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W S Allison

Publications and source records attributed to W S Allison.

At least 55 records · Page 3Linked to original sources
PubMed1989

Inhibition of the bovine-heart mitochondrial F1-ATPase by cationic dyes and amphipathic peptides.

The bovine heart mitochondrial F1-ATPase is inhibited by a number of amphiphilic cations. The order of effectiveness of non-peptidyl inhibitors examined as assessed by the concentration estimated to produce 50% inhibition (I0.5) of the enzyme at pH 8.0 is: dequalinium (8 microM), rhodamine 6G (10 microM), malachite green (14 microM), rosaniline (15 microM) greater than acridine orange (180 microM) greater than rhodamine 123 (270 microM) greater than rhodamine B (475 microM), coriphosphine (480 microM) greater than safranin O (1140 microM) greater than pyronin Y (1650 microM) greater than Nile blue A (greater than 2000 microM). The ATPase activity was also inhibited by the following cationic, amphiphilic peptides: the bee venom peptide, melittin; a synthetic peptide corresponding to the presence of yeast cytochrome oxidase subunit IV (WT), and amphiphilic, synthetic peptides which have been shown (Roise, D., Franziska, T., Horvath, S.J., Tomich, J.M., Richards, J.H., Allison, D.S. and Schatz, G. (1988) EMBO J. 7, 649-653) to function in mitochondrial import when attached to dihydrofolate reductase (delta 11.12, Syn-A2, and Syn-C). The order of effectiveness of the peptide inhibitors as assessed by I0.5 values is: Syn-A2 (40 nM), Syn-C (54 nM) greater than melittin (5 microM) greater than WT (16 microM) greater than delta 11,12 (29 microM). Rhodamines B and 123, dequalinium, melittin, and Syn-A2 showed noncompetitive inhibition, whereas each of the other inhibitors examined (rhodamine 6G, rosaniline, malachite green, coriphosphine, acridine orange, and-Syn-C) showed mixed inhibition. Replots of slopes and intercepts from Lineweaver-Burk plots obtained for dequalinium were hyperbolic indicating partial inhibition. With the exception of Syn-C, for which the slope replot was hyperbolic and the intercept replot was parabolic, steady-state kinetic analyses indicated that inhibition by the other inhibitors was complete. The inhibition constants obtained by steady-state kinetic analyses were in agreement with the I0.5 values estimated for each inhibitor examined. Rhodamine 6G, rosaniline, dequalinium, melittin, Syn-A2, and Syn-C were observed to protect F1 against inactivation by the aziridinium of quinacrine mustard in accord with their experimentally determined I0.5 values.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Selectivity of modification when latent and activated forms of the chloroplast F1-ATPase are inactivated by 7-chloro-4-nitrobenzofurazan.

The characteristics and specificity of inactivation of the chloroplast F1-ATPase (CF1) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) have been investigated. Inactivation of the octylglucoside-dependent Mg2+-ATPase activity of latent CF1 by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. Following inactivation of CF1 with [14C]Nbf-Cl, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the majority of the radioactive reagent incorporated is present in the beta subunit. Treatment of the enzyme with [14C]Nbf-Cl following dithiothreitol heat activation, led to similar labeling of the beta subunit and substantial incorporation of 14C into the gamma subunit. On complete inactivation, about 4 mol of Nbf-S-Cys is formed per mole of dithiothreitol-heat-activated CF1. Incorporation of 14C into the gamma subunit is prevented by prior treatment of the latent CF1 or of the dithiothreitol-heat-activated CF1 with iodoacetamide. Following incubation of the dithiothreitol-heat-activated CF1 with iodoacetamide, complete inactivation of the octylglucoside-dependent Mg2+-ATPase activity by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. After stabilization of the [14C]Nbf-O-Tyr derivative by treatment with sodium dithionite, a labeled peptide was purified. Automatic Edman degradation of this peptide revealed the sequence V-X-V-P-A-D-(D). The majority of the radioactivity was cleaved in the second cycle, the position occupied in CF1 by Tyr-beta-328, which is homologous to Tyr-beta-311, the residue reactive with Nbf-Cl in the beef heart mitochondrial F1-ATPase. When CF1, modified at Tyr-beta-328 with Nbf-Cl, is incubated at pH 9.0, the Nbf-O-Tyr adduct is hydrolyzed, leading to concomitant recovery of the ATPase activity. In double labeling experiments, two-dimensional isoelectric focusing in the presence of urea followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that 2-azido-ADP, covalently bound at the tight ADP binding site, and the tyrosine modified by [14C]Nbf-Cl are located in different beta subunits.

Benzofurans↗
PubMed1989

Localization of sites modified during inactivation of the bovine heart mitochondrial F1-ATPase by quinacrine mustard using [3H]aniline as a probe.

The aziridinium of purified quinacrine mustard at 50 microM inactivates the bovine heart mitochondrial F1-ATPase with a pseudo-first order rate constant of 0.07 min-1 at pH 7.0 and 23 degrees C. An apparent Kd of 27 microM for the enzyme-reagent complex was estimated from the dependence of the rate of inactivation on the concentration of quinacrine mustard. The pH inactivation profile revealed that deprotonation of a group with a pKa of about 6.7 is necessary for inactivation. The amount of reagent incorporated into the protein increased linearly with the extent of inactivation. Complete inactivation was estimated to occur when 3 mol of reagent were incorporated/mol of F1. Enzyme, in which steady state ATPase was inactivated by 98% by quinacrine mustard, hydrolyzed substoichiometric ATP with zero order kinetics suggesting that residual activity is catalyzed by F1 in which at least one beta subunit is modified. By exploiting the reactivity of the aziridinium of covalently attached reagent with [3H] aniline, sites modified by quinacrine mustard were labeled with 3H. Isolation of radioactive cyanogen bromide peptides derived from F1 inactivated with the reagent in the presence of [3H]aniline which were identified by sequence analysis and sequence analyses of radioactive tryptic fragments arising from them have revealed the following. About two thirds of the radioactivity incorporated into the enzyme during inactivation is apparently esterified to one or more of the carboxylic acid side chains in a CNBr-tryptic fragment of the beta subunit with the sequence: 394DELSEEDK401. The remainder of the radioactivity is associated with at least two sites within the cyanogen bromide peptide containing residues 293-358 of the beta subunit. From these results it is concluded that inactivation of F1 by the aziridinium of quinacrine mustard is due, at least in part, to modification of one or more of the carboxylic acid side chains in the DELSEED segment of the beta subunit and possibly also to modification of unspecified amino acid side chains between residues 302-356 of the beta subunit.

Adenosine Triphosphate↗
PubMed1988

On the location and function of the noncatalytic sites on the bovine heart mitochondrial F1-ATPase.

Modification of Tyr-345 at a catalytic site in a single beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) by 5'-p-fluorosulfonylbenzoylinosine did not affect subsequent labeling of noncatalytic sites at Tyr-368 and His-427 in three copies of the beta subunit by 5'-p-fluorosulfonylbenzoyladenosine (FSBA). These results clearly show that the beta subunit contains at least parts of the catalytic and noncatalytic nucleotide binding sites. Inactivation of MF1 by 96% with FSBA was accompanied by a decrease in the endogenous ADP content from 1.86 to 0.10 mol per mol of MF1. Decrease in the endogenous ADP content during the inactivation of the enzyme with FSBA paralleled loss in activity in a manner which suggests that the reaction of FSBA with an open noncatalytic site promoted release of ADP from another noncatalytic site until the third site reacted with FSBA. Two pKa values of about 5.9 and 7.6 were observed on the acid side of the pH optimum in the pH-rate profile for ATP hydrolysis catalyzed by MF1 in neutral acid buffers. In contrast, a single pKa of 5.9 was present in the pH-rate profile for ITP hydrolysis catalyzed by the enzyme in the same buffers. The augmented rate observed for ATP hydrolysis at pH 8.0, over that observed at pH 6.5, was lost as the enzyme was inactivated by FSBA in a manner suggesting that modulation is lost as the third noncatalytic site is modified. This suggests that ATP hydrolysis by MF1 is modulated in a pH-dependent manner by ATP binding to an open noncatalytic site. Two other modulations associated with binding of adenine nucleotides to noncatalytic sites, ADP-induced hysteretic inhibition and apparent negative cooperativity reflected by the Hill coefficient for the hydrolysis of 50-3000 microM ATP at pH 8.0, also disappeared as the third noncatalytic site reacted with FSBA.

Adenosine↗
PubMed1988

Inactivation of the F1-ATPase from the thermophilic bacterium PS3 by 5'-p-fluorosulfonylbenzoylinosine at 65 degrees C is accompanied by modification of beta-tyrosine-364.

A major radioactive peptide, T1, was resolved by high-performance liquid chromatography from a tryptic digest prepared from the F1-ATPase from the thermophilic bacterium PS3 which had been inactivated with p-fluorosulfonylbenzoyl[3H]inosine. Two radioactive peptides, T1P1 and T1P2, were isolated from a peptic digest of T1 by high-performance liquid chromatography. The sequences of T1P1 and T1P2 were shown to be E-E-H-X-Q-V-A-R and E-E-H-X-Q, respectively, where X corresponds to derivatized Tyr-364 of the beta subunit.

Amino Acid Sequence↗
PubMed1988

Inhibition and photoinactivation of the bovine heart mitochondrial F1-ATPase by the cytotoxic agent, dequalinium.

The bovine heart mitochondrial F1-ATPase is inhibited in the dark by the amphipathic cation, dequalinium, with a I0.5 of about 12 microM at pH 7.5. When illuminated at 350 nm in the presence of 1.7 microM dequalinium, the F1-ATPase is inactivated with a pseudo-first order rate constant of 7.9 X 10(-3) min-1. The apparent Kd of the dequalinium-enzyme complex was estimated to be about 12.5 microM by examining the rate of inactivation of the ATPase with 1.7-16.7 microM dequalinium. ATP, ADP, Pi, and Mg2+, singly or in combination, protected the ATPase against photoinactivation, with Mg2+ plus Pi being the most effective.

Adenosine Triphosphate↗
PubMed1987

Evidence for functional heterogeneity among the catalytic sites of the bovine heart mitochondrial F1-ATPase.

The characteristics of ATP hydrolysis at a single catalytic site of the bovine heart F1-ATPase (MF1) as originally described by Grubmeyer et al. (Grubmeyer, C., Cross, R.L., and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12092-12100) were compared with those of various chemically modified preparations of MF1 in which the steady state activity was severely attenuated. Although it was not necessary to age our preparations of native MF1 in the presence of 2 mM Pi to observe the same characteristics of single site catalysis, such aging did shift the equilibrium of bound substrate and bound products at the single catalytic site in favor of ATP. After loading a single catalytic site on the enzyme with substoichiometric [alpha,gamma-32P]ATP, the addition of 5-20 microM ATP or ADP was effective in promoting both the hydrolysis of bound [alpha,gamma-32P]ATP and release of radioactive products. Under these conditions, the 5-20 microM ATP added as promoter was hydrolyzed at a rate commensurate with the turnover rate of the enzyme, whereas the promoted hydrolysis of the [alpha,gamma-32P]ATP, preloaded at a single catalytic site, was considerably slower. Therefore, the high affinity, single catalytic site loaded first does not directly contribute to steady state ATP hydrolysis. That the single, high affinity catalytic site is not a "normal" catalytic site is supported by the properties of enzyme modified by 5'-p-fluorosulfonylbenzoyladenosine which exhibits only slightly altered characteristics of single site catalysis and promoted single site catalysis, despite exhibiting severely attenuated steady state turnover. Other modified forms of the enzyme in which the steady state activity was severely attenuated by derivatization with 5'-p-fluorosulfonylbenzoylinosine, 7-chloro-4-nitrobenzofurazan, or 1,5-difluoro-2,4-dinitrobenzene also bound substoichiometric ATP at a single catalytic site. However, the characteristics of single site hydrolysis by these modified forms of the enzyme differed considerably from those of native MF1.

Adenosine↗
PubMed1986

Inactivation of the bovine heart mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoyl[3H]inosine is accompanied by modification of tyrosine 345 in a single beta subunit.

The inactivation of the bovine heart mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoylinosine (FSBI) proceeds with pseudo-first order kinetics. The rate of inactivation increased from pH 7 to 9 revealing a pKa of about 8.2. When a tryptic digest of the enzyme which had been inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine ([3H]FSBI) was submitted to reversed phase high pressure liquid chromatography, a single major peak of radioactivity, T1, was resolved. Amino acid sequence analysis of purified peptide fragments derived from T1 showed that the modification of beta-Tyr-345 is responsible for inactivation of the enzyme. Complete inactivation of the enzyme by [3H]FSBI is estimated to proceed with modification of 0.8 mol of beta-Tyr-345/mol of enzyme. Another notable observation is that inosine triphosphatase (ITPase) activity catalyzed by F1 from bovine heart mitochondria is much more sensitive to inactivation by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) than is ATPase activity. Whereas complete inactivation of ATPase activity by FSBA has been shown to proceed with the mutually exclusive modification of Tyr-368 or His-427 in all three copies of the beta subunit (Bullough, D. A., and Allison, W. S. (1986) J. Biol. Chem. 261, 5722-5730), it is shown here that complete inactivation of ITPase activity by FSBA is accompanied by modification of these residues in only one copy of the beta subunit. Inactivation of both the ATPase and ITPase activities of the enzyme by FSBI proceeds with modification of Tyr-345 in a single copy of the beta subunit.

Adenosine↗
PubMed1986

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3.

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with [3H]ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. [3H]ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with [3H]ADP in 30 min with a Kd of 30 microM. [3H]ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of [3H]ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. [3H]ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits. It has also been demonstrated that enzyme-bound ATP is formed when the TF0.F1 complex containing bound ADP was incubated with Pi, Mg2+, and 50% dimethyl sulfoxide.

Adenosine Diphosphate↗
PubMed1986

Three copies of the beta subunit must be modified to achieve complete inactivation of the bovine mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoyladenosine.

The modification of both beta-Tyr-368 and beta-His-427 can be correlated with the loss of activity observed when the bovine mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoyl[3H]adenosine ([3H]FSBA). At pH 8.0, where the rate of inactivation is fast, beta-Tyr-368 is modified predominantly, while at pH 6.0, where the rate of inactivation is slow, beta-His-427 is modified predominantly. At pH 7.0, the 2 residues are modified with about equal efficiency. When the F1-ATPase was inactivated by 80% at pH 6.5, 7.0, and 7.5, the sum of radioactivity incorporated into beta-Tyr-368 and beta-His-427 was 1.99, 1.87, and 1.82 mol of label incorporated per mol of enzyme, respectively. Examination of the rate of inactivation of the enzyme by FSBA as a function of pH revealed two pKa values, one of about 7.6 associated with the modification of beta-Tyr-368 and the other of about 5.8 associated with the modification of beta-His-427. The inactivation of the F1-ATPase by FSBA exhibited an initial fast rate followed by a slower rate in triethanolamine-HCl, pH 7.0. In contrast, only a single rate, equivalent to the fast phase of inactivation in the absence of phosphate, was observed in 0.2 M phosphate, pH 7.0. The dependence of this stimulation on phosphate concentration is sigmoidal with half-maximal stimulation occurring at approximately 160 mM. The ratio of 3H incorporated into beta-Tyr-368 to that incorporated into beta-His-427 was approximately the same during the fast and slow phases of inactivation in triethanolamine-HCl, pH 7.0. Approximately the same ratio was observed when the enzyme was modified during the single phase of inactivation exhibited in the presence of 0.2 M phosphate, pH 7.0. The sum of the 3H incorporated into beta-Tyr-368 and beta-His-427 during inactivation of the F1-ATPase from bovine heart mitochondria by [3H]FSBA in the presence and absence of phosphate was linear and extrapolated to a value of about 2.6 residues modified on complete inactivation of the enzyme. From these data, it is concluded that FSBA binds to a single binding site on the beta subunits of the enzyme where it reacts with either beta-Tyr-368 or beta-His-427 in mutually exclusive reactions. All three beta subunits must be modified in this manner for complete inactivation to be observed.

Adenosine↗
PubMed1986

The use of dithionite reduction to identify the essential tyrosine residue in the F1-ATPase from the thermophilic bacterium, PS3, that reacts with 7-chloro-4-nitrobenzofurazan.

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by greater than 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.4, 1.4 mol of [14C]Nbf were incorporated per mol of enzyme. After pepsin digestion of the labeled enzyme at pH 3.0, a single, major peak of radioactivity was resolved by reversed-phase high-performance liquid chromatography under acidic conditions were peptidyl Nbf-O-tyrosine is stable. This radioactive peak, designated RP-1, eluted with a retention time of 95 min. When the material in RP-1 was subjected to reversed-phase high-performance liquid chromatography under the same conditions after treatment with sodium dithionite, a single, major peak of radioactivity, designated RP-2, was resolved with a retention time of 52 min. Automatic Edman degradation of this material revealed that it has the amino acid sequence I-Y*-V-P-A-D-(D), where Y* presumably represents peptidyl [14C]Nbf-O-tyrosine. These results provide the basis for a facile method to purify peptides containing [14C]Nbf-O-tyrosine in which the labeled residues can be identified by amino acid sequence analysis using the Edman degradation.

4-Chloro-7-nitrobenzofurazan↗
PubMed1986

Sequence of the radioactive tryptic peptide obtained after inactivating the F1-ATPase of the thermophilic bacterium PS3 with 5'-p-fluorosulfonylbenzoyl[3H]adenosine at 65 degrees C.

Following a lag of about 30 min, the F1-ATPase from the thermophilic bacterium, PS3 (TF1), was inactivated slowly by 0.8 mM 5'-p-fluorosulfonylbenzoyladenosine (FSBA) at 23 degrees C and pH 7.0. When the enzyme was treated with 0.2 mM FSBA at pH 7.0 and 23 degrees C for 15 min and gel-filtered, no enzyme activity was lost. However, the lag in inactivation was abolished when the enzyme was subsequently incubated with 2.0 mM FSBA at 23 degrees C in the pH range from 6.8 to 10.0. The pH-inactivation profile obtained under these conditions revealed a pK alpha of about 9.3 which was associated with the inactivation. When pretreated TF1 was inactivated at 23 degrees C with [3H]FSBA by about 90%, greater than 20 mol of [3H]SBA was incorporated per mole of enzyme. TF1 was inactivated rapidly by 0.8 mM FSBA at pH 6.4 and 65 degrees C, and no lag was observed. Following inactivation of TF1 with 0.8 mM [3H]FSBA at 65 degrees C and pH 6.4, about 10 mol of [3H]SBA was incorporated per mole of enzyme. When a tryptic digest of the labeled enzyme was fractionated by reversed-phase high-performance liquid chromatography, a single major radioactive peptide was isolated. When subjected to automatic Edman degradation, this peptide was shown to have the amino acid sequence: A-L-A-P-E-I-V-G-E-E-H-X-Q-V-A-R, where X indicates that a phenylthiohydantoin derivative was not detected in cycle 12. However, from the DNA sequence of the gene encoding the subunit of TF1 (Y. Kagawa, M. Ishizuka, T. Saishu, and S. Nakao (1985) Abstracts International Symposium on Energy Transducing ATPases, Kobe, Japan, p. 84), this position has been shown to be occupied by tyrosine. This tyrosine is homologous with beta-Tyr-368 of the bovine mitochondrial F1-ATPase (MF1) the modification of which is responsible for the inactivation MF1 by FSBA.

Adenosine↗
PubMed1985

The use of [3H]aniline to identify the essential carboxyl group in the bovine mitochondrial F1-ATPase that reacts with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.

The inactivation of the bovine heart mitochondrial F1-ATPase with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of [3H]aniline at pH 7.0 led to the covalent incorporation of 3H into the enzyme. When the ATPase was inactivated by 94% with 0.9 mM EEDQ in the presence of 3.6 mM [3H]aniline in a large-scale experiment in which the protein concentration was 21 mg/ml, 4.2 mol [3H]anilide were formed per mol enzyme, of which 0.35 mol was incorporated per mol of the alpha subunit and 1.0 mol was incorporated per mol of the beta subunit. Examination of a tryptic digest of the isolated alpha subunit revealed that the majority of the 3H was contained in a single tryptic peptide, which, when purified, was shown to contain the [3H]anilide of a glutamic acid residue which corresponds to alpha-Glu-402 of the Escherichia coli F1-ATPase. This residue was labeled to the extent of about 1.0 mol/mol enzyme. Analysis of tryptic peptides purified from the isolated beta subunit showed that 0.8 and 1.5 mol, respectively, of the [3H]anilides of beta-Glu-341 and beta-Glu-199 were formed per mol MF1 during the inactivation of the enzyme at 21 mg/ml. When the ATPase was inactivated by 90% at a protein concentration of 1.7 mg/ml by 0.9 mM EEDQ in the presence of 1.7 mM [3H]aniline, 3.1 mol [3H]anilide were formed per mol enzyme. From the analysis of the radioactive peptides purified from a tryptic digest of the labeled ATPase from this experiment it was estimated that 0.7 mol of the [3H]anilide of alpha-Glu-402, 0.3 mol of the [3H]anilide of beta-Glu-341, and 1.5 mol of the [3H]anilide of beta-Glu-199 were formed per mol F1-ATPase. Since beta-Glu-199 is labeled to the same extent in the two experiments while alpha-Glu-402 and beta-Glu-341 were not, suggests that the modification of beta-Glu-199 is responsible for inactivation of the enzyme by EEDQ.

Amino Acid Sequence↗
PubMed1985

The varied responses of different F1-ATPases to chlorpromazine.

The effects of chlorpromazine on various properties of the F1-ATPases from bovine heart mitochondria (MF1), the plasma membranes of Escherichia coli (EF1), and plasma membranes of the thermophilic bacterium PS3 (TF1) have been examined. While chlorpromazine inhibited MF1 with an I0.5 of about 50 microM and EF1 with an I0.5 of about 150 microM at 23 degrees C, the ATPase activity of TF1 was stimulated by chlorpromazine concentrations up to 0.6 mM at this temperature. Maximal activation of about 20% was observed at 0.2 mM chlorpromazine at 23 degrees C. Chlorpromazine concentrations greater than 0.6 mM inhibited TF1 at 23 degrees C. At 37 degrees C the ATPase activity of TF1 was doubled in the presence of 0.5 mM chlorpromazine, the concentration at which maximal stimulation was observed at this temperature. Chlorpromazine inhibited the rate of inactivation of EF1 by dicyclohexylcarbodiimide (DCCD) at 23 degrees C and pH 6.5. Concentrations of chlorpromazine which inhibited the ATPase activity of TF1 at pH 7.0 accelerated the rate of inactivation of the enzyme by DCCD at pH 6.5, while lower concentrations of the phenothiazine, which stimulated the ATPase, had no effect on DCCD inactivation. Chlorpromazine concentrations up to 1.0 mM had no effect on the rate of inactivation of TF1 by DCCD at 37 degrees C and pH 6.5. Chlorpromazine at 0.5 mM accelerated the rate of inactivation of MF1 by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), while it slowed the rate of inactivation of EF1 by FSBA. The inactivation of TF1 by FSBA in the absence of chlorpromazine was complex and was not included in this comparison. Chlorpromazine protected MF1 and EF1 against cold inactivation. Whereas 100 microM chlorpromazine afforded about 90% stabilization of MF1 at 4 degrees C, only about 30% stabilization of EF1 was observed under the same conditions in the presence of 400 microM chlorpromazine. Each of the ATPases was inactivated by the structural analog of chlorpromazine, quinacrine mustard. Whereas 5 mM ATP and 5 mM ADP protected MF1 and TF1 against inactivation by 0.5 mM quinacrine mustard, the rate of inactivation of EF1 by quinacrine mustard was accelerated fourfold by 5 mM ATP and slightly accelerated by 5 mM ADP.

Adenosine↗
PubMed1984

Identification of the lysine residue to which the 4-nitrobenzofurazan group migrates after the bovine mitochondrial F1-ATPase is inactivated with 7-chloro-4-nitro[14C]benzofurazan.

When bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, was inactivated by greater than 90% with a 4-fold molar excess of 7-chloro-4-nitro[14C]benzofurazan at pH 7.4, 1.15 mol of 4-nitrobenzofurazan [14C]Nbf were incorporated per mol of enzyme. Reactivation of a sample of the modified enzyme with dithiothreitol removed 0.82 mol of [14C]Nbf/mol of the F1-ATPase indicating that, of the 1.15 mol of [14C]Nbf incorporated, 0.82 mol were present on tyrosine residues and 0.33 mol on lysine residues. Incubation of the modified enzyme at pH 9.0 for 18 h at 23 degrees C led to an increase of 0.64 mol of [14C]Nbf-N'-Lys/mol of the F1-ATPase which occurred as a consequence of an O----N migration. About 15% enzyme reactivation occurred simultaneously with the migration indicating that the fraction of the [14C]Nbf group originally present on tyrosine which did not migrate was lost by hydrolysis. Examination of a tryptic digest of the labeled enzyme after the O----N migration by reversed-phase high-pressure liquid chromatography revealed a single major radioactive peptide. The labeled tryptic fragment was purified and subjected to automatic Edman degradation. This analysis revealed that Lys-beta-162 was specifically labeled during the O----N migration of the [14C]Nbf group.

Amino Acids↗
PubMed1984

Identification of an essential lysine residue in the beta subunit of the F1-ATPase from the thermophilic bacterium, PS3, using 7-chloro-4-nitro[14C]benzofurazan.

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.3 and then gel-filtered, 1.25 mols of [14C]Nbf-O-Tyr and less than 0.1 mol of Nbf-N-Lys were formed per mol of enzyme. After adjusting the pH of the gel-filtered, modified enzyme to 9.0 and incubating it for 14 hrs. at 23 degrees C to promote O----N migration, 0.68 mol of Nbf-N-Lys were formed per mol of enzyme while about 16% of the original activity reappeared. Isolation of the subunits after the O----N migration showed that 90% of the incorporated 14C was present in the beta subunit, which contained 0.21 mols of [14C]Nbf-N-Lys per mol. A tryptic peptide which contained the majority of the 14C incorporated into the beta subunit was isolated and subjected to automatic amino acid sequence analysis contained 38 residues. The amino acid sequence immediately around the lysine residue labeled with [14C]Nbf-, K*, was found to be: ...I-G-L-F-G-G-A-G-V-G-K*-T-V-L-I-G... .

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Identification of the essential tyrosine residue in the beta subunit of bovine heart mitochondrial F1-ATPase that is modified by 7-chloro-4-nitro[14C]benzofurazan.

Inactivation of the bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, with a 4-fold molar excess of 7-chloro-4-nitro[14C]benzofurazan at pH 7.5, led to the incorporation of 1.42 g atoms of 14C/mol. Treatment of the inactivated enzyme with dithiothreitol removed 0.99 g atom of 14C/mol of enzyme which was accompanied by reactivation of the ATPase. Therefore, of the 1.42 mol of 7-chloro-4-nitro-[14C]benzofurazan incorporated per mol of bovine heart mitochondrial F1-ATPase, 0.43 mol was present on lysine residues and 0.99 mol was present on tyrosine residues. When the inactivated enzyme was treated with 10 mM sodium dithionite at pH 6.0, 10% of the activity was recovered which was accompanied by a 10% loss in covalently bound 14C. Following dithionite treatment, that part of the 14C which remained covalently bound could not be removed by subsequent treatment of the labeled enzyme with dithiothreitol. It is presumed that dithionite reduces the 4-nitro group of the covalently bound reagent, converting it to 4-amino[14C]benzofurazan derivatives at lysine and tyrosine residues. The moles of 4-amino[14C]benzofurazan incorporated per mol of the isolated subunits were: alpha, 0.18; beta, 0.30; gamma, 0.03; and delta plus epsilon, less than 0.01. Gel filtration of a cyanogen bromide digest of the labeled beta subunit on Sephadex G-75 resolved a major 14C peak which contained 83% of the 14C recovered. The major, radioactive tryptic fragment derived from this peak was purified by gel filtration on Sephadex G-75 followed by reversed phase high performance liquid chromatography. Automatic Edman degradation of this peptide showed that the 14C was released at the position occupied by beta-Tyr-311.

4-Chloro-7-nitrobenzofurazan↗