ATPase Preparations Research Articles

Antisecretory actions of Baccharis trimera (Less.) DC aqueous extract and isolated compounds: Analysis of underlying mechanisms

Baccharis trimera (Less.) DC. (Asteraceae) is a species native to South America used in Brazilian folk medicine to treat gastrointestinal and liver diseases, kidney disorders and diabetes. Previous studies from this laboratory confirmed the antacid and antiulcer activities of the plant aqueous extract (AE) in rat and mouse models. To investigate the mechanisms involved in the antacid action of AE and isolated compounds from Baccharis trimera. AE was assayed in vivo in cold-restraint stress gastric ulcers and in pylorus-ligated mice. Nine fractions (F2-F10) previously isolated from AE were assayed in vitro on acid secretion measured as [(14)C]-aminopyrine ([(14)C]-AP) accumulation in rabbit gastric glands, and on gastric microsomal H(+), K(+)-ATPase preparations. Chlorogenic acids (F2, F3, F6, F7), flavonoids (F9), an ent-clerodane diterpene (F8) and a dilactonic neo-clerodane diterpene (F10) have been identified in these fractions. Intraduodenal injection of AE (1.0 and 2.0 g/kg) in 4h pylorus-ligated mice decreased the volume (20 and 50%) and total acidity (34 and 50%) of acid secretion compared to control values. Administered orally at the same doses AE protected against gastric mucosal lesions induced in mice by restraint at 4°C. Exposure of isolated rabbit gastric glands to fractions F8 (10-100 μM) and F9 (10-300 μg/ml) decreased the basal [(14)C]-AP uptake by 50 and 60% of control (Ratio=6.2±1.1), whereas the remaining fractions were inactive. In the presence of the secretagogues F2 and F4 (30-300 μg/ml) decreased the [(14)C]-AP uptake induced by histamine (His) with a 100-fold lower potency than that of ranitidine. F5 and F6 reduced the [(14)C]-AP uptake stimulated by carbachol (CCh), but they were 10 to 20-fold less potent than atropine. F8 (diterpene 2) and F9 (flavonoids) decreased both the His- and CCh-induced [(14)C]-AP uptake, whereas F10 (diterpene 1) was inactive against the [(14)C]-AP uptake stimulated by secretagogues. Diterpene 2 was the most active of all tested compounds being 7-fold less potent than ranitidine and equipotent to atropine in reducing acid secretion in vitro. This compound also reduced the gastric H(+), K(+)-ATPase activity by 20% of control, while the remaining fractions were inactive on the proton pump in vitro. The results indicate that Baccharis trimera presents constituents that inhibit gastric acid secretion by acting mainly on the cholinergic regulatory pathway. The plant extract also contains compounds that exert moderate inhibition of the histaminergic regulatory pathway of acid secretion and the gastric proton pump. Altogether these active constituents appear to provide effective inhibition of acid secretion in vivo, which may explain the reputed antiulcer activity of the plant extract.

Dynamic lipid–protein stoichiometry on E1 and E2 conformations of the Na +/K +-ATPase

Annular lipid–protein stoichiometry in native pig kidney Na +/K +-ATPase preparation was studied by [ 125I]TID-PC/16 labeling. Our data indicate that the transmembrane domain of the Na +/K +-ATPase in the E 1 state is less exposed to the lipids than in E 2, i.e. the conformational transitions are accompanied by changes in the number of annular lipids but not in the affinity of these lipids for the protein. The lipid–protein stoichiometry was 23 ± 2 (α subunit) and 5.0 ± 0.4 (β subunit) in the E 1 conformation and 32 ± 2 (α subunit) and 7 ± 1 (β subunit) in the E 2 conformation.

Ivermectin is a nonselective inhibitor of mammalian P-type ATPases

Ivermectin is a large spectrum antiparasitic drug that is very safe at the doses actually used. However, as it is being studied for new applications that would require higher doses, we should pay attention to its effects at high concentrations. As micromolar concentrations of ivermectin have been reported to inhibit the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), we decided to investigate its putative inhibitory effect on other two important P-type ATPases, namely the Na(+) , K(+)-ATPase and H(+)/K(+)-ATPase. We first extended the data on SERCA, using preparations from rat enriched in SERCA1a (extensor digitorum longus) and 1b (heart) isoforms. Secondly, we tested the effect of ivermectin in two preparations of rat Na(+), K(+)-ATPase in order to appreciate its putative selectivity towards the alpha(1) isoform (kidney) and the alpha(2)/alpha(3) isoforms (brain), and in an H(+)/K(+)-ATPase preparation from rat stomach. Ivermectin inhibited all these ATPases with similar IC(50) values (6-17 microM). With respect to the inhibition of the Na(+), K(+)-ATPase, ivermectin acts by a mechanism different from the classical cardiac glycosides, based on selectivity towards the isoforms, sensibility to the antagonistic effect of K(+) and to ionic conditions favoring different conformations of the enzyme. We conclude that ivermectin is a nonselective inhibitor of three important mammalian P-type ATPases, which is indicative of putative important adverse effects if this drug were used at high doses. As a consequence, we propose that novel analogs of ivermectin should be developed and tested both for their parasitic activity and in vitro effects on P-type ATPases.

Inhibition of mammal Na(+)K(+)-ATPase by diterpenes extracted from the Brazilian brown alga Dictyota cervicornis.

The dolastane diterpenes 4-acetoxy-9,14-dihydroxydolast-1(15),7-diene (1) and 4,7-diacetoxy-14-hydroxydolast-1(15),8-diene (2) were isolated from specimens of the alga Dictyota cervicornis collected from the Rio de Janeiro coast, Brazil. Chemical structures of the diterpenes were assigned by 1D and 2D NMR spectral data for the first time. Both substances inhibited Na(+)K(+)-ATPase preparations from guinea-pig brain or kidney, with the same inhibitory potency towards enzyme isoforms. The maximal inhibition obtained for 1 was 40% at a concentration of 0.5 mm in the incubation mixture, while it reached 80% for compound 2 at this concentration. Ouabain insensitive ATPases were inhibited by 1, but not by 2. Data comparing the inhibitory potency of these compounds with that of ouabain and oleic acid suggest a higher degree of selectivity of 2 towards the Na(+)K(+)-pump. Cardiac glycosides such as ouabain are used classically in the treatment of heart failure, but alterations of Na(+)K(+)-pump activity are also involved in several other diseases. Therefore, the study of compounds interfering with this pump activity is gaining further importance.

Inhibition of gastric acid secretion by a standardized aqueous extract of Cecropia glaziovii Sneth and underlying mechanism

Cecropia glazioui Sneth (Cecropiaceae) is used in folk medicine in tropical and subtropical Latin America as cardiotonic, diuretic, hypotensive, anti-inflammatory and anti-asthmatic. The hypotensive/antihypertensive activity of the plant aqueous extract (AE) and isolated butanolic fraction (BuF) has been confirmed and putatively related to calcium channels blockade in vascular smooth musculature [Lapa, A.J., Lima-Landman, M.T.R., Cysneiros, R.M, Borges, A.C.R., Souccar, C., Barreta, I.P., Lima, T.C.M., 1999. The Brazilian folk medicine program to validate medicinal plants - a topic in new antihypertensive drug research. In: Hostettman, K., Gupta, M.P., Marston, A. (Eds.), Proceedings Volume, IOCD/CYTED Symposium, Panamá City, Panamá, 23-26 February 1997. Chemistry, Biological and Pharmacological Properties of Medicinal Plants from the Americas. Harwood Academic Publishers, Amsterdam, pp. 185-196; Lima-Landman, M.T., Borges, A.C., Cysneiros, R.M., De Lima, T.C., Souccar, C., Lapa, A.J., 2007. Antihypertensive effect of a standardized aqueous extract of Cecropia glaziovii Sneth in rats: an in vivo approach to the hypotensive mechanism. Phytomedicine 14, 314-320]. Bronchodilation and antidepressant-like activities of both AE and BuF have been also shown [Delarcina, S., Lima-Landman, M.T., Souccar, C., Cysneiros, R.M., Tanae, M.M., Lapa, A.J., 2007. Inhibition of histamine-induced bronchospasm in guinea pigs treated with Cecropia glaziovi Sneth and correlation with the in vitro activity in tracheal muscles. Phytomedicine 14, 328-332; Rocha, F.F., Lima-Landman, M.T., Souccar, C., Tanae, M.M., De Lima, T.C., Lapa, A.J., 2007. Antidepressant-like effect of Cecropia glazioui Sneth and its constituents -in vivo and in vitro characterization of the underlying mechanism. Phytomedicine 14, 396-402]. This study reports the antiulcer and antisecretory gastric acid activities of the plant AE, its BuF and isolated compounds with the possible mechanism involved. Both AE and BuF were assayed on gastric acid secretion of pylorus-ligated mice, on acute models of gastric mucosal lesions, and on rabbit gastric H(+), K(+)-ATPase preparations. Intraduodenal injection of AE or BuF (0.5-2.0g/kg, i.d) produced a dose-related decrease of the basal gastric acid secretion in 4-h pylorus-ligated mice. At 1.0g/kg, BuF decreased the volume (28%) and total acidity (33%) of the basal acid secretion, and reversed the histamine (2.5mg/kg, s.c.)- or bethanecol (1.0mg/kg, s.c.)-induced acid secretion to basal values, indicating inhibition of the gastric proton pump. Pretreatment of mice with the BuF (0.05-0.5g/kg, p.o.) protected against gastric mucosal lesions induced by 75% ethanol, indomethacin (30mg/kg, s.c.) or restraint at 4 degrees C. BuF also decreased the gastric H(+), K(+)-ATPase activity in vitro proportionately to the concentration (IC(50)=58.8microg/ml). The compounds isolated from BuF, consisting mainly of cathechins, procyanidins and flavonoids [Tanae, M.M., Lima-Landman, M.T.R., De Lima, T.C.M., Souccar, C., Lapa, A.J., 2007. Chemical standardization of the aqueous extract of Cecropia glaziovii Sneth endowed with antihypertensive, bronchodilator, antacid secretion and antidepressant-like activities. Phytomedicine 14, 309-313], inhibited the in vitro gastric H(+), K(+)-ATPase activity at equieffective concentrations to that of BuF. The results indicate that C. glazioui constituents inhibit the gastric proton pump; this effect may account for the effective antisecretory and antiulcer activities of the standardized plant extract.

The Molecular Basis for Cyclopiazonic Acid Inhibition of the Sarcoplasmic Reticulum Calcium Pump

The sarcoplasmic reticulum Ca(2+)-ATPase is essential for calcium reuptake in the muscle contraction-relaxation cycle. Here we present structures of a calcium-free state with bound cyclopiazonic acid (CPA) and magnesium fluoride at 2.65 A resolution and a calcium-free state with bound CPA and ADP at 3.4A resolution. In both structures, CPA occupies the calcium access channel delimited by transmembrane segments M1-M4. Inhibition of Ca(2+)-ATPase is stabilized by a polar pocket that surrounds the tetramic acid of CPA and a hydrophobic platform that cradles the inhibitor. The calcium pump residues involved include Gln(56), Leu(61), Val(62), and Asn(101). We conclude that CPA inhibits the calcium pump by blocking the calcium access channel and immobilizing a subset of transmembrane helices. In the E2(CPA) structure, ADP is bound in a distinct orientation within the nucleotide binding pocket. The adenine ring is sandwiched between Arg(489) of the nucleotide-binding domain and Arg(678) of the phosphorylation domain. This mode of binding conforms to an adenine recognition motif commonly found in ATP-dependent proteins.

Inhibition of the gastric H +,K +-ATPase by plectrinone A, a diterpenoid isolated from Plectranthusbarbatus Andrews

This work assessed the mechanism underlying the antisecretory gastric acid effect of Plectranthus barbatus Andrews (Lamiaceae) and active constituents. Popularly known as “false-boldo”, this plant is used in Brazilian folk medicine to treat gastrointestinal and hepatic ailments. The plant aqueous extract (AE) and isolated compounds were assayed in vivo in pylorus-ligated mice, and in vitro on acid secretion measured as [ 14C]-aminopyrine ([ 14C]-AP) accumulation in rabbit gastric glands and gastric H +,K +-ATPase preparations. Injected into the duodenal lumen, the AE of the plant leaves (0.5 and 1.0 g/kg) decreased the volume (62 and 76%) and total acidity (23 and 50%) of gastric acid secretion in pylorus-ligated mice. Bioguided purification of the AE yielded an active fraction (IC 50 = 24 μg/ml) that inhibited acid secretion in rabbit gastric glands with a potency 10 to 18 times greater than that of the originating extract, on both the basal and stimulated acid secretion by histamine (His) (1 μM) or bethanechol (100 μM). At the same concentrations the gastric H +,K +-ATPase activity was also inhibited. The active constituent was chemically identified as the abietanoid dienedione plectrinone A which reduced the H +,K +-ATPase activity with IC 50 = 171 μM. The results indicate that inhibition of the gastric proton pump by this diterpenoid may account for the antisecretory acid effect and reputed antiulcer activity of Plectranthus barbatus.

Purification of Na+,K+-ATPase Expressed in Pichia pastoris Reveals an Essential Role of Phospholipid-Protein Interactions

Na+,K+-ATPase (porcine alpha/his10-beta) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-beta-maltoside and purified to 70-80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 degrees C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-beta-maltoside, appear to be mainly stable monomers (alpha/beta) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 degrees C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The beta subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 degrees C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the beta subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.

Isolation of a complete A1AO ATP synthase comprising nine subunits from the hyperthermophile Methanococcus jannaschii.

Archaeal A(1)A(O) ATP synthase/ATPase operons are highly conserved among species and comprise at least nine genes encoding structural proteins. However, all A(1)A(O) ATPase preparations reported to date contained only three to six subunits and, therefore, the study of this unique class of secondary energy converters is still in its infancy. To improve the quality of A(1)A(O) ATPase preparations, we chose the hyperthermophilic, methanogenic archaeon Methanococcus jannaschii as a model organism. Individual subunits of the A(1)A(O) ATPase from M. jannaschii were produced in E. coli, purified, and antibodies were raised. The antibodies enabled the development of a protocol ensuring purification of the entire nine-subunit A(1)A(O) ATPase. The ATPase was solubilized from membranes of M. jannaschii by Triton X-100 and purified to apparent homogeneity by sucrose density gradient centrifugation, ion exchange chromatography, and gel filtration. Electron micrographs revealed the A(1) and A(O) domains and the central stalk, but also additional masses which could represent a second stalk. Inhibitor studies were used to demonstrate that the A(1) and A(O) domains are functionally coupled. This is the first description of an A(1)A(O) ATPase preparation in which the two domains (A(1) and A(O)) are fully conserved and functionally coupled.

Fifty years of cardiovascular science together.

Circulation Research began 50 years ago, as I entered medical school in St Louis, and we have grown up together. I have luckily worked during a golden age for cardiovascular medicine, when biological and engineering sciences have prospered and greatly benefited our patients. This remarkable half-century testifies to the power of science to promote health. It is a vindication of the commitment my colleagues and I made to medical science so long ago and a tribute to the mission of Circulation Research . I will trace some of the scientific milestones, reflected by my Cardiology career. Having majored in history, I entered medical school in St Louis seriously deficient in science. I fortunately soon fell into the hands of Earl Sutherland, a physician who studied with Carl Cori and by 1952 was part of the Biochemistry faculty. He was trying to understand epinephrine action on glycogen metabolism, which subsequently led him to discover adenylyl cyclase and cAMP as the prototype “second messenger system,” winning the Nobel Prize in 1971. I worked half-time in his laboratory, purifying phosphorylase and absorbing his enthusiasm for science. During my second-year Pharmacology course, I was taught by Robert Furchgott, who received the Nobel Prize in 1998 for his work on nitric oxide. Medical school was exciting. During internship interviews with the prestigious Chairmen of Medicine, I was surprised that they grilled me on my research, not my clinical skills. So I returned to school and with two other students embarked on my first independent research effort to develop an antibody to lupus protein for immunolocalization. I collected plasma from a patient with severe systemic lupus erythematosus by withdrawing whole blood and returning packed red cells. Surprisingly, the patient immediately went into remission, possibly the first therapeutic plasmapheresis.1 Several supportive faculty provided the resources I …

Contribution of lipid dynamics on the inhibition of bovine brain synaptosomal Na+-K+-ATPase activity induced by 4-hydroxy-2-nonenal.

The effects of lipid hydroperoxide degradation products, such as 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA), on bovine brain synaptosomal ATPase activities and their membrane lipid organization were examined. When the synaptosomes were treated with HNE, this resulted in the decrease of Na(+)-K(+)-ATPase activity with the loss of sulfhydryl (SH) groups in the membrane proteins. In contrast, MDA treatment of the synaptosomes did not induce an appreciable decrease in the ATPase activity or a loss of SH groups. The decreases in ATPase activity and SH content by treatment with HNE were also observed, as a Na+-K+-ATPase preparation was used in place of the synaptosomes. On the other hand, HNE had very little effect on synaptosomal Ca2+- and Mg2+-ATPase activities. The results of the kinetic analysis of the Na+-K+-ATPase activity indicated that the decrease in the activity by HNE-modification is due to a decreased affinity for the substrate. ATP completely protected the ATPase from the HNE attack. Modification of the synaptosomes with HNE caused a decrease in the membrane lipid fluidity near the lipid/water interface, not the lipid layer interior. In addition, it was found that there is a good relationship between the lipid fluidity and the Na+-K+-ATPase activity under the presence of various concentrations of HNE, suggesting that the lipid dynamics are closely related to HNE-induced inhibition of the ATPase activity. On the other hand, MDA did not induce change in the membrane lipid fluidity. HNE and MDA are mainly incorporated into the lipid and protein fractions in the synaptosomal membranes, respectively. Based on these results, we proposed a possible mechanism of HNE-induced inhibition of synaptosomal Na+-K+-ATPase activity associated with alterations in the membrane lipid organization.

Large-scale preparation of sodium-potassium ATPase from kidney outer medulla

Large amounts of Na,K-ATPase are needed for studies involving protein chemistry. Preparation of Na,K-ATPase from kidney by the widely used, rapid procedure of Jørgensen (Biochim Biophys Acta 356:36-52, 1974; Methods Enzymol 156:29-43, 1988) includes labor-intensive dissection of tissue from the outer medulla and centrifugation into a step gradient of sucrose solution. In a large-scale modification presented here, tissue was dissected with a surgical instrument, a rongeur, and centrifugation was simply a five times repeated differential centrifugation. The procedure took seven days and 68 person-hours of work. The yield of activity from 26 kg of whole kidneys was 6600 units (micromol Pi/min) in one preparation. The amount of protein was 240 mg and the specific activity was 28 micromol Pi/min per mg protein. There is a significant saving of labor to obtain a product with a specific activity similar to that commonly obtained. The microsomal fraction may be useful for preparing other membrane proteins from the outer medulla.

Ca(2+ )transport by the sarcoplasmic reticulum Ca(2+)-ATPase in sea cucumber (Ludwigothurea grisea) muscle.

In muscle cells, the excitation-contraction cycle is triggered by an increase in the concentration of free cytoplasmic Ca(2+). The Ca(2+)-ATPase present in the membrane of the sarcoplasmic reticulum (SR) pumps Ca(2+) from the cytosol into this intracellular compartment, thus promoting muscle relaxation. The microsomal fraction derived from the longitudinal smooth muscle of the body wall from the sea cucumber Ludwigothurea grisea retains a membrane-bound Ca(2+)-ATPase that is able to transport Ca(2+) mediated by ATP hydrolysis. Immunological analyses reveal that monoclonal antibodies against sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA1 and SERCA2a) cross-react with a 110 kDa band, indicating that the sea cucumber Ca(2+)-ATPase is a SERCA-type ATPase. Like the mammalian Ca(2+)-ATPase isoforms so far described, the enzyme also shows a high affinity for Ca(2+) and ATP, has an optimum pH of approximately 7.0 and is sensitive to thapsigargin and cyclopiazonic acid, specific inhibitors of the SERCA pumps. However, unlike the mammalian SERCA isoforms, concentrations of ATP above 2 mmol l(-1) inhibit Ca(2+) transport, but not ATP hydrolysis, in sea cucumber vesicles, suggesting that high ATP concentrations uncouple the Ca(2+)-ATPase. Another unique feature observed with the sea cucumber Ca(2+)-ATPase is the high dependence of maximal activity on K(+) or Na(+). Similar activation promoted by these cations was observed with various mammalian Ca(2+)-ATPase preparations when they were incubated in the presence of low concentrations of sulphated polysaccharides. In control experiments, K(+) and Na(+) have almost no effect on Ca(2+) transport, but in the presence of heparin or fucosylated chondroitin sulphate, the activity of the different mammalian Ca(2+)-ATPases is inhibited and they are activated by either K(+) or Na(+) in a manner similar to the native sea cucumber ATPase. These results led us to investigate the possible occurrence of a highly sulphated polysaccharide on vesicles from the SR of sea cucumber smooth muscle that could act as an 'endogenous' Ca(2+)-ATPase inhibitor. In fact, SR vesicles derived from the sea cucumber, but not from rabbit muscle, contain a highly sulphated polysaccharide. After extraction and purification of these polysaccharide molecules, their effect was tested on vesicles obtained from rabbit muscle. This compound inhibited Ca(2+) uptake in rabbit SR vesicles, at concentrations lower than heparin, and restored the dependence on monovalent cations. These results strongly suggest that the sea cucumber Ca(2+)-ATPase is activated by monovalent cations because of the presence of endogenous sulphated polysaccharides.

Quantitation of Plasma Membrane Calcium Pump Phosphorylated Intermediates by Electrophoresis

P-ATPases are characterized by the formation of acid-stable phosphorylated intermediates ( EP) during their reaction cycle. We have developed a microscale method to determine EP that involves the phosphorylation of the enzyme using [γ- 32P]ATP and precipitation with TCA; separation of the sample by SDS-PAGE, and measurement of the enzyme protein and 32P-labeled EP by digital analysis of both the stained gel and its autoradiogram, respectively. The principal advantages of this method over typical procedures (filtration and centrifugation) are the low amount of enzyme required and the substantial decrease in the blank values and data scattering produced by unspecific phosphorylation and nonquantitative recovering of the enzyme. Application of this new method to a purified preparation of the plasma membrane calcium ATPase (PMCA) results in overcoming the difficulties of measuring EP at high ATP concentrations. A biphasic behavior of the substrate curve for EP was observed when the study was extended to ATP levels within the physiological range. Since, in principle, the method does not require the use of highly purified preparations, it could be helpful for the study of phosphorylated intermediates especially under conditions in which small amounts of protein are available, e.g., mutated variants of P-ATPases.

Apoptolidin, a selective cytotoxic agent, is an inhibitor of F 0F 1-ATPase

Background: Apoptolidin is a macrolide originally identified on the basis of its ability to selectively kill E1A and E1A/E1B19K transformed rat glial cells while not killing untransformed glial cells. The goal of this study was to identify the molecular target of this newly discovered natural product. Results: Our approach to uncovering the mechanism of action of apoptolidin utilized a combination of molecular and cell-based pharmacological assays as well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action. Cell killing induced by apoptolidin was independent of p53 status, inhibited by BCL-2, and dependent on the action of caspase-9. PARP was completely cleaved in the presence of 1 μM apoptolidin within 6 h in a mouse lymphoma cell line. Together these results suggested that apoptolidin might target a mitochondrial protein. Structural comparisons between apoptolidin and other macrolides revealed significant similarity between the apoptolidin aglycone and oligomycin, a known inhibitor of mitochondrial F 0F 1-ATP synthase. The relevance of this similarity was established by demonstrating that apoptolidin is a potent inhibitor of the F 0F 1-ATPase activity in intact yeast mitochondria as well as Triton X-100-solubilized ATPase preparations. The K i for apoptolidin was 4–5 μM. The selectivity of apoptolidin in the NCI-60 cell line panel was found to correlate well with that of several known anti-fungal natural products that inhibit the eukaryotic mitochondrial F 0F 1-ATP synthase. Significance: Although the anti-fungal activities of macrolide inhibitors of the mitochondrial F 0F 1-ATP synthase such as oligomycin, ossamycin and cytovaricin are well-documented, their unusual selectivity toward certain cell types is not widely appreciated. The recent discovery of apoptolidin, followed by the demonstration that it is an inhibitor of the mitochondrial F 0F 1-ATP synthase, highlights the potential relevance of these natural products as small molecules to modulate apoptotic pathways. The mechanistic basis for selective cytotoxicity of mitochondrial ATP synthase inhibitors is discussed.

Identification and Functional Expression of Four Isoforms of ATPase II, the Putative Aminophospholipid Translocase: EFFECT OF ISOFORM VARIATION ON THE ATPase ACTIVITY AND PHOSPHOLIPID SPECIFICITY

ATPase II, a vanadate-sensitive and phosphatidylserine-dependent Mg(2+)-ATPase, is a member of a subfamily of P-type ATPase and is presumably responsible for aminophospholipid translocation activity in eukaryotic cells. The aminophospholipid translocation activity plays an important physiological role in the maintenance of membrane phospholipid asymmetry that is observed in the plasma membrane as well as the membranes of certain cellular organelles. While the preparations of ATPase II from different sources share common fundamental properties, such as substrate specificity, inhibitor spectrum, and phospholipid dependence, they are divergent in several characteristics. These include specific ATPase activity and phospholipid selectivity. We report here the identification of four isoforms of ATPase II in bovine brain. These isoforms are formed by a combination of two major variations in their primary sequences and show that the structural variation of these isoforms has functional significance in both ATPase activity and phosholipid selectivity. Furthermore, studies with the phosphoenzyme intermediate of ATPase II and its recombinant isoforms revealed that phosphatidylserine is essential for the dephosphorylation of the intermediate. Without phosphatidylserine, ATPase II would be accumulated as phosphoenzyme in the presence of ATP, resulting in the interruption of its catalytic cycle.

Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: evidence that the reaction mechanism involves all of the sites.

The phosphorylation capacity of Na+,K+-ATPase preparations in common use is much less than expected on the basis of the molecular weight of the enzyme deduced from cDNA sequences. This has led to the popularity of half-of-the-sites or flip-flop models for the enzyme reaction mechanism. We have prepared Na+,K+-ATPase from nasal salt glands of salt-adapted ducks which has a phosphorylation capacity and specific activity near the theoretical maxima. Preparations with specific activities of >60 micromol (mg of protein)-1 min-1 at 37 degrees C had phosphorylation capacities of >60 nmol/mg of protein, and the rate of turnover of the enzyme was 9690 min-1, within the range reported for the enzyme from other sources. The fraction of the maximal specific activity of the enzyme compared well with the fraction of the protein on SDS-PAGE which was alpha and beta chains, especially at the highest specific activity which indicates that all of the alphabeta protomers are active. The gels of the most reactive preparations contained only alpha and beta chains, but less active preparations contained a number of extraneous proteins. The major contaminant was actin. The preparation did not contain any protein which migrated in the molecular weight range of the gamma subunit. The subunit composition of the enzyme was alpha1 and beta1 only. This is the first report of a pure, homogeneous, fully active preparation of the protein. Reaction models which incorporate a half-of-the-sites or flip-flop mechanism do not apply to this enzyme.

ATP and Acetyl Phosphate Induces Molecular Events near the ATP Binding Site and the Membrane Domain of Na+,K+-ATPase: THE TETRAMERIC NATURE OF THE ENZYME

The addition of ATP to Mg2+-Na+-bound-probe labeled Na+,K+-ATPase preparations containing approximately 0.5 mol of pyridoxal 5'-diphospho-5'-adenosine (AP2PL) probe at Lys-480 and approximately 0.9 mol of fluorescein 5'-isothiocyanate (FITC) probe at Lys-501 showed a decrease and an increase in the AP2PL fluorescence intensity with neither significant ATP-dependent phosphorylation nor FITC fluorescence change. The rate constants for the fluorescence change increased nearly linearly with increasing ATP concentrations. The substitution of AcP for ATP decreased the FITC fluorescence rather monophasically, 8.5/s, which was followed by the half-site phosphorylation with same amount of components with different rate constant, 7.2 and 4.6/s, followed by a much slower increase in the two components of AP2PL fluorescence, 1.4 and 0.2/s. The addition of Na+ with increasing concentrations of ATP to the K+-bound AP2PL-FITC enzymes induced accelerations in the decrease and an increase in the AP2PL fluorescence intensity with two different increases in the FITC fluorescence intensity, showing that the same concentration of ATP is capable of inducing four different fluorescence changes. The addition of ATP to the Mg2+-Na+-bound enzymes modified with N-[p-(2-benzimidazolyl)phenyl]-maleimide (BIPM) at Cys-964 and retaining full Na+,K+-ATPase activity induced two different increases in BIPM fluorescence intensity. Each rate constant for the BIPM fluorescence change versus concentrations of ATP gave two intersecting straight lines. These data and the stoichiometries of fluorescence probe bindings and ATP- and AcP-dependent phosphorylation provide strong support for the conclusion that the functional membrane-bound Na+,K+-ATPase is a tetramer.

Half-site Modification of Lys-480 of the Na+,K+-ATPase α-Chain with Pyridoxal 5′-Diphospho-5′-adenosine Reduces ATP-dependent Phosphorylation Stoichiometry from Half to a Quarter

Pig and dog kidney Na+,K+-ATPase preparations, irrespective of specific activity, showed approximately 0.5 mol of maximum phosphorylation/mol alpha-chain for ATP or acetyl phosphate (AcP) at steady state conditions. Pyridoxal 5'-diphospho-5'-adenosine (AP2PL)-treated pig kidney enzymes containing approximately 0.5 mol of AP2PL probe at Lys-480/mol (Tsuda, T., Kaya, S., Funatsu, H., Hayashi, Y., and Taniguchi, K. (1998) J. Biochem. (Tokyo) 123, 169-174) showed a quarter-site phosphorylation by ATP and half-site phosphorylation from AcP. The addition of 10 microM ATP to the Mg2+-Na+-bound AP2PL enzyme induced rapid quarter-site phosphorylation (47/s), followed by two different AP2PL fluorescence changes, a rapid decrease (29/s) and a slow increase (1.1/s). The addition of 1 mM AcP to the Mg2+-Na+-bound AP2PL enzyme induced a slow half-site phosphorylation (3/s), followed by a monophasic AP2PL fluorescence increase (1.2/s). After treatment of the AP2PL enzyme with fluorescein 5'-isothiocyanate to modify Lys-501 fully, the Mg2+-Na+-dependent phosphorylation capacity from ATP of the resulting AP2PL-fluorescein 5'-isothiocyanate enzyme was reduced to approximately 6% without significant changes in half-site phosphorylation capacity with respect to AcP, dynamic AP2PL fluorescence change by ATP and change by AcP. These data and others support the hypothesis that the functional membrane-bound Na+, K+-ATPase has tetrameric properties.

Effect of a Low Molecular Weight Factor from Na+, K+‐ATPase Preparations on Ouabain Bindinga

Effect of a Low Molecular Weight Factor from Na⁺, K⁺‐ATPase Preparations on Ouabain Binding^a