bluuebunny/crossref_metadata_2025 huggingface dataset:
Hemocyanin–antibody labeling of rhodopsin in mouse retina for a scanning electron microscope study
Authors: Jan Lily Yeh , Revel Jean‐Paul
Published: May 2005

To reveal the presence of rhodopsin on the surface of the mouse retina, a scanning electron microscope study of the immunolabeling of rhodopsin was attempted. The glutaraldehyde‐fixed mouse retina was treated first with rabbit antibodies specific against bovine rhodopsin and then with hemocynin‐labeled goat antibodies specific against rabbit antibody. The distribution of hemocyanin label on mouse retina and the technique used for labeling are discussed.
An activation mechanism for ATP cleavage in muscle
Authors: Harrington William F. , Reisler Emil , Burke Morris
Published: May 2005

Evidence for a proposed activation mechanism is summarized. The low rate of ATP cleavage in the resting state of muscle is considered to result from the formation of a stable ring structure involving the two essential sulfhydryl groups on each myosin head and MgATP. Activation is thought to occur by interaction of actin in the vicinity of one of the essential sulfhydryl groups, Thus opening the stable ring leading to rapid dissociation of split products. This idea is consistent with the kinetic scheme of ATP cleavage developed recently by other workers and allows a prediction of the shift in population of intermediate states with changes in solvent conditions. It is also supported by our recent studies on the spatial geometry of the ring. The possibility that other nucleophilic groups may replace the sulfhydryl groups in other contractile systems is considered. The relevance of the ring structure to the tension generating event is discussed on the basis of recent measurements of the rate of contraction of modified (SH1‐blocked) actomyosin threads. Results indicate that the ability to form the ring structure is an essential requirement of the contractile process in these systems, and, moreover, that single, modified heads of myosin can act independently to produce the same rate of contraction as native myosin. This latter finding suggests that the myosin duplex exhibits some type of negative cooperativity in the contractile process.
Segmental flexibility in the myosin molecule: Evidence from binding studies of myosin fragments with actin
Authors: Peller Leonard
Published: May 2005

From comparative studies of the association with polymeric actin of the bifunctional species heavy meromyosin and its monofunctional constituents, information about the relative freedom of these paired elements can be derived. An isotherm for the former binding process is presented which involves, as an experimentally determinable parameter, the local concentration of a second segment after the first of a pair is attached to the lattice. From combined data for these two association reactions a value of 10−4 M is obtained for this quantity. The large degree of segmental flexibility reported for the free heavy meromyosin is still manifested in the association with actin.
Conformational flexibility and structure of creatine kinase
Authors: Haugland Richard P.
Published: May 2005

The 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

We 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

EPR 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

According 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

Epithelial 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

The 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

Tightly 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

The 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

We 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

A 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

The 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

Simian 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

The 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.
Hexagonal packing of lipid acyl chains and membrane plasticity
Authors: Dorset D. L. , Hui S. W. , Strozewski C. M.
Published: May 2005

Electron microscope and electron diffraction observations on microcrystals of pure lipids (a phosphatidylethanolamine, two phosphatidylcholines, a phosphatidic acid, and a galactocerebroside) reveal an extreme flexibility of lipid layers when the acyl chains are hexagonally packed (d100 = 4.17 Å). This is corroborated by similar observations on wet bilayers of a lecithin. It is shown that more “crystalline” polymethylene packings do not impart such plasticity to lipid layers and are therefore an unsuitable structural matrix for dynamic biological membranes.
Trapping at low temperature of oriented chloroplasts: Application to the study of antenna pigments and of the trap of photosystem‐1
Authors: Vermeglio A. , Breton J. , Mathis P.
Published: May 2005

A technique is described for the preparation of oriented samples from spinach chloroplasts whose linear dichroism is then studied by (flash) absorption spectroscopy. The chloroplasts are suspended in a glycerol‐containing medium, oriented in a magnetic field, and slowly cooled in the magnet until the medium is rigid enough to avoid disorientation effects.

The absorption spectra in polarized light have been measured at −50° and −170°C. They allow the orientation of chlorophyll b to be resolved, and the red transition moment is found to be tilted out of the membrane plane.

A study of the flash‐induced absorption changes linked to Photosystem‐1 activity reveals a progressive evolution of the difference spectra and of the linear dichroism with decreasing temperatures. At −170°C, the difference spectrum of P700 in the red is well resolved. All transition moments are found to be largely parallel to the membrane plane.

The potential use of the technique for other experiments by differential absorption spectroscopy and by EPR techniques is discussed.
Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction
Authors: Brown Susan , Levinson Warren , Spudich James A.
Published: May 2005

Treatment of chick embryo fibroblasts with 0.5% Triton® X‐100 extracts most of the cell protein, leaving an organized part of the cell structure attached to the tissue culture dish. This “Triton cytoskeleton” consists largely of intermediate‐sized filaments and bundles of microfilaments. SDS polyacrylamide gel electrophoresis reveals that this cytoskeleton is made up of three main proteins. One protein component is 42,000 daltons and co‐migrates with muscle actin. The other two components are 52,000 and 230,000 daltons and remain quantitatively associated with the cytoskeleton during the detergent extraction. The possible identity of these three protein components and their organization into a supramolecular structure is discussed.
Localization and organization of microfilaments and related proteins in normal and virus‐transformed cells
Authors: Goldman Robert D. , Yerna Marie‐Jeanne , Schloss Jeffery A.
Published: May 2005

The localization and organization of actin‐like microfilaments in normal, SV‐40 and adenovirus transformed cells are determined by the coordinated use of light optical, electron optical and biochemical techniques. In adenovirus‐type 5 transformed hamster embryo cells, microfilament meshworks appear to be the predominant organizational form of cellular actin, while in normal hamster cells, microfilament bundles are prevalent. Differences between 3T3 and SV‐40 transformed 3T3 cells are less apparent and may be related to the packing and intracellular distribution of microfilament bundles. Attempts at relating these ultrastructural changes in transformed cells to the images obtained following reaction with fluorescein‐labelled myosin fragments and indirect immunofluorescence with smooth muscle myosin antibody are discussed. In several instances the fluorescence microscope images do not correspond to the ultrastructural observations. The results are discussed in terms of the possible relationships between alterations in cytoplasmic contractile elements and the abnormal behavior of transformed cells.
Fine structural localization of concanavalin a binding sites on hamster spermatozoa
Authors: Kinsey William H. , Koehler James K.
Published: May 2005

The plasma membrane of epididymal spermatozoa of the golden hamster (Mesocricetus auratus) exhibits morphological differences over various parts of the head and tail as detected by air‐dried replicas and freeze‐etching techniques. In an attempt to ascertain whether any topographical differences exist in the number or distribution of carbohydrate moieties associated with the cell surface, cells were labeled with Concanavalin A and marked with hemocyanin.

It was found that while the plasma membrane over the acrosomal region differed from that of the postacrosomal region in membrane components revealed by freeze fracturing, there was no apparent difference in the distribution or density of Con A binding sites detectable by hemocyanin localization. The tail regions exhibited differences in both fracture face appearance and the distribution of detectable carbohydrate moieties.

It was also found that binding sites for Concanavalin A exist on the inner and outer acrosomal membranes in addition to those on the plasma membrane.
Microtubles and actin filaments in teleost visual cone elongation and contraction
Authors: Burnside Beth
Published: May 2005

Teleost retinal cones contract in light and elongate in darkness. This paper describes the disposition of microtubules and cytoplasmic filaments in cone cells of 2 species of fish (Haemulon sciurus and Lutjanus griseus). In Haemulon, the neck‐like “myoid” region of the cone changes in length from 5 μ to 75 μ. Maximal observed rates of elongation and contraction are comparable to that of chromosome movement in mitosis (2–3 μ/min). Microtubules presumably participate in cone elongation, since numerous longitudinal microtubules are present in the myoid region, and colchicine blocks dark‐induced elongation.

Myoid shortening, on the other hand, appears to be an active contractile process. Disruption of microtubules in dark‐adapted cones does not produce myoid shortening in the absence of light, and light‐induced myoid shortening is blocked by cytochalasin‐B. Cone cells possess longitudinally‐oriented thin filaments which bind myosin subfragment‐1 to form arrowhead complexes typical of muscle actin. Myoid thin filaments are clearly observed in negatively stained preparations of isolated cones which have been disrupted with detergent after attachment to grids. These myoid filaments are not, however, generally preserved by conventional fixation, though bundles of thin filaments are preserved in other regions of the cell. Thus, actin filaments are poorly retained by fixation in precisely the region of the cone cell where contraction occurs. Cone cells also possess longitudinally‐oriented thick filaments 130–160Å in diameter. That these thick filaments may be myosin is suggested by the presence of side‐arms with approximately 150 Å periodicity. The linear organization of the contractile apparatus of the retinal cone cell makes this cell a promising model for morphological characterization of the disposition of actin and myosin filaments during contraction in a nonmuscle cell.
Cytoskeletal functions of cytoplasmic contractile proteins
Authors: Pollard Thomas D.
Published: May 2005

This is a review of the evidence that the cytoplasmic contractile proteins function as a cytoskeletal system inthe cytoplasmic matrix. Biochemical experiments show that cycoplasmic actin filaments can form a solid gel under conditions likely to exist in living cells. The actin filaments are associated with other proteins which may stabilize the gel and which are involved with motile force generation like myosin. Ultrastructural studies show that actin filaments are difficult to preserve, but that under stabilizing conditions networks of actin filaments are found throughout the cytoplasmic matrix.
Genetic control of membrane mosaicism
Authors: Satir Birgit
Published: May 2005

The mosaic and dynamic character of cellular membranes is illustrated by the specific intramembrane particle array, the fusion rosette, found to be essential for memberane fusion and secretion in the ciliated protozoa, Tetrahymena and Paramecium. The rosette is not a permanent site within the membrane. When secretion of mucocysts is synchronized by treating cells with the local anesthetic dibucaine, all rosettes disappear, only to reassemble as new mucocysts mature. Assembly of the functional rosette is under genetic control. A series of secretory mutants of Paramecium, blocked in various stages of the secretory cycle, has been studied (11). Mutants that do not secrete lack the fusion rosette, although other intramembrane particle components of the fusion site are present. Certain properties of the rosett, in particular its particle partition coefficient, are temperature‐dependent, which may affect the ability of the rosette particles to assemble. A temperature‐sensitive mutant, nd9, secretes normally, and has rosettes at 18°C, but fails to secrete attached trichocysts at 27°C.
Comparison of the structural and chemical composition of giant T‐even phage heads
Authors: Aebi U. , Bijlenga R. K. L. , ten Heggeler B. , Kistler J. , Steven A. C. , Smith P. R.
Published: May 2005

A study has been made of the structure of the capsids of T4D giant phage produced from mutants in gene 23 and temperature‐sensitive mutants in gene 24, and T4D and T2L giant phage formed by the addition of L‐canavanine followed by an L‐arginine chase in the growth medium.

All the giant phage capsids have been shown to be built according to the same geometrical architecture. This consists of a near‐hexsagonal surface net, lattice constant 129.5 Å, folded into a left‐hand T = 13 prolate icosahedron elongated along one of its fivefold symmetry axes. Their only apparent difference from wild‐type T‐even phage capsids is their abnormally elongated tubular part.

A comparison of the capsomere morphologies and protein compositions of the giant phage capsids showed that all T4D giants are indentical but differ from T2L: The T4D capsomere has a complex (6+6+1)‐type morphology, whereas the T2L has a simple 6‐type. T2L phage, however, lack two capsid proteins, “soc” and “hoc”, present in T4D. The difference in capsomere morphology can therefore be related to the difference in the protein compositions of these two phage.

Possible differences between the initiation and means of length regulation of giant phage heads and the aberrant polyheads are discussed.
Involvement of microtubules and microfilalments in the action of vasopressin in canine renal medulla
Authors: Iyengar Ravi , Lepper Kenneth G. , Mailman David S.
Published: May 2005

Vasopressin‐stimulated cyclic AMP content and the uptake of 3H2O and 22Na into canine renal medullary slices were measured. Cyclic AMP was increased threefold by 9 × 10−9M vasopressin in isotonic (290 mOsm/kg H2O) Krebs‐Ringers bicarbonate. A significant increase in vasopressin‐stimulated 3H2O uptake began at 2.75 min after hormone addition and lasted until 5.00 min. Colchicine (1 × 10−5 M) inhibited the vasopressin‐ stimulated 3H2O uptake. This effect required a minimum preincubation period of 30–40 min in colchicine‐containing medium. Colchicine had no effect on basal or vasopressin‐stimulated cyclic AMP (10 mM). Lumicolchicine (10−5M) had no effect on either vasopression‐ or dibutyryl cyclic AMP‐stimulated 3H2O uptake. 14 C‐colchicine bound predominantly to the cytosol fraction enriched in microtubules, while virtually no binding was observed on plasma membranes. Loght‐microscopic examinations of cross sections of tissue slices showed that a majority of vasopressin‐treated collecting tybulels and some control tubules had occluded lumens. Colchicine‐treated cells, in the presence of vasopressin, had open lumens indicating a blockage of the vasopressin‐induced water transport. Cells treated with cytochalasin B (1 μgm/ml) also had open lumens in the presence of vasopressin. Cytochalasin B also blocked vasopressin and dibutyryl cyclic AMP‐stimulated 3H2O uptake into collecting duct cells but had no effect on vasopressin stimulated cyclic AMP levels. It was concluded that microtubules and possibley microfilaments are involved in the subcellular mechanism by which vasopresssin increases the permeability of the collecting duct to water.
Intracellular regulation of cell shape and motility in naegleria. First insights and a working hypothesis
Authors: Fulton Chandler
Published: May 2005

Amoebae of Naegleria gruberi differentiate to temporary flagellates that have a regular, asymmetric, streamlined body contour. During the hour‐long differentiation, amoeboid movement gradually ceases and as a consequence the cells round up. Subsequent elongation to flagellate shape includes the formation of a microtubular cytoskeleton. Both the loss of amoeboid motility and the formation of the flagellate shape require prior transcription and translation, suggesting the possibility that specific syntheses of RNA and protein may be required for each shape change. Flagellates can “revert” to motile amoebae within 20 sec after a suitable stimulus, indicating that the amoeboid motility system remains latent in flagellates. A cell‐produced chemical factor extracted from Naegleria, Ψ, triggers a reproducible sequence of rapid shape changes in flagellates when added to their environment. Cells respond to the presence of external Ψ only “transiently,” and the reaction of flagellates to added Ψ requires extracellular Ca+2. Ionophore A23187 produces shape changes in flagellates similar to those produced by Ψ, supporting the conclusion that Ψ is involved in the movement of Ca+2. Normally Ψ is intracellular, and the intracellular distribution of Ψ changes during differentiation.

These results lead to and support a working hypothesis to explain the rapid changes in shape and motility in Naegleria. Four elements are postulated: Ca+2; an actin‐based amoeboid motility system that depends on free Ca+2 for functioning; a tubulin‐based cytoskeleton that assembles and remains assembled only when free Ca+2 is low; and Ψ. The factor Ψ is postulated to regulate the intracellular release of Ca+2. According to the hypothesis, intracellular free Ca+2 is constantly swept up into Ca‐reservoirs. Motility of amoebae depends on local release of Ca+2 from these reservoirs, which in turn is caused by the intracellular release of Ψ. During differentiation, Ψ is “compartmentalized” as part of the developmental program, and as a consequence intracellular Ca+2 is swept up into Ca‐reservoirs but not released. As free Ca+2 becomes limiting, amoeboid movement stops, and the cells round up. Subsequently, in a process that depends on low free Ca+2, the microtubular cytoskeleton is assembled, and the flagellate shape is formed. During reversion of flagellates to amoebae, release of Ψ from its “compartments” permits local release of Ca+2, which then causes both disassembly of the flagellate cytoskeleton and immediate resumption of amoeboid movement. This testable hypothesis has implications for the study of cell shape, motility, and differentiation.
Isolation of the alanine carrier from the membranes of a thermophilic bacterium and its reconstitution into vesicles capable of transport
Authors: Hirata Hajime , Sone Nobuhito , Yoshida Masasuke , Kagawa Yasuo
Published: May 2005

A carrier protein mediatine alanine transport was purified from the membranes of the thermophilic bacterium PS3, by ion exchange chromatography in the presence of both Triton X‐100 and urea.

The alanine carrier was recovered in the nonadsorbed fraction from either DEAE‐or CM‐cellulose columns, suggesting that its isoelectric point was in the neutral pH region.

The final preparation contained virtually no electron transfer components, ATPase, or NADH dehydrogenase. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the final preparation consisted of two major protein components with molecular weights of 36,000 and 9,400.

Active transport of alanine after incorporation of the alanine carrier into reconstituted proteoliposomes was driven not only by an artificial membrane potential generated by potassium ion diffusion via valinomycin but also by mitochondrial cytochrome oxidase incorporated into the same liposomes and supplemented with both cytochrome c and ascorbic acid.

The membrane‐integrated portion (TF0) of the ATPase complex uncoupled alanine transport by conducting protons across the membrane.
The transport of lysosomal enzymes
Authors: Neufeld Elizabeth F. , Sando Gloria N. , Garvin A. Julian , Rome Leonard H.
Published: May 2005

This paper reviews the experimental evidence for the proposal that hydrolytic enzymes are introduced into lysosomes of cultured fibroblasts only after secretion and receptormediated recapture.
Role of the membrane potential in serum‐stimulated uptake of amino acid in a diploid human fibroblast
Authors: Villereal Mitchel L. , Cook John S.
Published: May 2005

The Na+‐dependent accumulation of α‐aminoisobutyric acid (AIB), measured in normal growing and quiescent (serum‐deprived) HSWP cells (human diploid fibroblast), was found to be twofold higher (AIBin/AIBout = 20–25) under the normal growing conditions. Serum stimulation of quiescent cells increases their AIB concentrating capacity by approximately 70% within 1 hr. These observations suggest that the driving forces for AIB accumulation may be reversibly influenced by the serum concentration of the growth medium. Addition of valinomycin (Val) to cells preequilibrated with AIB causes an enhanced accumulation of AIB, suggesting that the membrane potential can serve as a driving force for AIB accumulation. After preequilibration with AIB in 6 mM K+, transfer to 94 mM K+ with Val results in a marked and rapid net loss of AIB. The effect of Val on the accumulation of AIB is greatest in quiescent cells, with the intracellular AIB concentrations reaching those seen both in Val‐stimulated normal cells and in Val‐stimulated serum‐stimulated cells. By adjusting [K+]0, in the presence of Val, the membrane potential of growing cells can be matched to that of quiescent cells or vice versa. When this is done, the two accumulate AIB to the same extent. Hence the AIB accumulating capacity is characteristic of the membrane potential rather than of the growth state. In summary, these data suggest that the accumulation of AIB in HSWP cells is influenced by changes in membrane potential and that a serum‐associated membrane hyperpolarization could be responsible for the increased capacity for AIB accumulation in serumstimulated cells.
Hormonal regulation of hepatic amino acid transport
Authors: Kilberg Michael S. , Neuhaus Otto W.
Published: May 2005

The transport of 2‐aminoisobutyric acid (AIB) into liver tissue was increased by both insulin and glucagon. We have now shown that these hormones do not stimulate the same transport system. Glucagon, possibly via cAMP, increased the hepatic uptake of AIB by a mechanism which resembled system A. This glucagon‐sensitive system could be monitored by the use of the model amino acid MeAIB. In contrast, the insulin‐stimulated system exhibited little or no affinity for MeAIB and will be referred to as system B. On the basis of other reports that the hepatic transport of AIB is almost entirely Na+ dependent and the present finding that the uptake of 2‐aminobicyclo [2,2,1] heptane‐2‐carboxylic acid (BCH) was not stimulated by either hormone, we conclude that system B is Na+ dependent. Furthermore, insulin added to the perfusate of livers from glucagon‐pretreated donors suppressed the increase in AIB or MeAIB uptake. Depending upon the specificities of systems A and B, both of which are unknown for liver tissue, the insulin/glucagon ratio may alter the composition of the intracellular pool of amino acids.
Distribution of a major surface‐associated glycoprotein, fibronectin, in cultures of adherent cells
Authors: Mosher Deane F. , Saksela Olli , Keski‐Oja Jorma , Vaheri Antti
Published: May 2005

Fibronectin was present in media and cell layers of cultures of adherent cells from human skin, kidney, lung, chest wall, liver, and heart. Cell‐surface fibronectin, visualized by immunofluorescence, was in dense fibrillar (cultures from lung), discrete fibrillar (e.g., cultures from skin), or punctate (some cultures from kidney) structures. The subunit sizes of cell‐surface fibronectin and fibronectin soluble in medium appeared identical in sodium dodecyl sulfate‐polyacrylamide gels. To explain the polymorphism of cell‐surface fibronectin, there must be chemical differences among the fibronectins synthesized by different cell strains or factors in the cell layer which influence fibronectin binding and aggregation.