bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conformational flexibility and structure of creatine kinase
Authors: Haugland Richard P. 
Published: May 2005

AbstractThe structural flexibility of creatine kinase has been investigated with the covalent hydrophobic probe 2‐[4′‐(2″‐iodoacetamido) phenyl] aminonaphthalene‐6‐sulfonic acid (IAANS) which reacts at vastly different rates with the two subunits to give a protein conjugate with fluorescence characteristic of reaction with a site in a hydrophobic cleft. Binding of purine nucleotides greatly enhances the probe fluorescence while pyrimidine nucleotides quench the fluorescence. Small anions bind to nucleotide‐free creatine kinase near the location of the transferable phosphoryl group and quench both the IAANS fluorescence of modified creatine kinase and the tryptophan fluorescence of native creatine kinase. Chloride and nitrate non‐competitively inhibit MgADP binding both with and without creatine. Fluorescence energy transfer demonstrates that the active sites of creatine kinase are well separated and become further apart after the nucleotide‐induced conformational change.
Partial purification of active delta and epsilon subunits of the membrane ATPase from escherichia coli
Authors: Smith Jeffrey B. , Sternweis Paul C. , Heppel Leon A. 
Published: May 2005

AbstractWe have partially purified active delta and epsilon subunits of the E. coli membranebound Mg2+ ‐ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (α, β, and γ) of the enzyme, but the two minor subunits (δ and ϵ), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five‐subunit ATPase and the enzyme deficient in the δ subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two‐subunit enzyme, consisting chiefly of the α and β subunits, was insensitive to the ATPase inhibitor, suggesting that the γ subunit may be required for inhibition by epsilon.The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase‐depleted membranes and to reconstitute ATP‐dependent transhydrogenase. Previously we reported (Biochem. Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1.
Magnetic resonance and kinetic studies of the mechanism of membrane‐bound sodium and potassium ION‐activated adenosine triphosphatase
Authors: Grisham Charles M. , Mildvan Albert S. 
Published: May 2005

AbstractEPR and water proton relaxation rate (1/T1) studies of partially (40%) and “fully” (90%) purified preparations of membrane‐bound (Na++K+) activated ATPase from sheep kidney indicate one tight binding site for Mn2+ per enzyme dimer, with a dissociation constant (KD = 0.88 μM) in agreement with the kinetically determined activator constant, identifying this Mn2+‐binding site as the active site of the ATPase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant.Inorganic phosphate and methylphosphonate bind to the enzyme‐Mn2+ complex with similar high affinities and decrease l/T1 of water protons due t o a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme‐bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with HPO and CH3PO as a function of pH indicates that Na+ induces the phosphate monoanion t o interact with enzyme‐bound Mn2+, while K+ causes the phosphate dianion to interact with the enzyme‐bound Mn2+. Thus protonation of an enzyme‐bound phosphoryl group would convert a K+‐binding site to a Na+‐binding site. Dissociation constants for K+ and Na+, estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding t o the active site.Parallel 32Pi‐binding studies show negligible formation (< 7%) of a covalent E–P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.5 and t o 106% at pH 6.1, produced further decreases in l/T 1 of water protons. Preliminary 31P‐relaxation studies of CH3PO in the presence of ATPase and Mn2+ yield an Mn to P distance (6.9 ± 0.5 Å) suggesting a second sphere enzyme‐Mn‐ligand‐CH3PO complex.Previous kinetic studies have shown that T1+ substitutes for K+ in the activation of the enzyme but competes with Na+ at higher levels. From the paramagnetic effect of Mn2+ at the active site on the enzyme on I/T1 of 205T1 bound at the Na+ site, a Mn2+ to T1+ distance of 4.0 ± 0.1 Å is calculated, suggesting the sharing of a common ligand atom by Mn2+ and T1+ on the ATPase. Addition of P. increases this distance to 5.4 Å consistent with the insertion of P between Mn2+ and T1+. These results are consistent with a mechanism for the \documentclass{article}\pagestyle{empty}\begin{document}$ (\mathop {\rm N}\limits^{\rm i} {\rm a}^{\rm + } {\rm + K}^ +) $\end{document}‐ATPase and for ion transport in which the ionization state of Pi at a single enzyme active site controls the binding and transport of Na+ and K+, and indicate that the transport site for monovalent cations is very near the catalytic site of the ATTase. Our mechanism also accounts for the order of magnitude weaker binding of Na+ compared to K+.
The quantitative measurement of rotational motion of the subfragment‐1 region of myosin by saturation transfer EPR spectroscopy
Authors: Thomas David D. , Seidel John C. , Gergely John , Hyde James S. 
Published: May 2005

AbstractAccording to current models of muscle contraction (Huxley, H. E., Science 164: 1356–1366 [19691]), motion of flexible myosin crossbridges is essential t o the contractile cycle. Using a spin‐label analog of iodoacetamide bound to the subfragment # 1 (S1) region of myosin, we have obtained rotational correlation times (τ2) for this region of the molecule with the ultimate goal of making quantitative measurements of the motion of the crossbridges under conditions comparable to those in living, contracting muscle. We used the newly developed technique of saturation transfer electron paramagnetic resonance spectroscopy (Hyde, J.S., and Thomas, D.D., Ann. N.Y. Acad. Sci. 222:680–692 [1973]), which is uniquely sensitive t o rotational motion in the range of 10−7–10−3 sec. Our results indicate that the spin label is rigidly bound to S1 (τ2 for isolated S1 is 2 × 10−7 sec) and that the motion of the label reflects the motion of the S1 region of myosin. The value of τ2 for the S1 segment of myosin is less than twice that for isolated S1, while the molecular weights differ by a factor of 4, indicating flexibility of myosin in agreement with the conclusions of Mendelson et aL (Biochemistry 12:2250–2255 [1973]). Adding F‐actin increases τ2 in either myosin or isolated S1 by a factor of nearly 103, indicating rigid immobilization of S1 by actin. Formation of myosin filaments (at an ionic strength of 0.15 or less) increases τ2 by a factor of 10–30, depending o n the ionic strength, indicating a decrease of the rotational mobility of S1 in these aggregates. The remaining motion is at least a factor of 10 faster than would be expected for the filament itself, suggesting motion of the S1 region independent of the filament backbone but slower than in a single molecule. F‐actin has a strong immobilizing effect on labeled S l in myosin filaments (in 0.137 M KC1), but the immobilization is less complete than that observed when F‐actin is added t o labeled myosin monomers (in 0.5 M KC1). A spin‐label analog of maleimide, attached to the SH‐2 thiol groups of S1, is immobilized to a much lesser extent by F‐actin than is the label on SH‐1 groups. The maleimide label also was attached directly to F‐actin and was sufficiently immobilized to suggest rigid binding to actin.
Surface modifications evoked by antidiuretic hormone in isolated epithelial cells: Evidence from lectin probes
Authors: Pietras Richard J. , Naujokaitis Paul J. , Szego Clara M. 
Published: May 2005

AbstractEpithelial cells (80–90% “granular” type) were isolated from urinary bladders of Bufo marinus and Rana catesbiana. The inhibitory effect of α‐methyl‐D‐mannoside on fluorescein‐labeled concanavalin A (Con A) binding to these cells indicates that they possess specific binding sites for Con A. The lectin also mediates adsorption of erythrocytes to these cells. Both Con A binding and Con A‐mediated hem‐adsorption to epithelial cells are depressed at 4°C, as compared with cells maintained at 22°C. Elevation of temperature to 37°C, however, enhances hemadsorption independently of alterations in lectin binding. Treatment of cells with antidiuretic hormone (ADH) at 22°C followed by 15 min of incubation at 22° or 37°C before exposure of cells to Con A promotes increments in Con A‐mediated hemadsorption, but not in lectin binding, at 22° or 37°C. These hormonal effects are not significant when hemadsorption is assayed at 4°C. Treatment of cells with another octapeptide, angiotensin, elicits a small, but significant, increment in hemadsorption to epithelial cells which is likewise uninfluenced by quantitative changes in lectin binding. Collectively, these data and other independent observations suggest that treatment with octapeptide hormones acts to enhance the redistribution and aggregation of lectin‐binding proteins in the membranes of granular epithelial cells from amphibian urinary bladder. Such changes, in turn, may contribute to the alterations in membrane transport properties which characterize the hormonal response.
Ultrastructural deformation studies on biological membranes
Authors: Wong W. M. , Geil P. H. 
Published: May 2005

AbstractThe ultrastructural deformation technique has been used to study various membranes, including several claimed to show subunit structure. With localized deformation fibers ca. 100–300 Å in diameter and up to 4,000 Å long were found extending across the cracks parallel to the draw direction in all membranes. Lipid extraction and proteolytic enzyme (papain) treatment of membranes has shown that the fibers are protein in nature. Deformation of membranes while still we showed no significant change in the appearance of the fibers compared with those obtained from deformation of membranes after air drying, indicating that formation of fibers is not an artifact due to dehydration. There must be extensive interaction between the protein molecules and thus we indicate that the results do not agree with current suggestions that membranes are composed of individual protein subunits immersed in a lipid bilayer.
Physical and enzymatic properties of nucleotide‐depleted beef heart mitochondrial adenosine triphosphatase
Authors: Garrett Neil E. , Penefsky Harvey S. 
Published: May 2005

AbstractTightly bound adenine nucleotides are removed from multiple binding sites on beef heart mitochondrial ATPase (F1) by chromatography on columns of Sephadex equilibrated with 50% glycerol. Release of nucleotides from the enzyme is associated with large decreases in sedimentation velocity (from 11.9 S to 8.4 S) which may be observed in concentrated solutions of polyols. Polyol‐induced conformational changes are reversed when the enzyme is returned to dilute buffers. The nucleotide‐depleted enzyme restores oxidative phosphorylation in F1 ‐deficient submitochondrial particles. Reconstitution of nucleotide‐depleted F1 with the ATP analog (adenylyl‐imidodiphosphate) (AMP‐PNP), almost 5 moles of AMP‐PNP per mole of enzyme, results in preparations with substantially inhibited ATPase activity which nevertheless restores oxidative phosphorylation and the 32Pi‐ATP exchange reaction in F1 ‐deficient submitochondrial particles. Incubation of the analog‐labeled enzyme with ATP and Mg++ results in partial displacement of the analog and a time‐dependent recovery of ATPase activity.
The size of adenylate cyclase and guanylate cyclase from the rat renal medulla
Authors: Neer Eva J. 
Published: May 2005

AbstractThe size distribution of adenylate cyclase from the rate renal medulla solubilized with the nonionic detergents Triton X‐100 and Lubrol PX was determined by gel filtration and by centrifugation in sucrose density gradients made up in H2O or D2O. The physical parameters of the predominant from in Triton X‐100 are 220,w, 5.9 S; Stokes radius, 62 A; partial specific volume (v), 0.74 ml/g; mass, 159,000 daltons; f/f0, 1.6; axial ratio (prolate ellipsoid), 11. For the minor form the values are : 220,w, 3.0; Stokes radius, 28 A; mass, 38,000 daltons; f/f0, 1.2. The corresponding values determined in Lubrol PX are similar.The value of v for the enzyme indicates that it binds less than 0.2 mg detergent/mg protein. Since interactions with detergents probably substitute for interactions with lipids and hydrophobic amino acid side chains, these findings suggest that no more than 5% of the surface of adenylate cyclase is involved in hydrophobic interactions with other membrance components. Thus, most of the mass of the enzyme is not deeply embedded in the lipid bilayer of the plasma membrance.Similar studies have been performed on the soluble guanylate cyclase of the rate renal medulla. In the absence of detergent, the molecular properties of this enzyme are: s20,w, 6.3 S; Stokes radius, 54 A, v, 0.75 ml/g; mass, 154,000 daltons f/f0, 1.4; axial ratio, 7. The addition of 0.1% Lubrol PX to this soluble enzyme increases its activity two‐ to fourfold and changes the physical properties to : s20,w, 5.5 S; Stokes radius, 62 A; v, 0.74 ml/g; mass, 148,000 daltons; f/f0, 1.6; axial ratio, 11. These results show that Lubrol PX activates the enzyme by causing a conformational change with unfolding on the polypeptide chain.Guanylate cyclase from the particulate cell fraction can be solubilized with Lubrol PX but has properties quite different from those of the enzyme in the soluble cell fraction. It is a heterogeneous aggregrate with s20,w, 10 S; Stokes radius, 65 A; mass about 300,000 daltons. The conditions which solubilize guanylate cyclase also solubilize adenylate cyclase and the two activities can be separated on the same sucrose gradient.
Fibroblast surface antigen (SF): Molecular properties, distribution in vitro and in vivo, and altered expression in transformed cells
Authors: Vaheri Antti , Ruoslahti Erkki , Linder Ewert , Wartiovaara Jorma , Keski‐Oja Jorma , Kuusela Pentti , Saksela Olli 
Published: May 2005

AbstractWe have recently described a cell type‐specific surface (SF) antigen that is deleted in chick fibroblasts transformed by Rous sarcoma virus. SF antigen is a major surface component and makes up about 0.5% of the total protein on normal cultured fibroblasts. The antigen is shed from normal cells and is present in circulation (serum, plasma), and in vivo, also, in tissue boundary membranes. The molecular equivalents of both cellular and serum SF antigen are distinct, large polypeptides, one of which (SF210, MW 210,000) is glycosylated and, on the cell surface, highly susceptible to proteases and accessible to surface iodination. Immunofluorescence and scanning electron microscopy have indicated that the antigen is located in fibrillar structures of the cell surface, membrane ridges, and processes.Human SF antigen is present in human fibroblasts and in human serum. We have recently shown that human SF antigen is identical to what has been known as the “cold‐insoluble globulin” and that it shows affinity toward fibrin and fibrinogen. Our results also indicate that loss of the transformation‐sensitive surface proteins is due not to loss of synthesis but to lack of insertion of the protein in the neoplastic cell surface. Both normal and transformed cells produce the SF antigen, but the latter do not retain it in the cell surface.The loss of SF antigen, a major cell surface component, from malignant cells creates an impressive difference between the surface properties of normal and malignant cells. The possible significance of SF antigen to the integrity of the normal membrane and its interaction to surrounding structures is discussed.
Chromosomally depleted interspecific hybrid cell clones selected with cytotoxic antisera: Utilization in the study of control of murine leukemia virus host‐range
Authors: Collins Jeffrey J. , Destree Antonia T. , Marshall Christopher J. , Bernstein Alan , Macpherson Ian A. 
Published: May 2005

AbstractA chromosomally stable mouse‐Chinese hamster hybrid cell line was subjected to five rounds of selection with cytotoxic antisera raised in rabbits against either the parental mouse 3T3 cells or the parental Chinese hamster Wg‐1 cells. Routine karyological analysis of clones isolated at each stage of serum selection revealed that treatment with either serum resulted in a limited loss of chromosomes (compared to the untreated hybrid cell cultured in parallel) and that the pattern of chromosome loss could not be correlated with the particular antiserum used for selection. However, more detailed analysis with the SSC‐formamide C‐banding technique, which identifies chromosomes containing a mouse centromere region, demonstrated that while large‐scale chromosome loss was not achieved as a result of antiserum selection, the limited loss of chromosomes did, in fact, reflect a specific depletion of chromosomes in response to treatment with cytotoxic antiserum. Specific chromosomal elimination was shown to occur as early as the first round of antiserum treatment. Antigenic analysis of the serum‐selected clones revealed a quantitative decrease in the expression of the species‐specific surface antigens selected against, but no qualitative loss of antigens was detected. The results suggest that treatment with cytotoxic antiserum may select for clones that have lost specific chromosomes bearing genes regulating the expression of species‐specific surface antigens, rather than for those demonstrating large‐scale depletion of chromosomes bearing the corresponding structural genes. Some of these chromosomally depleted hybrid cell clones have been used (along with pseudotype viruses containing the genome of vesicular stomatitis virus within the envelope of murine leukemia virus, VSV [MuLV]), to study the mechanisms regulating MuLV replication in Chinese hamster cells. The results indicate that the restriction of MuLV replication in Chinese hamster cells operates at two levels: (a) an inability to adsorb to or penetrate Chinese hamster cells; and (b) an additional intracellular block which is dominant in the mouse‐Chinese hamster hybrid cell clones examined. This latter block is presently under study.
The effect of transformation‐defective avian oncornavirus mutants on tumor antigen expression
Authors: Kurth R. , Wyke J. A. , Friis R. R. , Rohrschneider L. , Bauer H. 
Published: May 2005

AbstractThe recent isolation of conditional (temperature sensitive) and nonconditional transformation‐defective mutants of avian sarcoma virus strains has facilitated the investigation of the effect of virus transformation on the cell's phenotype, e.g., with respect to morphology, growth pattern, or cell surface antigenicity. Special emphasis was laid on elucidating the correlation between transformed phenotype and tumor antigen expression.All of the tested nontransforming deletion mutants and the majority of the temperature‐sensitive mutants were unable to induce tumor antigens in phenotypically untransformed cells. However, 3 temperature‐sensitive mutants were found which were able to support the expression of tumor specific surface antigens even at restrictive temperature, when cells otherwise exhibited a normal phenotype. The theoretical and practical implications of this association between normal phenotype and tumor antigen expression are discussed.
Regulation of SV40‐induced cell division and tumor antigen by dibutyryl adenosine 3′‐5′‐monophosphate
Authors: Robb James A. 
Published: May 2005

AbstractSimian virus 40 (SV40) induces cell division in microcultures of sparsely plated nongrowing mouse BALB/3T3 cells during acute infection at moderate multiplicities of infection (MOI = 10–100). The infected cells are killed when a MOI of 1,000 is used. SV40 tumor (T) antigen is synthesized in the infected cells, but viral DNA, virion antigen, and progeny virions are not synthesized (abortive infection). The addition of exogenous dibutyryl adenosine 3′‐5′‐monophosphate (dbcAMP) at the time of infection stimulates the SV40‐induced cell division at all MOI and inhibits SV40‐induced cell death at high MOI. The percentage of T antigen‐positive cells, as monitored by immunofluorescence, is also increased by the addition of dbcAMP at the time of infection. This regulation of SV40‐induced cell division and T antigen formation by exogenous dbcAMP occurs within the first 6 hr after infection at 37° C and is dependent upon both the MOI and the concentration of added dbcAMP. The addition of dbcAMP to productively infected TC7 monkey cells has little effect on the SV40‐induced cell death or T antigen formation.
The regulatory control of β‐receptor dependent adenylate cyclase
Authors: Levitzki Alexander , Sevilla Nehama , Steer Michael L. 
Published: May 2005

AbstractThe characteristics of the β‐receptor in turkey erythrocyte adenylate cyclase were studied using both kinetics of enzyme activation and direct binding measurement of the β‐agonists and antagonists to the β‐receptor. The regulatory ligands Gpp(NH)p and Ca2+ do not have any direct effect on the β‐receptor, but modulate the enzyme activity through the interaction with specific regulatory sites.It was found that the role of the catecholamine hormone is to facilitate the activation of the enzyme by the guanyl nucleotide. The regulatory guanyl nucleotide binds to its allosteric site in the absence of hormone, but the activation of the enzyme is slow in the absence of hormone. This role of the hormone can be described by the scheme: magnified image Where R is the receptor, E the enzyme, G the guanyl nucleotide, H the hormone, and E′ the activated form of the enzyme. The binding steps are fast and reversible but the conversion of the inactive enzyme E to its active form occurs with a k∼1.0 min−1 In the absence of the β‐agonist (l‐catecholamine) at the β‐receptor and at physiological free Mg2+ concentrations, the activation of the enzyme is insignificant. Thus the presence of a guanyl nucleotide at the allosteric site is obligatory but not sufficient to induce the conversion of the inactive enzyme to its active form. At high (nonphysiological) Mg2+ concentration the conversion of E to E′ occurs slowly in the absence of hormone probably by another pathway.There are two classes of Gpp(NH)p regulatory sites: tight sites and loose sites, both of which can be identified kinetically. We have also identified the tight sites by direct binding studies using 3H‐Gpp(NH)p. It is not clear, however, whether these are two distinct classes of sites or whether their existence reflects the presence of negative cooperativity among the guanyl nucleotide regulatory sites.Calcium was found to be a negative allosteric inhibitor of adenylate cyclase. The inhibitory effect of Ca2+ is exerted on the nonactivated enzyme as well as on the Gpp(NH)p preactivated enzyme.