Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A facile and high-yield synthesis of a new ATP analogue, 2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate (NANTP), is described. NANTP and ATP are hydrolyzed by skeletal myosin subfragment 1 (SF1) at comparable rates in the presence of Ca2+, Mg2+, or NH4+-EDTA. NANTP is also cleaved but less readily by mitochondrial F1-ATPase and by (Na+ + K+)-ATPase from dog brain and hog kidney. F-Actin markedly activates NANTP cleavage by SF1 in the presence of Mg2+, suggesting that the diphosphate product NANDP is slow to be released from the enzyme. [alpha-32P]NANDP binds to a single site on SF1 (KA = 1 X 10(6) M-1) with an affinity identical with that of ADP. The absorption maximum of NANDP was shifted from 474 to 467 nm upon binding to SF1, suggesting that the purine binding site has a dielectric constant of about 45. NANDP was trapped in nearly stoichiometric amounts at the active site by cross-linking SH1 and SH2 with N,N'-p-phenylenedimaleimide (pPDM) or by chelation with cobalt (III) phenanthroline [Wells, J., & Yount, R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966]. The trapped [beta-32P]NANDP X SF1 complex, like the comparable ADP X SF1 complex, was stable for days at 0 degree C and could be purified free of extraneous analogue by ammonium sulfate precipitation and gel filtration. Photolysis of the purified complex gave greater than 50% covalent incorporation of the trapped NANDP into the 95-kilodalton (kDa) heavy chain of SF1. Limited trypsinization and analysis by gel electrophoresis showed that greater than 95% of the bound label was associated with the 25-kDa NH2-terminal peptide. Without trapping, NANDP labeling of SF1 was nonspecific and was not prevented by addition of a large excess of ATP. This new approach of trapping photoaffinity analogues by cross-linking agents before photolysis may prove to be of general usefulness in increasing the specificity and extent of labeling of enzymes that undergo substrate-induced conformation changes.
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PMID:2-[(4-Azido-2-nitrophenyl)amino]ethyl triphosphate, a novel chromophoric and photoaffinity analogue of ATP. Synthesis, characterization, and interaction with myosin subfragment 1. 407 91

(1) Mitochondrial ATPase (F1) is influenced by specific nucleotides in its kinetic behavior towards its substrates. In this work, initial hydrolysis rates, as well as continuous reaction progress, were measured by recording proton production (equivalent to triphosphate hydrolysis). (2) After preincubation with ATP, F1 hydrolyzes MgITP partly as if it were MgATP, with respect to temperature dependence and 2,4-dinitrophenol inhibition/stimulation. (3) Acetyl ATP is a competitive inhibitor versus ATP on the F1-ATPase. With F1 which has been freed of ambient ATP by repeated precipitations with ammonium sulfate the Ki of acetyl ATP is 400 nM. (4) F1-ATPase which was depleted of bound nucleotides in the presence of glycerol (Garret, N.E. and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647) was preincubated with ADP and acetyl ATP. These preparations were assayed for hydrolytic activity with MgITP as substrate. Compared to a nonpreincubated control enzyme, the hydrolysis with these preparations was first stimulated, then inhibited. This stimulation/inhibition effect is most pronounced at 10 degrees C, but is also observed at 20 degrees C. (5) When nucleotide-depleted enzyme is preincubated with acetyl AMP, its ability to hydrolyze MgITP slowly decreases to approx. 50% after 60 min. This effect is reversed by further preincubation with acetyl ATP. It is speculated that under appropriate conditions AMP may exist or arise in a buried position on F1-ATPase, and act there as an inhibitor of MgITP hydrolysis.
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PMID:ATPase of bovine heart mitochondria. Modulation of ITPase activity by ATP, ADP, acetyl ATP and acetyl AMP. 613 89

Four stable murine hybridoma clones producing homogeneous antibodies against pig heart mitochondrial F1-ATPase have been established. All antibodies exhibit specific binding to F1-ATPase. Three of them interact with the beta subunit and one binds strongly to the alpha and barely to the beta subunit. All of them exhibit linear Scatchard plots and high binding affinities, with dissociation constants between 4.7 X 10(-8) M and 6.5 X 10(-10) M. The minimal number of moles of IgG bound at saturation per mole of immobilized F1-ATPase is 2.2 for the anti-alpha-subunit antibody and 2.5 for one anti-beta-subunit antibody. This suggests that more than two copies of the alpha and beta subunits are present in the enzyme. Two antibodies seem to recognize F1-ATPase equally before or after denaturation with sodium dodecyl sulfate. The two other antibodies exhibited a much higher affinity for the nondissociated enzyme, indicating that they are very sensitive to a specific conformation of the enzyme.
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PMID:Characterization of monoclonal antibodies against mitochondrial F1-ATPase. 619 26

A previously found yeast-mitochondrial protein fraction stabilizing the inactivated complex between mitochondrial ATPase and intrinsic ATPase inhibitor (Hashimoto, T., et al. (1983) J. Biochem. 94, 715-720) was separated into two proteins by high performance liquid chromatography on a cation exchanger. The molecular weights of the factors were estimated to be 9,000 and 15,000 daltons by sodium dodecyl sulfate (SDS)-gel electrophoresis. Both factors were required to stabilize a complex of inhibitor and proton-translocating ATPase (F1F0-ATPase) either in its purified form or in mitochondrial membranes. On the other hand both factors together could not stabilize a complex of the inhibitor and F1-ATPase, suggesting that both factors act together with the F0-portion. The factors also facilitated very efficiently the binding of ATPase inhibitor to F1F0-ATPase in the presence of ATP and Mg2+. Both the 15,000 and 9,000 dalton stabilizing factors were hardly distinguishable from delta- and epsilon-subunit, respectively, on an SDS-gel electrophoregram, but immuno-diffusion assay showed that neither factor was present in the purified F1-ATPase containing the delta- and epsilon-subunit.
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PMID:Purification and properties of factors in yeast mitochondria stabilizing the F1F0-ATPase-inhibitor complex. 620 Apr 68

(1) The natural ATPase inhibitor (IF1) from beef heart mitochondria has a tendency to form aggregates in aqueous solutions. The extent of aggregation and the structure of the aggregates were assessed by gel filtration and small-angle neutron scattering. IF1 polymerization was found to depend on the salt concentrations, pH of the medium and concentration of IF1. The higher the salt concentration, the lower the aggregation state. Aggregation of IF1 was decreased at slightly acidic pH. It increased with the concentration of IF1 as expected from the law of mass action. (2) Neutron scattering showed the aggregation of IF1 in 2 M ammonium sulfate solutions. The predominant species is the dimer which has a somewhat elongated shape. (3) The Sephadex G-50 chromatography that is supposed to deprive beef heart submitochondrial particles of loosely bound IF1 (Racker, E. and Horstman, L.L. (1967) J. Biol. Chem. 242, 2547-2551) was shown to have a limited effectiveness as a trap for IF1. The reason was that IF1 released from the particles formed high molecular weight aggregates that were not separated from the membrane vesicles by Sephadex G-50 chromatography. (4) The above observations provide the basis for a simple method of purification of beef heart IF1 which combines the recovery of the supernatant from submitochondrial particles with the last three steps of the IF1 preparation described by Horstman and Racker (J. Biol. Chem. (1970) 265, 1336-1344). The particles recovered in the sediment were deprived of IF1 and could therefore be used for preparation of F1-ATPase. The advantage of this method is that both IF1 and F1-ATPase can be prepared from the same batch of mitochondria.
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PMID:Spontaneous aggregation of the mitochondrial natural ATPase inhibitor in salt solutions as demonstrated by gel filtration and neutron scattering. Application to the concomitant purification of the ATPase inhibitor and F1-ATPase. 621 74

Upon incubation with trypsin, the adenosine-5'-triphosphatase (ATPase) activity of the nucleotide-depleted F1 is first rapidly and slightly activated and then slowly inactivated. The first phase is simultaneous with the conversion of the alpha subunit into an alpha' fragment which migrates between alpha and beta on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The second phase is related to the proteolysis of the three main subunits, alpha', beta, and gamma. Preincubation of the enzyme with low concentrations of adenosine 5'-diphosphate (ADP) or adenosine 5'-triphosphate (ATP) does not modify the slight increase of activity but efficiently prevents the inactivation induced by trypsin. The alpha leads to alpha' conversion is not affected whereas the further proteolysis of alpha', beta, and gamma does not occur. On the contrary, even high concentrations of GDP only slightly lower the trypsin-induced inactivation. The presence of endogenous tightly bound nucleotides also partially lowers the sensitivity to trypsin since F1 is less rapidly inactivated and proteolyzed than the nucleotide-depleted F1. Phosphate, at high concentrations, both slows down the first phase of activation and simultaneous alpha leads to alpha' conversion and prevents the second phase of inactivation and proteolysis of the main subunits. Pretreatment of the nucleotide-depleted F1 with trypsin under conditions where the ATPase activity is largely inhibited only slightly modifies, however, the hysteretic behavior of the enzyme: the ADP binding and the concomitant hysteretic inhibition of the residual activity are not markedly diminished. The purified ATPase-ATP synthase complex binds very few ADP's and is not hysteretically inhibited. Its ATPase activity is rapidly activated but not further inhibited by trypsin. Preincubation of the complex with ADP does not modify the effects of trypsin.
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PMID:Use of trypsin to monitor conformational changes of mitochondrial adenosinetriphosphatase induced by nucleotides and phosphate. 622 Jul 37

One of the mitochondrically coded components of the yeast mitochondrial ATPase complex (subunit 6) can be resolved into two components on certain polyacrylamide gels in the presence of sodium dodecyl sulfate. Purification of the ATPase complex from commercially processed yeast as well as immunoprecipitation of the holo-enzyme from cells labeled in vivo with 14C-labeled amino acids demonstrate that both forms of subunit 6 are physically associated with the assembled enzyme and present in two copies each per complex. One-dimensional papain-generated peptide maps of the two components are identical except for the mobility of a single fragment. It is concluded that the two components of subunit 6 are different forms of a single protein and are present on an average of two copies each per complex.
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PMID:Presence and stoichiometry of two forms of subunit 6 of the mitochondrial ATPase complex of yeast. 622 89

The soluble F1 moiety of the rat liver mitochondrial proton ATPase dissociates into two easily separable fractions when cold treated and then warmed. One fraction is soluble in potassium phosphate buffer, pH 7.4, whereas the other is insoluble. Neither of these two fractions alone can catalyze ATP hydrolysis under assay conditions optimal for the native F1-ATPase. The insoluble fraction when resolved via sodium dodecyl sulfate--polyacrylamide gel electrophoresis is shown to be composed of only alpha and gamma subunits. When this fraction is chromatographed on Sephadex G-75, it is resolved into an alpha gamma complex and into the alpha subunit alone. The soluble fraction when resolved in the same electrophoretic system is shown to contain the remaining subunits, beta, delta, epsilon, and some gamma. This fraction is resolved into two major components by chromatography on Sepharose CL-6B, a beta gamma complex and beta subunit alone. The cold-dissociated enzyme can be readily associated when the temperature is raised to 20 degrees C. In the presence of either ATP or MgATP the enzyme completely regains its original ATPase specific activity. In contrast, Mg2+ is only about 15% effective in restoring ATPase activity. The results presented here define conditions for the dissociation and reassociation of the major subunits comprising the F1-ATPase of rat liver and thus provide a unique system among mammalian enzymes for testing the function of individual subunits. In addition, they strongly indicate that neither the alpha nor beta subunits, nor complexes of these subunits with the gamma subunit, are capable of catalyzing ATP hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The proton adenosinetriphosphatase complex of rat liver mitochondria. Temperature-dependent dissociation-reassociation of the F1-ATPase subunits. 623 51

A sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system was used to investigate certain properties of the subunits of the beef heart mitochondrial ATPase, (native F1, nF1). By examining the affects of urea concentration and acrylamide concentration upon the electrophoretic mobilities of the polypeptides comprising the nF1 enzyme, we have obtained conditions under which all five subunits are simultaneously resolved when the discontinuous buffer system of Laemmli is used (U. K. Laemmli (1970) Nature (London) 277, 680-685). The determination of the apparent molecular weights by analysis of Ferguson plots (K. A. Ferguson (1964) Metabolism 13, 985-1002) revealed that the addition of urea to the SDS gels resulted in a decrease in the apparent molecular weight of the beta subunit. A dramatic increase in the apparent molecular weight of the delta subunit was also brought about by the presence of urea in the SDS gels. In addition, the apparent molecular weight of both the alpha and the beta subunits was dependent upon the acrylamide concentration used, indicating that these subunits contain either areas highly resistant to denaturation by the combined action of urea and SDS, or covalent modifications leading to anomalous electrophoretic mobility. The results of experiments in which urea analogs were used indicate that the interactions of urea with the beta subunit involve the formation of hydrogen bonds between urea and regions of this subunit. On the other hand, the interactions of urea with the delta subunit are primarily of a hydrophobic nature, suggesting that these interactions could involve domains of the delta subunit required for binding of the coupling factor to the mitochondrial membrane.
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PMID:Hydrophobic and ionic effects upon the electrophoretic mobilities of the subunits of coupling factor 1 from mitochondria. 623 67

Polyacrylamide gel electrophoresis in the presence of a cationic detergent, tetradecyltrimethylammonium bromide (TDAB) has been compared to electrophoresis in the presence of an anionic detergent, sodium dodecyl sulfate (SDS). Although, in both systems, the peptides generally migrated as a function of their molecular weight, the TDAB electrophoresis permitted us to obtain a much better resolution of several peptides of the mitochondrial F0-F1-ATPase, especially for the alpha and beta subunits and for the oligomycin sensitivity conferring protein (OSCP). The differences between the two electrophoretic profiles have been used to devise a new technique of two-dimensional electrophoresis using successively anionic and cationic detergents. This method could be very useful in the case of membrane proteins, which are generally soluble only in the presence of powerful ionic detergents. It has been particularly successful in resolving the small peptides of the F0-F1-ATPase which were difficult to differentiate by other techniques in one- or two-dimensional polyacrylamide gel electrophoresis.
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PMID:Two-dimensional gel electrophoresis of membrane proteins using anionic and cationic detergents. Application to the study of mitochondrial F0-F1-ATPase. 623 56


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