Biomedical subjects

W S Allison

Publications and source records attributed to W S Allison.

At least 37 records · Page 2Linked to original sources

PubMed1994

ADP tethered to tyrosine-beta 345 at the catalytic site of the bovine heart F1-ATPase is converted to tethered AMP by Mg(2+)-dependent hydrolysis when the enzyme is photoinactivated with 2-N3-ADP.

Comparison of profiles of radioactive peptides resolved by HPLC from tryptic digests of the bovine heart F1-ATPase depleted of nucleotides (nd-MF1) which had been photoinactivated with 2-N3-[beta-32P]ADP, on the one hand, and 2-[8-3H]ADP, on the other, shows that the beta phosphate of ADP tethered to tyrosine-beta 345 is slowly hydrolyzed in the presence of Mg2+. When nd-MF1 was photoinactivated with 2-N3-[8-3H]ADP in the absence of Mg2+, hydrolysis of the beta phosphate from ADP tethered to tyrosine-beta 345 was not observed. Subsequent addition of Mg2+ initiated conversion of ADP tethered to tyrosine-beta 345 to tethered AMP suggesting that functional groups at the catalytic site participate in the hydrolytic reaction.

Adenosine Diphosphate↗

PubMed1994

Inhibition and inactivation of the F1 adenosinetriphosphatase from Bacillus PS3 by dequalinium and activation of the enzyme by lauryl dimethylamine oxide.

The F1-ATPase from Bacillus PS3 (TF1) hydrolyzes 50 microM ATP in three kinetic phases. An initial burst rapidly decelerates to a partially inhibited, intermediate phase, which, in turn, gradually accelerates to an uninhibited, final steady-state rate. Lauryl dimethylamine oxide (LDAO) stimulates the final rate over 4-fold. The stimulatory effect saturates at about 0.1% LDAO. Under these conditions, the intermediate phase is nearly absent. Dequalinium inhibits TF1 reversibly in the dark in the presence or absence of LDAO. The apparent affinity of TF1 for dequalinium increases in the presence of LDAO. Dixon plots of the initial rates of the intermediate phase and the final rates against dequalinium concentration at a series of fixed ATP concentrations in the presence and absence of 0.03% LDAO indicate noncompetitive inhibition in each case. Replots of the slopes of the Dixon plots for the initial rate of the intermediate phase and the final rate against 1/[ATP] reveal apparent Km values of 770 microM and 144 microM, respectively, when obtained in the absence of LDAO. The apparent Km values determined from the data obtained in the presence of LDAO for the same phases are 303 microM and 163 microM, respectively. These results suggest that LDAO stimulates ATPase activity either by increasing the affinity of noncatalytic sites for ATP, which promotes release of inhibitory MgADP from a catalytic site, or by directly promoting release of MgADP from the affected catalytic site. Dequalinium retards this process without affecting the affinity of noncatalytic sites for ATP. When irradiated in the presence of dequalinium, TF1 is rapidly inactivated with an apparent Kd of 12.5 microM in the presence or absence of LDAO.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Hysteretic inhibition of the bovine heart mitochondrial F1-ATPase is due to saturation of noncatalytic sites with ADP which blocks activation of the enzyme by ATP.

Prior incubation of the bovine heart mitochondrial F1-ATPase depleted of endogenous nucleotides (nd-MF1) with saturating ADP in the presence or absence of Mg2+ induces inhibition of hydrolysis of 2 mM ATP or ITP. After incubation of nd-MF1 with free ADP, inhibition develops hysteretically which is characterized by an uninhibited initial rate which decelerates to an inhibited, steady-state rate. When prior incubation of nd-MF1 is performed with ADP in the presence of Mg2+, the enzyme is partially inhibited when diluted into assay medium and more extensive inhibition develops hysteretically during turnover. Correlation of binding of [14C]ADP, in the presence or absence of Mg2+, with the extent of hysteretic inhibition induced suggests that maximal inhibition occurs when at least two noncatalytic sites are filled with ADP. Hysteretic inhibition is also induced by prior incubation of the enzyme with 2-N3-ADP. Prior incubation of nd-MF1 with increasing concentrations of 2-N3-[beta-32P]ADP, in the presence or absence of Mg2+, increases the extent of induced inhibition which correlates with increasing derivatization of tyrosine beta 368 following irradiation of loaded enzyme. This demonstrates that binding of ADP to noncatalytic sites is, in part, responsible for induction of hysteretic inhibition. After inducing inhibition by prior incubation with ADP, the steady-state kinetic behavior of nd-MF1 differs from that of uninhibited enzyme. Lineweaver-Burk plots of steady-state rates of inhibited enzyme as a function of ATP concentration are linear rather than biphasic which is observed for uninhibited enzyme. The composite results suggest that prior saturation of noncatalytic sites of nd-MF1 with ADP prevents activation of the enzyme by blocking the binding of ATP to these sites which is necessary to promote dissociation of inhibitory MgADP from a catalytic site.

Adenosine Diphosphate↗

PubMed1994

Probing the specificity of nucleotide binding to the F1-ATPase from thermophilic Bacillus PS3 and its isolated alpha and beta subunits with 2-N3-[beta, gamma-32P]ATP.

Irradiation of the F1-ATPase from Bacillus PS3 (TF1) in the presence of 134 microM 2-N3-[beta, gamma-32P]ATP plus Mg2+ for 90 min led to 95% inactivation of the ATPase activity which was accompanied by exclusive labeling of the beta subunit. The isolated alpha and beta subunits were also treated separately with 2-N3-[beta, gamma-32P]ATP under similar conditions. Fractionation of a tryptic digest of photolabeled TF1 by reversed-phase HPLC resolved a major and a minor radioactive peptide. Sequence analyses showed that the major peptide contained labeled Tyr-beta 364, whereas the minor one contained labeled Tyr-beta 341, residues known to be part of noncatalytic and catalytic sites, respectively. Two closely eluting radioactive peptides were obtained when a tryptic digest of the photolabeled, isolated beta subunit was fractionated by HPLC. Sequence analyses revealed that both contained labeled Tyr-beta 341. Fractionation of a tryptic digest of the photolabeled, isolated alpha subunit by HPLC resolved two peptides which contained the majority of the radioactivity incorporated. When subjected to eight cycles of automatic Edman degradation, one gave the sequence APGVXDR, corresponding to residues 133-139, in which X is a gap and corresponds to Met-alpha 137, which presumably is the derivatized residue. Only the first five cycles yielded phenylthiohydantoin derivatives when the other radioactive peptide derived from the alpha subunit was submitted to automatic Edman degradation which revealed the sequence APGVM, suggesting that Asp-alpha 138 is derivatized. The overall results suggest that the isolated beta subunit is a useful model for studying binding of nucleotides to catalytic sites, whereas the isolated alpha subunit may be of limited value in modeling interactions of nucleotides with noncatalytic sites.

Adenosine Triphosphate↗

PubMed1993

In vivo affinity label of a protein expressed in Escherichia coli. Coenzyme A occupied the AT(D)P binding site of the mutant F1-ATPase beta subunit (Y307C) through a disulfide bond.

When Tyr-307 of the beta subunit of F1-ATPase from a thermophilic Bacillus strain PS3 is replaced by cysteine and expressed in Escherichia coli cells, about a half population of the mutant beta subunit are labeled by Coenzyme A at Cys-307 through a disulfide bond which is cleavable by reducing treatment. The mutant beta subunit can be reconstituted into the alpha 3 beta 3 complex of which ATPase activity is stimulated two-fold by reducing treatment either prior or after reconstitution. Since Tyr-307 has been supposed to be located at one of subdomains which form the ATP binding site of the beta subunit, Coenzyme A binds to the mutant beta subunit as an AT(D)P analogue in E. coli cells and then covalently attaches to Cys-307.

Adenosine Diphosphate↗

PubMed1993

Glutamine 170 to tyrosine substitution in yeast mitochondrial F1 beta-subunit increases catalytic site interaction with GDP and IDP and produces negative cooperativity of GTP and ITP hydrolysis.

Glutamine 170 to tyrosine mutation in the beta-subunit from Schizosaccharomyces pombe mitochondrial F1 was found to increase both affinity for ADP, apparent negative cooperativity of ATPase activity, and sensitivity to azide inhibition (Falson, P., Di Pietro, A., Jault, J.-M., Gautheron, D.C., and Boutry, M. (1989) Biochim. Biophys. Acta 975, 119-126). The mutation is shown here to increase the affinity for GDP, IDP, and guanosine 5'-(beta,gamma-imidotriphosphate), which are competitive inhibitors of GTPase and ITPase activities. Various fluorescence approaches also reveal an increased affinity of the catalytic site in mutant as compared with wild-type enzyme for GDP, IDP, and 2'(3')-N-methylanthraniloyl GDP. The mutation alters the maximal rates and pH dependence of GTPase and ITPase activities, whereas wild-type F1 exhibits single optima at pH 7.5-8.0. The pH activity profiles of the mutant enzyme for these substrates are biphasic, with optima at pH 8.5-9.0 and below 6.5. The mutation increases the sensitivity of GTPase and ITPase activities to azide inhibition, which increases with decreasing pH. At pH 6.0-7.0, an apparent negative cooperativity is observed when mutant F1 hydrolyzes GTP or ITP, whereas the wild-type enzyme shows Michaelian kinetics. Addition of bicarbonate at pH 7.0 substantially stimulates GTP or ITP hydrolysis and abolishes the apparent negative cooperativity by the mutant enzyme; on the contrary, the anion produces a slight inhibition of these activities catalyzed by wild-type F1. The overall results suggest that apparent negative cooperativity can be observed with GTP or ITP hydrolysis provided that the release of the respective diphosphate is a rate-limiting step.

Azides↗

PubMed1993

Photoinactivation of the bovine heart mitochondrial F1-ATPase by [14C]dequalinium cross-links phenylalanine-403 or phenylalanine-406 of an alpha subunit to a site or sites contained within residues 440-459 of a beta subunit.

Synthesis of [14C]dequalinium, 1,1'-(1,10-[1,10-14C]decanediyl)bis[4-amino-2-methylquinolinium ], is described, which photoinactivates the bovine heart mitochondrial F1-ATPase (MF1). Maximal photoinactivation occurs on incorporation of about 1.5 mol of [14C]dequalinium/mol of MF1. Three radioactive species were resolved when photoinactivated enzyme was submitted to polyacrylamide gel electrophoresis at pH 4.0 in the presence of tetradecyltrimethylammonium bromide, which correspond to the alpha and beta subunits and a cross-linked species with an M(r) of 116,000. Fractionation of a tryptic digest of photoinactivated enzyme by high-performance liquid chromatography led to isolation of a radioactive peptide which contains residues 399-420 of a alpha subunit. Two fragments containing equal amounts of radioactivity were obtained on fractionation of an endoproteinase Asp-N digest of the isolated radioactive tryptic peptide by high-performance liquid chromatography. Amino acid sequence analysis showed that both fragments contained residues 399-408 of the alpha subunit, but one was missing Phe-alpha 403 and the other was lacking Phe-alpha 406. Fractionation of a cyanogen bromide digest of photoinactivated enzyme followed by trypsin digestion of partially purified cyanogen bromide fragments and fractionation of the resulting radioactive tryptic fragments yielded several radioactive species comprised of residues 399-420 of the alpha subunit cross-linked to residues 440-459 of the beta subunit and a radioactive fragment containing residues 399-420 of the alpha subunit. Partial sequence analyses of the cross-linked fragments suggest that Phe-alpha 403 and Phe-alpha 406 participate in cross-links, whereas no information was obtained on the site or sites of cross-linking in the beta subunit fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Slow binding of ATP to noncatalytic nucleotide binding sites which accelerates catalysis is responsible for apparent negative cooperativity exhibited by the bovine mitochondrial F1-ATPase.

The bovine heart mitochondrial F1-ATPase depleted of nucleotides (nd-MF1) hydrolyzes 50 microM ATP in three kinetic phases at 30 degrees C. An initial "burst" rapidly transforms into an intermediate, slower rate, which slowly accelerates to the final, steady-state rate. The intermediate phase disappears progressively as the concentration of ATP in the assay medium is increased and is absent at 2 mM. Activation in the intermediate phase is lost when nd-MF1 is inactivated by 5'-p-fluorosulfonylbenzoyladenosine, which modifies three noncatalytic sites. Correlation of [3H]ATP binding to nd-MF1, after treatment either with 50 microM Mg[3H]ATP plus a regenerating system or 10 mM free [3H]ATP, with stimulation of the intermediate phase suggests that this phase is abolished when at least two noncatalytic sites are filled with ATP. Prior incubation of nd-MF1 with MgPPi stimulates hydrolysis of 30 microM to 2 mM ATP and abolishes the intermediate phase. Following incubation with Mg[32P]PPi, 3.3 mol of [32P]PPi/mol of enzyme are bound, 1 and 0.5 mol of which are released by cold chases with MgATP and MgITP, respectively. Since the cold chases diminish activation only slightly, the stimulatory effect is not caused by PPi binding to catalytic sites. A Lineweaver-Burk plot of initial rates of the intermediate phase for hydrolysis of 30 microM to 2 mM ATP by nd-MF1 is biphasic, extrapolating to apparent Km values of 120 and 440 microM. The latter value is the same as the apparent Kd determined from dependence of the rate of activation of the intermediate phase on ATP concentration in the assay medium. After prior incubation of nd-MF1 with MgPPi or free ATP, Lineweaver-Burk plots are linear with the highest Km disappearing. Thus, this Km reflects rate acceleration when ATP binds to noncatalytic sites. From these results it is concluded that slow binding of ATP to noncatalytic sites during hydrolysis of low concentrations of substrate, which accelerates catalysis, is responsible for apparent negative cooperativity exhibited by MF1.

Adenosine↗

PubMed1993

Study of the mechanism of MF1 ATPase inhibition by fluorosulfonylbenzoyl inosine, quinacrine mustard, and efrapeptin using intermediate 18O exchange as a probe.

The mitochondrial F1-ATPase (MF1) is known to be largely or totally inhibited by combination or reaction with one fluorosulfonylbenzoyl inosine (FSBI), quinacrine mustard, or efrapeptin per enzyme. Measurements were made with 18O in attempt to ascertain if the weak catalytic activity remaining after exposure to excess of these reagents was due to retention of some activity by the enzyme modified by these inhibitors. Any such activity could have different characteristics that might be revealed by the distribution of [18O]Pi isotopomers formed from [gamma-18O]ATP. The MF1 inhibited by FSBI showed progressive appearance of two new catalytic pathways as inhibition proceeded. Both pathways appeared to be operative in the enzyme after one beta subunit per enzyme had been modified by FSBI. A high exchange pathway showed no detectable change as ATP concentration was lowered. The lower exchange pathway showed an increase in the amount of exchange with lowering of the ATP concentration, similar to the cooperative behavior observed with the unmodified enzyme. With excess ATP more product was formed by the low exchange pathway, showing that compulsory alternation between two catalytic sites was not retained. The behavior can be explained by the ability of the modified beta subunit to undergo binding changes similar to those occurring in catalysis, with the other two beta subunits catalyzing sluggish hydrolysis by different pathways because of the asymmetry introduced by the modification. Inhibition by quinacrine mustard also resulted in the appearance of two new pathways, somewhat similar to those from FSBI inhibition. In contrast, activity remaining with excess efrapeptin present showed only one pathway like that of the native enzyme. This can be attributed to a low equilibrium concentration of free enzyme and total inhibition of MF1 combined with efrapeptin.

Affinity Labels↗

PubMed1993

The TF1-ATPase and ATPase activities of assembled alpha 3 beta 3 gamma, alpha 3 beta 3 gamma delta, and alpha 3 beta 3 gamma epsilon complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the alpha 3 beta 3, and alpha 3 beta 3 delta complexes.

The ATPase activity of the F1-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 microM where 70% stimulation is observed at 36 degrees C. Half maximal stimulation is observed at about 3 microM dye. At rhodamine 6G concentrations greater than 10 microM, ATPase activity declines with 50% inhibition observed at about 75 microM dye. The ATPase activities of the alpha 3 beta 3 gamma and alpha 3 beta 3 gamma delta complexes assembled from isolated subunits of TF1 expressed in E. coli deleted of the unc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF1. In contrast, the ATPase activities of the alpha 3 beta 3 and alpha 3 beta 3 delta complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 microM dye at 36 degrees C. The ATPase activity of TF1 is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF1 is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 microM dye at 30 degrees C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF1 stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.

Adenosine Triphosphatases↗

PubMed1992

Irradiation of the bovine mitochondrial F1-ATPase previously inactivated with 5'-p-fluorosulfonylbenzoyl-8-azido-[3H]adenosine cross-links His-beta 427 to Tyr-beta 345 within the same beta subunit.

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p'-fluorosulfonylbenzoyl-8-azidoadenosine (8-N3-FSBA) with an apparent Kd of 0.47 mM at pH 8.0 and 23 degrees C in the absence of light. Irradiation of dark-inactivated enzyme with long-wavelength UV light produced cross-linked dimers and, to a lesser extent, trimers made up of alpha and beta subunits. Two major radioactive peptides were resolved by high-performance liquid chromatography from tryptic digests of MF1 which had been inactivated with 8-N3-FSB[3H]A at pH 8.0 in the dark. Sequence analysis revealed that one contained Tyr-beta 368 and the other contained His-beta 427 which were labeled in the ratio of 18:15. Sequence analysis of radioactive tryptic peptides isolated from digests of irradiated MF1 derivatized with 8-N3-FSB[3H]A showed that photolysis induced cross-linking of His-427 to Tyr-345 within the same beta subunit in high yield. When MF1 derivatized with 8-N3-FSB[3H]A was irradiated in the presence of beta-mercaptoethanol, alpha-beta cross-links were eliminated, whereas those between His-beta 427 and Tyr-beta 345 were unaffected. Analysis of radioactive peptides in tryptic digests of MF1 derivatized with 8-N3-FSB[3H]A and then irradiated in the presence or absence of beta-mercaptoethanol showed that the nitrene generated from reagent attached to Tyr-beta 368 participates in formation of alpha-beta cross-links in the absence of beta-mercaptoethanol. Therefore, the nitrene generated from reagent tethered to His-beta 427 is shielded from solvent and reacts with the side chain of Tyr-beta 345. In contrast, the nitrene generated from reagent attached to Tyr-beta 368 is exposed to solvent, but in the absence of scavengers reacts with side chains present in the alpha subunit. Irradiation of MF1, partially inactivated with 8-N3-FSBA, led to loss of residual ATPase activity without affecting residual ITPase activity. The amount of photoinactivation was greater when partial dark inactivation was performed at pH 6.9, where modification of His-beta 427 predominates, than when performed at pH 8.0, where modification of Tyr-beta 368 predominates. This suggests that cross-linking of His-beta 427 to Tyr-beta 345, and not cross-linking of alpha and beta subunits, is responsible for the augmented inactivation induced by irradiation.

Adenosine↗

PubMed1992

Significant quantities of endogenous GDP and ADP are present on catalytic sites of the F1-ATPase isolated from M. lysodeikticus in the absence of added nucleotides.

The F1-ATPase from Micrococcus lysodeikticus is isolated in the absence of exogenous nucleotides. After removing loosely bound nucleotides from the isolated enzyme by gel permeation chromatography, analysis for tightly bound nucleotides revealed in 14 experiments 0.4 +/- 0.1 mol ADP, 0.5 +/- 0.2 mol GDP, and 0.8 +/- 0.2 mol ATP per mol of F1. Incubation of the isolated enzyme with Mg2+ or Ca2+ did not alter the endogenous nucleotide composition of the enzyme, indicating that endogenous ATP is not bound to a catalytic site. Incubation of the enzyme with P(i) decreased the amount of tightly bound ADP and GDP but did not effect the ATP content. Hydrolysis of MgATP in the presence of sulfite raised the tightly bound ADP and lowered tightly bound GDP on the enzyme. In the reciprocal experiment, hydrolysis of MgGTP in the presence of sulfite raised tightly bound GDP and lowered tightly bound ADP. Turnover did not affect the content of tightly bound ATP on the enzyme. These results suggest that endogenous ADP and GDP are bound to exchangeable catalytic sites, whereas endogenous ATP is bound to noncatalytic sites which do not exchange. The presence of endogenous GDP on catalytic sites of isolated F1 suggests that the F0F1-ATP synthase of M. lysodeikticus might synthesize both GTP and ATP under physiological conditions. In support of this hypothesis, we have found that plasma membrane vesicles derived from M. lysodeikticus synthesize [32P]GTP from [32P]P(i) using malate as electron donor for oxidative phosphorylation.

Adenosine Diphosphate↗

PubMed1992

Antipeptide antibodies to the carboxy terminal and the DCCD binding region of the human mitochondrial ATP synthase beta-subunit.

Antibodies to defined epitopes on the human ATP synthase would provide a powerful tool in the definition of the subunit composition of the enzyme complex and in the characterization of any defect in its assembly in diseases associated with mitochondrial disorders. Antibodies have been thus raised against synthetic peptides, corresponding to two regions on the human ATP synthase beta-subunit: the C-terminal region, and a region which includes the two dicyclohexylcarbodiimide (DCCD)-reactive glutamic acid residues suggested to be involved in the enzyme catalytic activity. The antibodies to the C-terminal peptide reacted with the ATP synthase beta-subunit in ELISA, in Western immunoblotting and in immunohistochemical experiments, and had the ability to immunoprecipitate the enzyme complex. The antibodies to the DCCD-binding region peptide did not react to the ATP synthase beta-subunit in its native configuration, although reacted well under Western immunoblotting conditions.

Amino Acid Sequence↗

PubMed1992

Functional sites in F1-ATPases: location and interactions.

This review focuses on the location and interaction of three functional sites in F1-ATPases. These are catalytic sites which are located in beta subunits, noncatalytic nucleotide-binding sites which are located at interfaces of alpha and beta subunits and modulate the hydrolytic activity of the enzyme, and a site that binds inhibitory amphipathic cations which is at an interface of alpha and beta subunits. The latter site may participate in transmission of conformational signals between catalytic sites in F1 and the proton-conducting apparatus of F0 in the intact ATP synthases.

Amino Acid Sequence↗

PubMed1991

Heterogeneous hydrolysis of substoichiometric ATP by the F1-ATPase from Escherichia coli.

The hydrolysis of 0.3 microM [alpha,gamma-32P]ATP by 1 microM F1-ATPase isolated from the plasma membranes of Escherichia coli has been examined in the presence and absence of inorganic phosphate. The rate of binding of substoichiometric substrate to the ATPase is attenuated by 2 mM phosphate and further attenuated by 50 mM phosphate. Under all conditions examined, only 10-20% of the [alpha,gamma-32P]ATP that bound to the enzyme was hydrolyzed sufficiently slowly to be examined in cold chase experiments with physiological concentrations of non-radioactive ATP. These features differ from those observed with the mitochondrial F1-ATPase. The amount of bound substrate in equilibrium with bound products observed in the slow phase which was subject to promoted hydrolysis by excess ATP was not affected by the presence of phosphate. Comparison of the fluxes of enzyme-bound species detected experimentally in the presence of 2 mM phosphate with those predicted by computer simulation of published rate constants determined for uni-site catalysis (Al-Shawi, M.D., Parsonage, D. and Senior, A.E. (1989) J. Biol. Chem. 264, 15376-15383) showed that hydrolysis of substoichiometric ATP observed experimentally was clearly biphasic. Less than 20% of the substoichiometric ATP added to the enzyme was hydrolyzed according to the published rate constants which were calculated from the slow phase of product release in the presence of 1 mM phosphate. The majority of the substoichiometric ATP added to the enzyme was hydrolyzed with product release that was too rapid to be detected by the methods employed in this study, indicating again that the F1-ATPase from E. coli and bovine heart mitochondria hydrolyze substoichiometric ATP differently.

Adenosine Triphosphate↗

PubMed1991

Separate beta subunits are derivatized with 14C and 3H when the bovine heart mitochondrial F1-ATPase is doubly labeled with 7-chloro-4-nitro[14C]benzofurazan and 5'-p-fluorosulfonylbenzoyl[3H]inosine.

Tyrosine residues 311 and 345 of the beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) are present on the same peptide when the enzyme is fragmented with cyanogen bromide. Maximal inactivation of MF1 with 7-chloro-4-nitro[14C]benzofurazan [( 14C]Nbf-Cl) derivatizes tyrosine-311 in a single beta subunit. Cyanogen bromide digests of MF1 containing the [14C]Nbf-O-derivative of tyrosine-beta 311 were submitted to reversed-phase HPLC, with and without prior reduction of the nitro group on the incorporated reagent with dithionite. The retention time of the radioactive cyanogen bromide peptide was shifted substantially by reduction. When a cyanogen bromide digest of MF1 inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine [( 3H]FSBI), which proceeds with derivatization of tyrosine-345 in a single beta subunit, was submitted to HPLC under the same conditions, the fragment labeled with 3H eluted with the same retention time as the [14C]Nbf-O-derivative before reduction. Doubly labeled enzyme was prepared by first derivatizing Tyr-beta 311 with [14C]Nbf-Cl and then derivatizing tyrosine-beta 345 with [3H]FSBI with and without reducing the [14C]Nbf-O-derivative of tyrosine-beta 311 with dithionite before modification with [3H]FSBI. The doubly labeled enzyme preparations were digested with cyanogen bromide and submitted to HPLC. The 14C and 3H in the cyanogen bromide digest prepared from doubly labeled enzyme not submitted to reduction eluted together. In contrast, the 14C and 3H in the digest prepared from doubly labeled enzyme which had been reduced eluted separately. From these results it is concluded that different beta subunits are derivatized when MF1 is doubly labeled with [14C]Nbf-Cl and [3H]FSBI.

Animals↗

PubMed1990

Tyrosine alpha 244 is derivatized when the bovine heart mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoylethenoadenosine.

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p-fluorosulfonylbenzoylethenoadenosine (FSB epsilon A) with pseudo-first order kinetics. The dependence of the rate of inactivation on the concentration of FSB epsilon A revealed an apparent Kd of 0.25 mM. ATP and ADP, and to a lesser extent, ITP and IDP provide partial protection against inactivation by the reagent. Isolation and sequence analysis of major radioactive fragments in peptic or cyanogen bromide digests of MF1 inactivated with [3H]FSB epsilon A indicate that modification of Tyr-alpha 244 is associated with the loss of activity observed. Assessment of the amount of Tyr-alpha 244 derivatized with [3H]FSB epsilon A at specific points during inactivation of the ATPase indicates that maximal inactivation is achieved on modification of this residue in slightly greater than one copy of the alpha subunit. The following characteristics of inactivation of MF1 by FSB epsilon A have also been determined. (a) The rate of inactivation of ITPase activity by FSB epsilon A is 1.4 times greater than that observed for inactivation of ATPase activity under identical conditions. (b) After maximally inactivating the capacity of MF1 to hydrolyze saturating ATP with FSB epsilon A, the modified enzyme retained its capacity to hydrolyze substoichiometric ATP. (c) Inactivation of the ATPase by FSB epsilon A is accelerated by Pi. In each of the above characteristics, MF1 modified by FSB epsilon A resembles enzyme inactivated with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) more than it does enzyme inactivated with 5'-p-fluorosulfonylbenzoylinosine (FSBI). Furthermore, prior inactivation of MF1 with FSBA completely prevents labeling of Tyr-alpha 244 with [3H]FSB epsilon A, whereas prior inactivation of the enzyme with FSBI does not. Since a single catalytic site is modified when FSBI inactivates MF1 whereas three noncatalytic sites are modified when it is maximally inactivated with FSBA, it is concluded that FSB epsilon A also modifies noncatalytic sites.

Adenosine↗

PubMed1990

The ATPase activity of the alpha 3 beta 3 complex of the F1-ATPase of the thermophilic bacterium PS3 is inactivated on modification of tyrosine 307 in a single beta subunit by 7-chloro-4-nitrobenzofurazan.

The catalytically active alpha 3 beta 3 complex, assembled as described (Miwa, K., and Yoshida, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6484-6487) from the isolated alpha and beta subunits of the F1-ATPase of the thermophilic bacterium PS3 (TF1), is inactivated by 7-chloro-4-nitrobenzofurazan (Nbf-Cl) with characteristics very similar to those observed when TF1, which has the subunit composition, alpha 3 beta 3 gamma delta epsilon, is inactivated by the reagent under the same conditions. Both native TF1 and the alpha 3 beta 3 complex are inactivated by 200 microM Nbf-Cl with a pseudo-first order rate constant of 3.7 x 10(-2) min-1 in the presence of 0.2 M Na2SO4 at pH 7.6 and 23 degrees C. The rate of increase in absorbance at 385 nm of reaction mixtures containing 200 microM [14C]Nbf-Cl and TF1, the wild-type alpha 3 beta 3 complex, or the mutant alpha 3(beta Y307----F)3 complex, each at 18 microM was also examined. Since the alpha 3(beta y307----F)3 complex is resistant to inactivation by Nbf-Cl, difference spectrophotometry revealed that inactivation of native TF1 and the wild-type alpha 3 beta 3 complex could be correlated with formation of about 1 mol of Nbf-O-Tyr/mol of enzyme or complex. Fractionation of peptic digests of the labeled enzyme and complexes by reversed-phase high performance liquid chromatography resolved a major radioactive peptide that was common to labeled TF1 and the labeled alpha 3 beta 3 complex but was absent in the digest of the labeled alpha 3(beta Y307----F)3 complex. This labeled peptide was shown to contain Tyr-beta 307 derivatized with [14C]Nbf-Cl by automatic amino acid sequence analyses. From these results, it is concluded that one-third of the sites' reactivity of Nbf-Cl with Tyr-beta 307 in TF1 or its equivalent in other F1-ATPases is not influenced by the presence of the gamma, delta, or epsilon subunits. It has also been shown that Tyr-307 is not modified to an appreciable extent when the isolated beta subunit is treated with [14C]Nbf-Cl under conditions in which this residue is nearly completely labeled in a single beta subunit when TF1 or the alpha 3 beta 3 complex is inactivated by the reagent.

Bacteria↗