Interaction between catalytic and regulatory sites of mitochondrial F1-ATPase as monitored by the differential effects of inhibitors and nucleotide analogs on the "hysteretic" behavior of the enzyme

Pietro, Attilio Di; Godinot, Catherine; Gautheron, D

doi:10.1021/bi00525a005

Cited by 42 publications

(7 citation statements)

References 28 publications

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“…The total inactivation produced by the complete modification of one Trp190 by NBS indicates an essential involvement of the noncatalytic regulatory site in the enzyme activity. This is consistent with its affinity labeling by various adenine nucleotide analogues which produced a large enzyme inactivation (Di Pietro et al, 1981;Fellous et al, 1984;Bullough & Allison, 1986;Cross et al, 1987;Verburg & Allison, 1990). Under the present conditions, exogenous ADP produces a very efficient protection against NBSdependent inactivation with a half-maximal concentration (around 3 #iM) similar to the Kd of radiolabeled ADP binding (2.2-2.7 ftM) to the noncatalytic site (Divita et al, 1992); this further supports the location of Trp190 within or very close to this site.…”

Section: Discussionsupporting

confidence: 88%

“…Under the present conditions, exogenous ADP produces a very efficient protection against NBSdependent inactivation with a half-maximal concentration (around 3 #iM) similar to the Kd of radiolabeled ADP binding (2.2-2.7 ftM) to the noncatalytic site (Divita et al, 1992); this further supports the location of Trp190 within or very close to this site. The fact that no endogenous ADP is released upon NBS modification of the single Trp190 indicates that the residue is located in a vacant asymmetric noncatalytic site, as previously characterized with other Fi preparations (Di Pietro et al, 1981; Kironde & Cross, 1986). Since the two nonexchangeable sites remain saturated by endogenous ADP, all three ADP sites appear to be occupied during the protection experiment in the presence of added ADP.…”

Section: Discussionsupporting

confidence: 56%

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Chemical modification of .alpha.-subunit tryptophan residues in Schizosaccharomyces pombe mitochondrial F1 ATPase: adenosine 5'-triphosphatase: Differential reactivity and role in activity

Divita¹,

Jault²,

Gautheron³

et al. 1993

Biochemistry

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Chemical modification of mitochondrial F1-ATPase from Schizosaccharomyces pombe by the tryptophan-specific reagent N-bromosuccinimide (NBS) at pH 5.0 in the presence of 20% glycerol produced a characteristic lowering in both enzyme absorbance at 280 nm and intrinsic fluorescence at 332 nm that varied with NBS/F1 molar ratio up to a value of 130. Fluorometric titration of tryptophans and correlation to residual ATPase activity showed that modification of three reactive residues among the seven present on alpha- and epsilon-subunits did not markedly modify the enzyme activity but efficiently released endogenous ATP and abolished the fluorescence quenching related to GDP or ATP binding to the catalytic site. Additional modification of one, less reactive, tryptophan altered both negative cooperativity of ATP hydrolysis and sensitivity to azide inhibition and produced a nearly complete inactivation at high NBS/F1 molar ratio. NBS-induced inactivation of F1 was favored by catalytic-site saturation with GDP or low ATP concentration and on the contrary was prevented by noncatalytic-site saturation with ADP or high ATP concentration. When reactive tryptophans were selectively modified by NBS in the presence of ADP, and subunits were isolated after guanidine hydrochloride dissociation by one-step purification on reversed-phase HPLC, the absorbance of alpha-subunit at 280 nm was decreased, whereas that of epsilon-subunit was unchanged. Cyanogen bromide cleavage of alpha-subunit and fragments separation by reversed-phase HPLC showed that one peptide of 3 kDa apparent molecular mass had decreased absorbance. N-Terminal sequencing allowed its identification to fragment 255-282 that contains tryptophan257.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionsupporting

confidence: 56%

Chemical modification of .alpha.-subunit tryptophan residues in Schizosaccharomyces pombe mitochondrial F1 ATPase: adenosine 5'-triphosphatase: Differential reactivity and role in activity

Divita¹,

Jault²,

Gautheron³

et al. 1993

Biochemistry

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“…High concentrations of GDP hardly modified the effects of trypsin and were not able to induce the hysteretic inhibition of the enzyme. Moreover, GDP binding did not prevent the fixation of ADP at site(s) responsible for the hysteretic inhibition, sites very selective for the adenine nucleotides (Harris et al, 1978;Baubichon et al, 1981;Di Pietro et al, 1981). Therefore, the conformational changes detected here by the trypsin effect were, indeed, due to ADP binding at those selective sites which can be considered as regulatory sites.…”

Section: Discussionmentioning

confidence: 75%

Use of trypsin to monitor conformational changes of mitochondrial adenosine triphosphatase induced by nucleotides and phosphate

Pietro¹,

Godinot²,

Gautheron³

1983

Biochemistry

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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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“…It is therefore concluded that the regulatory site must be located on the 0-subunit of the mitochondrial F,-ATPase. Di Pietro et al, 1981). On the contrary, all nucleoside triphosphates can be hydrolyzed at the catalytic site(s) (Schuster et al, 1975;Pedersen, 1975;Harris et al, 1978).…”

mentioning

confidence: 99%

Photolabeling on .beta.-subunit of the nucleotide site related to hysteretic inhibition of mitochondrial F1-ATPase

Fellous¹,

Godinot²,

Baubichon³

et al. 1984

Biochemistry

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Interaction between catalytic and regulatory sites of mitochondrial F1-ATPase as monitored by the differential effects of inhibitors and nucleotide analogs on the "hysteretic" behavior of the enzyme

Cited by 42 publications

References 28 publications

Chemical modification of .alpha.-subunit tryptophan residues in Schizosaccharomyces pombe mitochondrial F1 ATPase: adenosine 5'-triphosphatase: Differential reactivity and role in activity

Chemical modification of .alpha.-subunit tryptophan residues in Schizosaccharomyces pombe mitochondrial F1 ATPase: adenosine 5'-triphosphatase: Differential reactivity and role in activity

Use of trypsin to monitor conformational changes of mitochondrial adenosine triphosphatase induced by nucleotides and phosphate

Photolabeling on .beta.-subunit of the nucleotide site related to hysteretic inhibition of mitochondrial F1-ATPase

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