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1.
The process of ATP or GTP synthesis by bovine heart submitochondrial particles involves the binding of ADP or GDP to 3 exchangeable sites I, II, and III, and only upon substrate occupation of site III does rapid ATP or GTP synthesis take place. The dissociation constants determined for ADP were KADPI less than or equal to 10(-8) M, KADPII approximately 10(-7) M, and KADPIII (equivalent to apparent KADPm), approximately 3 x 10(-6) M in the low Km mode and KADPIII approximately 150 x 10(-6) M in the high Km mode. For GDP, these constants were KGDPI approximately 10(-6)-10(-5) M, KGDPII approximately 10(-4) M, and KGDPIII approximately 10(-3) M when NADH was the respiratory substrate (Matsuno-Yagi, A., and Hatefi, Y. (1990) J. Biol. Chem. 265, 82-88). Because of its low affinity for the above binding sites, GDP at micromolar concentrations does not lead to GTP synthesis. However, as shown in this paper, micromolar [GDP] undergoes phosphorylation in the presence of micromolar concentrations of ADP. Under these conditions, both ATP and GTP are synthesized. GDP inhibits ATP synthesis with KGDPi congruent to 7 microM, while ADP promotes GTP synthesis in a reaction that requires inorganic phosphate (apparent KPim = 2-3 mM) and is inhibited by uncouplers and inhibitors of the ATP synthase complex. The ADP-promoted GTP synthesis exhibited an "apparent" KGDPm = 4 microM and an "apparent" Vmax = 11 nmol of GTP (min.mg of protein)-1. These results were interpreted to mean that (a) micromolar [ADP] occupies sites I and II, allowing site III to bind and phosphorylate GDP, and (b) the KGDPm and Vmax calculated under these conditions represent values for the low Km-low Vmax mode of GTP synthesis, which in the absence of ADP is not detectable because of the positive cooperativity phase of GTP synthesis with the high KGDPII approximately 10(-4) M. 相似文献
2.
Henry A. Lardy Sheldon M. Schuster Richard E. Ebel 《Journal of cellular biochemistry》1975,3(3):214-221
Evidence is presented that mitochondrial ATPase has two types of sites that bind adenine nucleotides. The catalytic site, C, binds the substrates ATP, GTP, or ITP and the inhibitor guanylyl imidodiphosphate (GMP-PNP). A second type of site, R, binds ATP, ADP, adenylyl imidodiphosphate (AMP-PNP), and the chromium complexes of ATP or ADP. All of these substances binding to the R site inhibit the hydrolysis of ATP in a competitive manner; their inhibition of hydrolysis of ITP and GTP is noncompetitive. GMP-PNP inhibits oxidative phosphorylation in submitochondrial particles but AMP-PNP does not. The localization on mitochondrial membranes of sites for the binding of various antibiotics that inhibit oxidative phosphorylation is discussed. 相似文献
3.
Three serine kinases which phosphorylate the CTD of RNA polymerase II have been identified in Aspergillus nidulans. The kinases (KI, KII, KIII) were identified using a synthetic peptide containing four copies of the CTD consensus heptamer repeat, and differ in chromatographic behavior, and apparent molecular mass (KI approximately 60kDa; KII approximately 82kDa; KIII approximately 43 kDa). KIII utilized, in addition to peptide, histone H1 as substrate, whereas casein was not phosphorylated by any of the three kinases. The kinases appear to be unrelated to the p34cdc2 kinase, as judged by Western blot analysis and the position of serine phosphorylation of the synthetic CTD peptide. KI was highly purified and renaturation experiments have shown that it consists of a single polypeptide of 57 kDa. KI also phosphorylated RNA polymerase II associated in a preinitiation complex. 相似文献
4.
Succinyl coenzyme A synthetase of Pseudomonas aeruginosa with a broad specificity for nucleoside triphosphate (NTP) synthesis modulates specificity for NTP synthesis by the 12-kilodalton form of nucleoside diphosphate kinase
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Pseudomonas aeruginosa secretes copious amounts of an exopolysaccharide called alginate during infection in the lungs of cystic fibrosis patients. A mutation in the algR2 gene of mucoid P. aeruginosa is known to exhibit a nonmucoid (nonalginate-producing) phenotype and showed reduced activities of succinyl-coenzyme A (CoA) synthetase (Scs) and nucleoside diphosphate kinase (Ndk), implying coregulation of Ndk and Scs in alginate synthesis. We have cloned and characterized the sucCD operon encoding the alpha and beta subunits of Scs from P. aeruginosa and have studied the role of Scs in generating GTP, an important precursor in alginate synthesis. We demonstrate that, in the presence of GDP, Scs synthesizes GTP using ATP as the phosphodonor and, in the presence of ADP, Scs synthesizes ATP using GTP as a phosphodonor. In the presence of inorganic orthophosphate, succinyl-CoA, and an equimolar amount of ADP and GDP, Scs synthesizes essentially an equimolar amount of ATP and GTP. Such a mechanism of GTP synthesis can be an alternate source for the synthesis of alginate as well as for the synthesis of other macromolecules requiring GTP such as RNA and protein. Scs from P. aeruginosa is also shown to exhibit a broad NDP kinase activity. In the presence of inorganic orthophosphate (P(i)), succinyl-CoA, and either GDP, ADP, UDP or CDP, it synthesizes GTP, ATP, UTP, or CTP. Scs was previously shown to copurify with Ndk, presumably as a complex. In mucoid cells of P. aeruginosa, Ndk is also known to exist in two forms, a 16-kDa cytoplasmic form predominant in the log phase and a 12-kDa membrane-associated form predominant in the stationary phase. We have observed that the 16-kDa Ndk-Scs complex present in nonmucoid cells, synthesizes all three of the nucleoside triphosphates from a mixture of GDP, UDP, and CDP, whereas the 12-kDa Ndk-Scs complex specifically present in mucoid cell predominantly synthesizes GTP and UTP but not CTP. Such regulation may promote GTP synthesis in the stationary phase when the bulk of alginate is synthesized by mucoid P. aeruginosa. 相似文献
5.
Nucleotide-depleted F1-ATPase from Escherichia coli was reconstituted with F1-depleted membranes and shown to catalyze high rates of oxidative phosphorylation of ADP and GDP. Adenine nucleotide became bound to the nonexchangeable nucleotide sites on membrane-bound F1 during ATP synthesis, but binding of guanine nucleotides to nonexchangeable sites during GTP synthesis was not detectable. It was possible to reload the nonexchangeable sites on nucleotide-depleted F1 with radioactive adenine nucleotide prior to membrane reconstitution. The radioactive adenine nucleotide did not exchange significantly during oxidative phosphorylation of ADP or GDP. The amount of nonexchangeable adenine nucleotide found in membrane-bound F1 was the same when the nonexchangeable sites were reloaded either prior to membrane reconstitution of the F1 or after membrane reconstitution with nucleotide-free F1 followed by a burst of oxidative phosphorylation of ADP. The results showed that occupation of the nonexchangeable sites on F1 by tightly bound nucleotide is not required for oxidative phosphorylation of GDP (a physiological activity of F1 in the bacterial cell). Also, the results confirm directly that the adenine-specific nonexchangeable sites on F1 are noncatalytic sites. Using this experimental approach, it was possible to look for a regulatory effect of the nonexchangeable nucleotide on oxidative phosphorylation. Nucleotide-depleted F1 was first reloaded with (i) ATP, (ii) ADP, (iii) 5'-adenylyl imidodiphosphate, or (iv) zero nucleotide, and was then reconstituted with F1-depleted membranes. The reconstituted membranes were compared in respect to rates of oxidative phosphorylation of GDP and Km values of GDP and Pi. No regulatory role for the nonexchangeable nucleotide was evident.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
The first complete sequences of functionally rearranged VK genes (abbreviations ref. 1) of subgroups II and III are reported. The genes have been cloned from lymphoid cell lines synthesizing KII or KIII light chains as evidenced from immunochemical analyses with anti-VK subgroup-specific antisera. These data, together with the sequence of a KIV gene (described in the accompanying paper) and those of previously published KI genes make possible a comparison of genes representative of the four known V region subgroups of human K light chains. The VKII gene is distinguished from the VKI, VKIII, and VKIV genes by a much longer intron within the leader sequence: 426 bp vs ca. 120-220 bp. Blot hybridization experiments with human DNA digests using probes from the KII and KIII genes and from the respective upstream regions help to define subgroup specific probes and hybridization conditions. 相似文献
7.
Formation of a ternary initiation complex containing Met-tRNAf, GTP and eukaryotic initiation factor 2, is the first step in sequential assembly of the initiation complex. The concentration of GTP required for half maximal formation of the ternary complex is 2.5 with 10(-6) M. GDP is a potent competitive inhibitor of ternary complex formation with Ki = 3.4 with 10(-7) M. The nucleotide binding site on eukaryotic initiation factor 2 demonstrates relative specificity for GDP with KD(GDP) = 3.0 with 10(-8) M; 100-fold higher concentrations of GTP than GDP are required for displacement of either [(3)H]GDP or [(3)h]gtp from the necleotide binding site. An ATP-dependent stimulation of ternary complex formation observed in partially purified initiation factor preparations is due to nucleoside diphosphate kinase (EC 2.7.4.6) which serves to remove inhibitory levels of GDP by phosphorylation with ATP. Since GTP is hydrolyzed to GDP during protein synthesis, this provides a mechanism by which the ATP:ADP ratio may regulate the rate of initiation of protein synthesis. 相似文献
8.
Muscarinic agonists and adenyl nucleotides are noncompetitive modulators of sites labeled by [35S]GTP gamma S in washed cardiac membranes from Syrian golden hamsters. Specific binding of the radioligand and its inhibition by either GTP gamma S or GDP reveals three states of affinity for guanyl nucleotides. In the absence of adenyl nucleotide, carbachol promotes an apparent interconversion of sites from higher to lower affinity for GDP; the effect recalls that of guanyl nucleotides on the binding of agonists to muscarinic receptors. In the presence of 0.1 mM ATP gamma S, the binding of [35S]GTP gamma S is increased at concentrations up to about 50 nM and decreased at higher concentrations. At a radioligand concentration of 160 pM, binding exhibits a bell-shaped dependence on the concentration of both ATP gamma S and AMP-PNP; with ADP and ATP, there is a second increase in bound [35S]GTP gamma S at the highest concentrations of adenyl nucleotide. ATP gamma S and AMP-PNP also modulate the effect of GDP, which itself emerges as a cooperative process: that is, binding of the radioligand in the presence of AMP-PNP exhibits a bell-shaped dependence on the concentration of GDP; moreover, the GDP-dependent increase in bound [35S]GTP gamma S is enhanced by carbachol. The interactions among GDP, GTP gamma S, and carbachol can be rationalized quantitatively in terms of a cooperative model involving two sites tentatively identified as G proteins. Both GTP gamma S and GDP exhibit negative homotropic cooperativity; carbachol enhances the homotropic cooperativity of GDP and induces or enhances positive heterotropic cooperativity between GDP and [35S]GTP gamma S. An analogous mechanism may underlie the guanyl nucleotide-dependent binding of agonists to muscarinic receptors. The data suggest that the binding properties of G proteins and their associated receptors reflect cooperative effects within heterooligomeric arrays; agonist-induced changes in cooperativity may facilitate the exchange of GTP for bound GDP and thereby constitute the mechanism of G protein activation in vivo. 相似文献
9.
The binding parameters of a number of ADP or ATP analogs to the adenine nucleotide carrier in mitochondria and inside-out submitochondrial particles have been explored by means of two specific inhibitors, carboxyatractyloside and bongkrekic acid. The nucleotides tested fell into two classes depending on the shape of the binding curve. Curvilinear Scatchard plots were obtained for the binding of ADP, ATP, adenosine 5'-triphospho-gamma-1-(5-sulfonic acid)naphthylamidate [gamma-AmNS)ATP) and adenylyl (beta,gamma)-methylenediphosphate (p[CH2]ppA); on the other hand, rectilinear Scatchard plots were obtained in the case of naphthoyl-ADP (N-ADP) and 8-bromo ADP (8Br-ADP) binding. The total number of binding sites for N-ADP and 8Br-ADP could be extrapolated with good accuracy to 1.3-1.5 nmol/mg protein; this value corresponds to the number of carboxyatractyloside-binding sites in heart mitochondria (Block, M.R., Pougeois, R. and Vignais, P.V. (1980) FEBS Lett. 117, 335-340). On the other hand, because of the curvilinearity of the Scatchard plots for the binding of ADP, ATP, (gamma-AmNS)ATP and p[CH2]ppA, the total number of binding sites for these nucleotides could only be approximated to a value higher than 1 nmol/mg protein, the exact value being probably equal to that found for N-ADP and 8Br-ADP binding, i.e. 1.3-1.5 nmol/mg protein. Curvilinearity of Scatchard plots was discussed in terms of negative interactions between nucleotide-binding sites located on the same face of the adenine nucleotide carrier. A possible relationship between the features of the binding plots and the transportable nature of the nucleotide is discussed. Contrary to the enhancing effect of bongkrekic acid on [14C]ADP uptake observed essentially in nucleotide-depleted heart mitochondria (Klingenberg, M., Appel, M., Babel, W. and Aquila, H. (1983) Eur. J. Biochem. 131, 647-654), binding of bongkrekic acid to nondepleted heart mitochondria was found to partially displace previously bound [14C]ADP. These opposite effects of bongkrekic acid may be explained by assuming that bongkrekic acid is able to abolish negative cooperativity between external (cytosolic) ADP-binding sites. 相似文献
10.
The rat elongation factor eEF-2 catalyzes the translocation step of protein synthesis. Besides its well-characterized GTP/GDP binding properties, we have previously shown that ATP and ADP bind to eEF-2 [Sontag, B., Reboud, A. M., Divita, G., Di Pietro, A., Guillot, D., and Reboud, J. P. (1993) Biochemistry 32, 1976-1980]. However, whether the adenylic and guanylic nucleotide binding sites were different or not remained unclear. To further characterize these sites, eEF-2 was incubated in the presence of N-methylanthraniloyl (Mant) fluorescent derivatives of GTP, GDP, ATP, and ADP. This led to an increase in the probe fluorescence and to a partial quenching of eEF-2 tryptophans in each case. The Mant-derivatives and the unmodified corresponding nucleotides were shown to bind to eEF-2 with a similar affinity. Competition experiments between Mant-labeled and unmodified nucleotides suggested the presence of two different sites binding either guanylic or adenylic nucleotides. A F?rster's transfer between tryptophan residues and the Mant-probe is obtained with both the adenylic and the guanylic Mant-nucleotides, and comparison of the transfer efficiencies confirmed the presence of a second binding site specific for adenylic nucleotides. A sequence alignment of EF-Gs with eEF-2s from different species suggests the presence of potential Walker A and B motifs in an insert of the G-domain of eEF-2s from higher eukaryotes. Our results raise the possibility that a site specific for adenylic nucleotides and located in this insert has appeared in the course of evolution although its physiological function is still unknown. 相似文献
11.
Escherichia coli F1-ATPase contained 3 mol of tightly-bound adenine nucleotide/mol enzyme. A further 3 mol could be loaded by incubation of the enzyme with ATP. The unloaded enzyme was designated as a F1[2,1] type on the basis of the ability of GTP to displace 1 mol of adenine nucleotide/mol of F1 [Kironde, F.A.S., & Cross, R.L. (1986) J. Biol. Chem. 261, 12544-12549]. The loaded enzyme was designated F1[3,3] since GTP could displace 3 of the 6 mol of bound adenine nucleotide/mol of F1. Incubation of F1[2,1], F1[2,0], and F1[3,0] with phosphate in the presence of 30% (v/v) dimethyl sulfoxide led to the synthesis of ATP from endogenous bound ADP. Hydrolysis of newly synthesized ATP occurred on transfer of the F1 from 30% (v/v) dimethyl sulfoxide to an entirely aqueous medium. Thus, synthesis and hydrolysis of ATP can occur at GTP-nonchaseable adenine nucleotide binding sites, and these sites in dimethyl sulfoxide are not necessarily equivalent to noncatalytic sites. 相似文献
12.
Amino acid sequence of the N-terminal 139 residues of light chain derived from a homogeneous rabbit antibody 总被引:7,自引:4,他引:3
Jean-Claude Jaton 《The Biochemical journal》1974,141(1):1-13
The amino acid sequence of the N-terminal 139 residues of the L (light) chain derived from a homogeneous rabbit antibody (designated BS-1) to type III pneumococci was determined. A combination of methods involving tryptic cleavage restricted to the 2 arginine residues of the molecule and mild acid hydrolysis of a labile peptide bond between the V (variable) and C (constant) regions of the L chain (Fraser et al., 1972) allowed the isolation of two large peptides comprising the entire V region (residues 1-109); these peptides were suitable for automated Edman degradation. The complete sequence analysis of the V region was carried out with only 4mumol of L chain. This material was homogeneous, although minor variant sequences, if present at the 10% value, would not have been detected. The L chain contains 3 intrachain disulphide bridges, whose pairing was established by diagonal electrophoresis: there is one V-region bridge between positions 23 and 88 and one C-region bridge between positions 134 and 194; the third one connects V and C domains between positions 80 and 171. When compared with the basic sequence of human kappa chains, rabbit L chain BS-1 appears to be more similar to the V(KI) prototype sequence than to V(KII) or V(KIII) sequences, where V(KI), V(KII) and V(KIII) represent subgroups I, II and III respectively of V regions of kappa light chains. The V regions of rabbit heavy and light chains are homologous to each other. The presence of two clusters of 3 glycine residues in positions 94-96 and 99-101 respectively is remarkable. Residues 94-96 may be related to antibody complementarity whereas residues 99-101 function probably as a pivot permitting the combining region of the L chain to make optimal contact with the antigenic determinant (Wu & Kabat, 1970). 相似文献
13.
Banerji S Ngo M Lane CF Robinson CA Minogue S Ridgway ND 《Molecular biology of the cell》2010,21(23):4141-4150
Cholesterol and sphingomyelin (SM) associate in raft domains and are metabolically coregulated. One aspect of coordinate regulation occurs in the Golgi apparatus where oxysterol binding protein (OSBP) mediates sterol-dependent activation of ceramide transport protein (CERT) activity and SM synthesis. Because CERT transfer activity is dependent on its phosphatidylinositol 4 phosphate [PtdIns(4)P]-specific pleckstrin homology domain, we investigated whether OSBP activation of CERT involved a Golgi-associated PtdIns 4-kinase (PI4K). Cell fractionation experiments revealed that Golgi/endosome-enriched membranes from 25-hydroxycholesterol-treated Chinese hamster ovary cells had increased activity of a sterol-sensitive PI4K that was blocked by small interfering RNA silencing of OSBP. Consistent with this sterol-requirement, OSBP silencing also reduced the cholesterol content of endosome/trans-Golgi network (TGN) fractions containing PI4KIIα. PI4KIIα, but not PI4KIIIβ, was required for oxysterol-activation of SM synthesis and recruitment of CERT to the Golgi apparatus. However, neither PI4KIIα nor PI4KIIIβ expression was required for 25-hydroxycholesterol-dependent translocation of OSBP to the Golgi apparatus. The presence of OSBP, CERT, and PI4KIIα in the TGN of oxysterol-stimulated cells suggests that OSBP couples sterol binding or transfer activity with regulation of PI4KIIα activity, leading to CERT recruitment to the TGN and increased SM synthesis. 相似文献
14.
The KI and KII sites play a crucial role in kappa-chain gene rearrangement, which was investigated in mice deficient for these sites. Previously, we found that Pax-5 can bind to the KI and KII sites; however, the function of Pax-5 in kappa-chain gene rearrangement has not been investigated. Here, we have used an in vitro culture system in which differentiation from pre-B cells to immature B cells is induced by removing IL-7. We showed that, after the induction of differentiation, Pax-5 dissociated from the KI and KII revealed by EMSA analyses, and this dissociation occurred specifically at the KI and KII sites, but not at the Pax-5 binding site, in the CD19 promoter because of a lower binding affinity of Pax-5 for the KI and KII sites. During differentiation induced by removing IL-7, the underphosphorylated form of retinoblastoma preferentially associated with Pax-5, which caused dissociation of Pax-5 from KI and KII sites. These results suggest that the dissociation of Pax-5 from the KI and KII sites is important in the induction of kappa-chain gene rearrangement. 相似文献
15.
Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited. 相似文献
16.
The F1-ATPase from Micrococcus lysodeikticus is isolated in the absence of exogenous nucleotides. After removing loosely bound nucleotides from the isolated enzyme by gel permeation chromatography, analysis for tightly bound nucleotides revealed in 14 experiments 0.4 +/- 0.1 mol ADP, 0.5 +/- 0.2 mol GDP, and 0.8 +/- 0.2 mol ATP per mol of F1. Incubation of the isolated enzyme with Mg2+ or Ca2+ did not alter the endogenous nucleotide composition of the enzyme, indicating that endogenous ATP is not bound to a catalytic site. Incubation of the enzyme with P(i) decreased the amount of tightly bound ADP and GDP but did not effect the ATP content. Hydrolysis of MgATP in the presence of sulfite raised the tightly bound ADP and lowered tightly bound GDP on the enzyme. In the reciprocal experiment, hydrolysis of MgGTP in the presence of sulfite raised tightly bound GDP and lowered tightly bound ADP. Turnover did not affect the content of tightly bound ATP on the enzyme. These results suggest that endogenous ADP and GDP are bound to exchangeable catalytic sites, whereas endogenous ATP is bound to noncatalytic sites which do not exchange. The presence of endogenous GDP on catalytic sites of isolated F1 suggests that the F0F1-ATP synthase of M. lysodeikticus might synthesize both GTP and ATP under physiological conditions. In support of this hypothesis, we have found that plasma membrane vesicles derived from M. lysodeikticus synthesize [32P]GTP from [32P]P(i) using malate as electron donor for oxidative phosphorylation. 相似文献
17.
The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1. 相似文献
18.
Studies on the mechanism of oxidative phosphorylation. Catalytic site cooperativity in ATP synthesis
Oxidative phosphorylation catalyzed by bovine heart submitochondrial particles appears to exhibit negative cooperativity with respect to [ADP] and positive cooperativity in catalysis. Eadie-Hofstee plots (v/[S]versus v) of the kinetics of oxidative phosphorylation at the variable ADP concentration range of 1-1200 microM were curvilinear and could be analyzed for two apparent KmADP values differing by one order of magnitude, and two apparent Vmax values. The KmADP values with either NADH or succinate as the respiratory substrate were in the ranges of 10 and 100 microM, and the Vmax values in nmol of ATP formed X min-1 (mg of protein)-1 were, respectively, 500 and 1840 when NADH was the oxidizable substrate, and 550 and 100 when succinate was the energy source. Site-site cooperativity of the ATP synthase, which is a central feature of current theories for the mechanism of oxidative phosphorylation, has been well-documented for ATP hydrolysis by isolated F1-ATPase, but never before demonstrated for mitochondrial ATP synthesis. 相似文献
19.
F1I, the specific ATPase inhibitor protein, and the chromium(III) analogs of ATP and ADP, CrATP and CrADP, were used to study the inhibition of Pi goes to and comes from ATP exchange reaction catalyzed by beef heart submitochondrial particles. F1I was found to be an uncompetitive inhibitor of the exchange reaction. CrATP and CrADP, both competitive inhibitors of ATP hydrolysis in isolated F1 (Schuster, S. M., Ebel, R. E., and Lardy, H. A. (1975) ARch. Biochem. Biophys. 171, 656-661) were shown to be competitive and noncompetitive inhibitors of Pi goes to and comes from ATP exchange, respectively. Dual inhibitor studies were done using combinations of F1I and the chromium nucleotides, or the nucleotide analogs in combination. All cases show sets of intersecting Dixon plots indicative of interacting inhibitors. Upward curvature is also evident on some of the plots. This phenomenon was explained using the concept of multiple synergistic binding of the inhibitors. Binding mechanisms and their relevant kinetic equations were postulated to explain the results of the dual inhibitor studies. They support the notion that in addition to the catalytic site, there are two types of regulatory binding sites on F1, one specific for nucleotides and one specific for F1I. When one of these sites is occupied, other sites are either opened or other inhibitors become more potent. 相似文献
20.
The interaction of inorganic phosphate with native and nucleotide-depleted F1-ATPase was studied. F1-ATPase depleted of tightly bound nucleotides loses the ability to bind inorganic phosphate. The addition of ATP, ADP, GTP and GDP but not AMP, restores the phosphate binding. The nucleotides affecting the phosphate binding to F1-ATPase are located at the catalytic (exchangeable) site of the enzyme. The phosphate is thought to bind to the same catalytic site where the nucleotide is already bound. It is thought that ADP is the first substrate to bind to F1-ATPase in the ATP synthesis reaction. 相似文献