scholarly journals Isolation of α-subunits of factor F1 from submitochondrial particles and the reconstitution of active ATPase from isolated α-subunits and β-subunits bound to the mitochondrial membrane

1980 ◽  
Vol 192 (2) ◽  
pp. 483-488 ◽  
Author(s):  
I A Kozlov ◽  
Y M Milgrom ◽  
I S Tsybovski
Keyword(s):  
Atpase Activity ◽  
Active Site ◽  
Mitochondrial Membrane ◽  
Iodoacetic Acid ◽  
Reduced Form ◽  
Beta Subunits ◽  
Submitochondrial Particles ◽  
F1 Atpase ◽  
Alpha Subunits ◽  
Α Subunits

The alpha-subunits of factor-F1 ATPase are removed by extraction of submitochondrial particles with 1.75 M-LiCl, with the consequent loss of ATPase activity. ATPase activity is reconstituted by incubation of LiCl-extracted particles with purified alpha-subunits, and the reconstituted ATPase activity is oligomycin-sensitive. Reconstitution is enhanced by maintenance of the alpha-subunits in reduced form by dithiothreitol or NaBH4 and by modification of the alpha-subunits by p-chloromercuribenzoate, iodoacetic acid or N-ethylmaleimide. Experiments with the mixed anhydride of ATP and mesitylene-carboxylic acid, which was previously shown to interact with the F1 active site, localized on the beta-subunits, indicate that the active site of ATPase is shielded by the alpha-subunits.

Mitochondrial F1-ATPase activity of canine myocardium: effects of hypoxia and stimulation

1994 ◽  
Vol 266 (6) ◽  
pp. H2396-H2403 ◽  
Author(s):  
T. D. Scholz ◽  
R. S. Balaban
Keyword(s):  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Citrate Synthase ◽  
Submitochondrial Particles ◽  
F1 Atpase ◽  
Baseline Levels ◽  
Ph Buffer ◽  
Phenylephrine Infusion ◽  
Intact Heart ◽  
Canine Myocardium

Recent studies have suggested that modifications in mitochondrial F1-adenosinetriphosphatase (ATPase) activity may play an important role in the regulation of myocardial oxidative phosphorylation. The goal of the present study was to develop and characterize an assay of F1-ATPase activity that could be performed repeatedly on an intact heart under various physiological states. With the use of submitochondrial particles prepared from biopsy samples of canine myocardium, we found reproducible F1-ATPase activity when normalized to the activity of the intramitochondrial enzyme citrate synthase. The oligomycin-sensitive component of the ATPase activity was found to be mainly F1-ATPase. F1-ATPase activity of normal myocardium increased by incubation in high salt-pH buffer, suggesting baseline inhibition. Five minutes after global ischemia, F1-ATPase activity decreased to 60% of baseline. Hypoxia for 10 min resulted in no significant change in F1-ATPase activity. With phenylephrine infusion, myocardial oxygen consumption more than doubled, whereas F1-ATPase activity increased by approximately 30%. Both returned to baseline levels after discontinuation of the drug. With the use of an assay developed to measure F1-ATPase activity of intact myocardium, changes of the enzyme activity were found during both ischemia and at increased work loads. These data suggest that alterations of F1-ATPase activity may contribute to the regulation of myocardial oxidative phosphorylation.

scholarly journals Spermine binding to submitochondrial particles and activation of adenosine triphosphatase

1984 ◽  
Vol 218 (2) ◽  
pp. 495-499 ◽  
Author(s):  
G Solaini ◽  
B Tadolini
Keyword(s):  
Atpase Activity ◽  
Coupling Factor ◽  
Hill Coefficient ◽  
Atpase Inhibitor ◽  
Binding Studies ◽  
Submitochondrial Particles ◽  
Physiological Concentration ◽  
F1 Atpase ◽  
Atpase Assay ◽  
Hill Plots

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.

scholarly journals Conformational states of the (Na+ + K+)-transporting ATPase. Formation of 240000-Mr and 116000-Mr polypeptides in the presence of a bifunctional thiol probe

1984 ◽  
Vol 218 (2) ◽  
pp. 331-339 ◽  
Author(s):  
W E Harris ◽  
W L Stahl
Keyword(s):  
Atpase Activity ◽  
Adenine Nucleotide ◽  
Conformational State ◽  
Cross Linking ◽  
Beta Subunits ◽  
Specific Antibodies ◽  
Alpha Subunits ◽  
Phosphorylated Form ◽  
Kidney Enzyme ◽  
Thiol Reagent

Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4′-difluoro-3,3′-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.

scholarly journals Properties of membranes from mutant strains of Escherichia coli in which the β-subunit of the adenosine triphosphatase is abnormal

1979 ◽  
Vol 180 (1) ◽  
pp. 111-118 ◽  
Author(s):  
A E Senior ◽  
D R Fayle ◽  
J A Downie ◽  
F Gibson ◽  
G B Cox
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Beta Subunit ◽  
Beta Subunits ◽  
F1 Atpase ◽  
Mutant Strains ◽  
Membrane Bound ◽  
Phenotypic Variations

Five uncoupled mutant strains of Escherichia coli carrying mutations in the uncD gene have been studied. In each of these mutant strains the beta-subunit of the F1 portion of the membrane-bound adenosine triphosphatase is abnormal. In one of the mutant strains (carrying the uncD12 allele) in F1-ATPase aggregate was formed which was purified and found to have low ATPase activity. ATPase activity was absent in the other four strains and the abnormal beta-subunits were tightly bound to the membranes. However, membranes from these strains exhibited various proton permeabilities as indicated by NADH-dependent atebrin-fluorescence quenching and bound different amounts of normal F1-ATPase. The amounts of reconstitution of energy-linked reactions after the addition of normal F1-ATPase also varied depending on the mutant allele. It is apparent that considerable phenotypic variations can occur between strains carrying mutations in the same unc gene.

scholarly journals Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

1991 ◽  
Vol 280 (3) ◽  
pp. 659-662 ◽  
Author(s):  
J Martín ◽  
A Slade ◽  
A Aitken ◽  
R Arche ◽  
R Virden
Keyword(s):  
Active Site ◽  
Tertiary Structure ◽  
Thiol Group ◽  
Iodoacetic Acid ◽  
Penicillin Acylase ◽  
Protein Product ◽  
Side Chain ◽  
Serine Residue ◽  
Conserved Sequence ◽  
Ester Substrate

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2′-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

scholarly journals A kinetic analysis of the changes in fluorescence on the interaction of 8-anilinonaphthalene-l-sulphonate with submitochondrial particles

1976 ◽  
Vol 158 (2) ◽  
pp. 295-305 ◽  
Author(s):  
N Gains ◽  
A P Dawson
Keyword(s):  
Kinetic Analysis ◽  
Mitochondrial Membrane ◽  
Antimycin A ◽  
Submitochondrial Particles ◽  
Submitochondrial Particle ◽  
Fluorescence Change

A comparison of the fluorescence change on the addition of 8-anilinonaphthalene-1-sulphonate to succinate-energized submitochondrial particles with that on the addition of succinate to submitochondrial particles incubated with 8-anilinonaphthalene-1-sulphonate shows that these changes in fluorescence may be explained solely in terms of 8-anilinonaphthalene-1-sulphonate binding. This comparison does not support the proposal of an 8-anilinonaphthalene-1-sulphonate-monitored change in the conformation of submitochondrial-particle membranes [Brocklehurst, Freedman, Hancock & Radda (1970) Biochem. J.116, 721-731]. The biphasic nature of the decrease in fluorescence, which was found to follow the addition of uncoupler to submitochondrial particles incubated with ATP or succinate, or of antimycin A to submitochondrial particles incubated with succinate, does not support the existence of ‘aplectic’ and ‘symplectic’ states of the mitochondrial membrane [Barrett-Bee & Radda (1972) Biochim, Biophys. Acta 267, 211-215].

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