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)

Peptides generated from enzymatic hydrolysis of chicken enolase and the alpha- and beta-subunits of bovine F1-ATPase were analyzed by mass spectrometry to determine the nature of their modified N-termini. In the case of chicken enolase, a peptide was isolated from a Staphylococcus aureus proteinase digest by HPLC and analyzed directly by fast atom bombardment mass spectrometry (FABMS). In conjunction with mass spectral evidence obtained from the methyl ester derivative and a secondary tryptic peptide, a structure is proposed containing an N-acetyl serine at the N-terminus. The alpha-subunit of bovine mitochondrial ATPase was chromatographed by HPLC after S. aureus proteinase digestion and a single peak was analyzed on the basis of predicted retention times. A Mr 716 was determined by FABMS and pyrrolidone carboxylic acid was deduced on the basis of its amino acid composition and partial Edman sequence data. The beta-subunit of ATPase produced a series of closely eluting peaks on HPLC after limited digestion with trypsin of the alpha 2 beta 2 complex. These peptides were analyzed by both Edman degradation and FABMS. These data showed the N-terminus to be frayed with N-terminal sequences beginning in pyro-Glu-Ala-Ser, Gln-Ala-Ser, Glu-Ala-Ser, Ala-Ser, and Ser but with no N-acetyl-Ser as was previously thought.
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PMID:Structural elucidation of N-terminal post-translational modifications by mass spectrometry: application to chicken enolase and the alpha- and beta-subunits of bovine mitochondrial F1-ATPase. 289 18

The alpha-subunit of Escherichia coli F1-ATPase contains an adenine-specific noncatalytic nucleotide-binding domain. A recent proposal (Maggio, M. B., Pagan, J., Parsonage, D., Hatch, L., and Senior, A. E. (1987) J. Biol. Chem. 262, 8981-8984) suggested that this domain is formed by residues 160-340, approximately, in alpha-subunit. Within this proposed domain is a sequence Gly-X-X-X-X-Gly-Lys which is conserved in a large and diverse group of nucleotide-binding proteins and is thought to interact with phosphate groups of bound nucleotide. In this work, residue alpha Lys-175, the terminal residue of the above conserved sequence in F1-alpha-subunit, was mutagenized to Ile or Glu. The specific activity of purified mutant F1-ATPase was reduced by 2.5-fold (Ile) or 3-fold (Glu). Apparent binding of ATP to alpha-subunit, as measured by the centrifuge column procedure, was strongly impaired and ATP-induced conformational change in alpha-subunit, as measured by protection against trypsin proteolysis, was nearly abolished in both mutants. The results suggest that residue alpha Lys-175 is located within the nucleotide-binding domain of alpha-subunit, and that this residue is functionally involved in nucleotide binding. The results support previous suggestions that the alpha-subunit nucleotide-binding site is not involved, directly or indirectly, in catalysis.
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PMID:Directed mutagenesis of the strongly conserved lysine 175 in the proposed nucleotide-binding domain of alpha-subunit from Escherichia coli F1-ATPase. 290 46

Polar membrane in Campylobacter jejuni has been visualized on membrane vesicles. It was composed of doughnut-shaped particles 5-6 nm in diameter, with stalks, arranged in a hexagonal array. This structure was stabilized on the membrane by a high ionic strength buffer in the presence of 2-mercaptoethanol. Histochemical staining indicated localized ATPase activity at the poles of the cells. An ATPase with distinctive properties has been isolated and purified from this organism; it gives a specific activity of approximately 0.3 units/mg of protein. Electron microscopy showed doughnut-shaped particles 5-6 nm in diameter. Nondissociating and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed, respectively, a single band with ATPase activity and a molecular weight of ca. 75,000 Da. The enzyme was cold labile and activity was abolished by trypsin. Dicyclohexylcarbodiimide inhibited the membrane-bound form of the enzyme, but did not inhibit the soluble form. Oligomycin had no inhibitory activity on either form of the enzyme. The enzyme specifically hydrolysed ATP, but other nucleotide substrates were not degraded. The enzyme was activated by Mg2+ and inhibited by Ca2+, whereas other ions had no effect on activity. Antibodies prepared to this enzyme bound to the polar regions of whole cells as shown by protein A - colloidal gold immunoelectron microscopy. The antibodies to this ATPase cross reacted (shown by Western blotting) with four proteins from a whole-cell extract of this organism, two proteins in Aquaspirillum serpens MW5, and three proteins from Escherichia coli K12. They did not cross-react with any proteins from Spirillum volutans, Methanococcus voltae, Vibrio cholerae, or rat liver mitochondria. Antibodies raised against the F1-ATPase of E. coli K12 cross reacted with six proteins in a whole-cell extract of this organism, and one protein species in each of the whole-cell extracts of V. cholera, A. serpens MW5, S. volutans, and rat liver mitochondria. These antibodies did not recognize any whole cell proteins from either C. jejuni or M. voltae. These results along with the ATPase activity localized by histochemical staining suggest that polar membrane is an assembly of ATPase molecules at the poles of the cell and that the ATPase isolated from C. jejuni is serologically and structurally unusual.
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PMID:The ultrastructure and ATPase nature of polar membrane in Campylobacter jejuni. 297 56

1. Oligomycin and dicyclohexylcarbodiimide-sensitive ATPase was isolated from beef-heart mitochondria and treated with 3.5 M NaBr in order to remove F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecylsulfate-polyacrylamide-gel electrophoresis. Two of them correspond to the oligomycin-sensitivity-conferring protein and coupling factor F6, with apparent molecular weights of 21,000 and 9,400, respectively. Three additional polypeptides of molecular weights 23,000, 10,500 and 8,600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide-binding protein. 2. F0 induced an oligomycin and dicyclohexylcarbodiimide-sensitive enhancement of K+ + valinomycin-driven proton translocation across the membrane of artificial phospholipid vesicles. 3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1-ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 in both an oligomycin-sensitive and an oligomycin-insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F0/F1 ratios favoured in oligomycin-sensitive type of binding, indicating that F1 binds preferentially to oligomycin-sensitivity-conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin-sensitivity-conferring binding sites and a diminished ability to lower the specific activity an cold lability of F1. 4. Reconstitution of F0 treated with trypsin and F1, oligomycin-sensitivity-conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin-sensitivity-conferring protein and F6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity. 5. The effect of the order of addition of F1, oligomycin-sensitivity-conferring protein and F6 to F0 on the reconstitution of oligomycin-sensitive ATPase activity, and of F1 and oligomycin-sensitivity-conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components.
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PMID:Reconstitution of mitochondrial oligomycin and dicyclohexylcarbodiimide-sensitive ATPase. 610 10

The complement fixing antigen of the inner mitochondrial membrane previously shown to be associated with the mitochondrial ATPase could be further purified by subjecting the ATPase extracted from beef heart and brown fat mitochondria to ion exchange and gel filtration chromatography. Although the ATPase activity could be clearly dissociated from the complement fixing activity, subunits of the F1-ATPase complex were always found in the purified fractions. The alpha, gamma, delta and epsilon subunits of the complex could be excluded with high probability as target antigens in contrast to the beta band which was always found in association with the antigen activity. These findings imply that the active centre of the ATPase enzyme is not involved in the antibody reaction but molecules of the ATPase complex may have antigen binding capacity. Treatment of ATPase associated antigen with trypsin did not markedly affect the complement binding, while SMP's treated in the same way lost their antigen activity indicating that sera from patients with primary biliary cirrhosis (PBC) may have mitochondrial antibodies of different specificities reacting with trypsin sensitive as well as trypsin insensitive components of the inner membrane. The purified antigen reacted exclusively with sera from patients with PBC and may be therefore used as a marker antigen.
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PMID:Mitochondrial antibodies in primary biliary cirrhosis. VI. Association of the complement fixing antigen with a component of the mitochondrial F1-ATPase complex. 618 57

Adrenal chromaffin granules are known to possess an F1-ATPase which according to biochemical criteria is very similar to the mitochondrial one. To find a morphological equivalent for this enzyme chromaffin granules from bovine adrenal medullar were subjected to negative staining and freeze-etching. With both methods globular particles of 8 to 9 min diameter could be demonstrated on the surface of these organelles. A single granule possessed on average 22 particles. In negative staining the particles appeared separated from the membrane by a stalk of 8 nm. This typical morphological appearance was independent from a great variety of experimental procedures. After freeze-etching the particles were closely apposed to the membrane without any evidence for an interposed stalk. Pretreatment of chromaffin granules with pronase or trypsin led to a time dependent disappearance of the surface particles. In negative staining the stalked of chromaffin granules were found to be very similar in structure and size to those of mitochondria which have already been identified as F1-complexes. Based on this observation and other lines of evidence we suggest that the stalk particles found on the surface of chromaffin granules represent the F1-complex of the proton-pumping ATPase of these organelles.
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PMID:Adrenal chromaffin granules: evidence for an ultrastructural equivalent of the proton-pumping ATPase. 621 56

The coupling-factor ATPases from photosynthetically grown Rhodopseudomonas palustris and Rhodopseudomonas sphaeroides were purified by the same procedure to homogeneity. Gel chromatography on Sephacryl S-300 Superfine shortened the process of purification and improved its yield. Solubilization of the ATPase from both bacteria was found to be dependent on a specific sonication treatment of the cell suspensions, indicating a very weakly bound F1-ATPase in R. palustris. Depleted chromatophores could be restored in photophosphorylation and membrane-bound ATPase activities by adding the solubilized ATPase protein. The purified enzymes did not show a markedly trypsin-stimulated or dithiothreitol-stimulated activity. Isoelectric focusing and chromatofocusing revealed isoelectric points of 5.0 for both F1-ATPases. The molecular weights were determined by gel chromatography plus high-performance liquid chromatography. Hence, we calculated a molecular weight of 350000 for both F1-ATPases. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed five subunits for both enzymes. Kinetic parameters, regarding substrate specificity, the effect of divalent cations, Km and Ki values for the membrane-bound and solubilized ATPases were determined.
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PMID:Purification and properties of the coupling-factor ATPases F1 from Rhodopseudomonas palustris and Rhodopseudomonas sphaeroides. 621 69

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

Soluble beef-heart mitochondrial F1-ATPase modified in its alpha-subunit by mild trypsin treatment (alpha'-F1) can no longer bind oligomycin-sensitivity conferring protein (OSCP) but is still capable of binding to F1-depleted submitochondrial particles, giving rise to a maximally oligomycin-sensitive ATPase, provided the particles contain their native complement of OSCP. When OSCP is removed from the particles, alpha'-F1 can still bind to the particles, but added OSCP induces only a low degree of oligomycin sensitivity. The possible role of OSCP in the functional coupling of the catalytic (F1) and H+-translocating (Fo) moieties of mitochondrial ATPase is discussed. The results suggest a functional similarity between the OSCP component of mitochondrial ATPase and the delta-subunit of E. coli ATPase, which is in accordance with the structural homology recently found to exist between the two polypeptides.
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PMID:Lack of ability of trypsin-treated mitochondrial F1-ATPase to bind the oligomycin-sensitivity conferring protein (OSCP). 622 83

Studies on the effects of polyamines on oligomycin-sensitive ATPase activity of ox heart submitochondrial particles showed that, of the polyamines tested, only spermine affected the enzyme activity. Spermine within the physiological concentration range increased the Vmax. of the enzyme, but the Km for ATP was virtually unaffected. Binding studies of [14C]spermine to submitochondrial particles, under the same conditions as used for the ATPase assay, showed that the spermine binds to submitochondrial particles in a co-operative way; Hill plots of the data gave a Hill coefficient of 2 and a Kd of 8 microM. When submitochondrial particles were treated with trypsin, ATPase was not stimulated by spermine and the amount of spermine bound concomitantly was drastically decreased. The ATPase activity of isolated F1-ATPase was not affected by spermine. Removal of the natural protein ATPase inhibitor did not suppress either the stimulation of the ATPase activity by spermine or the spermine binding to the particles. The results obtained suggested that the polyamine binds and acts at the level of the liaison between the coupling factor F1 and the membrane sector F0 of the ATPase complex.
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PMID:Spermine binding to submitochondrial particles and activation of adenosine triphosphatase. 623 25


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