High Concentrations Of ATP Research Articles

ATP is a potent stimulator of the activation and formation of rodent osteoclasts.

1. There is increasing evidence that extracellular ATP acts directly on bone cells via P2 receptors. In normal rat osteoclasts, ATP activates both non-selective cation channels and Ca2+-dependent K+ channels. In this study we investigated the action of ATP on the formation of osteoclasts and on the ultimate function of these cells, namely resorption pit formation. 2. We found that ATP stimulated resorption pit formation up to 5.6-fold when osteoclast-containing bone cell populations from neonatal rats were cultured for 26 h on ivory discs, with a maximum effect occurring at relatively low concentrations (0.2-2 microM). The stimulatory effect of ATP was amplified greatly when osteoclasts were activated by culture in acidified media (pH 6.9-7.0). Pit formation by acid-activated osteoclasts in the absence of ATP was inhibited by apyrase, an ecto-ATPase and by suramin, an antagonist of P2 receptors. 3. Over the same concentration range at which rat osteoclast activation occurred (0.2-2 microM), ATP also enhanced osteoclast formation in 10 day mouse marrow cultures, by up to 3.3-fold, with corresponding increases in resorption pit formation. Higher concentrations of ATP (20-200 microM) reduced or blocked osteoclast formation. Adenosine, a P1 receptor agonist, was without effect on either osteoclast activation or formation. 4. These results suggest that low levels of extracellular ATP may play a fundamental role in modulating both the resorptive function and formation of mammalian osteoclasts.

Regulation of M-Type Potassium Current by Intracellular Nucleotide Phosphates

The effects of intracellular application of various concentrations of adenine nucleoside phosphates and nucleotide analogs on the M-type K current (IM) of single neurons isolated from sympathetic ganglia were studied. With 1 mM MgATP intracellularly IM decreased to 25% of its initial level 39 min after the start of whole-cell recording. In the absence of ATP the current decreased more rapidly. Addition of glucose and pyruvate extracellularly was equivalent to adding 1 mM MgATP intracellularly. AMP-PNP, a nonhydrolyzable ATP analog, at a concentration of 1 or 3 mM was unable to maintain IM in the absence of ATP. When ATP and AMP-PNP were combined in the pipette, however, the maintenance of IM was prolonged. A series of nucleotides and analogs have been combined with ATP to test for their ability to maintain IM and to alter calcineurin phosphatase activity. There was a positive correlation between the ability of a nucleotide to prevent the rundown of IM and its ability to inhibit calcineurin phosphatase activity. These findings show that the amplitude of IM is dually regulated by cellular levels of adenine nucleotide diphosphates and triphosphates. A hydrolyzable form of ATP is necessary to maintain the M current. The maintenance of IM is further enhanced by the simultaneous presence of ADP or other adenine nucleotides that alter calcineurin activity, but not by higher concentrations of ATP alone. These results are consistent with regulation of IM by phosphorylation events that maintain IM and dephosphorylation events that lead to current rundown.

ATP and energy charge regulate glycogen phosphorylase from Mytilus galloprovincialis mantle

Different ATP inhibition patterns are reported for both phosphorylated and unphosphorylated forms of glycogen phosphorylase from Mytilus galloprovincialis mantle. The inhibition of the `b' form conforms to the hyperbolic mixed (i.e. partial uncompetitive) model with respect to substrates, glycogen and inorganic phosphate (P i). At low AMP concentrations, ATP and other nucleotides (ADP and IMP) have an activating effect on the unphosphorylated form. The inhibition of the `a' form conforms to the hyperbolic mixed model with respect to glycogen, but to the simple competitive model with respect to P i. The effects of ATP on the phosphorylated form appear to reverse the effect of enzyme phosphorylation. The influence of energy status on the activity of glycogen phosphorylase was also studied. Activity was maximal at high AMP and ATP concentrations at low and high energy charges (ECs), respectively. At low EC, the enzyme was activated by AMP, strongly inhibited by ADP and weakly inhibited by ATP. These results support the hypothesis that seasonal variations in EC control glycogen mobilization in Mytilus, related to gametogenic development and to basal metabolism in sexual resting periods.

NO is one of the triggers of BBB permeability for norepinephrine (3HNE) during cold stress

The isoquinolinesulfonamide H-89, an inhibitor of cyclic AMP-dependent protein kinases (EC 2.7.1.37, cAPrK), inhibited the Ca2+ -ATPase activity of cardiac and skeletal muscle sarcoplasmic reticulum (SR) with concentrations giving half-maximal inhibition of 8.1 ± 1.3 and 7.2 ± 0.9 μmol/L, respectively. The effect of H-89 on cardiac SR Ca2+ -ATPase (EC 3.6.1.38) was the same irrespective of the presence or absence of inhibitors of cAPrK and furthermore, was not affected by a neutralising monoclonal antibody raised against phospholamban. Thus, the action of H-89 in inhibiting SR Ca2+ -ATPase would not appear to be mediated by inhibition of cAPrK to reduce the phosphorylation state of phospholamban. In both cardiac and skeletal muscle SR, the inhibition by H-89 was noncompetitive with respect to ATP at a low concentration of ATP (< 1 mmol/L) and of a mixed pattern at high concentrations of ATP. H-89 produced a decrease in affinity of the SR Ca2+ pump to Ca2+ with an increase in the Km for Ca from 0.52 ± 0.01 to 0.94 ± 0.03 μmol/L (P < 0.05) in cardiac SR and from 0.39 ± 0.01 to 0.79 ± 0.02 μmol/L (P < 0.05) in skeletal muscle SR. These results suggest that H-89 inhibits SR Ca2+ -ATPase by a direct action on the SR Ca2+ pump to decrease its affinity to Ca2+. Such an action may contribute to the pharmacological effect of H-89.

Effects of cytosolic ATP and other nucleotides on Ca 2+-activated K + channels in cultured bovine adrenal chromaffin cells

The effects of cytosolic ATP on Ca 2+-dependent K + (K Ca) channel activation in cultured bovine adrenal chromaffin cells were investigated by using single-channel recording patch-clamp techniques. Application of ATP to the intracellular surface of excised inside-out patches activated K Ca channels in a dose-dependent manner at 30 μM to 10 mM. The K Ca channels also were activated by 3 mM of adenosine 5′- O-(3′-thiotriphosphate) (ATP γS), a non-hydrolyzable analogue of ATP, but not by 5′-adenylylimidodiphosphate (AMP-PNP) (from 300 μM to 3 mM). Furthermore, other nucleotides also activated K Ca channels in inside-out patches. This modulation took place without addition of exogenous protein kinase and was dependent on the presence of Mg 2+ in the bathing solution. Staurosporine, a non-specific kinase inhibitor, or H-89 ( N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), a cAMP-dependent protein kinase inhibitor, was unable to alter ATP-mediated K Ca channel activation. Following complete removal of Mg 2+, a higher concentration of ATP (10 mM) and other nucleotides was required to activate K Ca channels; however, Mg 2+ was ineffective in altering the activation of K Ca channels by itself. It is concluded that intracellular ATP and other nucleotides activate K Ca channels directly. These nucleotides may regulate catecholamine release by changing the cell membrane potential in adrenal chromaffin cells.

APS Kinase fromArabidopsis thaliana:Genomic Organization, Expression, and Kinetic Analysis of the Recombinant Enzyme

The gene encoding 5′-adenylylsulfate (APS) kinase (EC 2.7.1.25) (APK) was cloned fromArabidopsis thaliana.There is a singleAPKlocus inA. thaliana.The coding sequence of the gene is composed of 7 exons, interrupted by 6 introns. A transcriptional initiation site was detected 120 bp 5′ of the initiation codon.APKmRNA is slightly more abundant in leaves than in roots ofA. thalianaand its level does not change in response to sulfur starvation. TheAPKprotein, synthesized in vitro, is able to enter isolated intact chloroplasts. Recombinant APS kinase shows maximal activity at 10 μM APS with 5 mM ATP, but it is inhibited at APS concentrations above 10 μM. The inhibition is alleviated at higher ATP concentrations. Reciprocal plot analysis showed that the theoretical Vmaxis ∼1.2 μmol min−1mg−1at 25°C, pH 8.0; the Kmvalues are 3.6 μM APS and 1.8 mM ATP.

Abscisic acid maintains S-type anion channel activity in ATP-depleted Vicia faba guard cells

The plant hormone abscisic acid (ABA) regulates important developmental and stress responses. Recent data show that ABA activates phosphorylation events, but whether dephosphorylation events are post-translationally regulated by ABA or whether these are constitutive remains unknown. Slow anion channels in the plasma membrane of guard cells have been proposed to play an important role during ABA-induced stomatal closing. Anion channels are deactivated by removal of cytosolic ATP. However, when guard cells were treated with ABA and depleted of ATP, anion currents remained active. Subsequent removal of extracellular ABA caused deactivation of currents. Deactivation of currents was reversed by reintroduction of cytosolic MgATP. These data show that anion channels are regulated by ABA even in the absence of cytosolic ATP required for kinase-induced phosphorylation events and that anion channel activity is maintained by ABA under conditions that favor dephosphorylation-induced deactivation. Furthermore, channel activation proceeded at high ATP concentrations with nanomolar cytosolic Ca 2+ showing a Ca 2+-independent final step in anion channel activation.

Processivity of the motor protein kinesin requires two heads.

A single kinesin molecule can move for hundreds of steps along a microtubule without dissociating. One hypothesis to account for this processive movement is that the binding of kinesin's two heads is coordinated so that at least one head is always bound to the microtubule. To test this hypothesis, the motility of a full-length single-headed kinesin heterodimer was examined in the in vitro microtubule gliding assay. As the surface density of single-headed kinesin was lowered, there was a steep fall both in the rate at which microtubules landed and moved over the surface, and in the distance that microtubules moved, indicating that individual single-headed kinesin motors are not processive and that some four to six single-headed kinesin molecules are necessary and sufficient to move a microtubule continuously. At high ATP concentration, individual single-headed kinesin molecules detached from microtubules very slowly (at a rate less than one per second), 100-fold slower than the detachment during two-headed motility. This slow detachment directly supports a coordinated, hand-over-hand model in which the rapid detachment of one head in the dimer is contingent on the binding of the second head.

Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of creatine kinase in skeletal muscle.

The AMP-activated protein kinase (AMPK) is activated by a fall in the ATP:AMP ratio within the cell in response to metabolic stresses. Once activated, it phosphorylates and inhibits key enzymes in energy-consuming biosynthetic pathways, thereby conserving cellular ATP. The creatine kinase-phosphocreatine system plays a key role in the control of ATP levels in tissues that have a high and rapidly fluctuating energy requirement. In this study, we provide direct evidence that these two energy-regulating systems are linked in skeletal muscle. We show that the AMPK inhibits creatine kinase by phosphorylation in vitro and in differentiated muscle cells. AMPK is itself regulated by a novel mechanism involving phosphocreatine, creatine and pH. Our findings provide an explanation for the high expression, yet apparently low activity, of AMPK in skeletal muscle, and reveal a potential mechanism for the co-ordinated regulation of energy metabolism in this tissue. Previous evidence suggests that AMPK activates fatty acid oxidation, which provides a source of ATP, following continued muscle contraction. The novel regulation of AMPK described here provides a mechanism by which energy supply can meet energy demand following the utilization of the immediate energy reserve provided by the creatine kinase-phosphocreatine system.

Differential characterization of binding sites for adenine and uridine nucleotides in membranes from rat lung as possible tools for studying P2 receptors in lung

Nucleotide receptors (P2 receptors) are involved in stimulating Cl − secretion in airway epithelia. These receptors may play a key role in development of new therapeutic strategies in the treatment of cystic fibrosis. However, the diversity of nucleotide binding sites in lung tissue has not yet been clarified. Here we studied the characteristics of various nucleotide binding sites in rat lung membranes by equilibrium binding analysis of several P2 receptor specific ligands. Displacement studies revealed a recognition site for adenosine 5′-O-(l-thiotriphosphate) ([ 35S]ATPαS; K d 243 nM). From this site the ligand is readily displaced by adenosine 5′-O-(2-thiodiphosphate) (ADPβS), a typical agonist for P2Y 1 receptors and also by α,β-methylene adenosine 5′-triphosphate (α,β-MeATP), a typical agonist for P2X receptors. [ 3H]α,β,β-MeATP labelled specific binding sites (K d 56 nM) in rat lung membranes. Analysis of binding of [ 3H]UTP to lung membranes revealed a high-affinity binding site (K d 44 nM). Membrane-bound [ 3H]UTP was not displaced even by high concentrations of ATP, indicating no common binding site for UTP and ATP. Furthermore, specific binding of P-1,P-4-di(adenosine 5′)tetraphosphate ([ 3H]Ap 4A; K d 91 nM) was found in lung membranes. Thus, we demonstrate at least four distinct types of nucleotide binding sites in lung membranes: Two have characteristics comparable to P2X and P2Y 1 receptors, while two further sites still have to be identified, one binding Ap 4A and the other binding UTP very specifically.

Cytolytic P2X purinoceptors.

Anecdoctal evidence accumulated over almost 20 years has shown that many different cell types are killed by sustained exposure to high concentrations of extracellular ATP. The plasma membrane receptors involved have been pharmacologically characterized and cloned during the last 3 years, and named purinergic P2X. P2X receptors share an intriguing structural relatedness with Caenorhabditis elegans degenerins and mammalian amiloride-sensitive Na channels (ENaCs). Depending on the ATP dose, length of stimulation and receptor subtype, P2X receptor stimulation may cause necrosis or apoptosis. The intracellular pathways activated are poorly known, but the perturbation in intracellular ion homeostasis clearly plays a major role. ICE proteases (caspases) are also triggered, nonetheless their activation is not requested for ATP-dependent cell death. The physiological meaning of P2X receptor-dependent cytotoxicity is not understood, but an involvement in immune-mediated reactions is postulated.

Engineering Src family protein kinases with unnatural nucleotide specificity

Protein kinases play a central role in controlling diverse signal transduction pathways in all cells. The identification of the direct cellular substrates of individual protein kinases remains the key challenge in the field. We describe the protein engineering of v-Src to produce a kinase which preferentially uses an ATP analog, N6-(benzyl) ATP, as a substrate, rather than the natural v-Src substrate, ATP. The sidechain of a single residue (Ile338) controls specificity for N6-substituted ATP analogs in the binding pocket of v-Src. Elimination of this sidechain by mutation to glycine produces a v-Src kinase which preferentially utilizes N6-(benzyl) ATP as a phosphodonor substrate. Our engineering strategy is generally applicable to the Src family kinases: mutation of the corresponding residue (Thr339 to glycine) in the Fyn kinase confers specificity for N6-(benzyl) ATP on Fyn. The v-Src tyrosine kinase has been engineered to exhibit specificity for an unnatural ATP analog, N6-(benzyl) ATP, even in a cellular context where high concentrations of natural ATP are present (1-5 mM), where preferential use of the ATP analog by the mutant kinase is essential. The mutant v-Src transfers phosphate more efficiently with the designed unnatural analog than with ATP. As the identical mutation in the Src-family kinase Fyn confers on Fyn the ability to recognize the same unnatural ATP analog, our strategy is likely to be generally applicable to other protein kinases and may help to identify the direct targets of specific kinases.

Nucleotide metabolism by gastric glands and H(+)-K(+)-ATPase-enriched membranes.

alpha-Toxin-permeabilized gastric glands represent a functional model in which acid secretion can be elicited by either adenosine 3',5'-cyclic monophosphate (cAMP) or ATP, with proven morphological and functional transition between resting and secretory states [X. Yao, S. M. Karam, M. Ramilo, Q. Rong, A. Thibodeau, and J. G. Forte. Am. J. Physiol. 271 (Cell Physiol. 40): C61-C73, 1996.] In this study we use alpha-toxin-permeabilized rabbit gastric glands to study energy metabolism and the interplay between nucleotides to support acid secretion, as indicated by the accumulation of aminopyrine (AP). When permeabilized glands were treated with a phosphodiesterase inhibitor, the secretory response to cAMP was inhibited, whereas the secretory response to ATP was potentiated. This implied that 1) ATP provided support not only as an energy source but also as substrate for adenylate cyclase, 2) activation of acid secretion by cAMP needed ATP, and 3) ATP and cAMP exchanged rapidly inside parietal cells. To address these issues, we tested the action of adenine nucleotides in the presence and absence of oxidizable substrates. All adenine nucleotides, including AMP, ADP, ATP, and cAMP, could individually enhance the glandular AP accumulation in the presence of substrates, whereas only a high concentration of ATP (5 mM) was able to support secretory activity in substrate-free buffer. Moreover, ATP could maintain 75-80% of maximal secretory activity in phosphate-free buffer; cAMP alone could not support secretion in phosphate-free buffer. In glands and in H(+)-K(+)-adenosinetriphosphatase-rich gastric microsomes, we showed the operation of adenylate kinase, creatine kinase, and ATP/ADP exchange activities. These enzymes, together with endogenous adenylate cyclase and phosphodiesterase, provide the recycling of nucleotides essential for the viability of alpha-toxin-permeabilized gastric glands and imply the importance of nucleotide recycling for energy metabolism in intact parietal cells.

In vitro rejoining of DNA double strand breaks: a comparison of genomic-DNA with plasmid-DNA-based assays

Purpose: To evaluate potential similarities between the enzymatic activities required to rejoin DNA double strand breaks (dsb) in an in vitro assay based on genomic DNA and an in vitro assay based on plasmid DNA. Because the latter assay is simpler and faster, it should be preferred for the characterization of repair factors if both assays are found to probe for the same activities. If, however, the enzymatic requirements for dsb rejoining are different between the two assays, both should be used as they are likely to play complementary roles in the characterization of repair factors. Materials and methods : A cell-free assay has been used, developed to study rejoining DNA dsb induced by radiation in 'naked' DNA prepared from agarose embedded cells using an extract of HeLa cells as a source of enzymes. Also employed was an in vitro assay using the ligation of linearized plasmid DNA to model dsb rejoining. Results : Evidence is presented that, under the conditions employed, different sets of activities are involved in the ligation of linearized plasmid DNA and in the rejoining of dsb in 'naked' genomic DNA. Optimal rejoining of dsb induced in genomic DNA is observed with cytoplasmic cell extract at 37 C, whereas optimal ligation of plasmid DNA is observed with nuclear extract at 25 C. Rejoining of dsb in genomic DNA comes to a near halt at 14 C, but plasmid DNA ligation proceeds at significant rates at this temperature. Furthermore, the activities required for the rejoining of dsb induced in genomic DNA are partly stable to heating at 50 C for 1 h, whereas activities required for the ligation of plasmid DNA are completely inactivated by a similar treatment. Both reactions require ATP for optimal performance, and in both, DNA joining is inhibited at high ATP concentrations. Conclusions : These observations indicate that the plasmid-and the genomic-DNA-based assays probe for, at least partly, different sets of activities and therefore are expected to play complementary roles in the purification and characterization of activities involved in dsb rejoining.

Fructose 2,6-bisphosphate biosynthesis and regulation of carbohydrate metabolism inAspergillus oryzae

The biosynthesis and role of fructose 2,6-bisphosphate (Fru-2,6-P2) in carbohydrate metabolism during induction of an amylolytic system in Aspergillus oryzae was studied. Fluctuations in Fru-2,6-P2were not dependent on the external glucose concentration during induction, whereas the level of Fru-2,6-P2increased significantly when the oxygen concentration was diminished. Phosphofructokinase II (PFK II) of A. oryzae was sensitive to phosphorylation in vitro by the catalytic subunit of cyclic AMP dependent protein kinase, which increased the Vmax(twofold), although the Km(0.7 mM) remained unchanged. Phosphofructokinase I was neither activated by micromolar Fru-2,6-P2nor inhibited by high ATP concentrations. The activity of fructose-1,6-bisphosphatase (FBPase) was subject to strong inhibition by Fru-2,6-P2. Addition of glucose to cultures under gluconeogenic conditions caused a decrease of approximately 40% in the FBPase activity within 4 min. These results indicate that the effect of Fru-2,6-P2in A. oryzae could preferentially control gluconeogenesis. The addition of 0.1 M glucose under gluconeogenic culture conditions also showed that Fru-2,6-P2fluctuations appeared to be, at least in short term, more closely related to temporal changes in the hexose-6-phosphate concentration.Key words: Aspergillus oryzae; fructose-2,6-bisphosphate; phosphofructokinase II (PFK II); cyclic AMP; gluconeogenesis control.

ATP in Hamster Embryos Developing from 2-Cell to Blastocyst Stage In Vitro and at the 2-Cell Block.

It is well known that hamster embryos cannot complete the second cleavage division in vitro (2-cell block), if cultured in glucose and inorganic phosphate (Glu/Pi) containing medium. It is unclear how the block to development in vitro is caused in the presence of Glu/Pi. In the present study, the ATP in hamster embryos at the 2-cell block in M199 supplemented with 5% heated calf serum was examined and compared to the ATP in 2-cell embryos just recovered from oviducts and preimplantation hamster embryos developing in vitro from 2-cell to blastocyst stages in Glu/Pi-free medium (HECM3). ATP in hamster embryos was measured by an ATP-dependent luciferin - luciferase bioluminescence assay. The average ATP content and SEM was 44.5 ± 7.4 fmol/embryo at the 2-cell stage, 13.1 ± 1.2 fmol/embryo at the 4-cell stage, 72.3 ± 21.0 fmol/embryo at the 8-cell stage and 22.7 ± 6.6 fmol/embryo at the blastocyst stage. A significant decrease in ATP content was observed at the 4-cell stage and blastocyst stage as compared to the 8-cell stage (P<0.05). The other hand, the average amount of ATP and SEM in the 2-cell block embryos was 98.3 ± 17.1 fmol/embryo and was much higher than that in the preimplantaion embryos developing in vitro. We therefore suggest that the 2-cell block of hamster embryos may be due, in part to the high ATP content, that is, low levels of cytochrome activity and low levels of ATP synthesis.

Selenocompounds Induce a Redox Modulation of Protein Kinase C in the Cell, Compartmentally Independent from Cytosolic Glutathione: Its Role in Inhibition of Tumor Promotion

Since selenite and other redox-active selenocompounds can modify protein kinase C (PKC) in the test tube, we have determined whether or not this redox regulation occurs inside the cell despite having high concentrations of GSH and the role of this regulation in the inhibition of tumor promotion. By using phorbol ester-promoted JB6 epidermal cell transformation assay, the concentrations of selenite, selenocystine, and selenodiglutathione which are optimal for chemopreventive activity were determined. At such concentrations (0.5 to 2 μm) in the cells treated with these agents, only a slight but transient decrease in PKC activity was observed when measured with a low (5 μm), but not with a high (100 μm) concentration of ATP. However, when the cells were serum starved or pretreated with 2-deoxyglucose, there was a pronounced but transient inactivation of PKC when assayed with both low and high concentrations of ATP. The inactivation was reversed in the cell by an endogenous mechanism or by treatment with thiol agents in the test tube. In spite of a substantial (90%) depletion of GSH in the cells by pretreatment with buthionine sulfoximine, there was no further increase in the redox modification of PKC by selenite as well as no change in the inhibitory effect of selenite on the phorbol ester-stimulated induction of ornithine decarboxylase, which is an intermediate marker related to cell transformation. While GSH is known to influence certain actions of selenium, it may not be required to mediate the effects of selenite tested in this study. The water-soluble cytosolic GSH did not interfere with the redox modification of PKC probably due to the shielding of the cysteine-rich region of the enzyme by a weak hydrophobic association with the membrane. Due to the presence of cofactors in the crude cell extracts, PKC was more sensitive to selenite than in the purified form and was inactivated by low concentrations of selenite (IC50= 0.05 μm). This modification was reversed by thiol agents as well as by NADPH. A protein disulfide reductase, which can regenerate PKC, was present in the homogenate. Conceivably, selenite and other selenocompounds induce a redox modification of cellular PKC, compartmentally independent from the cytosolic GSH, but intimately connected to a NADPH-dependent reductase system, to mediate, at least in part, some of the cancer-preventive actions.

RNA-unwinding and RNA-folding activities of RNA helicase II/Gu--two activities in separate domains of the same protein.

The human RNA helicase II/Gu protein (RH-II/Gu) is a member of the D-E-A-D box protein family. It is a unique enzyme, which possesses an ATP-dependent RNA-unwinding activity and has an RNA-folding activity that introduces an intramolecular secondary structure in single-stranded RNA. This report shows that these two enzymatic activities are distinct. ATP[S], GTP and low concentrations of ATP enhance the RNA-folding activity of RH-II/Gu but not the RNA-helicase activity. High concentrations of ATP are required for the helicase activity but are inhibitory to the RNA-folding activity. Mg2+ is required for the helicase activity but not for the RNA-folding reaction. Affinity-purified anti-(RH-II/Gu) polyclonal Ig inhibit the RNA-unwinding activity but not the folding activity. Mutations of the DEVD sequence, which corresponds to the DEAD box, and the SAT motif enhanced RNA-folding activity of RH-II/Gu but completely inhibited the RNA-helicase activity. A mutant that lacks the COOH-terminal 76 amino acid residues, including the four FRGQR repeats, had unwinding activity but did not catalyze the folding of a single-stranded RNA. The two enzymatic activities of RH-II/Gu reside in distinct domains. Amino acids 1-650 are active in the RNA-unwinding reaction but lack RNA-folding activity. Amino acids 646-801 fold single-stranded RNA but lack helicase activity. This report shows distinct RNA-unwinding and RNA-folding activities residing in separate domains within the same protein.

Binding of steroid modulators to recombinant cytosolic domain from mouse P-glycoprotein in close proximity to the ATP site.

We recently found that recombinant NBD1 cytosolic domain corresponding to segment 395-581 of mouse mdr1 P-glycoprotein bound fluorescent 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP) with high affinity [Dayan, G., Baubichon-Cortay, H., Jault, J.-M., Cortay, J. -C., Deléage, G., & Di Pietro, A. (1996) J. Biol. Chem. 271, 11652-11658]. The present work shows that a longer 371-705 domain (extended-NBD1), including tryptophan-696 as an intrinsic probe, which bound MANT-ATP with identical affinity, also interacted with steroids known to modulate anticancer drug efflux from P-glycoprotein-positive multidrug-resistant cells. Progesterone, which is not transported, its hydrophobic derivatives medroxyprogesterone acetate and megestrol acetate, and Delta6-progesterone produced nearly a 50% saturating quenching of the domain intrinsic fluorescence, with dissociation constants ranging from 53 to 18 microM. The even more hydrophobic antiprogestin RU 486 produced a complete quenching of tryptophan-696 fluorescence, in contrast to more hydrophilic derivatives of progesterone containing hydroxyl groups at positions 11, 16, 17, and 21 and known to be transported, which produced very little quenching. A similar differential interaction was observed with full-length purified P-glycoprotein. The steroid-binding region within extended-NBD1 appeared distinct from the nucleotide-binding site as the RU 486-induced quenching was neither prevented nor reversed by high ATP concentrations. In contrast, MANT-ATP binding was efficiently prevented or displaced by RU 486, suggesting that the hydrophobic MANT group of the bound nucleotide analogue overlaps, at least partially, the adjacent steroid-binding region revealed by RU 486.

Specific inhibition of cardiac and skeletal muscle sarcoplasmic reticulum Ca 2+ pumps by H-89

The isoquinolinesulfonamide H-89, an inhibitor of cyclic AMP-dependent protein kinases (EC 2.7.1.37, cAPrK), inhibited the Ca 2+ -ATPase activity of cardiac and skeletal muscle sarcoplasmic reticulum (SR) with concentrations giving half-maximal inhibition of 8.1 ± 1.3 and 7.2 ± 0.9 μmol/L, respectively. The effect of H-89 on cardiac SR Ca 2+ -ATPase (EC 3.6.1.38) was the same irrespective of the presence or absence of inhibitors of cAPrK and furthermore, was not affected by a neutralising monoclonal antibody raised against phospholamban. Thus, the action of H-89 in inhibiting SR Ca 2+ -ATPase would not appear to be mediated by inhibition of cAPrK to reduce the phosphorylation state of phospholamban. In both cardiac and skeletal muscle SR, the inhibition by H-89 was noncompetitive with respect to ATP at a low concentration of ATP (< 1 mmol/L) and of a mixed pattern at high concentrations of ATP. H-89 produced a decrease in affinity of the SR Ca 2+ pump to Ca 2+ with an increase in the K m for Ca from 0.52 ± 0.01 to 0.94 ± 0.03 μmol/L ( P < 0.05) in cardiac SR and from 0.39 ± 0.01 to 0.79 ± 0.02 μmol/L ( P < 0.05) in skeletal muscle SR. These results suggest that H-89 inhibits SR Ca 2+ -ATPase by a direct action on the SR Ca 2+ pump to decrease its affinity to Ca 2+. Such an action may contribute to the pharmacological effect of H-89.