Biomedical subjects

R A Capaldi

Publications and source records attributed to R A Capaldi.

At least 73 records · Page 4Linked to original sources

PubMed1995

Conformational changes in the gamma and epsilon subunits are integral to the functioning of the Escherichia coli H(+)-pumping ATPase (ECF1F0).

ATP synthesis and ATP hydrolysis by F1F0-type ATPases involve conformational changes transmitted from the catalytic site regions to the proton channel, a distance of more than 100 A. Our studies focus attention on the gamma and epsilon subunits that provide a part of the stalk region in the energy-coupling process within the complex. There are conformational changes in the gamma subunit, and translocations of the epsilon unit, linked to nucleotide-binding changes in catalytic sites, which might be expected to alter the interaction of this subunits with c subunits and, hence, be linked to proton translocation.

Adenosine Triphosphate↗

PubMed1994

Developmental regulation of tissue-specific isoforms of subunit VIa of beef cytochrome c oxidase.

The switching of the subunit VIa isoforms of cytochrome c oxidase has been followed in heart tissue during bovine development both by transcript levels and in terms of the incorporation of L- (liver) and H- (heart) polypeptides into mitochondria. In early fetuses, e.g., 60-days development, there are high levels of VIaL transcript and high levels of the VIaL polypeptide incorporated into mitochondria. In late fetuses (after 200 days), the levels of VIaL transcript are still high, with less but still significant amounts of VIaL polypeptide present in comparison to adult heart in which the amount of this isoform is negligible. As the proportion of VIaL transcript is reduced, the proportion of VIaH transcript increases along with the amount of the VIaH isoform in mitochondria. These data indicate isoform switching during late fetal development. The presence of COLBP (cytochrome oxidase liver isoform binding protein) (Preiss, T. and Lightowlers, R.N. (1993) J. Biol. Chem. 268, 10659-10667) was examined at different developmental stages. COLBP binding activity was observed in hearts of late fetuses but not found in adult heart tissue, providing a correlation between the presence of this factor and the presence of the VIaL polypeptide in mitochondria.

Aging↗

PubMed1994

ATP binding causes a conformational change in the gamma subunit of the Escherichia coli F1ATPase which is reversed on bond cleavage.

ATP hydrolysis by the Escherichia coli F1 ATPase (ECF1) induces a conformational change in the gamma subunit. This change can be monitored by fluorescence changes in N-[4-[7-(diethylamino)-4-methyl]coumarin-3-yl)]maleimide (CM) bound at a cysteine introduced by site-directed mutagenesis into the gamma subunit at position 106 [Turina, P., & Capaldi, R. A. (1994) J. Biol. Chem. 269, 13465-13471]. In studies reported here, the magnitude of the fluorescence change has been determined with the noncleavable nucleotide analogue AMP-PNP and by rapid measurements using the slowly cleavable ATP gamma S. The data indicate that maximal fluorescence change occurs with binding of 1 mol of nucleotide triphosphate per mole of ECF1. During unisite catalysis, ATP binding causes a fluorescence enhancement from CM bound at position 106, which is then followed by fluorescence quenching. The kinetics of these fluorescence changes have been measured using both ATP and ATP gamma S as substrate. With ATP gamma S, these kinetics can be simulated using rate constants similar to those for ATP except for an approximately 30-fold slower rate of the bond cleavage and resynthesis steps, i.e., k+2 and k-2. The observed rates and amplitudes of the fluorescence changes on hydrolysis of ATP and ATP gamma S were analyzed by simulations in which the bond cleavage or the Pi release step was responsible for fluorescence quenching. The results indicate that ATP or ATP gamma S binding causes the fluorescence enhancement of CM bound to the gamma subunit and that this conformational change is reversed upon bond cleavage to yield ADP.Pi or ADP.PiS in catalytic sites.

Adenosine Triphosphate↗

PubMed1994

The cDNA sequence of beef heart CII-3, a membrane-intrinsic subunit of succinate-ubiquinone oxidoreductase.

We provide the first full-length cDNA and amino acid sequences for beef heart CII-3, one of two hydrophobic subunits that bind succinate dehydrogenase to the mitochondrial inner membrane to form succinate-ubiquinone oxidoreductase (EC 1.3.99.1). Other low molecular weight proteins present in preparations of the isolated complex, including three possible forms of the second anchor polypeptide CII-4, have been identified by amino terminal sequencing.

Amino Acid Sequence↗

PubMed1994

A cryoelectron microscopy study of the interaction of the Escherichia coli F1-ATPase with subunit b dimer.

A complex between the Escherichia coli F1-ATPase and a truncated form of the ECF0-b subunit was formed and examined by cryoelectron microscopy in amorphous ice. Image analysis of single particles in the hexagonal projection revealed that the polar domain of the b subunit interacts with a beta subunit different from the one which interacts with the epsilon subunit. The cavity in the enzyme, visible in the hexagonal projection, is not filled by the b polypeptide, therefore leaving enough room for extensive conformational changes of the gamma and epsilon subunits within the native F1F0 complex.

Amino Acid Sequence↗

PubMed1994

Regulation of cytochrome c oxidase by interaction of ATP at two binding sites, one on subunit VIa.

Cytochrome c oxidase isolated from a wild-type yeast strain and a mutant in which the gene for subunit VIa had been disrupted were used to study the interaction of adenine nucleotides with the enzyme complex. At low ionic strength (25 mM potassium phosphate), in the absence of nucleotides, the cytochrome c oxidase activity of the mutant enzyme lacking subunit VIa was higher than that of the wild-type enzyme. Increasing concentrations of ATP, in the physiological range, enhanced the cytochrome c oxidase activity of the mutant much more than the activity of the wild-type strain, whereas ADP, in the same concentration range, had no significant effect on the activity of the cytochrome c oxidase of either strain. These results indicate an interaction of ATP with subunit VIa in the wild-type enzyme that prevents the stimulation of the activity observed in the mutant enzyme. The stimulation of the mutant enzyme implies the presence of a second ATP binding site on the enzyme. Quantitative titrations with the fluorescent adenine nucleotide analogues 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) confirmed the presence of two binding sites for adenine nucleotides per monomer of wild-type cytochrome c oxidase and one binding site per monomer of mutant enzyme. Covalent photolabeling of yeast cytochrome c oxidase with radioactive 2-azido-ATP further confirmed the presence of an ATP binding site on subunit VIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1994

TFPACD, a novel bifunctional reagent for reacting with DCCD sites in proteins: studies using Escherichia coli ATP synthase.

A novel cross-linker, 1-[6-(4-azido-2,3,5,6-tetrafluorobenzamido)hexyl]-3-cyclohexylc arbodiimide (TFPACD), has been synthesized and tested by reaction with the Escherichia coli ATP Synthase (ECF1F0). The reagent has a carbodiimide as one reactive group, which is shown to react with ECF1F0 in a similar way to 1,3-dicyclohexylcarbodiimide (DCCD) and modify the beta subunit of the ECF1 part and the c subunit of the F0 part. Reaction with both the ECF1 and F0 parts of the complex inhibited ATPase activity. The second reactive group in the reagent is the photoactivatable tetrafluorophenylazide moiety. Subsequent UV photolysis of TFPACD--modified ECF1 and ECF1F0 led to generation of cross-linked products in significant yields, one between beta and alpha subunits; the second, dimers of the c subunit of the F0 part.

Azides↗

PubMed1994

ATP hydrolysis-driven structural changes in the gamma-subunit of Escherichia coli ATPase monitored by fluorescence from probes bound at introduced cysteine residues.

Four mutants of the Escherichia coli F1ATPase, gamma S8-C, gamma T106-C, gamma S179-C, and gamma V286-C, which have a cysteine introduced at different sites in the gamma-subunit by site-directed mutagenesis, were reacted with the fluorescent reagent N-(4-7-(diethylamino)4-methylcoumarin-3-yl)-maleimide (CM) under conditions that selectively label the introduced Cys residue. With each mutant the effect of nucleotide binding on the fluorescence of the probe has been monitored. The results obtained with the mutants gamma S8-C and gamma T106-C are similar. In both cases, there was a spectral shift and change in fluorescence intensity on adding AMP.PNP or ATP to enzyme emptied of nucleotide from catalytic sites, while no change in the fluorescence spectrum was observed upon adding ADP. The fluorescence spectral changes obtained with ATP were transient and involved an initial rapid fluorescence enhancement followed by a subsequent fluorescence quenching. The kinetics of these ATP-induced fluorescence changes and the kinetics of ATP hydrolysis as monitored by the rates of ATP binding and of Pi formation were the same under conditions of unisite catalysis, indicating that the conformational changes in the gamma-subunit being measured by the fluorescent probe are driven by ATP hydrolysis in catalytic sites. No nucleotide-dependent fluorescence changes were observed with CM bound at a Cys at position 179. Nucleotide-dependent changes in fluorescence were seen with CM bound at position 286, but these appear to reflect structural changes due to binding of ADP or ATP in noncatalytic sites. The fluorescence changes observed in mutants gamma S8-C and gamma T106-C were not seen in subunit epsilon-free E. coli F1ATPase, although such enzyme preparations are highly active ATPases. We conclude that the structural changes monitored by the fluorescent probe are a part of the conformational coupling, whereby catalytic site events are linked to proton channeling.

Adenosine Triphosphate↗

PubMed1994

Structure of the gamma subunit of Escherichia coli F1 ATPase probed in trypsin digestion and biotin-avidin binding studies.

The arrangement and functional role of the gamma subunit of the Escherichia coli F1ATPase (ECF1) has been probed by protease digestion and avidin-biotin labeling experiments using wild-type enzyme and four mutants, gamma S8C, gamma T106C, gamma S179C, and gamma V286C, respectively. Trypsin was found to cleave the gamma subunit at four sites, Arg70, Lys199, Lys201, and Lys212. Cleavage at these four sites did not greatly reduce the high ATPase activity of the enzyme that is obtained when the epsilon subunit is removed by the protease treatment. However, prolonged trypsin cleavage led to loss of inhibition by epsilon subunit added back to the trypsin-treated enzyme. Endoproteinase-Lys-C cleaves the gamma subunit of ECF1 at three of the four sites, i.e. Lys199, Lys201, and Lys212, but not at Arg70. The enzyme was activated by treatment with this protease because of degradation and release of the epsilon subunit, but added pure epsilon subunit still caused inhibition of ATPase activity. Therefore, cleavage at Arg70 by trypsin is responsible for the loss of response to the epsilon subunit inhibition. Biotin was reacted with Cys residues at positions 8, 106, 179, and 286 in different gamma subunit mutants and the accessibility of the biotin to avidin monitored in the intact ECF1 from the different mutants. Avidin was able to react with biotin when incorporated at position 106, not at 8, 179, or 286. The four trypsin cleavage sites, Arg70, Lys199, Lys201, and Lys212, as well as Thr106 are in regions of the gamma subunit predicted to be mainly beta-sheet and beta-turn structures.

Amino Acid Sequence↗

PubMed1994

Coupling between catalytic sites and the proton channel in F1F0-type ATPases.

F1F0-type ATPases catalyse both ATP-driven proton translocation and proton-gradient-driven ATP synthesis. Recent cryoelectronmicroscopy and low-resolution X-ray studies provide a first glimpse at the structure of this complicated membrane-bound enzyme. The F1 part is roughly globular and linked to the membrane-intercalated F0 part by a narrow stalk domain, which contains the gamma-, delta- and epsilon-subunits along with domains of the b-subunit of the F0 part. Here, we review evidence that conformational and positional changes in the gamma- and epsilon-subunits provide the coupling between catalytic sites and proton translocation within the F1F0 complex.

Binding Sites↗

PubMed1994

F1-ATPase in a spin.

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Adenosine Triphosphate↗

PubMed1994

Asymmetry and structural changes in ECF1 examined by cryoelectronmicroscopy.

The Escherichia coli ATPase (ECF1) has been studied by cryoelectronmicroscopy and an intrinsic asymmetry of the molecule in the hexagonal projection identified. The three beta subunits could be distinguished. One, which we have called beta 1, has a greater density in projection than the other two; the second, beta 2, is of intermediate density in projection, while the third, beta 3, is smeared out in density. These different features of the beta subunits were used to orient images, and the positions of the gamma and epsilon subunits then established. The location of the gamma subunit, as monitored by the central mass, was not fixed. This subunit could be found in positions that followed an arc from close to beta 2 to close to beta 3, a shift of around 10A, with respect to the center of the mass. The location of the epsilon subunit was monitored after reconstituting a complex of epsilon subunit-depleted ECF1 with a mutant epsilon subunit in which His at residue 38 had been replaced by Cys, and this Cys labeled with an approximately 14A gold particle. The epsilon subunit was found in positions described by an arc between an alpha subunit (alpha 1) and the neighboring beta subunit (beta 1), a shift of around 20A, with respect to the center of the gold particle. A nucleotide dependence of the position of the gamma subunit has been established by Gogol, E.P., Johnston, E., Aggeler, R. and Capaldi, R.A. (1990) Proc. Natl. Acad. Sci. USA 87, 9585-9589. A nucleotide dependence of the position of the epsilon subunit is shown here.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1993

Tissue distribution of cytochrome c oxidase isoforms in mammals. Characterization with monoclonal and polyclonal antibodies.

Monoclonal and polyclonal antibodies specific to the two isoforms of subunit VIa of bovine cytochrome c oxidase were generated and used to study the tissue distribution of this subunit pair in beef, human and rat. The so-called H-(heart) form was found exclusively in heart and skeletal muscle, whereas the so-called L-(liver) form was the only isoform present in brain, kidney, liver and smooth muscle. Little or no L-form was detected in skeletal muscle. In bovine heart no subunit VIa-L was detected, while in human heart the subunit VIa-H and VIa-L isoforms were present in roughly equal proportions. These results imply that, in humans, the deficiency of a subunit VIa isoform may have a different effect on the physiology of heart then on the physiology of skeletal muscle.

Amino Acid Sequence↗

PubMed1993

The gamma subunit of the Escherichia coli F1-ATPase can be cross-linked near the glycine-rich loop region of a beta subunit when ADP + Mg2+ occupies catalytic sites but not when ATP + Mg2+ is bound.

A mutant of the Escherichia coli F1-ATPase, gamma S8C, has been reacted with a novel bifunctional reagent, N-maleimido-N'-(4-azido-2,3,5,6-tetrafluorobenzamido) cystamine (TFPAM-SS1). Modification of Cys-8 via the maleimide, followed by photolysis to convert the azido group to a reactive nitrene, led to cross-linking of the gamma subunit to a beta subunit. When this cross-linking was conducted with ADP + Mg2+ in catalytic sites, the predominant cross-linked product had a M(r) of 108,000. If cross-linking was done with uncleaved ATP + Mg2+ in catalytic sites, cross-linked products of 102,000 and 84,000 were formed. Cross-linking under both conditions led to inhibition of ATPase activity. TFPAM-SS1 could be cleaved by using reducing agents to break the disulfide bond that links the malemide and tetrafluorophenylazide moieties. Cleavage of this disulfide bond after formation of 102,000 and 84,000 species led to full recovery of ATPase activity. When the 108-kDa cross-linked product was cleaved, full activity was not restored, presumably because of insertion of the tetrafluorophenylazide into a functionally important site on the beta subunit. After cleavage of the disulfide bond, the free thiols could be reacted with [14C]N-ethylmaleimide, thereby radioactively tagging the sites of insertion of the tetrafluorophenylnitrene moiety. In this way, the site of cross-linking from Cys-8 of gamma to the beta subunit in the presence of ADP + Mg2+ was localized to within the sequence Val 145-Lys-155, which contains the glycine-rich loop. This loop region is a part of the catalytic site of the enzyme.

Adenosine Diphosphate↗

PubMed1993

Subunit VIa of yeast cytochrome c oxidase is not necessary for assembly of the enzyme complex but modulates the enzyme activity. Isolation and characterization of the nuclear-coded gene.

COX13, the nuclear gene for cytochrome c oxidase subunit VIa of Saccharomyces cerevisiae, has been isolated in two steps. First, the partial amino acid sequence information of the subunit was used to design two degenerate oligodeoxynucleotide primers to amplify part of the gene in a polymerase chain reaction. Next, the amplified product was used to screen a yeast genomic library in order to obtain the entire gene and its flanking sequences. COX13 is present as a single copy gene per haploid genome. Alignment of the N-terminal sequence of mature, subunit VIa with the amino acid sequence deduced from the DNA sequence indicates that subunit VIa is synthesized as a precursor comprised of a leader sequence of 9 amino acid residues and a mature polypeptide of 120 amino acid residues. The mature polypeptide shares 34% identical amino acid residues with the human subunit isoform VIa-L. Sequence analysis of the 3'-flanking region of COX13 revealed that the gene is located 599 base pairs downstream of CDC55, a gene which has been mapped to the left arm of chromosome VII. Null mutants of COX13, generated by gene replacement, showed a slightly reduced growth rate on nonfermentable carbon sources. Heme spectra and analysis of immunopurified cytochrome c oxidase from a null strain demonstrated that the enzyme is fully assembled without subunit VIa. At low ionic strength, cytochrome c oxidase missing subunit VIa was more active, whereas at high ionic strength, it was less active than the enzyme complex in which subunit VIa was present. In addition, distinct effects of ATP on the activity of the null and wild type enzyme were found. The results suggest that ATP interacts specifically with subunit VIa and thereby modulates the cytochrome c oxidase activity.

Adenosine Triphosphate↗

PubMed1993

ATP hydrolysis-linked structural changes in the N-terminal part of the gamma subunit of Escherichia coli F1-ATPase examined by cross-linking studies.

A mutant of Escherichia coli F1-ATPase (ECF1) in which the serine residue in position 8 of the gamma subunit has been replaced by a cysteine residue (gamma S8C) has been used to study nucleotide-dependent cross-linking of the gamma subunit to a beta subunit. When examined in the presence of ADP+Mg2+, either supplied directly or as produced during catalytic turnover of ATP+Mg2+, the main cross-linked product generated using the heterobifunctional, photoactivatable, cross-linker tetrafluorophenylazide maleimide-6 had a M(r)(app) of 108,000. When ATP hydrolysis was inhibited, either by cold or by reaction with sodium azide, or when ATP hydrolysis was prevented by the use of adenyl-5'-yl beta,gamma-imidodiphosphate, the main cross-linked products were species with M(r)(app) of 102,000 and 84,000. The nucleotide-dependent switching from one cross-linking pattern to another could only be observed when the epsilon subunit was bound to ECF1; it was not seen in ECF1*, an enzyme preparation missing delta and epsilon subunits, but was observed in preparations selectively depleted of the delta subunit. We conclude that the changes detected in these cross-linking experiments are occurring during the hydrolysis of ATP when the beta-gamma phosphate bond is cleaved and that they are related to the coupling of ATP hydrolysis to proton translocation.

Adenosine Diphosphate↗

PubMed1993

The cysteine introduced into the alpha subunit of the Escherichia coli F1-ATPase by the mutation alpha R376C is near the alpha-beta subunit interface and close to a noncatalytic nucleotide binding site.

Mutation of the alpha subunit of the Escherichia coli F1-ATPase to convert Arg-376 to a Cys (alpha R376C) lowers multisite ATPase activity 400-1,000-fold while affecting unisite catalysis only around 6-fold, suggesting that the mutation is in a region important for transmission of conformational changes between catalytic sites (Soga, S., Noumi, T., Takeyama, M., Maeda, M., and Futai, M. (1989) Arch. Biochem. Biophys. 268, 643-648; this study). To learn more of the structural features of the segment of the alpha subunit around Arg-376, mutant enzyme with a Cys at this position was modified with several maleimides. N-[14C]Ethylmaleimide reacted rapidly with this Cys in one of the three alpha subunits/F1 (2,500 M-1 s-1); more slowly with a second alpha subunit (390 M-1 s-1); and the same Cys in the third copy of the alpha subunit was completely unreactive to the reagent, indicating asymmetry of alpha subunits in the ECF1 complex. The photoactivatable cross-linker N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide++ +, when reacted via its maleimide to alpha Cys-376 of the mutant, covalently linked alpha to beta subunits upon photolysis, indicating that Cys-376 of alpha is close to an interface between the alpha and beta subunits. The EDTA-induced exchangeable noncatalytic site could be filled by TNP-ATP in both wild type and alpha R376C mutant ECF1. Occupancy of this site in the alpha R376C mutant altered the rate of reaction of the second-fastest reacting Cys-376 from 390 M-1 s-1 to below 130 M-1 s-1, suggesting that the two sites are on the same alpha subunit. TNP-ATP in the EDTA-induced exchangeable noncatalytic site was quenched by reacting Cys-376 with 4-maleimido-(2,2,6,6-tetramethylpiperidine-N(oxyl), indicating that the region around Cys-376, which is involved in transmission of conformational changes between alpha and beta, and noncatalytic sites are maximally 10-12 A from each other.

Adenosine Triphosphate↗

PubMed1993

Isolation of a cDNA specifying subunit VIIb of human cytochrome c oxidase.

Human cytochrome c oxidase (COX) is a complex of 13 subunits: three are encoded by mitochondrial DNA and ten by nuclear DNA. We have now isolated a full-length cDNA specifying subunit VIIb, the last remaining uncharacterized nuclear-encoded subunit cDNA of human COX. The cDNA encodes a deduced 80-aa polypeptide, including a 24-amino acid (aa) N-terminal leader sequence and a 56-aa mature polypeptide with 82% identity to mature bovine COX VIIb. Southern blot hybridization of human muscle genomic DNA showed multiple hybridizing bands, implying the presence of a large coxVIIb gene family, including a potential processed pseudogene.

Amino Acid Sequence↗