗
Differential regulation of superoxide dismutase in copper-deficient rat organs.
The effects of dietary copper deprivation on the activities, immunoreactive protein concentrations, and mRNA abundance of copper/zinc- and manganese-superoxide dismutase (Cu/Zn- and Mn-SOD) were examined in liver, heart, and brain of weanling rats fed a Cu-deficient diet for 4 weeks. Hepatic Cu/Zn-SOD activity, enzyme content, and mRNA abundance were significantly reduced, and, conversely, the activity, protein, and mRNA levels of Mn-SOD were significantly elevated in Cu-deficient rats. In Cu-deficient heart, the activity and protein content for Cu/Zn-SOD were reduced, whereas those for Mn-SOD were increased; the levels of mRNAs for these two enzymes was unaffected. Dietary Cu deficiency was without effect on the activities, enzyme contents, and mRNA abundance of brain Cu/Zn- and Mn-SOD. These results indicate that SODs from liver, heart, and brain exhibit differential sensitivities to dietary Cu deprivation, and that different mechanisms (transcriptional, posttranscriptional, or posttranslational) may be involved in their regulation.
Animals↗