A rapid and sensitive assay for the detection of eukaryotic ribosome dissociation factors.

A rapid and sensitive assay has been developed for the factor-dependent dissociation of eukaryotic ribosomes. This assay takes advantage of the observation that initiation factor eIF-2 will bind Met-tRNA_f^met to 40 S subunits but not to 80 S ribosomes. Incubation of wheat germ ribosomes at 1 mm Mg²⁺ results in their dissociation into 40 S subunits. These subunits spontaneously reassociate when the Mg²⁺ concentration is raised to 4 mm. However, if the incubation at 1 mm Mg²⁺ is carried out in the presence of an extract containing a ribosome dissociation factor, a certain portion of the subunits will fail to reassociate when the Mg²⁺ concentration is raised to 4 mm. The 40 S subunits remaining due to the presence of the dissociation factor can bind [³⁵S]Met-tRNA_f^met in the presence of wheat germ eIF-2. The [³⁵S]Met-tRNA_f^met bound to the 40 S subunits is readily detected by its retention on a Millipore filter.     

A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center

The ribosome is an ancient macromolecular machine responsible for the synthesis of all proteins in all living organisms. Here we demonstrate that the ribosomal peptidyl transferase center (PTC) is supported by a framework of magnesium microclusters (Mg²⁺-μc's). Common features of Mg²⁺-μc's include two paired Mg²⁺ ions that are chelated by a common bridging phosphate group in the form Mg_(a)²⁺–(O1P-P-O2P)–Mg_(b)²⁺. This bridging phosphate is part of a 10-membered chelation ring in the form Mg_(a)²⁺–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg_(a)²⁺. The two phosphate groups of this 10-membered ring are contributed by adjacent residues along the RNA backbone. Both Mg²⁺ ions are octahedrally coordinated, but are substantially dehydrated by interactions with additional RNA phosphate groups. The Mg²⁺-μc's in the LSU (large subunit) appear to be highly conserved over evolution, since they are unchanged in bacteria (Thermus thermophilus, PDB entry 2J01) and archaea (Haloarcula marismortui, PDB entry 1JJ2). The 2D elements of the 23S rRNA that are linked by Mg²⁺-μc's are conserved between the rRNAs of bacteria, archaea and eukarya and in mitochondrial rRNA, and in a proposed minimal 23S-rRNA. We observe Mg²⁺-μc's in other rRNAs including the bacterial 16S rRNA, and the P4–P6 domain of the tetrahymena Group I intron ribozyme. It appears that Mg²⁺-μc's are a primeval motif, with pivotal roles in RNA folding, function and evolution.     

Structural dynamics of bacterial ribosomes: II. Preparation and characterization of ribosomes and subunits active in the translation of natural messenger RNA

Ribosomes from Escherichia coli were tested for activity in initiation with R17 RNA as messenger. All vacant 70 S ribosomes but not all subunits were found to be active. The ability of 30 S and 50 S subunits to form a 70 S couple at Mg²⁺ concentrations above 4 mm is a stringent test for activity.Fresh extracts, prepared at 10 mm-Mg²⁺ from cells harvested after slow cooling contain up to 80% of the ribosomes in the form of vacant 70 S couples and 20% of free subunits. The proportion of subunits increases with standing as a result of the preferential inactivation of the 50 S particles. “Native” subunits are heterogeneous and consist mostly of active 30 S and inactive 50 S particles.In contrast to 50 S subunits, 30 S subunits prepared by exposure of 70 S ribosomes to low Mg²⁺ concentrations, are largely inactive and unable to reassociate with their active 50 S counterparts. However, both initiation and association activity can be restored by heating.The results imply that the structures necessary for subunit association are most critical for the biological activity of ribosomes, presumably because they are topologically closely related to the binding sites for messenger RNA, transfer RNA, and the protein factors for initiation, translocation and termination.     

Structural dynamics of bacterial ribosomes. I. Characterization of vacant couples and their relation to complexed ribosomes

Ribosomes not engaged in protein synthesis (vacant couples), in contrast to complexed ribosomes bearing nascent chains, dissociate during sedimentation in sucrose gradients at high g forces and at Mg²⁺ concentrations below 15 mm. As a result of this dissociation, a new peak between the 70 S complexed ribosomes and the free 50 S subunits is observed, the position of which shifts from about 55 S to 70 S as the Mg²⁺ concentration in the gradient is raised from 5 to 15 mm. The apparent 60 S peak consists of 50 S subunits produced during dissociation in the gradient. At low g forces, the sedimentation rate of complexed and vacant ribosomes is indistinguishable, even at 5 mm-Mg²⁺. These sedimentation properties are valid criteria to differentiate vacant and complexed ribosomes. This is shown by converting complexed ribosomes quantitatively into vacant couples by removing the nascent chains through termination release or with puromycin, or by converting vacant couples into initiation complexes with R17 RNA, fMet-tRNA and initiation factors.Ribosomes from cells harvested by slow cooling consist almost entirely of vacant couples, all of which are active in protein synthesis with natural messengers. The structural features responsible for the interaction between subunits are discussed.     

Role of polyamines in the binding of initiator tRNA to the 70S ribosomes of extreme thermophilic bacterium Calderobacterium hydrogenophilum

Slowly cooled cells of an extreme thermophilic eubacterium Calderobacterium hydrogenophilum possess ribosomes with weakly associated subunits. These ribosomal subunits are capable of association to 70S ribosomes either at higher Mg²⁺ concentrations (30–40 mM) or at 4–10 mM Mg²⁺ and in the presence of polyamines. The contribution of 30S and 50S subunits to the hydrodynamic stability of ribosomes was examined by forming hybrid 30S–50S couples from C. hydrogenophilum and Escherichia coli. At lower Mg²⁺ (4–10 mM) heterogeneous subunits containing 30S E. coli and 50S C. hydrogenophilum and homogeneous subunits of the thermophilic bacterium associated only in the presence of polyamines. Ribosomal subunits associated at 30 mM Mg²⁺ lose thermal stability and activity concerning poly(AUG)-dependent binding of f[³H]Met-tRNA to the P-site on 70S ribosomes or translation of poly(UG). Poly(AUG), deacylated-tRNA or initiator-tRNA have no valuable effect on association of 30S and 50S subunits. Protein synthesis initiation factor IF3 of C. hydrogenophilum prevents association of ribosomal subunits to 70S ribosomes at physiological temperature (70°C). The factor also stimulates dissociation of 70S ribosomes of E. coli at 37°C. The codon-specific binding of f[³H]Met-tRNA to homogeneous 70S ribosomes of C. hydrogenophilum at 70°C is dependent on the presence of initiation factors and concentrations of tri-pentaamines. However, excess of polyamines inhibited the reaction. Our results indicate that tri-pentaamines enhance conformational stability of 70S initiation complex at elevated temperatures.     

Plastid-associated Porphobilinogen Synthase from Toxoplasma gondii: KINETIC AND STRUCTURAL PROPERTIES VALIDATE THERAPEUTIC POTENTIAL*

Apicomplexan parasites (including Plasmodium spp. and Toxoplasma gondii) employ a four-carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast, a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin. The PBGS family exhibits a complex use of metal ions (Zn²⁺ and Mg²⁺) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ∼320-kDa octamer, and low levels of dimers but no hexamers were also observed. The enzyme displays a broad activity peak (pH 7–8.5), with a K_m for aminolevulinic acid of ∼150 μm and specific activity of ∼24 μmol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg²⁺ but not Zn²⁺ and shows two Mg²⁺ affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg²⁺-binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic, and structural features of apicomplexan PBGS offer scope for developing selective inhibitors of the parasite enzyme based on its quaternary structure characteristics.     

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca²⁺]_i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca²⁺]_i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9415-6) contains supplementary material, which is available to authorized users.     

The Hemocyanin of the Squid Sepioteuthis lessoniana: Structural Comparison with Other Cephalopod Hemocyanins

The subunit structure of the hemocyanin from the squid Sepioteuthis lessoniana has been characterized by analytical sedimentation. The protein exists free in the hemolymph as a 60S, multisubunit structure with a molecular mass of approximately 4.0 × 10⁶ gm/mol. At extremes of pH this can be dissociated into 10 monomers, of mass 3.8 × 10⁵ gm/mol each.At neutral pH, if 5 mM Mg²⁺ or less is present, the monomers associate to form a dimer of molecular mass 7.7 × 10⁵ gm/mol. In the pH range from 10.5 to 7.5, formation of the dimer is facilitated by the binding of one proton per monomer. If the magnesium concentration is raised to 25 mM or more, the dimers reversibly and completely associate into decamers. This process is shown to require the binding of 10 magnesium ions.In its size, subunit structure, and behavior with respect to association–dissociation processes, Sepioteuthis hemocyanin resembles the corresponding proteins of other squid much more than it does those of Octopus or Nautilus.     

Purification and Characterization of a Tripeptidase from Lactococcus lactis subsp. cremoris Wg2

A tripeptidase from a cell extract of Lactococcus lactis subsp. cremoris Wg2 has been purified to homogeneity by DEAE-Sephacel and phenyl-Sepharose chromatography followed by gel filtration over a Sephadex G-100 SF column and a high-performance liquid chromatography TSK G3000 SW column. The enzyme appears to be a dimer with a molecular weight of between 103,000 and 105,000 and is composed of two identical subunits each with a molecular weight of about 52,000. The tripeptidase is capable of hydrolyzing only tripeptides. The enzyme activity is optimal at pH 7.5 and at 55°C. EDTA inhibits the activity, and this can be reactivated with Zn²⁺, Mn²⁺, and partially with Co²⁺. The reducing agents dithiothreitol and β-mercaptoethanol and the divalent cation Cu²⁺ inhibit tripeptidase activity. Kinetic studies indicate that the peptidase hydrolyzes leucyl-leucyl-leucine with a K_m of 0.15 mM and a V_max of 151 μmol/min per mg of protein.     

Peptides Interfering 3A Protein Dimerization Decrease FMDV Multiplication

Nonstructural protein 3A is involved in relevant functions in foot-and-mouth disease virus (FMDV) replication. FMDV 3A can form homodimers and preservation of the two hydrophobic α-helices (α1 and α2) that stabilize the dimer interface is essential for virus replication. In this work, small peptides mimicking residues involved in the dimer interface were used to interfere with dimerization and thus gain insight on its biological function. The dimer interface peptides α1, α2 and that spanning the two hydrophobic α-helices, α12, impaired in vitro dimer formation of a peptide containing the two α-helices, this effect being higher with peptide α12. To assess the effect of dimer inhibition in cultured cells, the interfering peptides were N-terminally fused to a heptaarginine (R₇) sequence to favor their intracellular translocation. Thus, when fused to R₇, interference peptides (100 μM) were able to inhibit dimerization of transiently expressed 3A, the higher inhibitions being found with peptides α1 and α12. The 3A dimerization impairment exerted by the peptides correlated with significant, specific reductions in the viral yield recovered from peptide-treated FMDV infected cells. In this case, α2 was the only peptide producing significant reductions at concentrations lower than 100 μM. Thus, dimer interface peptides constitute a tool to understand the structure-function relationship of this viral protein and point to 3A dimerization as a potential antiviral target.     

Relaxation kinetics of E. coli ribosomes.

Relaxation kinetics measurements on two types of ribosome preparations were parformed by the pressure-jump and temperature-Jump techniques, using light scattered at 90° as detector. For freshly prepared tibosomes isolated as 70S tight coupled from 26 000 RPM sucrose gradint sedimentation in 10 mM Mg²⁺, surprisingly large reaction amplitudes were found in 10 mM Mg²⁺ wilh both techniques, leading to an overall formation constant for 70S couples approximately three orders of magnitude smaller than that reported fot tight couples. For pelleted, two-tunes salt-washed ribosomes, amplitude titration versus Mg²⁺ in the pressure-jump apparatus showed an amplitude maximum near 10 mM Mg²⁺ with a relaxation time near 20 ms, and a second amplitude maximum near 2.5 mM Mg²⁺ with a relaxation time near 25 s. Both types of preparation on reanalysis on sucrose gradients at 5 mM Mg²⁺ showed approximately 15% of subunits, with a distinct zone in the 50S region. 70S light couples recovered from a sucrose density gradient separation at 5 mM Mg²⁺ on pelleted two-times salt-washed ribosomes behaved in the same way as the original sample in pressure-jump experiments at 10 mM Mg²⁺. These findings have been interpreted as follows (I) the processes observed at 10 mM Mg²⁺ are due entirety to the relatively small loose couple content of the samples, even in the case of material isolated as 70S tight couples, (2) the processes observed at 2.5 mM Mg²⁺ are due almost entirely to the preponderant tight couple population of the material, and (3) samples isolated as 70S tight couples from sucrose gradients at 5 mM Mg²⁺ spontaneously revert within hours into micro-heterogeneous material containing about 15% loose couples, for both types of ribosomes.     

Mechanism of action of the wheat germ ribosome dissociation factor: Interaction with the 60 S subunit

Recently a ribosome dissociation factor that stimulates natural mRNA translation has been isolated from extracts of wheat germ. In this investigation, we have studied the subunit site of action of the purified ribosome dissociation factor (eucaryotic initiation), eIF-6. The following evidence strongly indicates that eIF-6 acts as a dissociation factor by binding to the 60 S ribosomal subunit and preventing its interaction with the 40 S subunit. Incubation of 60 S subunits with eIF-6 reduces the formation of 80 S monosomes when 40 S subunits are subsequently added at 5 mm Mg²⁺. The 40 S subunits preincubated with eIF-6 reassociate normally with 60 S subunits. ¹⁴C-labeled eIF-6 binds to 60 S subunits but not to 40 S subunits. Slight binding to 80 S ribosomes is also observed. The interaction of eIF-6 with the 60 S subunit requires an elevated temperature, and occurs rapidly at 37 °C.     

The distribution of ribosomes between different functional states in livers of fed and starved mice

1. To investigate the role of ribosome function in regulating protein synthesis, the activity, distribution and functional states of ribosomal particles were investigated in livers of mice fed ad libitum or starved overnight. 2. The distribution of protein-synthesizing activity between polyribosomes of different sizes was analysed after incorporation of radioactive leucine, and the quantitative distribution of ribosomes as native subunits, monomers and polyribosomes was analysed after incorporation of orotic acid. Precursors labelled with ³H or ¹⁴C were given separately to fed and starved mice, so that livers from the two groups of animals were processed together. 3. The former experiments showed that starvation has little effect on the distribution of protein-synthesizing activity across polyribosome sedimentation patterns, though the latter experiments showed that the proportion of ribosomes existing as monomers increased from 9.5% to 15.2%, whereas the proportion existing as polyribosomes decreased from 81.4% to 75.6%. Starvation had a negligible effect on the proportion of native subunits, which accounted for 9.1% and 9.2% of the ribosomes in fed and starved mice respectively. 4. The monomeric ribosome fraction was isolated and subjected to ionic conditions which selectively dissociate single ribosomes. Starvation increased the proportion of monomers that dissociated from 59% to 72%, so the monomers that accumulate in livers of starved animals are single ribosomes and not monoribosomes resulting from degradation of polyribosomes. 5. The fate of newly formed ribosomal particles was studied by measuring the specific radioactivity of native subunits, monomers and polyribosomes at different times after injection of radioactively labelled orotic acid. Starvation did not appear to affect equilibration between newly formed particles and polyribosomes, and the radioactivity of polyribosomes in both groups of mice reached about 90% of that in native subunits after 4h. The radioactive labelling of monomers proceeded at a slower rate, especially after starvation. At 4h, the radioactivity of monomers was 64% and 55% that of native subunits in fed and starved mice respectively.     

Characterization of ribosomes of a strain ofStreptomyces aureofaciens producing chlortetracycline

These studies were designated to investigate the effect of chlortetracycline on sedimentation properties of polysomes and ribosomes present in the chlortetracycline producing strain ofStreptomyces aureofaciens. In presence of chlortetracycline polysomes and ribosomes are more stable than the bacterial ones. At lower chlortetracycline concentrations (1–5 μg/ml) dissociation of polysomes into 70 S monomers was not observed. Ribosomes in higher concentration of chlortetracycline (400 μg/ml) form aggregates. A decrease of Mg²⁺ to 0.1mm caused dissociation of ribosomes to two subunits and in this state none of indicated concentrations of chlortetracycline caused aggregation. The exact sedimentation values of ribosomes and ribosomal subunits were calculated from extrapolation to infinite dilution. S_20,w for monomer form was 68.8, and for ribosomal subunits 49.8 and 31.2 respectively. Ribosomal RNA sedimentates as two Schlieren peaks of 16 S and 22 S. It was found that 30 S subunits contain 15 structural proteins, while 21 proteins were resolved from 50 S subunits.     

Effect of carbamazepine and gabapentin on excitability in the trigeminal subnucleus caudalis of neonatal rats using a voltage-sensitive dye imaging technique

Background

The antiepileptic drugs carbamazepine and gabapentin are effective in treating neuropathic pain and trigeminal neuralgia. In the present study, to analyze the effects of carbamazepine and gabapentin on neuronal excitation in the spinal trigeminal subnucleus caudalis (Sp5c) in the medulla oblongata, we recorded temporal changes in nociceptive afferent activity in the Sp5c of trigeminal nerve-attached brainstem slices of neonatal rats using a voltage-sensitive dye imaging technique.

Results

Electrical stimulation of the trigeminal nerve rootlet evoked changes in the fluorescence intensity of dye in the Sp5c. The optical signals were composed of two phases, a fast component with a sharp peak followed by a long-lasting component with a period of more than 500 ms. This evoked excitation was not influenced by administration of carbamazepine (10, 100 and 1,000 μM) or gabapentin (1 and 10 μM), but was increased by administration of 100 μM gabapentin. This evoked excitation was increased further in low Mg²⁺ (0.8 mM) conditions, and this effect of low Mg²⁺ concentration was antagonized by 30 μM DL-2-amino-5-phosphonopentanoic acid (AP5), a N-methyl-d-aspartate (NMDA) receptor blocker. The increased excitation in low Mg²⁺ conditions was also antagonized by carbamazepine (1,000 μM) and gabapentin (100 μM).

Conclusion

Carbamazepine and gabapentin did not decrease electrically evoked excitation in the Sp5c in control conditions. Further excitation in low Mg²⁺ conditions was antagonized by the NMDA receptor blocker AP5. Carbamazepine and gabapentin had similar effects to AP5 on evoked excitation in the Sp5c in low Mg²⁺ conditions. Thus, we concluded that carbamazepine and gabapentin may act by blocking NMDA receptors in the Sp5c, which contributes to its anti-hypersensitivity in neuropathic pain.     

Ribosomes of Brachionus plicatilis (Rotifera) – distribution in homogenate fractions and general properties

(1) The content of DNA, RNA and of proteins of Brachionus plicatilis was estimated and the distribution of RNA and of proteins of different homogenate fractions characterised. (2) Ribosomes were isolated from Brachionus plicatilis homogenates and were characterised by gradient centrifugation. (3) Unlike the RNA content, the yield of ribosomes from different homogenate fractions is strongly dependent on the concentration of Mg²⁺-ions in the buffers. Likewise resuspension of ribosomes is more effective in Mg²⁺- (or Ca²⁺-) free buffers. (4) Dissociation of ribosomes was brought about by centrifugation of ribosomes in gradients containing less than 4 mM Mg²⁺. In this case, beside the peaks of subunits, a peak in the region of 80 S remained which vanished only under conditions destroying ribosomal material altogether. (5) Proteins were isolated from ribosomal subunits and from undissociated ribosomes and were characterised by two-dimensional gel electrophoresis techniques. Patterns of 51 spots were regularly obtained from large subunits and patterns of 41 spots from small subunits. The undissociated ribosomes showed 83 spots, most of which could be attributed to the large or the small subunit. The ribosomal proteins have molecular masses of between 11000 and 56000 Da, while the molecular mass of the total protein content of Brachionus ribosomes was estimated to be 1.8 ±0.5) ×10⁶ Da.     

The sedimentation behaviour of ribonuclease-active and -inactive ribosomes from bacteria

1. The `30s' and `50s' ribosomes from ribonuclease-active (Escherichia coli B) and -inactive (Pseudomonas fluorescens and Escherichia coli MRE600) bacteria have been studied in the ultracentrifuge. Charge anomalies were largely overcome by using sodium chloride–magnesium chloride solution, I 0·16, made 0–50mm with respect to Mg²⁺. 2. Differentiation of enzymic and physical breakdown at Mg²⁺ concentrations less than 5mm was made by comparing the properties of E. coli B and P. fluorescens ribosomes. 3. Ribonuclease-active ribosomes alone showed a transformation of `50s' into 40–43s components. This was combined with the release of a small amount of `5s' material which may be covalently bound soluble RNA. Other transformations of the `50s' into 34–37s components were observed in both ribonuclease-active and -inactive ribosomes at 1·0–2·5mm-Mg²⁺, and also with E. coli MRE600 when EDTA (0·2mm) was added to a solution in 0·16m-sodium chloride. 4. Degradation of ribonuclease-active E. coli B ribosomes at Mg²⁺ concentration 0·25mm or less was coincident with the formation of 16s and 21s ribonucleoprotein in P. fluorescens, and this suggested that complete dissociation of RNA from protein was not an essential prelude to breakdown of the RNA by the enzyme. 5. As high Cs⁺/Mg²⁺ ratios cause ribosomal degradation great care is necessary in the interpretation of equilibrium-density-gradient experiments in which high concentrations of caesium chloride or similar salts are used. 6. The importance of the RNA moiety in understanding the response of ribosomes to their ionic environment is discussed.     

Studies on (NA + K)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule

ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na⁺ + K⁺)-ATPase in the absence of Mg²⁺ (EDTA present) with a homogeneous but 15-fold different affinity, the K_d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na⁺ + K⁺)-ATPase, respectively. The K_d value for ATP is equal to the K_m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na⁺ + K⁺)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg²⁺, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The K_d and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg²⁺ concentration. The K_d increases from 0.06 mM at 0.5 mM Mg²⁺ to a maximum of 0.26 mM at 2 mM Mg²⁺ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg²⁺ to 3.3 nmol/mg protein at 4 mM Mg²⁺. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg²⁺ concentration yields a maximal binding capacity at infinite Mg²⁺ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na⁺ + K⁺)-ATPase. The K_d for Mg²⁺ at the sites, where it exerts this effect, is 0.8 mM. The K_d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg²⁺ to a maximum of 4.2 μM at 2 mM Mg²⁺ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg²⁺ concentration. Hence, Mg²⁺ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na⁺ + K⁺)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.     

HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway

The HIV-1 dimerization initiation sequence (DIS) is a conserved palindrome in the apical loop of a conserved hairpin motif in the 5′-untranslated region of its RNA genome. DIS hairpin plays an important role in genome dimerization by forming a ‘kissing complex’ between two complementary hairpins. Understanding the kinetics of this interaction is key to exploiting DIS as a possible human immunodeficiency virus (HIV) drug target. Here, we present a single-molecule Förster resonance energy transfer (smFRET) study of the dimerization reaction kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the formation of the mature extended duplex complex that is ultimately required for virion packaging. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. The universally conserved A272 is essential for the formation of this intermediate, which is stabilized by Mg²⁺, but not by K⁺ cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps with two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg²⁺ ions.     

Ca-stimulated, Mg-independent ATP hydrolysis and the high affinity Ca-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m = 0.25 μM, V_max = 24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.