The ATP-Mediated Regulation of KaiB-KaiC Interaction in the Cyanobacterial Circadian Clock

The cyanobacterial circadian clock oscillator is composed of three clock proteins—KaiA, KaiB, and KaiC, and interactions among the three Kai proteins generate clock oscillation in vitro. However, the regulation of these interactions remains to be solved. Here, we demonstrated that ATP regulates formation of the KaiB-KaiC complex. In the absence of ATP, KaiC was monomeric (KaiC^1mer) and formed a complex with KaiB. The addition of ATP plus Mg²⁺ (Mg-ATP), but not that of ATP only, to the KaiB-KaiC^1mer complex induced the hexamerization of KaiC and the concomitant release of KaiB from the KaiB-KaiC^1mer complex, indicating that Mg-ATP and KaiB compete each other for KaiC. In the presence of ATP and Mg²⁺ (Mg-ATP), KaiC became a homohexameric ATPase (KaiC^6mer) with bound Mg-ATP and formed a complex with KaiB, but KaiC hexamerized by unhydrolyzable substrates such as ATP and Mg-ATP analogs, did not. A KaiC N-terminal domain protein, but not its C-terminal one, formed a complex with KaiB, indicating that KaiC associates with KaiB via its N-terminal domain. A mutant KaiC^6mer lacking N-terminal ATPase activity did not form a complex with KaiB whereas a mutant lacking C-terminal ATPase activity did. Thus, the N-terminal domain of KaiC is responsible for formation of the KaiB-KaiC complex, and the hydrolysis of the ATP bound to N-terminal ATPase motifs on KaiC^6mer is required for formation of the KaiB-KaiC^6mer complex. KaiC^6mer that had been hexamerized with ADP plus aluminum fluoride, which are considered to mimic ADP-Pi state, formed a complex with KaiB, suggesting that KaiB is able to associate with KaiC^6mer with bound ADP-Pi.     

Mechanisms Underlying Regulation of a Barium -sensitive K<Superscript>+ </Superscript>Conductance by ATP in Single Proximal Tubule Cells Isolated from Frog Kidney

K⁺ channels play an important role in pump-leak coupling and volume regulation in the renal proximal tubule. Previous experiments have identified a barium-sensitive K⁺ conductance (G_Ba) in proximal tubule cells isolated from frog kidneys. In this paper we examine the regulation of G_Ba by ATP. G_Ba was measured in single cells isolated from frog kidney using the whole-cell patch-clamp technique. G_Ba was activated by 2 mM intracellular ATP. This activation was enhanced by inhibition of protein kinase C and attenuated by inhibition of protein kinase A, indicating reciprocal regulation by these kinases. Activation by ATP was reduced in the presence of a hypertonic bath solution, suggesting that cell swelling was required. However, after activation to steady-state, G_Ba was not sensitive to cell-volume changes. Hypotonic shock-induced volume regulation was inhibited by barium and quinidine, inhibitors of G_Ba. The effect of maximal inhibitory concentrations of barium and quinidine on volume regulation was similar and addition of both blockers together did not augment the inhibitory response. G_Ba was also activated by ADP, via a mechanism dependent on the presence of Mg²⁺. However, the responses to ADP and ATP were not additive, suggesting that these nucleotides may share a common mechanism of activation. The regulation of G_Ba by ATP was biphasic, with a half-maximal activating concentration of 0.89 mM and a half maximal inhibitory concentration of 6.71 mM. The sensitivity to nucleotides suggests that G_Ba may be regulated by the metabolic state of the cell. Furthermore, the sensitivity to solution osmolality, coupled with the blocker profile of inhibition of volume regulation, suggests that G_Ba could play a role in volume regulation.     

Influence of free Mg2+ on the kinetics of human erythrocyte phosphofructokinase

The influence of Mg2+ on the reaction catalyzed by human erythrocyte phosphofructokinase has been investigated using kinetic methods. The catalytic activity of PFK is dependent upon the presence of Mg2+ which constitutes with ATP the true Mg-ATP2- substrate. Free Mg2+ has no influence on the affinity of the enzyme for Mg-ATP2- substrate. Erythrocyte PFK is more inhibited by ATP4- and uncomplexed citrate than it is by Mg-ATP2- and Mg-citrate. Free Mg2+ relieves the MgATP2- and Mg-citrate inhibition under conditions where free ATP4-is negligible. We can assume that uncomplexed Mg2+ acts as positive effector by direct binding to the enzyme. These results emphasize the role of Mg2+ in the regulation of PFK activity in the erythrocyte.     

Regulation of erythrocyte membrane shape by Ca2+

In the presence of 1.0 mM ATP and MgCl₂, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca²⁺ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca²⁺ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca²⁺ (1 to 5 μM) also inhibited Mg²⁺ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca²⁺ on viscosity and phosphorylation may be due to a membrane bound Ca²⁺ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg²⁺ dependent kinase activity.     

Functional inactivation of the human guanylyl cyclase C receptor: modeling and mutation of the protein kinase-like domain

Receptor guanylyl cyclases possess an extracellular ligand-binding domain, a single transmembrane region, a region with sequence similar to that of protein kinases, and a C-terminal guanylyl cyclase domain. ATP regulates the activity of guanylyl cyclase C (GC-C), the receptor for the guanylin and stable toxin family of peptides, presumably as a result of binding to the kinase homology domain (KHD). Modeling of the KHD of GC-C indicated that it could adopt a structure similar to that of tyrosine kinases, and sequence comparison with other protein kinases suggested that lysine(516) was positioned in the KHD to interact with ATP. A monoclonal antibody GCC:4D7, raised to the KHD of GC-C, did not recognize ATP-bound GC-C, and its epitope mapped to a region in the KHD of residues 491--568 of GC-C. Mutation of lysine(516) to an alanine in full-length GC-C (GC-C(K516A)) dramatically reduced the ligand-stimulated activity of mutant GC-C, altered the ATP-mediated effects observed with wild-type GC-C, and failed to react with the GCC:4D7 monoclonal antibody. ATP interaction with wild-type GC-C converted a high-molecular weight oligomer of GC-C to a smaller sized oligomer. In contrast, GC-C(K516A) did not exhibit an alteration in its oligomeric status on incubation with ATP. We therefore suggest that the KHD in receptor guanylyl cyclases provides a critical structural link between the extracellular domain and the catalytic domain in regulation of activity in this family of receptors, and the presence of K(516) is critical for the possible proper orientation of ATP in this domain.     

Properties of Neurospora crassa plasma membrane ATPase.

A variety of the biochemical properties of the electrogenic plasma membrane ATPase of Neurospora crassa are described. The enzyme catalyzes the hydrolysis of ATP, resulting in the formation of ADP and inorganic phosphate. Optimal activity is observed between pH 6 and 6.5. ATP hydrolysis approaches a maximum rate at an Mg-ATP concentration of 10–20 mm with a half-maximal velocity around 2 mm Mg-ATP. The enzyme requires a divalent cation for activity in the following order of preference at 10 mm: Mg²⁺, Co²⁺ > Mn²⁺ > Zn²⁺ > Fe²⁺, Ca²⁺, Cu²⁺. The enzyme is quite specific for ATP compared to the other nucleotides tested. Treatment of the plasma membranes with sodium deoxycholate inactivates the ATPase and the inactivation can be prevented by the addition of certain acidic phospholipids with the deoxycholate. Other classes of lipids cannot prevent the deoxycholate inhibition. The organic mercurials parachloromercuribenzoate and parachloromercuriphenylsulfonate are potent inhibitors of the ATPase, but N-ethylmaleimide at a similar concentration is not inhibitory. The organic mercurial inhibition is not reversed by mercaptoethanol. Under appropriate conditions, the inhibitory effect of p-chloromercuribenzoate is suppressed in the presence of ATP. Treatment of the plasma membranes with trypsin leads to a marked inhibition of the ATPase activity and this inhibition can be prevented by Mg-ATP. Neither the organic mercurial reactive site(s) nor the trypsin-sensitive site(s) are accessible from the outer surface of the plasma membranes. Some of the implications of the above findings are discussed.     

Summary

The plasma membrane H⁺-ATPase in higher plants has been implicated in nutrient uptake, phloem loading, elongation growth and establishment of turgor. Although a C-terminal regulatory domain has been identified, little is known about the physiological factors involved in controlling the activity of the enzyme. To identify components which play a role in the regulation of the plant H⁺-ATPase, a fusicoccin responsive yeast expressing Arabidopsis plasma membrane H⁺-ATPase AHA2 was employed. By testing the fusicoccin binding activity of yeast membranes, the C-terminal regulatory domain of AHA2 was found to be part of a functional fusicoccin receptor, a component of which was the 14–3-3 protein. ATP hydrolytic activity of AHA2 expressed in yeast internal membranes was activated by all tested isoforms of the 14–3-3 protein of yeast and Arabidopsis, but only in the presence of fusicoccin, and activation was prevented by a phosphoserine peptide representing a known 14–3-3 protein binding motif in Raf-1. The results demonstrate that the 14–3-3 protein is an activator molecule of the H⁺-ATPase and provides the first evidence of a protein involved in activation of plant plasma membrane H⁺-ATPase.     

Effects of Small Molecule Modulators on ATP Binding to Skeletal Ryanodine Receptor

Calcium release for muscle contraction in skeletal muscle is mediated in part by the ryanodine receptor 1, RyR1, Ca²⁺-channel and is strongly affected by intrinsic modulators like Ca²⁺, Mg²⁺ and ATP. We showed differential effects on ATP binding in the presence of Ca²⁺ or Mg²⁺ ions using ESR spectroscopy and a spin-labeled ATP analog, SL-ATP (Dias et al. Biochemistry 45: 9408–9415, 2006). We here report the effects of RyR1 modulators like ryanodine, caffeine and dantrolene on the ATP binding of RyR1 using the same technique. We present evidence that the exogenous effectors induce changes within RyR1 that lead to different ATP binding characteristics: In the presence of the activating modulator, caffeine, or in the presence of ryanodine, which causes a half-open state of the channel, binding of eight ATP per RyR1 was observed, even in the presence of inhibitory Ca²⁺, suggestive of a stable “open” channel conformation. In the presence of the inhibitory modulator dantrolene, ATP binding affinity decreased in the presence of activating Ca²⁺, while in the presence of inhibitory Ca²⁺, ATP binding affinity increased, but at the same time the number of accessible sites decreased to four, suggestive of a closed conformation of the channel. The results imply that modulation of ATP binding to RyR1 as well as the overall number of accessible ATP binding sites on the channel are crucial for regulation and are in direct correlation with the modified activity of the channel induced by pharmacological agents.     

The cytochemical assay of NAD+ kinase activity in the follicular cells of guinea-pig thyroid gland

Summary A quantitative cytochemical assay for NAD⁺ kinase-like activity in the guinea-pig thyroid gland is described. The NADP⁺ produced by the activity of the kinase was used to drive the NADP⁺-dependent enzyme glucose-6-phosphate dehydrogenase which is endogenous to the tissue. The activity of glucose-6-phosphate dehydrogenase is greatly in excess of that of the kinase and was unaffected by the constituents of the kinase incubation medium (ATP, Mg²⁺ and NAD⁺) either alone or in combination. Kinase activity was dependent both on ATP and Mg²⁺, with maximal activity seen when the Mg-ATP ratio was between 1:1 and 4:1. Free ATP inhibited the activity of the enzyme. Enzyme activity was exhibited over a broad pH range (7–9) with a peak at pH 8.2. The sulphhydryl-blocking agents,p-chloromercuribenzoate, iodoacetate and iodoacetamide (at 1 mM), completely abolished kinase activity but were without effect on glucose-6-phosphate dehydrogenase activity.N-ethylmaleimide and citrate (both at 1 mM) had no effect on either kinase or glucose-6-phosphate dehydrogenase activities.     

The Decay of the ATPase Activity of Light Plus Thiol-Activated Thylakoid Membranes in the Dark

Oxidized ATP synthase of spinach thylakoid membranes catalyzes high rates of ATP synthesis in the light, but very low rates of ATP hydrolysis in the dark. Reduction of the disulfide bond in the γ subunit of the ATP synthase in the light enhances the rate of Mg²⁺-ATP hydrolysis in the dark. The light plus thiol-activated state decays in a few minutes in the dark after illumination in Tris buffer, but not when Tricine was used in place of Tris. In this paper, it is shown that Tris in the assay mixture is an inhibitor of the light plus thiol-activated ATPase activity of thylakoids, but only after the activated membranes had incubated in the dark. Aminopropanediols and diethanolamine, also selectively inhibited ATPase activity of activated membranes after storage in the dark, whereas NH₄Cl and imidazole inhibit the ATPase activity of activated thylakoids almost equally whether they are added directly after the illumination or several minutes later. The fluorescence of 9-amino-6-chloro-2-methoxyacridine (ACMA) is quenched by the establishment of proton gradients by ATP-dependent proton uptake. Addition of ATP to activated membranes results in rapid quenching of ACMA fluorescence. If the activated membranes were incubated in the dark prior to ATP addition, a lag in the ATP-dependent ACMA fluorescence quenching as well as a similar lag in the rate ATP hydrolysis were seen. It is concluded that ADP rebinds to CF1 in the dark following illumination and inhibits the activity of the ATP synthase. Reactivation of the ATP synthase in the dark can occur by the slow generation of proton gradients by ATP hydrolysis in the dark. This reactivation takes place in Tricine buffer, but not in Tris because of its uncoupling action. Whether ADP binding plays a role in the regulation of the activity of the ATP synthase in situ remains to be established.     

Inhibition of ion pump ATPase activity by 3′-O-(4-Benzoyl)benzoyl-ATP (BzATP): assessment of BzATP as an active site-directed probe

The interaction of 3′-O-(4-Benzoyl)benzoyl-ATP (BzATP) with the rnal (Na⁺ + K⁺)-ATPase, the sarcoplasmic reticulum Ca-transport ATPase, and the gastric (H⁺ + K⁺)-ATPase has been investigated in order to determine whether BzATP is a suitable probe for the labeling and identification of a peptide from the ATP binding sites of these ion pumps. After ultraviolet irradiation BzATP inhibited the enzymatic hydrolysis of ATP by each of the ion pumps, and also was covalently incorporated into the 100 000 dalton polypeptides of each protein. The presence of excess ATP in the reaction solution did not prevent either the inactivation of ATPase activity or the labeling of the catalytic polypeptides by BzATP. Prior modification of the ATPases with fluorescein-5′-isothiocyanate (FITC), however, prevented much of the labeling of the 100 000 dalton polypeptides by BzATP. BzATP competitively inhibited the high-affinity binding of ATP to the ion pumps, but ATP did not block the high-affinity binding of BzATP by the enzymes. BzATP binds to the membrane-bound ATPases at a high-affinity site with a K_d of 0.8–1.2 μM and a B_max of 2–3 nmol/mg, and also binds to at least one low-affinity, high-capacity site on the membranes. HPLC separation of the soluble peptides from a tryptic digest of BzATP-labeled (Na⁺ + K⁺)-ATPase revealed the presence of several labeled peptides, none of which was protected by either ATP or FITC. Although BzATP can displace ATP from a high-affinity binding site on the ion pumps, it appears, therefore, that inactivation of enzymatic activity is the result of reactions between BzATP and the proteins at locations outside this site. Thus, it is concluded from these experiments that BzATP is not likely to be a useful probe for the ATP binding sites on the ion transport ATPases.     

The Bacteroides fragilis NAD-specific Glutamate Dehydrogenase Enzyme is Cell Surface-Associated and Regulated by Peptides at the Protein Level

Bacteroides fragilis has two enzymes with glutamate dehydrogenase (GDH) activity, namely, a dual cofactor NAD(P)H-dependent GDHA, and an NADH-specific GDHB. The presence of two enzymes with the same function is unusual and may play a role in the ability of this organism to survive a variety of environmental conditions. Here we report on the purification and characterisation of the GDHB protein expressed in Escherichia coli from the recombinant plasmid, pGDH15-1 carrying the gdh B gene. The recombinant protein was purified to electrophoretic homogeneity and had a subunit molecular mass of approximately 48 kDa. The temperature and pH activity optima were 38°C and 8.0, respectively, and GDHB enzyme activity was inhibited two-fold by the presence of divalent cations (Ca²⁺, Mg²⁺). The presence of monovalent cations (Na⁺, K⁺) or metabolites (ATP, AMP, ADP or GTP) did not affect enzyme activity. The regulation of GDHB activity was examined at the protein level and evidence of post-translational regulation of the protein in response to peptides but not ammonia was found. Localisation studies using cell fractions of B. fragilis grown under high peptide conditions showed that 79% of GDHB activity was expressed in the membrane fraction. This result was confirmed by immunogold labelling and electron microscopy of B. fragilis cells. It is possible that the GDHB enzyme might play an important role in bacterial survival during invasion of host tissue through its cell-surface location and its regulation via peptides produced by proteases.     

Activation and stabilization of cardiac adenylate cyclase by GTP analog and fluoride.

The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t_1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl₂. The activity was most labile in the presence of 50 m m Mg²⁺ and 1 m m ATP, having t_1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m, for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t_1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t_1/2 = 72min, than nonactivated enzyme. Preactivation with F? (30 min at 24 °C) also stabilized the activity; t_1/2 > 70 min in the absence or presence of Mg-ATP. The Mg²⁺ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F?activated enzyme.     

ATP Dependence of Na+/H+ Exchange : Nucleotide Specificity and Assessment of the Role of Phospholipids

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na⁺/H⁺ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pH_i) by microfluorimetry. Na⁺/H⁺ exchange activity was measured as the Na⁺-driven pH_i recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na⁺/H⁺ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na⁺/H⁺ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca²⁺ exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na⁺/H⁺ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na⁺/H⁺ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTPγS was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg²⁺ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the γ-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.     

Effect of the electrochemical proton gradient and anions on the ATPase activity of soybean submitochondrial particles

Submitochondrial particles from soybean (Glycine max L. cv Jupiter) hypocotyls with an ATPase activity of 0.3 to 1.0 micromole per minute per milligram were prepared by sonication with Mg-ATP. The particles catalyzed ATP synthesis with NADH and succinate; the ratios of ATP/O with these substrates were 1.0 and 0.1, respectively. As monitored by oxonol-VI, the particles built up and maintained a membrane potential that was higher with NADH than with succinate or Mg-ATP. The ATPase activity of the particles increased two to threefold by preincubation with 50 millimolar phosphate at a temperature of 38°C. The increase in ATPase activity became higher (five to sixfold) when particles were preincubated with Mg-ATP plus phosphate. Under the latter conditions, collapse of μH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone prevented the activation. An increase in ATPase activity of the particles was also observed with NADH and succinate, although activation was lower with succinate. With these substrates, phosphate did not increase ATPase activation. When particles were preincubated with Mg-ATP, anions that stimulate ATP hydrolysis (malate, malonate, and bicarbonate) had an activating effect similar to that of phosphate. The data suggest that the soybean mitochondrial ATPase can be activated by μH but that this activation is increased by the binding of certain anions to a conformation of the enzyme that appears during hydrolytic cycles.     

Modifications of the activity of nitrate reductase from cucumber roots

The regulatory properties of NADH-dependent nitrate reductase (NR) in desalted root extracts from hydroponically grown cucumber (Cucumis sativus L.) seedlings were examined. The lowest activity of NR was detected in extracts incubated with Mg²⁺ and ATP. An inhibitory effect of Mg-ATP was cancelled in the presence of staurosporine (the protein kinase inhibitor) and completely reversed after addition of ethylenediaminetetraacetate (EDTA) as well as AMP into reaction mixture. Reactivation of enzyme due to AMP presence, contrary to the chelator-dependent NR activation, was sensitive to microcystin LR (the protein phosphatase inhibitor). Above results indicated that the nitrate reductase in cucumber roots was regulated through reversible phosphorylation of enzyme protein. A drop in the activity of NR was also observed after incubation of enzyme at low pH. At low pH, the presence of ATP alone in the incubation medium was sufficient to inactivate NR, indicating that H⁺ can substitute the Mg²⁺ in formation of an inactive complex of enzyme. ATP-dependent inactivation of NR at low pH was prevented by staurosporine and reversed by AMP. However, AMP action was not altered by microcystin LR suggesting that in low pH the nucleotide induced reactivation of NR is not limited to the protein phosphorylation.     

Phosphofructokinase from lycopersicon esculentum fruits-I. Kinetic properties in relation to its substrates

The kinetic properties of phosphofructokinase with regard to its substrates are discussed. Free ATP is inhibitory to the enzyme while the Mg-ATP complex at a concentration up to 5 mM is not. The kinetics with respect to Mg-ATP follow simple Michaelis-Menten kinetics and this pattern is not affected by changes in concentration of the second substrate fructose-6-phosphate (F6P). The kinetics with respect to F6P showed apparent negative co-operative interactions in the presence of saturating levels of Mg²⁺ relative to ATP. In the presence of inhibitory levels of free ATP, the kinetics showed positive co-operative interactions. The relationship between the nature of the kinetics of the enzyme with F6P and the various molecular forms of PFK are discussed.     

ATPase activity of SopA, a protein essential for active partitioning of F plasmid

Summary The SopA, B, C genes of the F plasmid play an essential role in plasmid partitioning during cell division in Escherichia coli. In this paper, the products of the sopA and sopB genes were isolated and their biochemical activities studied. [-³²P]ATP was cross-linked to the SopA protein by UV irradiation; this cross-linking was observed only in the presence of magnesium ion, and was competitively inhibited in the presence of non-radioactive ATP, ADP and dATP, but not other NTPs or dNTPs. In contrast, no ATP binding activity was detected for the SopB protein. The SopA protein showed a modest magnesium ion-dependent ATPase activity and this activity was stimulated in the presence of DNA. The ATPase activity in the presence of DNA was further stimulated by addition of the SopB protein. However, the SopB protein alone failed to stimulate the ATPase activity.