Site-directed mutagenesis of the yeast V-ATPase B subunit (Vma2p)

The B subunit of the vacuolar (H+)-ATPase (V-ATPase) has previously been shown to participate in nucleotide binding and to possess significant sequence homology with the alpha subunit of the mitochondrial F-ATPase, which forms the major portion of the noncatalytic nucleotide binding sites and contributes several residues to the catalytic sites of this complex. Based upon the recent x-ray structure of the mitochondrial F1 ATPase (Abrahams, J.P., Leslie, A.G., Lutter, R., and Walker, J.E. (1994) Nature 370,621-628), site-directed mutagenesis of the yeast VMA2 gene has been carried out in a strain containing a deletion of this gene. VMA2 encodes the yeast V-ATPase B subunit (Vma2p). Mutations at two residues postulated to be contributed by Vma2p to the catalytic site (R381S and Y352S) resulted in a complete loss of ATPase activity and proton transport, with the former having a partial effect on V-ATPase assembly. Interestingly, substitution of Phe for Tyr-352 had only minor effects on activity (15-30% inhibition), suggesting the requirement for an aromatic ring at this position. Alteration of Tyr-370, which is postulated to be near the adenine binding pocket at the noncatalytic sites, to Arg, Phe, or Ser caused a 30-50% inhibition of proton transport and ATPase activity, suggesting that an aromatic ring is not essential at this position. Finally, mutagenesis of residues in the region corresponding to the P-loop of the alpha subunit (H180K, H180G, H180D, N181V) also inhibited proton transport and ATPase activity by approximately 30-50%. None of the mutations in either the putative adenine binding pocket nor the P-loop region had any effect on the ability of Vma2p to correctly fold nor on the V-ATPase to correctly assemble. The significance of these results for the structure and function of the nucleotide binding sites on the B subunit is discussed.     

ATP and ADP bind to cytochrome c oxidase and regulate its activity

By equilibrium dialysis of cytochrome c oxidase from bovine heart with [35S]ATPalphaS and [35S]ADPalphaS, seven binding sites for ATP and ten for ADP were determined per monomer of the isolated enzyme. The binding of ATP occurs in a time-dependent manner, as shown by a filtration method, which is apparently due to slow exchange of bound cholate. In the crystallized enzyme 10 mol of cholate were determined and partly identified in the high resolution crystal structure. Binding of ADP leads to conformational changes of the Tween 20-solubilized enzyme, as shown by a 12% decrease of the gamma-band. The conformational change is specific for ADP, since CDP, GDP and UDP showed no effects. The spectral changes are not obtained with the dodecylmaltoside solubilized enzyme. The polarographically measured activity of cytochrome c oxidase is lower after preincubation with high ATP/ADP-ratios than with low, in the presence of Tween 20. This effect of nucleotides is due to interaction with subunit IV, because preincubation of the enzyme with a monoclonal antibody to subunit IV released the inhibition by ATP. In the presence of dodecylmaltoside the enzyme had a 2 to 3-fold higher total activity, but this activity was not influenced by preincubation with ATP or ADP.     

Inhibition of the hepatitis C virus helicase-associated ATPase activity by the combination of ADP, NaF, MgCl2, and poly(rU). Two ADP binding sites on the enzyme-nucleic acid complex

Hepatitis C virus (HCV) helicase has an intrinsic ATPase activity and a nucleic acid (poly(rU))-stimulated ATPase activity. The poly(rU)-stimulated ATPase activity was inhibited by F- in a time-dependent manner during ATP hydrolysis. Inhibition was the result of trapping an enzyme-bound ADP-poly(rU) ternary complex generated during the catalytic cycle and was not the result of generating enzyme-free ADP that subsequently inhibited the enzyme. However, catalysis was not required for efficient inhibition by F-. The stimulated and the intrinsic ATPase activities were also inhibited by treatment of the enzyme with F-, ADP, and poly(rU). The inhibited enzyme slowly recovered (t1/2 = 23 min) ATPase activity after a 2000-fold dilution into assay buffer. The onset of inhibition by 500 microM ADP and 15 mM F- in the absence of nucleic acid was very slow (t1/2 > 40 min). However, the sequence of addition of poly(rU) to a diluted solution of ADP/NaF-treated enzyme had a profound effect on the extent of inhibition. If the ADP/NaF-treated enzyme was diluted into an assay that lacked poly(rU) and the assay was subsequently initiated with poly(rU), the treated enzyme was not inhibited. Alternatively, if the treated enzyme was diluted into an assay containing poly(rU), the enzyme was inhibited. ATP protected the enzyme from inhibition by ADP/NaF. The stoichiometry between ADP and enzyme monomer in the inhibited enzyme complex was 2, as determined from titration of the ATPase activity ([ADP]/[E] = 2.2) and from the number of radiolabeled ADP bound to the inhibited enzyme ([ADP]/[E] = 1.7) in the presence of excess NaF, MgCl2, and poly(rU). The Hill coefficient for titration of ATPase activity with F- (n = 2.8) or MgCl2 (n = 2.1) in the presence of excess ADP and poly(rU) suggested that multiple F- and Mg2+ were involved in forming the inhibited enzyme complex. The stoichiometry between (dU)18, a defined oligomeric nucleic acid substituting for poly(rU), and enzyme monomer in the inhibited enzyme complex was estimated to be 1 ([(dU)18/[E] = 1.2) from titration of the ATPase activity in the presence of excess ADP, MgCl2, and NaF.     

Nucleotide binding to the C-terminal nucleotide binding domain of ArsA. Studies with an ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine

ArsA protein, the catalytic component of the plasmid-encoded anion-translocating ATPase in Escherichia coli, contains two consensus nucleotide binding domains, A1 and A2, that are connected by a flexible linker. ATP has previously been shown to cross-link to the A1 domain upon activation with UV light but not to the A2 domain. The ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) was used to probe the nucleotide binding domains of ArsA. The covalently labeled protein was subjected to partial trypsin proteolysis, followed by Western blot analysis of the fragments with the anti-FSBA serum. The N-terminal amino acid sequence of the labeled fragment showed that FSBA binds preferentially to the C-terminal domain A2 both in the absence and the presence of antimonite. Occupancy of the two nucleotide binding sites was determined by protection from trypsin proteolysis. Trypsin cleaved the ArsA protein at Arg290 in the linker to generate a 32-kDa N-terminal and a 27-kDa C-terminal fragment. The 32-kDa fragment is compact and largely inaccessible to trypsin; however, the 27-kDa was cleaved further. Incubation with FSBA, which binds to the C-terminal domain, resulted in significant protection of the 27-kDa fragment. This fragment was not protected upon incubation with ATP alone, indicating that A2 might be unoccupied. However, upon incubation with ATP and antimonite, almost complete protection from trypsin was seen. ATP and FSBA together mimicked the effect of ATP and antimonite, implying that this fully protected conformation might be the result of both sites occupied with the nucleotide. It is proposed that the A1 site in ArsA is a high affinity ATP site, whereas the allosteric ligand antimonite is required to allow ATP binding to A2, resulting in catalytic cooperativity. Thus antimonite binding may act as a switch in regulating ATP binding to A2 and hence the ATPase activity of ArsA.     

7-Chloro-4-nitrobenzofurazan inactivates chloroplast H(2+)-ATPase by modification of different tyrosines, depending on the presence of ATP

The addition of 7-chloro-4-nitrobenzofurazan (NBD) to isolated CF1 at pH 7.5 leads to one tyrosine-bound NBD molecule per CF1 in one of the three beta-subunits, concomitantly with the inhibition of the ATPase activity. Addition of ADP prior to NBD-incubation protected the ATPase activity and reduced binding of NBD to beta-subunits. The addition of MgATP prior to modification did not result in protection against modification of the beta-subunit as well as preservation of activity. Cleavage of the NBD-labelled subunits with cyanogen bromide, followed by analysis of the labelled peptides, led to detection of a 14C-labelled peptide of 7 kDa in both cases (+/- ATP-preincubation). Sequence analysis of this peptide showed that in ATP-incubated CF1, tyr beta 328 was modified with NBD-Cl, whereas the ATP free sample contained no NBD bound to this tyrosine. Further digestion of the labelled peptide with protease V8 (Staphylococcus aureus) followed by sequence analysis of the radioactive labelled peptide, led to the detection of beta-tyr362 as the modified amino acid in case of ATP-free CF1. Both tyrosines are closely related to a proposed nucleotide binding region of beta.     

Effects of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin on nucleotide binding to the three F1-ATPase catalytic sites measured using specific tryptophan probes

Equilibrium nucleotide binding to the three catalytic sites of Escherichia coli F1-ATPase was measured in the presence of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin to elucidate mechanisms of inhibition. Fluorescence signals of beta-Trp-331 and beta-Trp-148 substituted in catalytic sites were used to determine nucleotide binding parameters. Azide brought about small decreases in Kd(MgATP) and Kd(MgADP). Notably, under MgATP hydrolysis conditions, it caused all enzyme molecules to assume a state with three catalytic site-bound MgATP and zero bound MgADP. These results rule out the idea that azide inhibits by "trapping" MgADP. Rather, azide blocks the step at which signal transmission between catalytic sites promotes multisite hydrolysis. Aurovertin bound with stoichiometry of 1.8 (mol/mol of F1) and allowed significant residual turnover. Cycling of the aurovertin-free beta-subunit catalytic site through three normal conformations was indicated by MgATP binding data. Aurovertin did not change the normal ratio of 1 bound MgATP/2 bound MgADP in catalytic sites. The results indicate that it acts to slow the switch of catalytic site affinities ("binding change step") subsequent to MgATP hydrolysis. Dicyclohexylcarbodiimide shifted the ratio of catalytic site-bound MgATP/MgADP from 1:2 to 1.6:1.4, without affecting Kd(MgATP) values. Like azide, it also appears to affect activity at the step after MgATP binding, in which signal transmission between catalytic sites promotes MgATP hydrolysis.     

Influence of ADP, AMP-PNP and of depletion of nucleotides on the structural properties of F1ATPase: a Fourier transform infrared spectroscopic study

Mitochondrial F1ATPase from beef heart was treated with different buffers in order to modulate the nucleotide content of the enzyme and then analysed by FT-IR spectroscopy. Treatment of F1ATPase with a buffer lacking nucleotides and glycerol led to the formation of two fractions consisting of an inactive aggregated enzyme deprived almost completely of bound nucleotides and of an active enzyme containing ATP only in the tight sites and having a structure largely accessible to the solvent and a low thermal stability. Treatment of F1ATPase with saturating ADP, which induced the hysteretic inhibition during turnover, or AMP-PNP did not affect remarkably the secondary structure of the enzyme complex but significantly increased its compactness and thermal stability. It was hypothesised that the formation of the inactive aggregated enzyme was mainly due to the destabilisation of the alpha-subunits of F1ATPase and that the induction of the hysteretic inhibition is related to a particular conformation of the enzyme, which during turnover becomes unable to sustain catalysis.     

Affinity labeling of rat liver carbamyl phosphate synthetase I by 5'-p-fluorosulfonylbenzoyladenosine

The ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA) has been used to study the interaction of MgATP with rat liver carbamyl phosphate synthetase I. Incubation of the enzyme with concentrations of FSBA as low as 0.025 mM produced considerable inactivation (41% at 120 min); identical rates and extents of reaction were produced by 0.5, 1, and 2 mM FSBA. Of the substrates for carbamyl phosphate synthetase I, only MgATP protected against FSBA inactivation. In the presence of a constant concentration of MgATP, increasing the FSBA concentration led to increased inhibition. Conversely, an increase in MgATP concentration led to decreased inhibition from a constant concentration of FSBA. Other nucleotide triphosphates provided no protection against FSBA inactivation. Addition of dithiothreitol to the FSBA-inactivated enzyme led to partial reactivation, suggesting that cysteine residue(s) were involved in the FSBA reaction. 5,5'-Dithiobis(2-nitrobenzoic acid) titration of the free sulfhydryl groups on the enzyme confirmed that cysteine residues were involved in reaction with FSBA; titration of the enzyme after incubation in the absence and presence of FSBA yielded values of 21 and 18(+/- 1), respectively. Binding studies with 5'-p-fluorosulfonylbenzoyl[2-3H]adenosine indicated that: 4 amino acid residues were involved in reaction with FSBA; 2 of these reaction sites were cysteine residues and 2 were noncysteine residues; MgATP protected one of the cysteine residues and one of the noncysteine residues from reaction with FSBA; the MgATP-protected noncysteine residue is essential for fully activity. These data strongly suggest that FSBA is an affinity label for two distinct MgATP sites on carbamyl phosphate synthetase I.     

Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria

1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside. 2. [35S]carboxyatractyloside and [3H]bongkrekic acid bind to potato mitochondria with high affinity (Kd = 10 to 20 nM, n=0.6-0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [3H]atractyloside binds to potato mitochondria with a much lower affinity (Kd=0.45 muM) than carboxyatractyloside or bongkrekic acid. 3. Bound [3H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [35S]carboxyatractyloside by bongkrekic acid and of bound [3H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP. 4. Bongkrekic acid competes with [35S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [35S]carboxyatractyloside binding. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.     

Conformational changes of the mitochondrial F1-ATPase epsilon-subunit induced by nucleotide binding as observed by phosphorescence spectroscopy

Changes in conformation of the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex as a result of nucleotide binding have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg2+ increases the flexibility of the protein structure surrounding the chromophore, nucleoside diphosphate acts in an opposite manner, enhancing the rigidity of this region of the macromolecule. Such changes in dynamic structure of the epsilon-subunit are evident at high ligand concentration added to both the nucleotide-depleted F1 (Nd-F1) and the F1 preparation containing the three tightly bound nucleotides (F1(2,1)). Since the effects observed are similar in both the F1 forms, the binding to the low affinity sites must be responsible for the conformational changes induced in the epsilon-subunit. This is partially supported by the observation that the Trp lifetime is not significantly affected by adding an equimolar concentration of adenine nucleotide to Nd-F1. The effects on protein structure of nucleotide binding to either catalytic or noncatalytic sites have been distinguished by studying the phosphorescence emission of the F1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F1(3,0)). Phosphorescence lifetime measurements on this F1 form demonstrate that the binding of Mg-NTP to catalytic sites induces a slight enhancement of the rigidity of the epsilon-subunit. This implies that the binding to the vacant noncatalytic site of F1(2,1) must exert the opposite and larger effect of enhancing the flexibility of the protein structure observed in both Nd-F1 and F1(2,1). The observation that enhanced flexibility of the protein occurs upon addition of adenine nucleotides to F1(2,1) in the absence of Mg2+ provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the epsilon-subunit is discussed.     

Selective interaction of homophtalazine derivatives with morphine

Binding of ATP to bovine serum albumin was shown by ultrafiltration and NMR. The binding was pH dependent. Scatchard analysis revealed that at pH 5.4, 6.4 and 7.4, dissociation constant Kd was 13, 40 and 120 microM, respectively, and no binding was observed at pH 8.4. The binding stoichiometry was 1:1 for all pH. Dimer of BSA did not bind ATP. From chemical shifts of 31P-NMR, Kd was estimated to be 15 microM at pH 5.4, which is very close to that determined by ultrafiltration. While adenosine did not interfere with the binding. GTP, dCTP, ADP, UTP, AMP, phosphate and pyrophosphate were competitive inhibitors and their inhibition constants Ki were 25, 32, 36, 50, 130, 1000 and 186 microM, respectively. Fatty acids such as lauric acid and palmitic acid did not interfere with the binding. Warfarin was a non-competitive inhibitor. Cl- competitively inhibited the binding, and the inhibition constant was 20 mM. The dissociation constants of the Cl- binding were reported to be 0.42 mM for the first binding site, 10-5 mM for the second and 303-143 mM for the third [G. Scatchard, W.T. Yap, J. Am. Chem. Soc., 86 (1964) 3434; G. Scatchard et al., J. Am. Chem. Soc. 79 (1957) 12]. This suggests that the ATP binding site may be the second Cl- binding site.     

Domain motion between the regulatory light chain and the nucleotide site in skeletal myosin

Resonance energy transfer probes were attached to skeletal myosin's nucleotide site and regulatory light chain (RLC) to examine nucleotide analog-induced structural transitions. A novel chemical modification of the RLC was developed for specific labeling of the basic N-terminus without affecting myosin ATPase activity. The modification allows attachment of a terbium chelate to rabbit skeletal RLC and was mapped by tryptic digestion to an amino group on the six N-terminal RLC residues. The use of terbium as a resonance energy transfer donor allowed the determination of the efficiency of energy transfer by sensitized emission lifetime measurements that practically eliminate background from unlabeled donor and acceptor sites as well as potential orientation factor artifacts in the calculation of the critical transfer distance. The nucleotide site was labeled with a functional CY3-labeled nucleotide as an energy transfer acceptor. Of the nucleotide states examined, ADP, ADP. vanadate, ADP. A1F4, and ADP. BeFx, the difference between the ADP and ADP. vanadate states was greatest (0.4-nm change), but was not considered to be statistically significant. The binding of actin to ADP-myosin also failed to produce a statistically significant change (0.3-nm change). These results are not consistent with a number of versions of the swinging lever arm hypothesis.     

Separation of tubulin subunits under nondenaturing conditions

The dissociation and separation of the tubulin alpha- and beta-subunits have been achieved by binding alpha-subunits to an immunoadsorbent gel and selectively inducing release of free beta-subunits. The immunoadsorbent gel was prepared by coupling the monoclonal antibody YL1/2 to Sepharose 4B which specifically recognizes the C-terminal end of tyrosinated alpha-subunits. Extensive tubulin subunit dissociation and separation occurred in Tris buffer at neutral pH but was greatly enhanced at basic pHs (8. 0-8.5). The binding of colchicine to heterodimeric tubulin resulted in a marked protection against dissociation. The dissociation of tubulin subunits was accompanied by loss of colchicine binding capacity, and ability to polymerize into microtubules. As shown by circular dichroism, loss of functional properties was not due to extensive denaturation of tubulin, as tubulin retained most of its secondary structure. Neither of the separated alpha- or beta-subunits was able to bind colchicine, but functional tubulin that was able to bind colchicine could be reconstituted from the dissociated subunits by changing the buffer to a neutral mixture of Tris and Pipes. The yield of reconstitution, as estimated from kinetic measurements of colchicine binding capacity, amounted to about 25%. Such a yield can probably be improved with minor changes in experimental conditions. The quantitative dissociation of tubulin into separated "native" alpha- and beta-subunits should provide a powerful tool for further studies on the properties of the individual tubulin subunits and the structure-function relationships of the tubulins.     

X-ray structure of nucleoside diphosphate kinase complexed with thymidine diphosphate and Mg2+ at 2-A resolution

We report the crystal structure of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum with thymidine diphosphate (dTDP) and Mg2+ bound at the active site. The structure has been refined to an R-factor of 18.3% at 2-A resolution. The base stacks on the aromatic ring of Phe 64 near the protein surface and is wedged between the side chains of Phe 64 and Val 116. The sugar and the pyrophosphate are deeper inside the protein and make numerous H-bonds with protein side chains. There is no backbone interaction with the nucleotide. A Mg2+ ion bridges the alpha- and beta-phosphates and interacts with the protein via water molecules. NDP kinase shows little specificity toward ribonucleotides and deoxyribonucleotides. This property, required by the enzyme biological function, can now be analyzed by comparing the crystal structures of free, ADP-ligated, and dTDP-ligated enzymes. The most significant differences are located in residues 60-64, which adapt their conformation to allow Phe 64 to stack on both types of bases. Nonspecific binding is achieved by the absence of polar interaction between the base and protein atoms. The ribose of ADP and the deoxyribose of dTDP occupy similar positions, their hydroxyl groups interacting with Lys 16 and Asn 119. The H-bond between Lys 16 and the O2' hydroxyl of ADP is replaced by a similar interaction with a water molecule in the dTDP complex. The beta-phosphate position is the same for ADP and dTDP, suggesting that the mechanism of phosphate transfer is the same for all substrates ofNDP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of free and ATP-bound magnesium and manganese ions on the ATPase activity of chaperonin GroEL14

Hydrolysis of ATP by the GroEL14 chaperonin oligomer is activated and modulated by Mg2+ or Mn2+ ions. Mg-ATP and Mn-ATP can serve as substrates of the reaction and bind in a positively cooperative manner to the same catalytic sites on GroEL14, with similar binding constants in the micromolar range. In addition, millimolar amounts of Mg2+ and Mn2+ ions can further activate the GroEL14-ATPase while interacting with low-affinity noncatalytic sites on the chaperonin. The extent of ATPase activation by Mn2+ is half of that by Mg2+ ions. When both Mg2+ and Mn2+ ions are present in the same reaction, Mn2+ behaves as a noncompetitive partial inhibitor of the Mg-dependent ATPase. This inhibition requires the presence of ADP in the catalytic site. The binding affinity of Mn-ADP to the site is significantly higher than that of Mg-ADP. A slower release of Mn-ADP from the catalytic site thus changes the rate-determining step of the GroEL14-ATPase cycle. In the cell, the concentrations of Mg2+ and Mn2+ ions are such that both divalent ions may modulate chaperonin activity.     

Interaction of thymidylate synthase with pyridoxal 5'-phosphate as studied by UV/visible difference spectroscopy and molecular modeling

Pyridoxal 5'-phosphate (PLP) is an effective inhibitor of Lactobacillus casei thymidylate synthase (TS), competitive with respect to the nucleotide substrate dUMP (Chen et al., 1989). The UV/vis difference spectra of TS-PLP complexes show lambda max at 328 nm due to the specific interaction between Cys 198 of TS and PLP to form a thiohemiacetal, and lambda min at 388 nm due to depletion of free PLP. At high concentrations of PLP a new absorbance at 430 nm forms due to nonspecific Schiff base formation between PLP and lysine residues of the enzyme. Using spectral titration at 328 nm, the binding constant of the specific TS-PLP complex was determined to be 0.5 microM, and the stoichiometry was 2 mol of PLP/mol of TS dimer. The 328-nm absorbance of the TS-PLP complex can be competitively and completely eliminated by addition of dUMP or dTMP; this serves as a convenient binding assay for molecules which bind to the active site of TS. Analogs of PLP which do not contain the phosphate or the aldehyde moieties of PLP bound poorly to the enzyme, thus demonstrating the importance of these functional groups for binding. When treated with PLP, C244T TS, which contains the active site Cys 198 as the sole cysteine residue, showed the same properties as the wild-type enzyme. Treatment of the C198A and C198S mutants with PLP did not produce the absorbance at 328 nm assigned to thiohemiacetal formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysine 129 of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) participates in the binding of ATP to inhibit the cyclic ADP-ribose hydrolase

CD38 catalyzes not only the formation of cyclic ADP-ribose (cADPR) from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD+ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.     

Growth-regulated formation of heteromeric complexes of the centromere and promoter factor, Cbf1p, in yeast

The gastrointestinal pathogen Aeromonas hydrophila strain A186 produces a collagen-binding protein (CNBP) which is found extracellularly and loosely associated with the cell surface. The cell-associated CNBP was purified by sequential ammonium sulphate precipitation, size-exclusion chromatography and ion-exchange chromatography, or by sequential ammonium sulphate precipitation and affinity chromatography with collagen-Sepharose. The purified CNBP was homogeneous in SDS-PAGE, and had a mol. wt of c. 98 kDa. Cyanogen bromide cleavage of the CNBP destroyed collagen-binding activity; however, enzymic digestion with Staphylococcus aureus V8 protease generated > 10 polypeptide fragments, from which a 30-kDa polypeptide contained the strongest collagen-binding activity. Binding of collagen by the CNBP was restricted to the alpha1 (I) chain of the collagen molecule and binding seemed to involve both the carbohydrate moieties and certain peptide sequences on the collagen. Collagen-saccharides generated by alkaline hydrolysis inhibited collagen binding by A. hydrophila. Also, glycosidase digestion and chemical alteration of the carbohydrate residues of collagen reduced its ability to be bound by the CNBP. Collagen-homologous synthetic peptides inhibited binding of 125I-collagen by the bacteria.     

The interactions of ATP, ADP, and inorganic phosphate with the sheep cardiac ryanodine receptor

The effects of ATP, ADP, and inorganic phosphate (Pi) on the gating of native sheep cardiac ryanodine receptor channels incorporated into planar phospholipid bilayers were investigated. We demonstrate that ATP and ADP can activate the channel by Ca2+-dependent and Ca2+-independent mechanisms. ATP and ADP appear to compete for the same site/s on the cardiac ryanodine receptor, and in the presence of cytosolic Ca2+ both agents tend to inactivate the channel at supramaximal concentrations. Our results reveal that ATP not only has a greater affinity for the adenine nucleotide site/s than ADP, but also has a greater efficacy. The EC50 value for channel activation is approximately 0.2 mM for ATP compared to 1.2 mM for ADP. Most interesting is the fact that, even in the presence of cytosolic Ca2+, ADP cannot activate the channel much above an open probability (Po) of 0.5, and therefore acts as a partial agonist at the adenine nucleotide binding site on the channel. We demonstrate that Pi also increases Po in a concentration and Ca2+-dependent manner, but unlike ATP and ADP, has no effect in the absence of activating cytosolic [Ca2+]. We demonstrate that Pi does not interact with the adenine nucleotide site/s but binds to a distinct domain on the channel to produce an increase in Po.     

A subunit interaction in chloroplast ATP synthase determined by genetic complementation between chloroplast and bacterial ATP synthase genes

F1F0-ATP synthases utilize protein conformational changes induced by a transmembrane proton gradient to synthesize ATP. The allosteric cooperativity of these multisubunit enzymes presumably requires numerous protein-protein interactions within the enzyme complex. To correlate known in vitro changes in subunit structure with in vivo allosteric interactions, we introduced the beta subunit of spinach chloroplast coupling factor 1 ATP into a bacterial F1 ATP synthase. A cloned atpB gene, encoding the complete chloroplast beta subunit, complemented a chromosomal deletion of the cognate uncD gene in Escherichia coli and was incorporated into a functional hybrid F1 ATP synthase. The cysteine residue at position 63 in chloroplast beta is known to be located at the interface between alpha and beta subunits and to be conformationally coupled, in vitro, to the nucleotide binding site > 40 A away. Enlarging the side chain of chloroplast coupling factor 1 beta residue 63 from Cys to Trp blocked ATP synthesis in vivo without significantly impairing ATPase activity or ADP binding in vitro. The in vivo coupling of nucleotide binding at catalytic sites to transmembrane proton movement may thus involve an interaction, via conformational changes, between the amino-terminal domains of the alpha and beta subunits.