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S Verjovski-Almeida

Publications and source records attributed to S Verjovski-Almeida.

At least 37 records · Page 2Linked to original sources
PubMed1989

Beneficial effects of anti-growth hormone antiserum in avian muscular dystrophy.

Human subjects and mice have been found to have a milder progression of muscular dystrophy when the disease is associated with genotypically determined dwarfism. In this paper we describe an experimental test for reducing growth hormone in dystrophic chickens that uses rabbit anti-chicken growth hormone anti-serum (anti-cGH). Antiserum was injected daily into dystrophic (line 413) male chickens from day 1 to day 8 after hatching. Dystrophic chickens injected with anti-cGH maintained a significantly higher score in the standardized test for righting ability (P less than 0.001-0.051) from 3 to 9 1/2 wk after hatching when compared with dystrophic controls. The observed prolongation of the functional ability of injected dystrophic animals suggests that growth hormone plays a role in potentiating the symptoms of dystrophy in chickens.

Animals↗
PubMed1988

Interaction of spin-labeled nucleotides with sarcoplasmic reticulum adenosinetriphosphatase.

Spin-labeled derivatives of AMP-PCP, ATP, and 2'-deoxy-ATP, with a nitroxide moiety attached to the ribose ring [3'-O-(1-oxy-2,2,5,5-tetramethylpyrroline-3-carbonyl)nucleotide], are used to study the nucleotide binding site stoichiometry of sarcoplasmic reticulum (SR) ATPase. With all derivatives, a maximal binding of 4.5 nmol/mg of SR protein is found, a value close to the number of phosphorylation sites obtained with ATP. The spin-labeled nucleotides cannot be utilized by the enzyme as substrates. Binding of spin-labeled nucleotides is inhibited by labeling the ATPase with fluorescein 5'-isothiocyanate, indicating that all the labeled nucleotide is located at the catalytic site. Additions of spin-labeled ATP to vesicle suspensions during steady turnover demonstrate competitive inhibition of both catalysis and the regulatory effect normally exhibited by ATP. As secondary binding of spin-labeled ATP is not detected at pertinent concentrations, it is suggested that both functions of ATP may be effected through a single site.

Adenosine Triphosphatases↗
PubMed1988

Stoichiometry and mapping of the nucleotide sites in sarcoplasmic reticulum ATPase with the use of UTP.

Purified sarcoplasmic reticulum ATPase was phosphorylated by either ATP or UTP under otherwise identical conditions. Calcium, pH, and nucleotide concentrations were adjusted to permit maximal steady-state accumulation of phosphoenzyme (EP). Either 4 or 8.5 nmol of EP/mg of protein were obtained with ATP or UTP, respectively. Tryptic digestion of phosphorylated ATPase followed by acid gel electrophoresis showed that EP from UTP was on fragment A1, similar to the report in the literature for EP from ATP. Phosphorylation with Pi in the absence of calcium gave EP levels similar to those obtained from UTP. Thus, comparison of EP levels from different substrates measured in parallel in the same preparation reveal that with ATP half of the sites are phosphorylated. Illumination of the ATPase with UV light in the presence of [3H]UTP caused photolabeling of the ATPase at a maximal level of 1 nmol of [3H]UTP incorporated/mg of ATPase. The UTP concentration dependence for photolabeling was the same as that for promoting catalysis. ATP when present in the illumination protected with a competitive pattern against photolabeling with UTP. Tryptic digestion and autoradiography of photolabeled ATPase revealed that UTP was covalently attached to tryptic fragment A2. The data indicate that a peptide sequence of fragment A2 is involved in the binding of the nucleoside moiety of UTP and possibly belongs to the nucleotide domain of the ATPase in addition to the sequence of fragment A1 which contains the phosphorylation residue.

Adenosine Triphosphatases↗
PubMed1988

Utilization of arylazido-ATP by sarcoplasmic reticulum ATPase in the absence of calcium.

The ATP analog arylazido-ATP 5'-triphosphate) (3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate) was shown to phosphorylate the calcium-ATPase from sarcoplasmic reticulum in the absence of calcium. Levels of 0.6 nmol of phosphoenzyme/mg of protein were attained. Calcium either at micromolar or millimolar concentrations did not affect the level of phosphoenzyme. A non-Michaelian dependence of the hydrolytic activity as a function of analog concentration was obtained in the absence of calcium. Calcium addition did not modify either the analog concentration dependence for the activation of hydrolysis or the maximal rate of hydrolysis. In the presence of micromolar calcium, arylazido-ATP promoted calcium accumulation inside the vesicles, and a steady-state level of 100 nmol of calcium/mg of protein was maintained. ESR spectra of spin-labeled ATPase showed that addition of the analog in the absence of calcium caused a spectral change, and the resulting spectral parameters were different from those obtained for ATP under similar conditions. Calcium addition did not cause any further modification of the spectra, which was clearly distinct from the change when ATP was used. The partition coefficient of the analog from a water medium into an organic phase was found to be 1 order of magnitude higher than that of ATP. It is suggested that it might be the hydrophobic nature of the analog which makes it bypass the calcium requirement for utilization of the substrate by the ATPase.

Adenosine Triphosphatases↗
PubMed1986

Pressure-induced dissociation of solubilized sarcoplasmic reticulum ATPase.

The effect of hydrostatic pressure on the self-association of sarcoplasmic reticulum ATPase solubilized by nonionic detergent was studied in the pressure range of 1 atm up to 2 kilobars. Polarization of intrinsic tryptophan fluorescence or of fluorescence of a pyrene probe covalently attached to the ATPase was measured. An increase in hydrostatic pressure promoted dissociation of the protein into monomers. For a midpoint dissociation pressure of 1.3 kilobars, the standard volume change in the dissociation reaction was delta Vop = -167 ml/mol. Full reversibility of the pressure effects was shown to occur, as seen by recovery of polarization. An increase in Ca2+ concentration from 50 microM to 5 mM and of pH from 6.9 to 8.6 were found to increase the midpoint dissociation pressure, indicating that these factors stabilize the dimeric state. The hydrolytic activity of the ATPase was measured under pressure. The activity was inhibited by pressure increase. It was found that an irreversible inactivation of the solubilized enzyme occurred during turnover and that increasing pressure added to this instability. Reversibility of the activity was critically dependent on the presence of 10 mM Ca2+ in the assay medium.

Adenosine Triphosphatases↗
PubMed1985

Labeling of a thiol residue in sarcoplasmic reticulum ATPase by pyrene maleimide. Solvent accessibility studied by fluorescence quenching.

Sarcoplasmic reticulum ATPase was specifically labeled by the fluorescent probe N-(1-pyrene)maleimide which modified 1 mol of a highly reactive thiol residue per mol of ATPase under appropriate conditions, when the probe concentration was varied in the range 0.1-1.5 microM. Addition of inorganic phosphate to the labeling medium increased both the rate of labeling and the number of modified thiol residues. Addition of ATP gave a marked kinetic protection from labeling, suggesting that the label was attached to a protein domain which is sensitive to changes at the catalytic site. Quenching of pyrene fluorescence emission of labeled ATPase by acrylamide and cesium chloride gave linear Stern-Volmer plots. The Stern-Volmer quenching constants of pyrene-ATPase fluorescence were 10 times lower than the constant obtained for acrylamide quenching of the fluorescent adduct of pyrene-maleimide-cystein used as a control, indicating that the pyrene moiety of the probe was considerably shielded from the medium solvent when covalently attached to the ATPase. The efficiency of quenching of pyrene-ATPase fluorescence increased by a significant amount upon addition of 100 microM Ca2+, when compared to the quenching in the presence of a Ca2+ chelator. It suggests that occupancy of the high affinity Ca2+ sites of the ATPase increases the accessibility of medium solvent into hydrophobic domains of the enzyme. The fluorescence lifetime of the solubilized pyrene-ATPase emission was 144-149 ns. The fluorescence polarization of pyrene-ATPase solubilized by nonionic detergent C12E8 was rho = 0.10 and it increased with an increase in the viscosity of the medium yielding a linear Perrin plot. The rotational correlation time for the soluble ATPase was 532 ns, corresponding to the overall rotation of a detergent-pyrene-ATPase particle with radius of 87A.

Acrylamide↗
PubMed1985

Monomer-dimer association constant of solubilized sarcoplasmic reticulum ATPase.

The monomer-dimer association constant of solubilized and delipidated sarcoplasmic reticulum ATPase was measured by large zone elution gel chromatography in the presence of a high concentration (18.6 mM) of the nonionic detergent dodecyloctaethylene glycol monoether (C12E8) and of different ATPase protein concentrations in the range of 0.74 (6.4 nM monomers) to 30 (0.26 microM monomers) microgram/ml. The association equilibrium constant (Ka) obtained from the concentration-dependent dissociation curve was 9.37 X 10(7) M-1 at 24 degrees C. The derived free energy change (delta G0) for the monomer-dimer association was -10.8 kcal/mo, reflecting a high degree of tightness between inter-subunit domains in soluble dimeric ATPase. A steep dissociation curve within a short natural logarithmic span (2.5 units) was obtained when the degree of dissociation increased from 0.1 to 0.9, suggesting that a conformational drift accompanies the dissociation of soluble dimeric ATPase. A unique leading boundary was formed in the large zone chromatographies, indicating a reversible equilibrium which was rapid when compared to the time taken for the chromatographic run. Enzymatic activity was continuously monitored in the eluate, revealing that soluble ATPase at different degrees of dissociation was active.

Adenosine Triphosphatases↗
PubMed1985

Stoichiometric photolabeling of two distinct low and high affinity nucleotide sites in sarcoplasmic reticulum ATPase.

Highly purified 3'-arylazido-ATP (aATP) was obtained by high performance liquid chromatography. In the dark, this photoactivatable ATP analog was a competitive inhibitor of ATP hydrolysis catalyzed by purified sarcoplasmic reticulum (SR) ATPase with a Ki of 10 microM. The analog itself was hydrolyzed by the enzyme in the dark. A biphasic curve of velocity of hydrolysis of the analog versus aATP concentration was obtained, indicating the presence of high and low affinity sites with K0.5 of approximately 10 microM and 300 microM, respectively. Upon irradiation with visible light, a biphasic curve was obtained for the level of covalent photolabeling of the enzyme versus [beta-32P]aATP concentrations. Levels of 6.5-9 nmol of analog/mg of protein and 20-22 nmol of analog/mg of protein were obtained when labeling with 20-30 or with 400 microM aATP, respectively, showing the existence of 1 mol of high affinity sites/mol of ATPase and 1-1.5 mol of low affinity sites/mol of enzyme. The rate of light-dependent incorporation of [beta-32P]aATP was decreased by the presence of ATP, Pi, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene-ATP, or Ca2+ in the illumination media. Photolabeling of the high affinity sites had little effect on the velocity of ATP hydrolysis but significantly inhibited the splitting of additional aATP added in the dark. Photolabeling the low affinity sites caused irreversible inhibition of the ATPase activity. The inhibition was prevented by having ATP in the illumination medium, which protected it from labeling. Gel filtration chromatography in the presence of detergent showed that radioactive photolabel was incorporated in the SR ATPase protein. The results indicate that aATP is a useful tool for stoichiometrically labeling and probing the nucleotide binding domains of the SR ATPase.

Adenosine Triphosphatases↗
PubMed1983

Lipid composition and catalytic properties of sarcoplasmic reticulum from normal and dystrophic chicken muscle.

The lipid composition of sarcoplasmic reticulum vesicles isolated from the pectoralis muscle of dystrophic chickens (line 413) and of the genetically related line of normal chickens (line 412) were studied. The total lipid content in the dystrophic preparation was increased by 12% when compared to the normal. The increase was restricted to the neutral lipids in the membrane, with a significant increase of 25% in the cholesterol and of 23% in the triacylglycerol contents. Identical phospholipid composition and content was found. Most of the fatty acid acyl chains from the phospholipids were similar, with an increase in the degree of saturation of phosphatidylcholine, phosphatidylserine and phosphatidylinositol. Similar profiles of the fatty acid acyl chains from triacylglycerol were found. The affinities of the ATPase enzyme for ATP and Ca2+ were similar in the normal and dystrophic preparations. The K0.5 for ATP in both preparations was 28 microM for the Mg2+-dependent activity, and 5-8 microM for the Ca2+-dependent activity which exhibited a secondary increase in the velocity of hydrolysis, in the millimolar ATP concentration range. The K0.5 for Ca2+ in both preparations was approximately 0.3 microM.

Animals↗
PubMed1981

Different degrees of cooperativity of the Ca2+-induced changes in fluorescence intensity of solubilized sarcoplasmic reticulum ATPase.

The tryptophan fluorescence emission of sarcoplasmic reticulum Ca2+-ATPase was studied both in purified ATPase vesicles and in ATPase solubilized with the nonionic detergent dodecyloctaethyleneglycolmonoether (C12E8). Fluorescence intensity changes in purified ATPase were titrated as a function of free Ca2+ in the medium. It exhibited a cooperative pattern, with a Hill number of 2.21 +/- 0.02 and K0.5 = 0.51 microM Ca2+. Upon solubilization of the ATPase, the cooperative pattern of fluorescence change was lost; the Hill number was 0.96 and K0.5 = 1.4 microM Ca2+. When solubilization was carried out in the presence of 0.5 or 1.0 mM CaCl2, followed by the titrations of fluorescence change in the micromolar Ca2+ range, the cooperative pattern was preserved under the same concentrations of C12E8 which would otherwise promote the loss in cooperativity. For the ATPase solubilized in millimolar Ca2+, the Hill number was 1.98 with a K0.5 = 1.5 microM Ca2+. The maximal amount of Ca2+ bound to the high affinity sites corresponded to approximately 1 mol of calcium/mol of polypeptide chains, both in purified ATPase vesicles and in the soluble ATPase. A model is suggested, which involves a minimum of 4 interacting Ca2+ sites (tetramers). Cooperativity is accounted for in the model by the predominance in the absence of Ca2+ of low affinity state (E') of the Ca2+ site (K'D = 5.7 x 10(-4) M), which would be congruent to 90 times more concentrated than (E), the high affinity state (KD = 1.9 x 10(-7) M). Simulations derived from this model fit the experimental data.

Animals↗
PubMed1979

Rapid kinetics of calcium ion transport and ATPase activity in the sarcoplasmic reticulum of dystrophic muscle.

Vesicular fragments of sarcoplasmic reticulum were isolated from pectoralis muscle of normal and dystrophic chicken. Purification of both preparations was equally satisfactory, as shown by a prominent ATPase band in electrophoresis gels. Measurements of ATPase phosphorylation, Ca2+ transport and Pi cleavage by rapid quench methods revealed a lower specific activity of the dystrophic vesicles with respect to all of these functions. On the other hand, Ca2+-independent ATPase activity was found to be increased in dystrophic vesicles. It is suggested that a fraction of ATPase units of dystrophic sarcoplasmic reticulum is not activated by Ca2+, owing to an altered protein assembly within the membrane bilayer. In fact, when the membrane structure is perturbed by detergents normal and dystropic preparations acquire an equally high Ca2+-dependent ATPase.

Adenosine Triphosphatases↗