The specific, mitochondrial ATP synthase protein (If1) was covalently cross-linked to its binding site on the catalytic sector of the enzyme (f1-ATPase). The cross-linked complex was selectively cleaved, leaving If1 i...
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The specific, mitochondrial ATP synthase protein (If1) was covalently cross-linked to its binding site on the catalytic sector of the enzyme (f1-ATPase). The cross-linked complex was selectively cleaved, leaving If1 intact to facilitate the subsequent purification of the f1 fragment to which If1 was cross-linked. This fragment was identified by sequence analysis as comprising residues 394-459 on the f1 beta-subunit, near the C-terminus. This finding is discussed in the light of secondary structure predictions for both If1 and the f1 beta-subunit, and sequence homologies between mitochondrial and other ATP synthases.
Stigmatellin is shown to raise the midpoint potential of the Rieske feS-center of isolated cytochrome b6 f complex from 320 to about 460 mV. This increase in potential is accompanied by a modified EPR spectrum, i.e., ...
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Stigmatellin is shown to raise the midpoint potential of the Rieske feS-center of isolated cytochrome b6 f complex from 320 to about 460 mV. This increase in potential is accompanied by a modified EPR spectrum, i.e., a shift of the gy line and a sharpening of the gz peak. An equivalent effect on the mitochondrial cytochrome bc1 complex has been observed earlier. Stigmatellin therefore inhibits both types of cytochrome complexes in the same way, by altering the feS center to an unfavorable reductant for cytochrome f or c1. The decreased reduction of cytochrome f by quinol in the presence of stigmatellin is accompanied by an increased transient reduction of cytochrome b6. This can be explained by an increased formation of semiquinone during quinol oxidation by a stronger oxidizing feS center.
Purified pea mitochondrial f1-ATPase reconstituted oxidative phosphorylation in both partially and completely f1-depleted bovine heart mitochondrial membranes. The isolated plant enzyme exhibited high rates of ATP syn...
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Purified pea mitochondrial f1-ATPase reconstituted oxidative phosphorylation in both partially and completely f1-depleted bovine heart mitochondrial membranes. The isolated plant enzyme exhibited high rates of ATP synthesis when combined with bovine heart membranes, suggesting great evolutionary conservation of the ATP synthase complex in mitochondria.
The effects of volume loading on a nociceptive reflex, arterial blood pressure and heart rate were studied in spontaneously hypertensive rats (SHRs), Wistar Kyoto normotensive rats (WKYs) and the f1 offspring of a SHR...
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The effects of volume loading on a nociceptive reflex, arterial blood pressure and heart rate were studied in spontaneously hypertensive rats (SHRs), Wistar Kyoto normotensive rats (WKYs) and the f1 offspring of a SHR .times. WKY cross. Volume loading resulted in significantly greater inhibition of the tail-flick reflex to painful radiant heat in SHRs compared to WKYs. The f1 offspring of a SHR .times. WKY cross showed levels of hypoalgesia to volume loading that were intermediate to those of SHRs and WKYs. There were no differences between these strains in their hypotensive and bradycardic responses to volume loading. These findings are discussed in terms of cardiovascular-somatosensory interactions.
To test whether ATP synthesis could occur via a mechanism of rotational catalysis in which the .alpha. and .beta. subunits off1 would rotate with respect to the minor subunits, we have measured the rate of ATP synthe...
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To test whether ATP synthesis could occur via a mechanism of rotational catalysis in which the .alpha. and .beta. subunits off1 would rotate with respect to the minor subunits, we have measured the rate of ATP synthesis after binding various masses of antibodies to f1. If the rotation was an essential feature of the mechanism, the rate of ATP synthesis should be inhibited either completely or proportionately to the load carried by f1. Bivalent immunoglobulins (IgG) or monovalent fab fragments of an anti-.alpha. monoclonal antibody (7B3) were bound to f1 present in electron-transport particles in a ratio of 2 fab or 2 IgG per f1. This binding similarly inhibited the rate of ATP synthesis by a maximum of about 50%. When anti-mouse immunoglobulins were added to the f1-7B3 (IgG) complex, no significant change in the rate of inhibition was observed. In conclusion, the rate of ATP synthesis was the same when f1 was loaded with 100 kDa (2 fab), 300 kDa (2 IGG, 7B3) or 900 kDa (2 IgG + 4 anti-mouse IgG). It is concluded that the rotation of the .alpha. subunits is extremely unlikely to play an essential role in the mechanism of ATP synthesis.
The α- and β-subunits of sweet potato mitochondrial f 1 ATPase were purified from the f 1 complex by gel filtration and ion-exchange high-performance liquid chromatography. Isoelectric focussing and N-terminal amino...
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The α- and β-subunits of sweet potato mitochondrial f 1 ATPase were purified from the f 1 complex by gel filtration and ion-exchange high-performance liquid chromatography. Isoelectric focussing and N-terminal amino acid sequencing indicated that the purified β-subunit contains at least two polypeptides similar to each other. The N-terminal 18 amino acid sequence of the β-subunit showed homology to the amino acid sequence of the tobacco mitochondrial f 1 ATPase β-subunit precursor deduced from the nucleotide sequence [(1985) EMBO J. 4, 2159-2165] between residues 56 and 73, suggesting that the N-terminal 55 amino acids of the tobacco precursor constitute the presequence required for mitochondrial targetting.
A quantitative method was developed to estimate the concentration of cytochrome (cyt) f in isolated thylakoids, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with a heme-specific...
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A quantitative method was developed to estimate the concentration of cytochrome (cyt) f in isolated thylakoids, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with a heme-specific reagent containing 3,3'',5,5''-tetramethylbenzidine and hydrogen peroxide. This densitometric technique was at least as sensitive as difference spectroscopy. Analysis of thylakoid preparations by densitometry of stained bands using cyt c as standard gave molar ratios of cyt/chlorophyll which were identical to ratios obtained by difference spectroscopy. Densitometric assays demonstrated that the molar ratio of cyt t/chlorophyll decreased during leaf aging in seven higher plants;however, there was a marked difference in the rate at which cyt f was lost from the leaves of different species.
Two ADP binding sites have been demonstrated on the reconstitutively active β-subunit, that was removed from the Rhodospirillum rubrum membrane-bound ATP synthase. One is a high affinity site ( K d = 0.7 μM) that do...
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Two ADP binding sites have been demonstrated on the reconstitutively active β-subunit, that was removed from the Rhodospirillum rubrum membrane-bound ATP synthase. One is a high affinity site ( K d = 0.7 μM) that does not require MgCl 2 and is unaffected by it. The second is a low affinity binding site ( K d = 80 μM) that is absolutely dependent on MgCl 2 . for stable binding of ADP to this site, MgCl 2 must be present not only during the binding step but also during the elution-centrifugation step used to separate the β-subunit bound [ 3 H]ADP from the free ligand. When MgCl 2 is removed together with the free ligand [ 3 H]ADP dissociates very rapidly from this second binding site.
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