ADP and ATP binding to noncatalytic sites of thiol-modulated chloroplast ATP synthase

A modified ‘cold chase’ technique was used to study tight [¹⁴C]ADP and [¹⁴C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF₀F₁). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF₀F₁ or CF₁ reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k _d of about 5 min⁻¹. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF₀F₁. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF₁ dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF₁ becomes bound to a thylakoid membrane.     

Regulation of monomeric dynein activity by ATP and ADP concentrations.

Axonemal dyneins are force-generating ATPases that produce ciliary and flagellar movement. A dynein has large heavy chain(s) in which there are multiple (4-6) ATP-binding consensus sequences (P-loops) as well as intermediate and light chains, constituting a very large complex. We purified a monomeric form of dynein (dynein-a) that has at least three light chains from 14S dyneins of Tetrahymena thermophila and characterized it. In in vitro motility assays, dynein-a rotated microtubules around their longitudinal axis as well as translocated them with their plus-ends leading. ATPase activity at 1 mM ATP was doubled in the presence of a low level of ADP (> or = 20 microM). Both ATPase activity and translocational velocities in the presence of ADP (> or = 20 microM) fit the Michaelis-Menten equation well. However, in the absence of ADP (< 0.1 microM), neither of the activities followed the Michaelis-Menten-type kinetics, probably due to the effect of two ATP-binding sites. Our results also indicate that dynein-a has an ATP-binding site that is very sensitive to ADP and affects ATP hydrolysis at the catalytic site. This study shows that a monomeric form of a dynein molecule regulates its activity by direct binding of ATP and ADP to itself, and thus the dynein molecule has an intramolecular regulating system.     

The calcium response of mouse sperm flagella: role of calcium ions in the regulation of dynein activity

Triton X-100-extracted mouse sperm treated with 0.1 mM ATP and 1.0 mM Ca(2+) exhibit an extremely coiled configuration that has been previously described as a curlicue. Sperm in the curlicue configuration exhibit a monotonically curved flagellum where the shear angle of the flagellum can reach a value as high as 14 radians at the flagellar tip. We utilized this strong reaction to Ca(2+) to elucidate the mechanism of the calcium response. The disintegration of the axoneme was facilitated by the use of an extraction procedure that removed the mitochondrial sheath without eliminating the calcium response. The order of emergence of the doublet microtubule outer dense fiber complexes was observed in the presence and absence of added Ca(2+). The identity of the emergent elements was confirmed by transmission electron microscopy. Ca(2+) altered the order of emergence of internal axoneme elements to favor the appearance of the elements of the 9-1-2 side of the axoneme. These elements are propelled baseward by the action of dyneins on doublets 1 and 2. It was also possible to establish that the motive force for maintaining the curlicue configuration is dynein-based. The curlicues were relaxed by inhibition with 50 μM NaVO(3) and were reestablished by disinhibiting the vanadate with 2.5 mM catechol.     

The regulation of motile activity in fish chromatophores

Chromatophores, including melanophores, xanthophores, erythrophores, leucophores and iridophores, are responsible for the revelation of integumentary coloration in fish. Recently, blue chromatophores, also called cyanophores, were added to the list of chromatophores. Many of them are also known to possess cellular motility, by which fish are able to change their integumentary hues and patterns, thus enabling them to execute remarkable or subtle chromatic adaptation to environmental hues and patterns, and to cope with various ethological encounters. Such physiological color changes are indeed crucial for them to survive, either by protecting themselves from predators or by increasing their chances of feeding. Sometimes, they are also useful in courtship and mutual communications among individuals of the same species, leading to an increased rate of species survival. Such strategies are realized by complex mechanisms existing in the endocrine and/or nervous systems. Current studies further indicate that some paracrine factors such as endothelins (ETs) are involved in these processes. In this review, the elaborate mechanisms regulating chromatophores in these lovely aquatic animals are described.     

Mechanoreception in motile flagella of Chlamydomonas

Ciliates and flagellates temporarily swim backwards on collision by generating a mechanoreceptor potential. Although this potential has been shown to be associated with cilia in Paramecium, the molecular entity of the mechanoreceptor has remained unknown. Here we show that Chlamydomonas cells express TRP11, a member of the TRP (transient receptor potential) subfamily V, in the proximal region of the flagella, and that suppression of TRP11 expression results in loss of the avoiding reaction. The results indicate that Chlamydomonas flagella exhibit mechanosensitivity, despite constant motility, by localizing the mechanoreceptor in the proximal region, where active bending is restricted.     

Modulation of the GTPase activity of the chloroplast F1-ATPase by ATP binding at noncatalytic sites

Although the binding of nucleotides at the noncatalytic sites of F1-ATPase has been regarded as probably having some type of regulatory function, only limited observations have been reported that support such a role. We present here results showing that the presence of ATP at noncatalytic sites can give a fivefold enhancement of the rate of GTP hydrolysis by the chloroplast F1-ATPase. Heat-activation of the chloroplast F1-ATPase in the presence of ATP, followed by column separation from the medium nucleotides gives an enzyme with two of the three noncatalytic sites filled with ATP. In contrast, heat-activation in the presence of ADP gives an enzyme with only one noncatalytic site filled with ADP. Such an enzyme with two noncatalytic sites empty catalyzes MgGTP hydrolysis only very slowly. The filling of a second noncatalytic site with ATP by exposure of the enzyme to ATP without Mg2+ present, followed by column separation, markedly increases the rate of GTP hydrolysis. A further increase occurs when a third noncatalytic site is filled by exposure to Mg2+ and ATP. The rate of MgATP hydrolysis is the same for the enzyme heat-activated in the presence of ATP or ADP, probably because MgATP, unlike MgGTP, rapidly binds to both catalytic and noncatalytic sites.     

Tight ATP and ADP binding in the noncatalytic sites of Escherichia coli F1-ATPase is not affected by mutation of bulky residues in the 'glycine-rich loop'

It is shown that ATP dissociates very slowly (koff less than 6.4 x 10(5) s-1, t1/2 greater than 3 h) from the three noncatalytic sites of E. coli F1-ATPase and that ADP dissociates from these three sites in a homogeneous fashion with koff = 1.5 x 10(-4) s-1 (t1/2 = 1.35 h). Mutagenesis of alpha-subunit residues R171 and Q172 in the 'glycine-rich loop' (Homology A) consensus region of the noncatalytic sites was carried out to test the hypothesis that unusually bulky residues at these positions are responsible wholly or partly for the observed tight binding of adenine nucleotides. The mutations alpha Q172G or alpha R171S,Q172G had no effects on ATP or ADP binding to or rates of dissociation from F1 noncatalytic sites. KdATP and KdADP of isolated alpha-subunit were weakened by approximately 1 order of magnitude in both mutants. The results suggest that neither residue alpha R171 nor alpha Q172 interacts directly with bound nucleotide, and show that the presence of bulky residues per se in the glycine-rich loop region of F1-alpha-subunit is not responsible for tight binding in the noncatalytic sites.     

The established and the predicted roles of dynein light chain in the regulation of mitochondrial apoptosis

The mitochondrial pathway of apoptosis is regulated by the interplay between the members of Bcl-2 family. Within this family, BH3-only proteins are the sensors of apoptotic stimuli and can trigger apoptosis either by inhibiting the anti-apoptotic Bcl-2-family proteins or by directly activating the effectors Bax and Bak. An expanding body of research suggests that a number of non-Bcl-2 proteins can also interact with Bcl-2 proteins and contribute to the decision of cell fate. Dynein light chain (LC8, DYNLL or DLC), a hub protein and a dimerizing engine has been proposed to regulate the pro-apoptotic activity of two BH3-only proteins, Bim and Bmf. Our recent work has provided insight into the mechanisms through which DLC1 (DYNLL1) modulates Bim activity. Here we discuss the present day understanding of Bim-DLC interaction and endeavor to evaluate this interaction in the light of information from studies of DLC with other binding partners.     

ATP binding to noncatalytic sites of chloroplast coupling factor CF1

A kinetic analysis of ATP binding to noncatalytic sites of chloroplast coupling factor CF₁ was made. The ATP binding proved to be unaffected by reduction of the disulfide bridge of the CF₁ -subunit. The first-order equation describing nucleotide binding to noncatalytic sites allowed for two vacant nucleotide binding sites different in their kinetics. As suggested by nucleotide concentration dependence of the rate of nucleotide binding, the tight binding was preceded by rapid reversible binding of nucleotides. Preincubation of CF₁ with Mg²⁺ resulted in a decreased rate of ATP binding. ATP dissociation from noncatalytic sites was described by the first order equation for similar sites with a dissociation rate constant k _d (ATP) 10^–3 min^–1. Noncatalytic sites of CF₁ were shown to be not homogeneous. One of them retained the major part of endogenous ADP after precipitation of CF₁ with ammonium sulfate. Its two other sites differed in kinetic parameters and affinity for ATP. Anions of phosphate, sulfite, and especially, pyrophosphate inhibited the interaction between ATP and the noncatalytic sites.     

ATP binding at noncatalytic sites of soluble chloroplast F1-ATPase is required for expression of the enzyme activity

The F1-ATPase from chloroplasts (CF1) lacks catalytic capacity for ATP hydrolysis if ATP is not bound at noncatalytic sites. CF1 heat activated in the presence of ADP, with less than one ADP and no ATP at non-catalytic sites, shows a pronounced lag in the onset of ATP hydrolysis after exposure to 5-20 microM ATP. The onset of activity correlates well with the binding of ATP at the last two of the three noncatalytic sites. The dependence of activity on the presence of ATP at non-catalytic sites is shown at relatively low or high free Mg2+ concentrations, with or without bicarbonate as an activating anion, and when the binding of ATP at noncatalytic sites is slowed 3-4-fold by sulfate. The latent CF1 activated by dithiothreitol also requires ATP at noncatalytic sites for ATPase activity. A similar requirement by other F1-ATPases and by ATP synthases seems plausible.     

Nucleotide binding to the chaperonin GroEL: non-cooperative binding of ATP analogs and ADP, and cooperative effect of ATP

Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by GroEL, which undergoes a large structural change by the ATP binding or hydrolysis. One of the main concerns of GroEL is the mechanism of the productive and cooperative structural change of GroEL induced by the nucleotide. We studied the cooperative nature of GroEL by nucleotide titration using isothermal titration calorimetry and fluorescence spectroscopy. Our results indicated that the binding of ADP and ATP analogs to a single ring mutant (SR1), as well as that to GroEL, was non-cooperative. Only ATP induces an apparently cooperative conformational change in both proteins. Furthermore, the fluorescence changes of pyrene-labeled GroEL indicated that GroEL has two kinds of nucleotide binding sites. The fluorescence titration result fits well with a model in which two kinds of binding sites are both non-cooperative and independent of each other. These results suggest that the binding and hydrolysis of ATP may be necessary for the cooperative transition of GroEL.     

ADP binding and ATP synthesis by reconstituted H-ATPase from chloroplasts

The H⁺-ATPase from chloroplasts, CF₀F₁, was isolated, purified and reconstituted into asolectin liposomes. The enzyme was brought either into the oxidized state or into the reduced state, and the rate of ATP synthesis was measured after energisation of the proteoliposomes with an acid—base transition ΔpH (pH_in = 5.0, pH_out = 8.5) and a K⁺/valinomycin diffusion potential, Δφ (K⁺_in = 0.6 mM, K⁺_out = 60 mM). A rate of 250 s⁻¹ was observed with the reduced enzyme (85 s⁻¹ in the absence of Δφ). A rate of 50 s⁻¹ was observed with the oxidized enzyme under the same conditions (15 s⁻¹ in the absence of Δφ). The reconstituted enzyme contained 2 ATP_bound per CF₀F₁ and 1 ADP_bound per CF₀F₁. Upon energisation the enzyme was activated and 0.9 ADP per CF₀F₁, was released. Binding of ADP to the active reduced enzyme was observed under different conditions. In the absence of phosphate the rate constant for ADP binding was 10⁵ M⁻¹·s⁻¹ under energized and de-energized conditions. In the presence of phosphate the rate of ADP binding drastically increased under energized conditions, and strongly decreased under de-energized conditions.     

Kinetics of ATP/ADP binding to the gp16 ATPase

The gp16 ATPase is the constituent subunit of the pentameric dsDNA (double-stranded deoxyribonucleic acid) translocation motor of the Bacillus subtilis Φ29 bacteriophage. Although recent single-molecule studies have provided tantalizing clues about the activity of this motor, the mechanism by which the gp16 subunits couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of dsDNA translocation remains unknown. To address this need, we have characterized the binding of fluorophore-labeled ATP and ADP to monomeric gp16 using a stopped-flow fluorescence assay. These experiments show that the binding of ATP/ADP occurs through a single-step mechanism with corresponding affinities of 523.8 ± 247.3 nM for ATP and a lower limit of 30 μM for ADP. When analyzed through the lens of changes in free energy of the system, this difference in binding affinities is reasonable for a cyclical process of binding, hydrolysis, and product release. In addition to answering questions about the activity of monomeric gp16, these results are also a necessary step in constructing a model for intersubunit communication within the pentameric gp16 motor.     

Conformational changes of the beta chain of the outer-arm dynein from sea urchin sperm flagella coupled with ATP hydrolysis

Conformational changes of the beta chain of the outer-arm dynein from sea urchin sperm flagella in relation to ATP hydrolysis was examined by tryptic digestion. Tryptic digestion of the beta chain in the presence of 2 mM ATP (ADP) and 100 microM vanadate (Vi) or in the presence of 4 mM ATP gamma S produced different polypeptides from in the case of no addition. The difference was similar to the result previously reported for 21S outer-arm dynein heavy chains [Inaba, K. & Mohri, H. (1989) J. Biol. Chem. 264, 8384-8388]. Unlike the tryptic digestion pattern of 21S dynein heavy chains, however, the 135-kDa polypeptide was consistently produced from the beta chain, even in the presence of ATP (ADP) and Vi. The tryptic digestion pattern of the 21S particle reconstituted from the separated a chain, the beta/IC1 complex and the IC2/IC3 complex [Tang, W.-J.Y., Bell, C.W., Sale, W.S., & Gibbons, I.R. (1982) J. Biol. Chem. 257, 508-515] was similar to that of intact 21S dynein; the 135-kDa polypeptide was only slightly produced in the presence of ATP and Vi. The digestion rate constant of the 135-kDa polypeptide from the beta chain in the presence of ATP and Vi was significantly decreased as compared with in the case of 21S dynein or that of the reconstituted 21S particle. These results suggest that the trypsin sensitivity of the 135-kDa region of the beta chain changes with the association of the beta/ICI complex with the alpha chain and the IC2/IC3 complex in the presence of ATP and Vi.     

Preparation of analogues of ATP, ADP and AMP suitable for binding to matrices and the enzymic interconversion of ATP and ADP in solid phase.

Alkylation of ATP with iodoacetic acid at pH 6.5 yielded 1-carboxymethyl-ATP which, after alkaline rearrangement, gave N-6-carboxymethyl-ATP. Condensation of this analogue with 1,6-diaminohexane in the presence of a water-soluble carbodiimide generated N-6-[(6-aminohexyl)carbamoylmethyl]-ATP in an overall yield of 40% based on the parent nucleotide ATP. The coenzymic activities of both N-6-adenine-substituted derivatives of ATP were tested with three kinases. Both derivatives showed coenzymic function against hexokinase with the "long" derivative having highest activity (95%) relative to unsubstituted ATP. Their activities towards the other two kinases tested was negligible except with the "long" analogue against glycerokinase (20%). The latter ATP analogue, when bound to Sepharose through its terminal amino group, could be dephosphorylated to the corresponding ADP analogue with soluble hexokinase yielding glucose 6-phosphate in an enzymic "solidphase" fashion. The Sepharose-bound ADP formed could subsequently be phosphorylated back to ATP using soluble acetate kinase. Sepharose-ATP preparations were also used in preliminary affinity chromatography studies using citrate synthase. Alkylation of ADP following the above procedure yielded the corresponding ADP analogue, N-6-[(6-aminohexyl)carbamoylmethyl]-ADP in an overall yield of 40%. Alkylation of AMP yielded the corresponding N-6-[(6-aminohexyl)carbamoylmethyl]-AMP in an overall yield of 45%.     

ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome

The 26S proteasome degrades polyubiquitinated proteins by an energy-dependent mechanism. Here we define multiple roles for ATP in 26S proteasome function. ATP binding is necessary and sufficient for assembly of 26S proteasome from 20S proteasome and PA700/19S subcomplexes and for proteasome activation. Proteasome assembly and activation may require distinct ATP binding events. The 26S proteasome degrades nonubiquitylated, unstructured proteins without ATP hydrolysis, indicating that substrate translocation per se does not require the energy of hydrolysis. Nonubiquitylated folded proteins and certain polyubiquitylated folded proteins were refractory to proteolysis. The latter were deubiquitylated by an ATP-independent mechanism. Other folded as well as unstructured polyubiquitylated proteins required ATP hydrolysis for proteolysis and deubiquitylation. Thus, ATP hydrolysis is not used solely for substrate unfolding. These results indicate that 26S proteasome-catalyzed degradation of polyubiquitylated proteins involves mechanistic coupling of several processes and that such coupling imposes an energy requirement not apparent for any isolated process.