PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities.

PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. We show that PARP14 is a dual-function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a marked increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13, and displays specific cellular phenotypes. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response.

Nanoscale Diamond-Based Formulation as an Immunomodulator and Potential Therapeutic for Lymphoma.

Integrative medicine practices, such as Ayurveda, are popular in India and many South Asian countries, yet basic research to investigate the concepts, procedures, and medical benefits of ayurvedic products has received little attention and is not fully understood. Here, we report a functional nanodiamond-based traditional Ayurvedic herbomineral formulation, Heerak Bhasma (Ayu_ND), for the treatment of solid tumors called Dalton's lymphoma generated in CD1 mice. Ayu_ND-mediated immunostimulation significantly reduces tumor cell proliferation and induces apoptosis aided by the active participation of dendritic cells. Immunomodulatory Ayu_ND treatment is highly immunostimulatory and drives dendritic cells to produce TNF-α. Treatment with Ayu_ND significantly reduces the tumor volume, inhibits metastasis in distant vascularized organs, and increases the life span of tumor-bearing animals compared with untreated littermates. These events were associated with elevated serum levels of the protective cytokines IFN-γ and TNF-α and downregulated the disease, exacerbating TGF-ß. Ayu_ND-mediated therapeutic success was also accompanied by the depletion of regulatory T cells and enhanced vaccine-induced T-cell immunity, guided by the restoration of the memory CD8+ T-cell pool and prevention of PD-1-mediated T cell exhaustion. The results provide a basis for further evaluation of ayurvedic formulations and drug efficacy in treating cancers.

AKAP79 Orchestrates a Cyclic AMP Signalosome Adjacent to Orai1 Ca2+ Channels.

To ensure specificity of response, eukaryotic cells often restrict signalling molecules to sub-cellular regions. The Ca2+ nanodomain is a spatially confined signal that arises near open Ca2+ channels. Ca2+ nanodomains near store-operated Orai1 channels stimulate the protein phosphatase calcineurin, which activates the transcription factor NFAT1, and both enzyme and target are initially attached to the plasma membrane through the scaffolding protein AKAP79. Here, we show that a cAMP signalling nexus also forms adjacent to Orai1. Protein kinase A and phosphodiesterase 4, an enzyme that rapidly breaks down cAMP, both associate with AKAP79 and realign close to Orai1 after stimulation. PCR and mass spectrometry failed to show expression of Ca2+-activated adenylyl cyclase 8 in HEK293 cells, whereas the enzyme was observed in neuronal cell lines. FRET and biochemical measurements of bulk cAMP and protein kinase A activity consistently failed to show an increase in adenylyl cyclase activity following even a large rise in cytosolic Ca2+. Furthermore, expression of AKAP79-CUTie, a cAMP FRET sensor tethered to AKAP79, did not report a rise in cAMP after stimulation, despite AKAP79 association with Orai1. Hence, HEK293 cells do not express functional active Ca2+-activated adenylyl cyclases including adenylyl cyclase 8. Our results show that two ancient second messengers are independently generated in nanodomains close to Orai1 Ca2+ channels.

The N terminus of Orai1 couples to the AKAP79 signaling complex to drive NFAT1 activation by local Ca2+ entry.

To avoid conflicting and deleterious outcomes, eukaryotic cells often confine second messengers to spatially restricted subcompartments. The smallest signaling unit is the Ca2+ nanodomain, which forms when Ca2+ channels open. Ca2+ nanodomains arising from store-operated Orai1 Ca2+ channels stimulate the protein phosphatase calcineurin to activate the transcription factor nuclear factor of activated T cells (NFAT). Here, we show that NFAT1 tethered directly to the scaffolding protein AKAP79 (A-kinase anchoring protein 79) is activated by local Ca2+ entry, providing a mechanism to selectively recruit a transcription factor. We identify the region on the N terminus of Orai1 that interacts with AKAP79 and demonstrate that this site is essential for physiological excitation-transcription coupling. NMR structural analysis of the AKAP binding domain reveals a compact shape with several proline-driven turns. Orai2 and Orai3, isoforms of Orai1, lack this region and therefore are less able to engage AKAP79 and activate NFAT. A shorter, naturally occurring Orai1 protein that arises from alternative translation initiation also lacks the AKAP79-interaction site and fails to activate NFAT1. Interfering with Orai1-AKAP79 interaction suppresses cytokine production, leaving other Ca2+ channel functions intact. Our results reveal the mechanistic basis for how a subtype of a widely expressed Ca2+ channel is able to activate a vital transcription pathway and identify an approach for generation of immunosuppressant drugs.

Control of NFAT Isoform Activation and NFAT-Dependent Gene Expression through Two Coincident and Spatially Segregated Intracellular Ca2+ Signals.

Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca2+-dependent transcription factor isoforms, NFAT1 and NFAT4, require distinct sub-cellular InsP3 and Ca2+ signals for physiologically sustained activation. NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.

Ca(2+) Channel Re-localization to Plasma-Membrane Microdomains Strengthens Activation of Ca(2+)-Dependent Nuclear Gene Expression.

In polarized cells or cells with complex geometry, clustering of plasma-membrane (PM) ion channels is an effective mechanism for eliciting spatially restricted signals. However, channel clustering is also seen in cells with relatively simple topology, suggesting it fulfills a more fundamental role in cell biology than simply orchestrating compartmentalized responses. Here, we have compared the ability of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels confined to PM microdomains with a similar number of dispersed CRAC channels to activate transcription factors, which subsequently increase nuclear gene expression. For similar levels of channel activity, we find that channel confinement is considerably more effective in stimulating gene expression. Our results identify a long-range signaling advantage to the tight evolutionary conservation of channel clustering and reveal that CRAC channel aggregation increases the strength, fidelity, and reliability of the general process of excitation-transcription coupling.

Distinct spatial Ca2+ signatures selectively activate different NFAT transcription factor isoforms.

Protein isoforms are widely expressed in biological systems. How isoforms that co-exist within the same sub-cellular domain are differentially activated remains unclear. Here, we compare the regulatory mechanism of two closely related transcription factor isoforms, NFAT1 and NFAT4, that migrate from the cytoplasm to the nucleus following the increase in intracellular Ca(2+) that accompanies the opening of store-operated Orai1/CRAC channels. We demonstrate that NFAT1 has a private line of communication with Orai1, activating in response to Ca(2+) microdomains near the open channels. By contrast, NFAT4 stimulation requires both local Ca(2+) entry and a nuclear Ca(2+) rise. We mapped differences in nuclear location to amino acids within the SP-3 motif of the NFAT regulatory domain. The different Ca(2+) dependencies enable agonists to recruit different isoform combinations as stimulus strength increases. Our study uncovers a mechanism whereby co-existing cytoplasmic transcription factor isoforms are differentially activated by distinct sub-cellular Ca(2+) signals.

Dynamic assembly of a membrane signaling complex enables selective activation of NFAT by Orai1.

NFAT-dependent gene expression is essential for the development and function of the nervous, immune, and cardiovascular systems and kidney, bone, and skeletal muscle. Most NFAT protein resides in the cytoplasm because of extensive phosphorylation, which masks a nuclear localization sequence. Dephosphorylation by the Ca(2+)-calmodulin-activated protein phosphatase calcineurin triggers NFAT migration into the nucleus. In some cell types, NFAT can be activated by Ca(2+) nanodomains near open store-operated Orai1 and voltage-gated Ca(2+) channels in the plasma membrane. How local Ca(2+) near Orai1 is detected and whether other Orai channels utilize a similar mechanism remain unclear. Here, we report that the paralog Orai3 fails to activate NFAT. Orai1 is effective in activating gene expression via Ca(2+) nanodomains because it participates in a membrane-delimited signaling complex that forms after store depletion and brings calcineurin, via the scaffolding protein AKAP79, to calmodulin tethered to Orai1. By contrast, Orai3 interacts less well with AKAP79 after store depletion, rendering it ineffective in activating NFAT. A channel chimera of Orai3 with the N terminus of Orai1 was able to couple local Ca(2+) entry to NFAT activation, identifying the N-terminal domain of Orai1 as central to Ca(2+) nanodomain-transcription coupling. The formation of a store-dependent signaling complex at the plasma membrane provides for selective activation of a fundamental downstream response by Orai1.

STIM proteins, Orai1 and gene expression.

Cytoplasmic Ca(2+) is an universal intracellular messenger that activates cellular responses over a broad temporal range, from neurotransmitter release to cell growth and proliferation. Inherent to the use of the multifarious Ca(2+) signal is the question of specificity: how can some Ca(2+)-dependent responses be activated in a cell and not others? A rise in cytoplasmic Ca(2+) can evoke a response either by binding directly to the target (as occurs with certain Ca(2+)-activated K(+) and Cl(-) channels, for example) or through recruitment of intermediary proteins, such as calmodulin and troponin C. A substantial body of evidence has now established that Ca(2+)-binding proteins differ both in their affinities for Ca(2+) and in their on- and off-rates for Ca(2+) binding/unbinding. Furthermore, different Ca(2+)-binding proteins often occupy distinct locations within the cell. Therefore, the size, kinetics and spatial profile of a cytoplasmic Ca(2+) signal are all important in determining which Ca(2+)-dependent response will be activated, when and for how long.

Different agonists recruit different stromal interaction molecule proteins to support cytoplasmic Ca2+ oscillations and gene expression.

Stimulation of cells with physiological concentrations of calcium-mobilizing agonists often results in the generation of repetitive cytoplasmic Ca(2+) oscillations. Although oscillations arise from regenerative Ca(2+) release, they are sustained by store-operated Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca(2+) oscillations, CRAC channel activity, and downstream Ca(2+)-dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FCεRI receptors evoked Ca(2+) oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca(2+) signals and downstream responses.

CRAC channels drive digital activation and provide analog control and synergy to Ca(2+)-dependent gene regulation.

Ca(2+)-dependent gene expression is critical for cell growth, proliferation, plasticity, and adaptation [1-3]. Because a common mechanism in vertebrates linking cytoplasmic Ca(2+) signals with activation of protein synthesis involves the nuclear factor of activated T cells (NFAT) family of transcription factors [4, 5], we have quantified protein expression in single cells following physiological Ca(2+) signals by using NFAT-driven expression of a genetically encoded fluorescent protein. We find that gene expression following CRAC channel activation is an all-or-nothing event over a range of stimulus intensities. Increasing agonist concentration recruits more cells but each responding cell does so in an essentially digital manner. Furthermore, Ca(2+)-dependent gene expression shows both short-term memory and strong synergy, where two pulses of agonist, which are ineffectual individually, robustly activate gene expression provided that the time interval between them is short. Such temporal filtering imparts coincidence detection to Ca(2+)-dependent gene activation. The underlying molecular basis mapped to time-dependent, nonlinear accumulation of nuclear NFAT. Local Ca(2+) near CRAC channels has to rise above a threshold level to drive gene expression, providing analog control to the digital activation process and a means to filter out fluctuations in background noise from activating transcription while ensuring robustness and high fidelity in the excitation-transcription coupling mechanism.

Selective activation of the transcription factor NFAT1 by calcium microdomains near Ca2+ release-activated Ca2+ (CRAC) channels.

NFATs are a family of Ca(2+)-dependent transcription factors that play a central role in the morphogenesis, development, and physiological activities of numerous distinct cell types and organ systems. Here, we visualize NFAT1 movement in and out of the nucleus in response to transient activation of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels in nonexcitable cells. We show that NFAT migration is exquisitely sensitive to Ca(2+) microdomains near open CRAC channels. Another Ca(2+)-permeable ion channel (TRPC3) was ineffective in driving NFAT1 to the nucleus. NFAT1 movement is temporally dissociated from the time course of the Ca(2+) signal and remains within the nucleus for 10 times longer than the duration of the trigger Ca(2+) signal. Kinetic analyses of each step linking CRAC channel activation to NFAT1 nuclear residency reveals that the rate-limiting step is transcription factor exit from the nucleus. The slow deactivation of NFAT provides a mechanism whereby Ca(2+)-dependent responses can be sustained despite the termination of the initial Ca(2+) signal and helps explain how gene expression in nonexcitable cells can continue after the primary stimulus has been removed.

m-Calpain-mediated cleavage of Na+/Ca2+ exchanger-1 in caveolae vesicles isolated from pulmonary artery smooth muscle.

Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have also been detected. Casein zymogram studies also revealed the presence of m-calpain in the caveolae vesicles. We have also identified Na(+)/Ca(2+) exchanger-1 (NCX1) in the caveolae vesicles. Purification and N-terminal sequence analyses of these two proteins confirmed their identities as m-calpain and NCX1, respectively. We further sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of NCX1 in the caveolae vesicles. Treatment of the caveolae vesicles with the calcium ionophore, A23187 (1 microM) in presence of CaCl(2) (1 mM) appears to cleave NCX1 (120 kDa) to an 82 kDa fragment as revealed by immunoblot study using NCX1 monoclonal antibody; while pretreatment with the calpain inhibitors, calpeptin or MDL28170; or the Ca(2+) chelator, BAPTA-AM did not cause a discernible change in the NCX protein profile. In vitro cleavage of the purified NCX1 by the purified m-calpain supports this finding. The cleavage of NCX1 by m-calpain in the caveolae vesicles may be interpreted as an important mechanism of Ca(2+) overload, which could arise due to inhibition of Ca(2+) efflux by the forward-mode NCX and that could lead to sustained Ca(2+) overload in the smooth muscle leading to pulmonary hypertension.

Characteristic properties of endoplasmic reticulum membrane m-calpain, calpastatin and lumen m-calpain: a comparative study between membrane and lumen m-calpains.

Previously, we reported that bovine pulmonary smooth muscle endoplasmic reticulum (ER) membrane possesses associated m-calpain and calpastatin and ER lumen contains only m-calpain. Herein, we report characteristic properties of ER membrane m-calpain (MCp), calpastatins and lumen m-calpain (LCp) and a brief comparative study between MCp and LCp. MCp containing 80 kDa large and 28 kDa small subunit is non-phosphorylated, whereas LCp containing only 80 kDa large subunit is phosphorylated. Optimum pH, Ca(2+) concentration and pI value of both MCp and LCp are 7.5, 5 mM and 4.5, respectively. MCp and LCp have similar kinetic parameters and circular dichroism (CD) spectra. Autolysis of MCp and LCp are different. Coimmunoprecipitation studies revealed that LCp is associated with ERp57 in the ER lumen, which suggests that the regulation of LCp differs from the regulation of MCp. In presence of Ca(2+), the activated LCp cleaves inositol 1,4,5-trisphosphate receptor-1 (IP(3)R1) in the ER lumen, whereas the activated MCp cleaves Na(+)/Ca(2+) exchanger-1 (NCX1) in the ER membrane. We have determined pI (4.6 and 4.7, respectively) and IC(50) (0.52 and 0.8 nM, respectively) values of 110 and 70 kDa calpastatins. For first time, we have determined the characteristic properties, regulation and functional activity of LCp in the ER lumen.

Identification, purification and partial characterization of a 70kDa inhibitor protein of Na(+)/K(+)-ATPase from cytosol of pulmonary artery smooth muscle.

AIMS: We sought to identify, purify and partially characterize a protein inhibitor of Na(+)/K(+)-ATPase in cytosol of pulmonary artery smooth muscle. MAIN METHODS: (i) By spectrophotometric assay, we identified an inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na(+)/K(+)-ATPase alpha(2)beta(1) and alpha(1)beta(1) isozymes for determining some characteristics of the inhibitor. KEY FINDINGS: We identified a novel endogenous protein inhibitor of Na(+)/K(+)-ATPase having an apparent mol mass of approximately 70kDa in the cytosolic fraction of the smooth muscle. The IC(50) value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the alpha(2)beta(1) and alpha(1)beta(1) isozymes of the Na(+)/K(+)-ATPase; (ii) it interacted reversibly to the E(1) site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na(+)/K(+)-ATPase exists as (alphabeta)(2) diprotomer. SIGNIFICANCE: The inhibitor binds to the Na(+)/K(+)-ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where alpha(2) is more sensitive than alpha(1).

Calcium-dependent cleavage of the Na(+)/Ca(2+) exchanger by m-calpain in isolated endoplasmic reticulum.

We have recently demonstrated the localization of associated m-calpain and calpastatin in the endoplasmic reticulum (ER) of bovine pulmonary artery smooth muscle. Herein, we sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of Na(+)/Ca(2+) exchanger (NCX) in the ER. Treatment of the ER with Ca(2+) (5 mM) dissociates m-calpain-calpastatin association leading to the activation of m-calpain, which subsequently cleaves the ER integral transmembrane protein NCX1 (116 kDa) to an 82 kDa fragment. Pre-treatment of the ER with calpain inhibitors, calpeptin (10 microM) or MDL28170 (10 microM), or Ca(2+) chelator, EGTA (10 mM) does not cleave NCX1. In vitro cleavage of the ER purified NCX1 by the ER purified m-calpain also supports our finding. Cleavage of NCX1 by m-calpain in the ER may be interpreted as the main cause of intracellular Ca(2+) overload in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.

Mitochondrial calpain system: an overview.

Calpain system is generally known to be comprised of three molecules: two Ca2+-dependent proteases: mu- and m-calpains, and their endogenous inhibitor, calpastatin. While calpains have previously been considered as the cytoplasmic enzymes, research in the recent past demonstrated that mu-calpain, m-calpain and calpain 10 are present in mitochondria, which play important roles in a variety of pathophysiological conditions including necrotic and apoptotic cell death phenomena. Although a number of original research articles on mitochondrial calpain system are available, yet to the best of our knowledge, a precise review article on mitochondrial calpain system has, however, not been available. This review outlines the key features of the mitochondrial calpain system, and its roles in several cellular and biochemical events under normal and some pathophysiological conditions.

Role of protein kinase C in NADPH oxidase derived O2*(-)-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca2+]i in pulmonary smooth muscle cells.

Treatment of bovine pulmonary smooth muscle cells with the TxA(2) mimetic, U46619 stimulated [Ca(2+)](i), which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K(V) channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca(2+)](i), indicating a role of K(V)-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K(V)-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca(2+)](i) in the cells. Calphostin C pretreatment also markedly reduced p(47phox) phosphorylation and translocation to the membrane and association with p(22phox), a component of Cyt.b(558) of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O(2)(-)-mediated regulation of K(V)-LVOCC axis leading to an increase in [Ca(2+)](i) by U46619 in the cells.

mu-Calpain mediated cleavage of the Na+/Ca2+ exchanger in isolated mitochondria under A23187 induced Ca2+ stimulation.

Treatment of bovine pulmonary artery smooth muscle mitochondria with the calcium ionophore, A23187 (0.2 microM) stimulates mu-calpain activity and subsequently cleaves Na(+)/Ca(2+) exchanger (NCX). Pretreatment of the A23187 treated mitochondria with the calpain inhibitors, calpeptin or MDL28170 or with Ca(2+) chelator, EGTA does not cleave NCX. Treatment of the mitochondria with A23187 increases Ca(2+) level in the mitochondria, which subsequently dissociates mu-calpain-calpastatin association leading to the activation of mu-calpain. Immunoblot study of the A23187 treated mitochondria with the NCX polyclonal antibody indicates the degradation of mitochondrial inner membrane NCX (110kDa) resulting in the doublet of approximately 54-56kDa NCX fragments. Moreover, in vitro cleavage of mitochondrial purified NCX by mitochondrial purified mu-calpain supports our conclusion. This cleavage of NCX may be interpreted as the main cause of Ca(2+) overload and could lay a key role in the activation of apoptotic process in pulmonary smooth muscle.

Solubilization, purification, and reconstitution of alpha 2 beta 1 isozyme of Na+/K+ -ATPase from caveolae of pulmonary smooth muscle plasma membrane: comparative studies with DHPC, C12E8, and Triton X-100.

We identified alpha(2), alpha(1), and beta(1) isoforms of Na(+)/K(+)-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase from caveolae vesicles were studied during solubilization and purification using the detergents 1,2-heptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C(12)E(8), whereas C(12)E(8) was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase elicited higher E1Na-E2 K transition compared with that of the C(12)E(8)- and Triton X-100-purified enzyme. The rate of Na(+) efflux in DHPC-DOPC-reconstituted isozyme was higher compared to the C(12)E(8)-DOPC- and Triton X100-DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase possessed more organized secondary structure compared to the C(12)E(8)- and Triton X-100-purified isozyme.