A massive stellar bulge in a regularly rotating galaxy 1.2 billion years after the Big Bang.

Cosmological models predict that galaxies forming in the early Universe experience a chaotic phase of gas accretion and star formation, followed by gas ejection due to feedback processes. Galaxy bulges may assemble later via mergers or internal evolution. Here we present submillimeter observations (with spatial resolution of 700 parsecs) of ALESS 073.1, a starburst galaxy at redshift [Formula: see text] when the Universe was 1.2 billion years old. This galaxy's cold gas forms a regularly rotating disk with negligible noncircular motions. The galaxy rotation curve requires the presence of a central bulge in addition to a star-forming disk. We conclude that massive bulges and regularly rotating disks can form more rapidly in the early Universe than predicted by models of galaxy formation.

A massive, dead disk galaxy in the early Universe.

At redshift z = 2, when the Universe was just three billion years old, half of the most massive galaxies were extremely compact and had already exhausted their fuel for star formation. It is believed that they were formed in intense nuclear starbursts and that they ultimately grew into the most massive local elliptical galaxies seen today, through mergers with minor companions, but validating this picture requires higher-resolution observations of their centres than is currently possible. Magnification from gravitational lensing offers an opportunity to resolve the inner regions of galaxies. Here we report an analysis of the stellar populations and kinematics of a lensed z = 2.1478 compact galaxy, which-surprisingly-turns out to be a fast-spinning, rotationally supported disk galaxy. Its stars must have formed in a disk, rather than in a merger-driven nuclear starburst. The galaxy was probably fed by streams of cold gas, which were able to penetrate the hot halo gas until they were cut off by shock heating from the dark matter halo. This result confirms previous indirect indications that the first galaxies to cease star formation must have gone through major changes not just in their structure, but also in their kinematics, to evolve into present-day elliptical galaxies.

Effects of age, education and gender in the Consortium to Establish a Registry for the Alzheimer's Disease (CERAD)-Neuropsychological Assessment Battery for Cantonese-speaking Chinese elders.

BACKGROUND: The Consortium to Establish a Registry for Alzheimer's Disease Neuropsychological Assessment Battery (CERAD-NAB) offers information on the clinical diagnosis of Alzheimer's disease (AD) and gives a profile of cognitive functioning. This study explores the effects of age, education and gender on participants' performance on eight subtests in the Chinese-Cantonese version of the CERAD-NAB. METHODS: The original English version of the CERAD-NAB was translated and content-validated into a Chinese-Cantonese version to suit the Hong Kong Chinese population. The battery was administered to 187 healthy volunteers aged 60 to 94 years. Participants were excluded if they had neurological, medical or psychiatric disorders (including dementia). Stepwise multiple linear regression analyses were performed to assess the relative contribution of the demographic variables to the scores on each subtest. RESULTS: The Cantonese version of CERAD-NAB was shown to have good content validity and excellent inter-rater reliability. Stepwise multiple regression analyses revealed that performances on seven and four out of eight subtests in the CERAD-NAB were significantly influenced by education level and age, respectively. Age and education had significant effects on participants' performance on many tests. Gender also showed a significant effect on one subtest. CONCLUSIONS: The preliminary data will serve as an initial phase for clinical interpretation of the CERAD-NAB for Cantonese-speaking Chinese elders.

Substitution of betaGlu(201) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 increases the affinity of catalytic sites for nucleotides.

In the crystal structure of bovine mitochondrial F(1)-ATPase (MF(1)) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the side chain oxygen of betaThr(163) interacts directly with Mg(2+) coordinated to 5'-adenylyl beta, gamma-imidodiphosphate or ADP bound to catalytic sites of beta subunits present in closed conformations. In the unliganded beta subunit present in an open conformation, the hydroxyl of betaThr(163) is hydrogen-bonded to the carboxylate of betaGlu(199). Substitution of betaGlu(201) (equivalent to betaGlu(199) in MF(1)) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 with cysteine or valine increases the propensity to entrap inhibitory MgADP in a catalytic site during hydrolysis of 50 microM ATP. These substitutions lower K(m3) (the Michaelis constant for trisite ATP hydrolysis) relative to that of the wild type by 25- and 10-fold, respectively. Fluorescence quenching of alpha(3)(betaE201C/Y341W)(3)gamma and alpha(3)(betaY341W)(3)gamma mutant subcomplexes showed that MgATP and MgADP bind to the third catalytic site of the double mutant with 8.4- and 4.4-fold higher affinity, respectively, than to the single mutant. These comparisons support the hypothesis that the hydrogen bond observed between the side chains of betaThr(163) and betaGlu(199) in the unliganded catalytic site in the crystal structure of MF(1) stabilizes the open conformation of the catalytic site during ATP hydrolysis.

Inhibitory Mg-ADP-fluoroaluminate complexes bound to catalytic sites of F(1)-ATPases: are they ground-state or transition-state analogs?

Schemes are proposed for coupling sequential opening and closing the three catalytic sites of F(1) to rotation of the gamma subunit during ATP synthesis and hydrolysis catalyzed by the F(o)F(1)-ATP synthase. A prominent feature of the proposed mechanisms is that the transition state during ATP synthesis is formed when a catalytic site is in the process of closing and that the transition state during ATP hydrolysis is formed when a catalytic site is in the process of opening. The unusual kinetics of formation of Mg-ADP-fluoroaluminate complexes in one or two catalytic sites of nucleotide-depleted MF(1) and wild-type and mutant alpha(3)beta(3)gamma subcomplexes of TF(1) are also reviewed. From these considerations, it is concluded that Mg-ADP-fluoroaluminate complexes formed at catalytic sites of isolated F(1)-ATPases or F(1) in membrane-bound F(o)F(1) are ground-state analogs.

Oxidation of the alpha(3)(betaD311C/R333C)(3)gamma subcomplex of the thermophilic Bacillus PS3 F(1)-ATPase indicates that only two beta subunits can exist in the closed conformation simultaneously.

In the crystal structure of the bovine heart mitochondrial F(1)-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the two liganded beta subunits, one with MgAMP-PNP bound to the catalytic site (beta(T)) and the other with MgADP bound (beta(D)) have closed conformations. The empty beta subunit (beta(E)) has an open conformation. In beta(T) and beta(D), the distance between the carboxylate of beta-Asp(315) and the guanidinium of beta-Arg(337) is 3.0-4.0 A. These side chains are at least 10 A apart in beta(E). The alpha(3)(betaD311C/R333C)(3)gamma subcomplex of TF(1) with the corresponding residues substituted with cysteine has very low ATPase activity unless it is reduced prior to assay or assayed in the presence of dithiothreitol. The reduced subcomplex hydrolyzes ATP at 50% the rate of wild-type and is rapidly inactivated by oxidation by CuCl(2) with or without magnesium nucleotides bound to catalytic sites. Titration of the subcomplex with iodo[(14)C]acetamide after prolonged treatment with CuCl(2) in the presence or absence of 1 mM MgADP revealed nearly two free sulfhydryl groups/mol of enzyme. Therefore, one pair of introduced cysteines is located on a beta subunit that exists in the open or partially open conformation even when catalytic sites are saturated with MgADP. Since V(max) of ATP hydrolysis is attained when three catalytic sites of F(1) are saturated, the catalytic site that binds ATP must be closing as the catalytic site that releases products is opening.

The adenine pocket of a single catalytic site is derivatized when the bovine heart mitochondrial F1-ATPase is photoinactivated with 4-amino-1-octylquinaldinium.

The bovine heart mitochondrial F1-ATPase (MF1) is reversibly inhibited in the dark by 4-amino-1-octylquinaldinium (AOQ) with an I0.5 value of 48 microM. When irradiated in the presence of AOQ, MF1 is photoinactivated with an apparent Kd of 12 microM. About 1.1 mol of [3H]AOQ were incorporated per mol of MF1 on complete photoinactivation. Fractionation of a cyanogen bromide digest of MF1 photolabeled with [3H]AOQ followed by fractionation of peptic digests of partially purified cyanogen bromide fragments led to isolation of two CNBr/peptic fragments labeled with 3H. Sequence analysis of the labeled peptides revealed that one contained residues 423-441 of the beta subunit. A gap in position 2 of the sequence indicates that beta Phe424 is derivatized. The phenyl side-chain of this residue is part of a pocket that binds the adenine moiety of ATP or ADP at catalytic sites. The other peptide, which was labeled to a greater extent, contained residues 342-358 of the beta subunit, but in this case, no gap was found in the sequence indicating that the derivatized amino-acid side-chain might not have survived the conditions of automatic Edman degradation. This peptide contains beta Tyr345, the side-chain of which is also a component of the pocket that binds the adenine moiety of ATP or ADP to catalytic sites. However, for the reason stated, there is no direct evidence that beta Tyr345 is labeled in this peptide.

The alpha 3(beta Y341W)3 gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 fails to dissociate ADP when MgATP is hydrolyzed at a single catalytic site and attains maximal velocity when three catalytic sites are saturated with MgATP.

The hydrolytic properties of the alpha3beta3gamma and mutant alpha3(betaY341W)3gamma subcomplexes of the TF1-ATPase have been compared. ATPase activity of the mutant is less sensitive to turnover-dependent inhibition by azide, less suppressed by increasing concentrations of Mg2+ during assay, and less stimulated by lauryl dimethylamine oxide (LDAO). Therefore, it has much lower propensity than wild-type to entrap inhibitory MgADP in a catalytic site during turnover. The fluorescence of the introduced tryptophans in the alpha3(betaY341W)3gamma subcomplex is completely quenched when catalytic sites are saturated with ATP or ADP with or without Mg2+ present. As reported for the betaY331W mutant of Escherichia coli F1 (Weber, J., Wilke-Mounts, S., Lee, R. S.-F., Grell, E., Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133), this provides a direct probe of nucleotide binding to catalytic sites. Addition of stoichiometric MgATP to the mutant subcomplex quenched one-third the tryptophan fluorescence which did not recover after 60 min. This was caused by entrapment of MgADP in a single catalytic site. Titration of catalytic sites of the alpha3(betaY341W)3gamma subcomplex with MgADP or MgATP revealed Kd's of < 50 nM, about 0.25 microM and about 35 microM. Titrations were not affected by azide, whereas LDAO lowered the affinities of catalytic sites 2 and 3 for MgADP by 5-fold and 2-fold, respectively. During titration with MgATP, LDAO slightly lowered affinity at ATP concentrations below 30 microM and had no effect at ATP concentrations above 30 microM. Maximal velocity was attained when the third catalytic site was titrated with MgATP in the presence or absence of LDAO. The same Kd's for binding MgATP to the (alphaA396C)3beta3(gammaA22C) mutant were observed before and after inactivating it by cross-linking alpha to gamma. This implies that the different affinities of catalytic sites for MgATP do not represent negative cooperativity, but rather represent heterogeneous affinities of catalytic sites dictated by the position of the coiled-coil of the gamma subunit within the central cavity of the (alpha beta)3 hexamer.

Mutations in the nucleotide binding domain of the alpha subunits of the F1-ATPase from thermophilic Bacillus PS3 that affect cross-talk between nucleotide binding sites.

Inactivation of MF1 (bovine mitochondrial F1-ATPase) with 5'-p-fluorosulfonylbenzoylethenoadenosine is caused by labeling alpha Y244 [Verburg, J. G., and Allison, W. S. (1990) J. Biol. Chem. 265, 8065-8074]. In the crystal structure [Abrahams, J.P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], alpha Y244 is hydrogen bonded to alpha R304 which is also hydrogen bonded to alpha Y300. The catalytic properties of mutant alpha 3 beta 3 gamma subcomplexes of the TF1-ATPase from the thermophilic Bacillus PS3 containing the alpha F244C, alpha R304C, and alpha Y300C substitutions have been examined. Each has unique features for hydrolyzing ATP and forming inhibitory ADP-fluoroaluminate complexes in catalytic sites. Unlike wild-type, the (alpha R304C)3 beta 3 gamma and (alpha Y300C)3 beta 3 gamma subcomplexes entrap inhibitory MgADP in a catalytic site during turnover which fails to dissociate when ATP binds to noncatalytic sites. Although the hydrolytic properties of the (alpha F244C)3 beta 3 gamma subcomplex and wild-type are similar, the mutant forms ADP-fluoroaluminate complexes 7 times faster than wild-type when Al3+ and F- are added to it in the presence of excess ADP and Mg2+. It also resists inhibition by high Mg2+ concentrations in the assay medium. At least one noncatalytic site of the (alpha F244C)3 beta 3 gamma subcomplex has increased affinity for ADP, indicating that the enhanced rate of formation of the ADP-fluoroaluminate complex reflects augmented cooperativity between noncatalytic and catalytic sites. The rate of formation of the ADP-fluoroaluminate complex in (alpha Y300C)3 beta 3 gamma increases only 40% when MgADP in bound to two catalytic sites rather than one, compared to a 9-fold increase exhibited by wild type. When Al3+ and F- are added to the (alpha Y300C)3 beta 3 gamma subcomplex after incubation with excess ADP and Mg2+, ADP-fluoroaluminate complexes are formed in three catalytic sites rather than two observed with the other subcomplexes. Reconciliation of the catalytic properties of the mutant subcomplexes in terms of the crystal structure suggests that alpha F244, alpha R304, and alpha Y300 of TF1 are part of a pathway that propagates conformational signals from one catalytic site to another.

ADP-fluoroaluminate complexes are formed cooperatively at two catalytic sites of wild-type and mutant alpha3beta3gamma subcomplexes of the F1-ATPase from the thermophilic Bacillus PS3.

Addition of Al3+ and F- to the alpha3beta3gamma subcomplex of the TF1-ATPase containing MgADP in one catalytic site causes slow, complete inactivation as the ADP-fluoroaluminate complex is formed. This conflicts with the "bisite" stochastic model suggested earlier (Issartel, J. P., Dupuis, A., Lunardi, J. & Vignais, P. V. (1991) Biochemistry 30, 4726-4733] on the finding that complete inactivation of the bovine mitochondrial F1-ATPase by Al3+, F-, Mg2+, and excess ADP occurs as ADP-fluoroaluminate complexes form in two catalytic sites. When Al3+ and F- were added to alpha3beta3gamma containing MgADP in two catalytic sites, inactivation accelerated 8-fold, indicating catalytic to catalytic site cooperativity. When added to alpha3beta3gamma containing MgADP bound to one or two catalytic sites prior to addition of Al3+ and F-, phosphate inhibits formation of the ADP-fluoroaluminate complex. When introduced after adding 200 microM ADP plus Mg2+ to alpha3beta3gamma, but before adding Al3+ and F-, phosphate accelerated formation of the ADP-fluoroaluminate complex 3-fold. Sulfite accelerated formation of the ADP-fluoroaluminate complex 9-fold when 200 microM ADP plus Mg2+ was added to alpha3beta3gamma before adding Al3+ and F-. The accelerations induced by phosphate or sulfite in the presence of excess ADP and Mg2+ suggest noncatalytic to catalytic site cooperativity. When Al3+ and F- were added to the (alphaD261N)3beta3gamma subcomplex containing MgADP in a single catalytic site, the ADP-fluoroaluminate complex formed at least 10-fold more slowly than observed with wild-type under the same conditions. Therefore, the catalytic site containing MgADP recognizes the alphaD261N substitution when noncatalytic sites are empty. Cross-linking alpha to gamma or beta to gamma by oxidizing the (alphaA396C)3beta3(gammaA22C) and alpha3(betaD390C)3(gammaS90C) subcomplexes, respectively, abolishes cooperative formation of ADP-fluoroaluminate complexes in two catalytic sites. ADP-fluoroaluminate complex formation is restricted to a single catalytic site in the oxidized double mutants. The alpha3beta3delta subcomplex does not form an inhibitory ADP-fluoroaluminate complex under any of the conditions examined for the alpha3beta3gamma subcomplexes.

Catalytic activity of the alpha3beta3gamma complex of F1-ATPase without noncatalytic nucleotide binding site.

A mutant alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 microM; Km(2), 135 microM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below approximately 300 microM.

Photoinactivation of the F1-ATPase from spinach chloroplasts by dequalinium is accompanied by derivatization of methionine beta183.

In contrast to the F1-ATPases from bovine mitochondria and the thermophilic Bacillus PS3, which are reversibly inhibited by dequalinium in the absence of irradiation, the Mg2+-ATPase activity of heat- or dithiothreitol-activated chloroplast F1 (CF1) from spinach chloroplasts is slightly stimulated by dequalinium. Conversely, dequalinium is a partial inhibitor (maximal inhibition is 85-90%) of the Ca2+-ATPase of CF1 activated by heat, dithiothreitol, or octylglucoside. The Mg2+- and Ca2+-ATPase activities of CF1 respond differently in the presence of lauryl dimethylamine oxide (LDAO) in the assay medium. Whereas the Mg2+-ATPase activity of heat- or dithiothreitol-activated CF1 is stimulated up to 14-fold by increasing concentrations of LDAO, the Ca2+-ATPase is inhibited in a biphasic manner by increasing concentrations of LDAO. In the presence of LDAO, dequalinium does not stimulate the heat-activated Mg2+-ATPase over that promoted by LDAO alone. That dequalinium slightly stimulates Mg2+-ATPase activity although it inhibits Ca2+-ATPase activity can be reconciled by assuming that dequalinium binds to two sites in CF1, a stimulatory site that also binds LDAO and an inhibitory site. By acting as a partial inhibitor of the Mg2+-ATPase activity that it activates, the combined effect of dequalinium is modest stimulation. Irradiation of heat- or dithiothreitol-activated CF1 or the alpha3beta3gamma subcomplex of CF1 in the presence of 12 microM dequalinium led to rapid photoinactivation. ATP and ADP, separately or in combination with Mg2+, protect against photoinactivation. After photoinactivating the alpha3beta3gamma subcomplex of CF1 with [14C]dequalinium, tryptic and peptic digests of the isolated, derivatized beta subunit were fractionated by high performance liquid chromatography. Sequencing of the isolated, radioactive tryptic and peptic peptides revealed that Metbeta183, which is at or near the catalytic site, is derivatized in a single beta subunit when CF1 is photoinactivated with [14C]dequalinium.

The alpha3beta3gamma subcomplex of the F1-ATPase from the thermophilic bacillus PS3 with the betaT165S substitution does not entrap inhibitory MgADP in a catalytic site during turnover.

The hydrolytic properties of the mutant alpha3(betaT165S)3gamma and wild-type alpha3beta3gamma subcomplexes of TF1 have been compared. Whereas the wild-type complex hydrolyzes 50 microM ATP in three kinetic phases, the mutant complex hydrolyzes 50 microM ATP with a linear rate. After incubation with a slight excess of ADP in the presence of Mg2+, the wild-type complex hydrolyzes 2 mM ATP with a long lag. In contrast, prior incubation of the mutant complex under these conditions does not affect the kinetics of ATP hydrolysis. The ATPase activity of the wild-type complex is stimulated 4-fold by 0. 1% lauryl dimethylamine oxide, whereas this concentration of lauryl dimethylamine oxide inhibits the mutant complex by 25%. Compared with the wild-type complex, the activity of the mutant complex is much less sensitive to turnover-dependent inhibition by azide. This comparison suggests that the mutant complex does not entrap substantial inhibitory MgADP in a catalytic site during turnover, which is supported by the following observations. ATP hydrolysis catalyzed by the wild-type complex is progressively inhibited by increasing concentrations of Mg2+ in the assay medium, whereas the mutant complex is insensitive to increasing concentrations of Mg2+. A Lineweaver-Burk plot constructed from rates of hydrolysis of 20-2000 microM ATP by the wild-type complex is biphasic, exhibiting apparent Km values of 30 microM and 470 microM with corresponding kcat values of 26 and 77 s-1. In contrast, a Lineweaver-Burk plot for the mutant complex is linear in this range of ATP concentration, displaying a Km of 133 microM and a kcat of 360 s-1.

Does the gamma subunit move to an abortive position of ATP hydrolysis when the F1.ADP.Mg complex isomerizes to the inactive F1*.ADP.Mg complex?

F1-ATPases transiently entrap inhibitory MgADP in a catalytic site during turnover when noncatalytic sites are not saturated with ATP. An initial burst of ATP hydrolysis rapidly decelerates to a slow intermediate rate that gradually accelerates to a final steady-state rate. Transition from the intermediate to the final rate is caused by slow binding of ATP to noncatalytic sites which promotes dissociation of inhibitory MgADP from the affected catalytic site. Evidence from several laboratories suggests that the gamma subunit rotates with respect to alpha/beta subunit pairs of F1-ATPase during ATP hydrolysis. The alpha 3 beta 3 and alpha 3 beta 3 delta subcomplexes of the TF1-ATPase do not entrap inhibitory MgADP in a catalytic site during turnover, suggesting involvement of the gamma subunit in the entrapment process. From these observations, it is proposed that the gamma subunit moves into an abortive position for ATP hydrolysis when inhibitory MgADP is entrapped in a catalytic site during ATP hydrolysis.

Inhibition of inducible nitric oxide synthase prevents myocardial and systemic vascular barrier dysfunction during early cardiac allograft rejection.

NO is produced during cardiac allograft rejection by expression of inducible NO synthase (iNOS) in the rejecting heart. Recent evidence indicates that NO modulates vascular permeability under both physiological and pathophysiological conditions. The present study explored the effects of early acute cardiac allograft rejection, and specifically the effects of NO, on myocardial and systemic vascular barrier function using a quantitative double-tracer permeation method in a rat cardiac transplant model. Early allograft rejection increased albumin permeation twofold to fivefold in the allograft heart and systemic vasculature (brain, lung, sciatic nerve, diaphragm, retina, muscle, kidney, and uvea) compared with isografts and controls. There were no detectable differences in regional blood flow or hemodynamics, suggesting that increased albumin permeation resulted from increased vascular permeability. iNOS mRNA was expressed in the allograft heart and native lung and was associated with increased serum nitrite/nitrate levels. iNOS inhibition with aminoguanidine prevented or attenuated allograft heart and systemic vascular barrier dysfunction and reduced allograft serum nitrite/nitrate levels to isograft values. Aminoguanidine did not affect the mild histological changes of rejection present in allografts. These data demonstrate the novel observations that (1) endothelial barrier function is compromised in the systemic vasculature, particularly in the brain, remote from the site of allograft rejection; (2) allograft vascular barrier dysfunction is associated with increased NO production and iNOS mRNA expression in the affected tissues (eg, native lung and grafted heart); and (3) inhibition of NO production by iNOS prevents vascular barrier dysfunction in the allograft heart and systemic vasculature.

An attempt to convert noncatalytic nucleotide binding site of F1-ATPase to the catalytic site: hydrolysis of tethered ATP by mutated alpha subunits in the enzyme.

The alpha and beta subunits of F1-ATPase are homologous in primary structure and have similar folding topologies. The position of the essential Glu residue in the catalytic sites which reside in the beta subunits is occupied by a Gln residue in the noncatalytic nucleotide binding sites which reside in the alpha subunits. To test if an exchange of catalytic and noncatalytic binding sites is possible, we have replaced the Gln-Lys sequence in the noncatalytic binding site of the alpha subunit with Glu-Arg and, reciprocally, the Glu in the catalytic site of the beta subunit with Gln. The resultant mutant alpha3beta3gamma complex lost steady-state ATPase activity. However, HPLC analysis of tryptic digests of the mutant alpha3beta3gamma complex which had been photolabeled with 2-N3-[8-3H]ATP revealed that ATP tethered to the noncatalytic binding side was hydrolyzed, indicating that a primitive catalytic ability was generated at the alpha subunit by the introduced Glu.

The alpha 3 beta 3 gamma complex of the F1-ATPase from thermophilic Bacillus PS3 containing the alpha D261N substitution fails to dissociate inhibitory MgADP from a catalytic site when ATP binds to noncatalytic sites.

ATP hydrolyses by the wild-type alpha 3 beta 3 gamma and mutant (alpha D261N)3 beta 3 gamma subcomplexes of the F1-ATPase from the thermophilic Bacillus PS3 have been compared. The wild-type complex hydrolyzes 50 microM ATP in three kinetic phases: a burst decelerates to an intermediate phase, which then gradually accelerates to a final rate. In contrast, the mutant complex hydrolyzes 50 microM or 2 mM ATP in two kinetic phases. The mutation abolishes acceleration from the intermediate phase to a faster final rate. Both the wild-type and mutant complexes hydrolyze ATP with a lag after loading a catalytic site with MgADP. The rate of the MgADP-loaded wild-type complex rapidly accelerates and approaches that observed for the wild-type apo-complex. The MgADP-loaded mutant complex hydrolyzes ATP with a more pronounced lag, and the gradually accelerating rate approaches the slow, final rate observed with the mutant apo-complex. Lauryl dimethylamide oxide (LDAO) stimulates hydrolysis of 2 mM ATP catalyzed by wild-type and mutant complexes 4- and 7.5-fold, respectively. The rate of release of [3H]ADP from the Mg[3H]ADP-loaded mutant complex during hydrolysis of 40 microM ATP is slower than observed with the wild-type complex. LDAO increases the rate of release of [3H]ADP from the preloaded wild-type and mutant complexes during hydrolysis of 40 microM ATP. Again, release is slower with the mutant complex. When the wild-type and mutant complexes are irradiated in the presence of 2-N3-[3H]ADP plus Mg2+ or 2-N3-[3H]ATP plus Mg2+ and azide, the same extent of labeling of noncatalytic sites is observed. Whereas ADP and ATP protect noncatalytic sites of the wild-type and mutant complexes about equally from labeling by 2-N3-[3H]ADP or 2-N3-[3H[ATP, respectively, AMP-PNP provides little protection of noncatalytic sites of the mutant complex. The results suggest that the substitution does not prevent binding of ADP or ATP to noncatalytic sites, but rather that it affects cross-talk between liganded noncatalytic sites and catalytic sites which is necessary to promote dissociation of inhibitory MgADP.

Lowered temperature or binding of pyrophosphate to sites for noncatalytic nucleotides modulates the ATPase activity of the beef heart mitochondrial F1-ATPase by decreasing the affinity of a catalytic site for inhibitory MgADP.

Lineweaver-Burk plots for ATP hydrolysis catalyzed by bovine heart mitochondrial F1-ATPase (MF1) at 30 degrees C are biphasic, whereas they are linear at 15 degrees C. The rate of inactivation of the enzyme at 23 degrees C by 5'-[(p-fluorosulfonyl)benzoyl]adenosine (FSBA), which derivatizes noncatalytic nucleotide binding sites, is about 4 times faster when loss of activity is monitored at 15 degrees C as opposed to 30 degrees C. This suggests that maximal loss of ATPase monitored at 15 degrees C is observed when a single noncatalytic site is derivatized, whereas maximal inactivation at 30 degrees C requires modification of three noncatalytic sites. Prior incubation of MF1 depleted of endogenous nucleotides (nd-MF1) with pyrophosphate (PPi) stimulates ATPase activity 2-fold when assayed at 30 degrees C and pH 8.0. This stimulation correlates with binding of [32P]PPi to the second and third binding sites for PPi to be filled. Prior binding of PPi to nd-MF1 increases the rate of inactivation of the enzyme by FSBA at 23 degrees C about 4-fold when loss of activity is monitored at 30 degrees C and pH 8.0, whereas it does not affect the rate of inactivation when loss of ATPase is monitored at 15 degrees C or loss of ITPase is monitored at 30 degrees C. This indicates that the accelerated rate of inactivation induced by PPi when assays are conducted at 30 degrees C is not due to an increased rate of derivatization of noncatalytic sites. After 85% inactivation with FSBA, nd-MF1 retains the capacity to bind 2.8 mol of [32P]PPi per mole.(ABSTRACT TRUNCATED AT 250 WORDS)