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P Graceffa

Publications and source records attributed to P Graceffa.

36 records · Page 2Linked to original sources
PubMed1990

Caldesmon from rabbit liver: molecular weight and length by analytical ultracentrifugation.

Although smooth muscle caldesmon migrates as a 140- to 150-kDa protein during sodium dodecyl sulfate-gel electrophoresis, its molecular mass is around 93 kDa as determined by sedimentation equilibrium (P. Graceffa, C-L. A. Wang, and W. F. Stafford, 1988, J. Biol. Chem. 263, 14,196-14,202). Nonmuscle caldesmon migrates during electrophoresis with a molecular mass close to 77 kDa, about half that of the muscle isoform. However, it is controversial whether the molecular weight of nonmuscle caldesmon is the same or much less than that of the muscle protein. Therefore we have now determined the molecular mass of rabbit liver caldesmon by sedimentation equilibrium and found a value of 66 +/- 2 kDa, a value much smaller than that of muscle caldesmon. This new value of the molecular weight, together with a sedimentation coefficient of 2.49 +/- 0.02 S. yields an apparent length of 53 +/- 2 nm and a diameter of 1.7 nm for the liver protein. We previously estimated a length of 74 nm and a diameter of 1.7 nm for the muscle caldesmon. We have also determined the amino acid composition of liver caldesmon and found it to be similar to that of the muscle protein. In conclusion, muscle and nonmuscle caldesmons appear to have similar overall amino acid composition and tertiary structure with the smaller nonmuscle protein having a correspondingly smaller length. The difference in molecular weight between the two caldesmons is consistent with the nonmuscle protein lacking a central peptide of the muscle isoform, as suggested by E. H. Ball, and T. Kovala, (1988, Biochemistry 27, 6093-6098).

Amino Acids↗
PubMed1989

In-register homodimers of smooth muscle tropomyosin.

Gizzard smooth muscle tropomyosin dimer molecules were dissociated by guanidinium chloride and reassociated by dialysis against 1 M NaCl. Several properties of the protein were changed by this treatment. There was a large decrease in tropomyosin's low-salt viscosity, owing to reduced end-to-end polymerization, the helix unfolding profile changed from a one-step to a two-step process, and the ability to form intramolecular, interchain, disulfide-cross-linked homodimers increased dramatically. Thus, the native molecule, though to exist predominantly as by the beta gamma heterodimer which cannot form disulfide cross-links [Sanders, C., Burtnick, L.D., & Smillie, L. B. (1986) J. Biol. Chem. 261, 12774-12778], reassembles, after dissociation, to form predominantly parallel, in-register beta beta and gamma gamma homodimers able to form disulfide cross-links. This suggests that the physical properties, including the end-to-end interaction, of gizzard tropomyosin homodimers differ considerably from those of the heterodimer. This is a first step toward a molecular understanding of the end-to-end interaction of smooth muscle tropomyosin.

Animals↗
PubMed1988

Caldesmon. Molecular weight and subunit composition by analytical ultracentrifugation.

A wide range of values has been reported for the subunit and molecular weights of smooth muscle caldesmon. There have also been conflicting reports concerning whether caldesmon is a monomer or dimer. We attempted to resolve these uncertainties by determining the molecular weight of chicken gizzard smooth muscle caldesmon using the technique of sedimentation equilibrium in the analytical ultracentrifuge. Unlike previous methods that have been used to estimate the molecular weight of caldesmon, the molecular weight determined by equilibrium sedimentation does not depend upon assumptions about the shape of the molecule. We concluded that caldesmon in solution is monomeric with a molecular mass of 93 +/- 4 kDa, a value that is much less than those previously reported in the literature. This new value, in conjunction with sedimentation velocity experiments, led to the conclusion that caldesmon is a highly asymmetric molecule with an apparent length of 740 A in solution. The mass of a cyanogen bromide fragment, with an apparent mass of 37 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was determined to be 25.1 +/- 0.6 kDa using sedimentation equilibrium. These results imply that the reported molecular weights of other fragment(s) of caldesmon have also been overestimated. We have determined an optical extinction coefficient for caldesmon (E1%(280 nm) = 3.3) by determining its concentration from its refractive index which was measured in the analytical ultracentrifuge. From the above values of the molecular weight and the extinction coefficient, we redetermined that the caldesmon molecule has two cysteines and recalculated the stoichiometric molar ratio of actin/tropomyosin/caldesmon in the smooth muscle thin filament to be 28:4:1.

Amino Acids↗
PubMed1988

Spin trapping the cysteine thiyl radical with phenyl-N-t-butylnitrone.

The cysteine thiyl radical has been detected in a variety of biological systems by means of the ESR spectrum of the adduct between the radical and nitrone spin traps. 5,5-Dimethyl-1-pyroline N-oxide (DMPO) is the spin trap of choice in these studies for several reasons. However, we show here that the adduct between the cysteine thiyl radical and phenyl-N-t-butylnitrone (PBN) spin trap can be observed under certain oxidizing conditions where the adduct with DMPO is not detected. This suggests the use of PBN in searching for the thiyl radical under such conditions.

Cyclic N-Oxides↗
PubMed1988

Binding of deferoxamine to asbestos fibers in vitro and in vivo.

We studied the binding of tritium-labeled deferoxamine, a strong iron chelator, to crocidolite asbestos fibers in vitro and in vivo. In aqueous suspension of asbestos, deferoxamine binding was rapid and strong, suggesting specific binding to iron. For the in vivo experiments, diffusion chambers containing native asbestos fibers or deferoxamine-washed asbestos were implanted in the peritoneal cavities of mice. Five days after parenteral injection of tritiated deferoxamine chambers were removed and the asbestos counted. More than twice as much label (2206 +/- 348 c.p.m./100 mg asbestos) was bound to the native asbestos as compared to the deferoxamine-washed asbestos (1080 +/- 201 c.p.m./100 mg asbestos), suggesting specific binding in vivo. Since deferoxamine can inhibit asbestos toxicity in vitro, these experiments suggest the feasibility of testing whether deferoxamine can prevent asbestos-related disease in vivo.

Animals↗
PubMed1987

Evidence for interaction between smooth muscle tropomyosin and caldesmon.

The viscosity of chicken gizzard smooth muscle tropomyosin is enhanced 4.7-fold in the absence of salt and 1.43-fold in 0.1 M salt by the presence of stoichiometric amounts of gizzard caldesmon, indicating that the two proteins interact under these conditions. Since the thin filament regulation of smooth muscle contraction by caldesmon requires the presence of tropomyosin, these results suggest that the direct interaction between tropomyosin and caldesmon on the thin filament plays a role in this regulation.

Actin Cytoskeleton↗
PubMed1986

Tropomyosin's end-to-end polymerization is irreversibly lost on exposure to urea or cyanate.

Tropomyosin's low-salt viscosity which is due to end-to-end polymerization, is irreversibly lost upon incubation in 8M urea at room temperature. This effect is due to the chemical modification of lysine residues by cyanate in the urea. In the absence of urea, cyanate alone has the same effect. A loss in tropomyosin binding to actin accompanies the loss in viscosity, consistent with the view that tropomyosin's end-to-end interaction is necessary for strong binding to actin. During column chromatography in 8M urea, used to separate the alpha and beta chains of tropomyosin, the loss of viscosity can be minimized by using freshly-prepared urea and by reducing the time during which the protein and urea are in contact.

Actins↗
PubMed1985

Conformation of spin-labeled tropomyosin in reconstituted muscle thin filaments in response to calcium ion and heavy meromyosin.

Tropomyosin (TM) exists in thermal equilibrium between a highly structured N state, a partially unfolded X state, and a completely unfolded D state, i.e., N in equilibrium X in equilibrium D. The strongly immobilized electron spin resonance (ESR) spectral component of spin-labeled TM corresponds to TM in the N state and the weakly immobilized component to TM in the X state below the main unfolding transition and to TM in the D state above this transition [Graceffa, P., & Lehrer, S. S. (1984) Biochemistry 23, 2606-2612]. The addition of actin, troponin (TN), and heavy meromyosin (HMM) to spin-labeled TM reduces the ratio of weakly to strongly immobilized labels, indicating a shift in the N in equilibrium X in equilibrium D equilibrium toward the N state. At 37 degrees C, for spin-labeled TM alone K (=X/N) greater than 1.0 with some TM in the D state, K = 0.8 for spin-labeled TM bound to actin, and K less than 0.05 for spin-labeled TM bound to actin + TN +/- Ca2+, actin + HMM + TN +/- Ca2+, and actin + HMM. Thus, actin + TN dramatically shifts the TM structure to the N conformation with little further effect upon addition of Ca2+ or HMM. The temperature at which spin-labeled TM begins to dissociate from a protein complex was determined from the temperature dependence of the ESR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1984

Dynamic equilibrium between the two conformational states of spin-labeled tropomyosin.

Tropomyosin was labeled with a maleimide nitroxide spin-label attached to cysteine-190 via a succinimido ring which was subsequently opened by incubation at alkaline pH. Electron spin resonance (ESR) spectra showed a temperature-dependent equilibrium, below the main unfolding transition of tropomyosin, between labels which were restricted in their motion (strongly immobilized), predominating at low temperatures, and those which were highly mobile (weakly immobilized), predominating at higher temperatures. These label states were associated with two protein states from a comparison of the ESR spectral changes with the thermal unfolding profile of tropomyosin. The strongly immobilized labels were associated with the completely folded molded and the weakly immobilized labels with a partially unfolded (in the cysteine-190 region) state which is an intermediate in the thermal unfolding of tropomyosin. A spectral subtraction technique was used to measure the concentration ratio of strongly and weakly immobilized labels from which an equilibrium constant, K, was determined at different temperatures. A linear van't Hoff plot was obtained, indicating that the spin-labeled protein is in thermal equilibrium between these two conformational states with delta H = 17 kcal/mol, delta S = 56 cal/(deg X mol), and K = 1.0 at 34 degrees C. An upper limit of 10(7) s-1 for the conformational fluctuation was estimated from the shapes and separation of the two ESR spectral components. In contrast to the label with the opened succinimido ring, the spin-label with an intact succinimido ring remained strongly immobilized on the protein, indicating that in the partially unfolded state the molecule retains structure in the cysteine-190 region.

Animals↗
PubMed1984

Comparison of the fluorescence and conformational properties of smooth and striated tropomyosin.

In contrast to previous conformational studies with rabbit skeletal and cardiac tropomyosins, (i) when the cysteine side chains of chicken gizzard tropomyosin were reacted with 5,5'-dithiobis(2-nitrobenzoate), an interchain disulfide cross-link was not produced, (ii) when they were labeled with pyrenylmaleimide , excimer fluorescence was not observed, and (iii) when they were labeled with didansylcystine , a long-lived fluorescence component did not appreciably contribute to the fluorescence decay over a large temperature range including the major unfolding transition. In addition, the temperature dependence of the ellipticity at 222 nm did not reveal a pretransition prior to the main helix unfolding transition. This indicates that gizzard tropomyosin does not exhibit a localized chain-open state in the region of its cysteine residues, analogous to that seen with cardiac and skeletal tropomyosins, nor in any other region of the molecule. As a consequence, these observations suggest that gizzard tropomyosin is more rigid than striated tropomyosin.

Animals↗
PubMed1984

Asbestos catalyzes hydroxyl and superoxide radical generation from hydrogen peroxide.

To understand chemical characteristics of the asbestos minerals which might contribute to tissue damage, the catalytic properties of three different varieties were studied. Using spin trapping techniques it was determined that crocidolite, chrysotile, and amosite asbestos were all able to catalyze the generation of toxic hydroxyl radicals from a normal byproduct of tissue metabolism, hydrogen peroxide. The iron chelator desferroxamine inhibits this reaction, indicating a major role for iron in the catalytic process, and suggesting a possible mechanism by which asbestos toxicity might be reduced.

Asbestos↗
PubMed1983

Spin labeling of protein sulfhydryl groups by spin trapping a sulfur radical: application to bovine serum albumin and myosin.

Reaction of sulfhydryl-containing compounds, RSH, with Ce4+ in the presence of the spin trap phenyl-N-t-butylnitrone results in the appearance of a nitroxide ESR spectrum, which is greatly diminished if the sulfhydryl group is blocked prior to reaction. The spectra have short lifetimes which can be increased two- to fivefold to half-lives of 5-60 min by prior flushing of the solutions with nitrogen. For small molecules, such as cysteine, N-acetylcysteine, glutathione, and 2-mercaptoethanol, the spectrum is that of a freely rotating nitroxide while for the proteins, bovine serum albumin and myosin, the spectrum is characteristic of a strongly immobilized nitroxide spin label rigidly attached to the protein. Since Ce4+ is reported to oxidize the sulfhydryl group via the thiyl radical, RS, the following reactions are proposed to account for the formation of the nitroxide: (formula; see text) These reactions permit the spin labeling of sulfhydryl proteins such that the nitroxide is much closer to the point of attachment than when using conventional spin-labeling methods.

Animals↗
PubMed1980

The excimer fluorescence of pyrene-labeled tropomyosin. A probe of conformational dynamics.

Rabbit skeletal and cardiac tropomyosin were specifically labeled at their cysteine side chains with N-(1-pyrene)-maleimide. A high degree of intramolecular excimer formation due to interaction between adjacent pyrenes on Cys 190 of each chain was observed by fluorescence techniques. This modification produced low values of specific viscosity at low salt, indicating an inhibition of the salt-dependent polymerizability. Despite the loss of polymerizability, the excimer fluorescence of pyrene-tropomyosin increased with a similar salt dependence as the decrease in viscosity of an unlabeled control. Transient and steady state fluorescence measurements on the labeled tropomyosin indicated the presence of two states of labeled tropomyosin, an excimer-forming state (State B) and a nonexcimer-forming state (State A), in equilibrium with each other. The increase in excimer with increasing temperature and salt concentration can be explained by a shift in equilibrium toward State B. Steric considerations suggest that, in order for the pyrenes to form an excimer, localized chain separation in State B is required.

Animals↗
PubMed1979

Enzymatic activities and ATP-induced fluorescence enhancement of myosin from fast and slow skeletal and cardiac muscles.

The maximal ATP-induced enhancement of fluorescence and the dependence of this enhancement on ATP concentration were determined for myosins from fast and slow skeletal and cardiac muscle of the rabbit. With myosins from slow and cardiac muscle modifications in the preparative procedure and chromatography on DEAE-Sephadex were required to obtain preprations which were free of actin, which exhibited the maximal fluorescence enhancement and which bound two moles of ATP per mole of myosin. Since the fluorescence enhancement of cardiac and slow muscle myosins is labile at slightly alkaline pH, it was also necessary to minimize incubation at pH greater than 7 in order to attain the maximal enhancement. With fast muscle myosin the changes in preparative procedure, together with chromatography, led to a 50 to 100% increase in the steady-state rate of ATP hydrolysis and fluorescence enhancement, without changing the maximal binding of ATP. From a comparison of the rate of steady-state hydrolysis of ATP with the rate of decay of the enhanced fluorescence, it appears that for all three myosins, both ATP binding sites have the same enzymatic activity, the steady-state rate per site being slower for cardiac and slow muscle myosins than for fast muscle myosin.

Adenosine Triphosphatases↗
PubMedDate not supplied

Phytic acid, an iron chelator, attenuates pulmonary inflammation and fibrosis in rats after intratracheal instillation of asbestos.

Reactive oxygen species, especially iron-catalyzed hydroxyl radicals (.OH) are implicated in the pathogenesis of asbestos-induced pulmonary toxicity. We previously demonstrated that phytic acid, an iron chelator, reduces amosite asbestos-induced .OH generation, DNA strand break formation, and injury to cultured pulmonary epithelial cells (268[1995, Am. J. Physiol.(Lung Cell. Mol. Physiol.) 12:L471-480]). To determine whether phytic acid diminishes pulmonary inflammation and fibrosis in rats after a single intratracheal (it) instillation of amosite asbestos, Sprague-Dawley rats were given either saline (1 ml), amosite asbestos (5 mg; 1 ml saline), or amosite treated with phytic acid (500 microM) for 24 hr and then instilled. At various times after asbestos exposure, the rats were euthanized and the lungs were lavaged and examined histologically. A fibrosis score was determined from trichrome-stained specimens. As compared to controls, asbestos elicited a significant pulmonary inflammatory response, as evidence by an increase (approximately 2-fold) in bronchoalveolar lavage (BAL) cell counts at 1 wk and the percentage of BAL neutrophils (PMNs) and giant cells at 2 wk (0.1 vs 6.5% and 1.3 vs 6.1%, respectively; p < 0.05). Asbestos significantly increased the fibrosis score at 2 wk (0 +/- 0 vs 5 +/- 1; p < 0.05). The inflammatory and fibrotic changes were, as expected, observed in the respiratory bronchioles and terminal alveolar duct bifurcations. The increased percentage of BAl PMNs and giant cells persisted at 4 wk, as did the fibrotic changes. Compared to asbestos alone, phytic acid-treated asbestos elicited significantly less BAL PMNs (6.5 vs 1.0%; p < 0.05) and giant cells (6.1 vs 0.2%; p < 0.05) and caused significantly less fibrosis (5 vs 0.8; p < 0.05) 2 wk after exposure. We conclude that asbestos causes pulmonary inflammation and fibrosis in rats after it instillation and that phytic acid reduces these effects. These data support the role of iron-catalyzed free radicals in causing pulmonary toxicity from asbestos in vivo.

Animals↗