bluuebunny/crossref_metadata_2025 huggingface dataset:
Microtubule assembly: Some possible regulatory mechanisms
Authors: Olmsted J. B. , Marcum J. M. , Johnson K. A. , Allen C. , Borisy G. G.
Published: May 2005

Microtubule polymerization in vitro was examined using material purified from porcine brain tissue by a reversible temperature dependent assembly procedure, and was characterized by electron microscopy, viscometry, and sedimentation. The reaction was endothermic, colchicine sensitive, and occurred at neutral pH and moderate ionic strength. Divalent cations (calcium, magnesium) were inhibitory at millimolar concentrations, but stimulated polymerization at the micromolar level. Nucleoside triphosphates were required for assembly of purified subunits. As determined by quantitative sedimentation analyses, the reaction was an equilibrium process. Below a critical concentration of tubulin no assembly occurred. Analytical ultracentrifugation studies indicated that tubulin species with sO20, w of 6S and 30S were in equilibrium with each other, and that both were incorporated into microtubules. Electron microscopic analyses suggested that disc (or ring) structures might be intermediates in assembly, and that they were primarily utilized early in the polymerization process. Assembly could be seeded by mixing microtubular fragments from brain or flagella with brain microtubule subunits; depending on conditions of temperature and protein concentration, addition of subunits occurred either with unipolar or biased polar directionality. The possible significance of these properties of the polymerization reaction for control of assembly is discussed.
A molecular model of membrane excitability
Authors: Baumann Gilbert , Mueller Paul
Published: May 2005

Alamethicin, monazomycin, or EIM induce electrical excitability in lipid bilayers. The voltage‐dependent gating displays all the characteristics observed in excitable cells and its basic features can be quantitatively described by the Hodgkin‐Huxley equations.

A common molecular mechanism of membrane excitation has been postulated. It assumes that in the absence of an electrical field the channel‐forming molecules lie at the surface of the membrane. An applied potential tilts them from the surface into the hydrocarbon region of the bilayer. Once in this position the molecules diffuse laterally and form aggregates which act as channels for the flow of ions.

In the case of alamethicin we assume that the molecule forms an elongated ellipsoid with two glutamic residues at one end, and a metal ion in four‐ or five‐fold coordination with peptide carbonyl oxygens at the other. An applied field pulls the cationic end through the membrane to the other side, while the glutamic residues hold the other end attached to the original surface. The molecules now span the membrane and aggregate, forming oligomeric channels in which most of the peptide carbonyls face toward the center, and the methyl groups outward.

Monomers and dimers do not conduct and an individual channel can have different conductance values depending on the number of monomers in the aggregate and the resulting channel diameter. A quantitative description of this process matches observed gating kinetics, gating currents, and the single channel conductance increments. Without additional assumptions, inactivation follows directly from the aggregation process because with proper rate constants, the average degree of polymerization and therefore number of open channels goes through a maximum in time.

The model may also apply to the excitation process of higher cells.
Calcium efflux from internally dialyzed squid axons: The influence of external and internal cations
Authors: Blaustein M. P. , Russell J. M. , De Weer P.
Published: May 2005

Internal dialysis techniques have been used to examine the influence of external and internal cations on Ca efflux from ATP‐depleted squid axons. The main observation is that Ca efflux is promoted by external Na and inhibited by internal Na. The Na0 ‐dependent Ca efflux appears to be a function of [Na], and is also affected by the membrane potential; a 25 mV depolarization may cause as much as an e‐fold decrease in Ca efflux. These data are consistent with a counter‐transport exchange of 3Na+–for–1Ca2+. A Ca0‐dependent Ca efflux has also been observed; it is prominent in Na sea water or Le sea water, and is markedly diminished in choline sea water. This flux is consistent with the idea of a Ca‐Ca exchange diffusion process. Taken together, the Na0 ‐ and the Ca0 ‐dependent Ca effluxes fit a two‐site model for carrier‐mediated Ca transport; one site binds two Na+ or one Ca2+, while the second site can bind either one Na+ or one Li+. The data reported here suggest that both sites must be filled on the inward journey, but that only the Ca‐binding site need be occupied on the outward journey of the carrier. A mechanism of this type could derive sufficient energy from the Na and voltage gradients to maintain a [Ca2+]0/[Ca2+]i concentration ratio of about 104 in the absence of ATP. The present experiments do not, however, rule out the possible participation of a metabolically driven Ca transport mechanism in vivo.
Physical properties of the lipid phase of membranes from cultured animal cells
Authors: Wisnieski Bernadine J. , Huang Yoshie O. , Fox C. Fred
Published: May 2005

ESR analysis of membranes from cultured animal cells reveals a more complex lipid phase behavior than that displayed by ideal binary lipid systems. When endoplasmic reticulum membranes from LM cells are spin labeled with a nitroxide derivative of decane, 5N10, and scanned by ESR at 1° C‐intervals, the partitioning of 5N10 between the hydrocarbon and aqueous portions of the membrane suspension undergoes thermotropic changes at characteristic temperatures of 9°, 16°, 22°, 32°, and 38° C. Lipids extracted from these same membranes, however, exhibit only two characteristic temperatures, 16° and 35° C, and in this respect resemble binary lipid systems. The phase behavior of lipids in animal cell membranes is suggestive of an organized distribution of lipid which is disrupted by extraction. In support of this, mathematical treatment of the partitioning data indicates that four of these characteristic temperatures can define the boundaries (i.e., the t1 and th ) of two independent phase transitions in endoplasmic reticulum membranes. These results are similar to those of a physical treatment of data from plasma membranes of both mouse and chick cells in which the two monolayers appear to exist as independent physical entities with different physical properties. The most probable phase boundaries for the two monolayers of the endoplasmic reticulum membranes studied here are 16° and 32° C for one monolayer and 22° and 38° C for the other.
The effect of membrane‐lipid phase transitions on membrane structure and on the growth of acholeplasma laidlawii B
Authors: McElhaney Ronald N.
Published: May 2005

The physical state of the membrane lipids, as determined by fatty acid composition and environmental temperature, has a marked effect both on the temperature range within which A. laidlawii can grow and on the temperature coefficient of growth within the permissible temperature range. The minimum growth temperature under certain conditions is clearly defined by the lower boundary of the gel–to–liquid‐crystalline phase transition of the membrane lipids. The physical state of the membrane lipids can also influence the optimum and maximum growth temperatures. An a brupt increase in the temperature coefficient of growth is noted at temperatures between the phase transition boundaries. Both the absolute rates and the temperature coefficients of cell growth are similar for cells whose membrane lipids exist entirely or predominantly in the liquid‐crystalline state, but absolute growth rates decline rapidly and temperature coefficients increase when most of the membrane lipids become solidified. Some cell growth, however, can continue at temperatures at which less than 10% of the total lipid remains in the fluid state. Conversion of the membrane lipid from the liquid‐crystalline to the gel state is accompanied by a progressive aggregation of intramembranous protein particles. An appreciable heterogeneity in the physical state of the membrane lipids can apparently be tolerated by this organism without a detectable loss of membrane function.
Effect of membrane lipid composition and microtubule structure on lectin interactions of mouse lm cells
Authors: Rittenhouse Harry G. , Williams Robert E. , Fox C. Fred
Published: May 2005

Concanavalin A (Con A) binding and Con A‐mediated hemadsorption to LM cells were found to decrease significantly at both 5–7°C and 15–19°C. The higher of these critical temperatures responds to a change in state of the membrane lipids and can be increased or decreased in cells where the membrane phospholipids contain less or more double bonds, respectively. The lower critical temperature for Con A binding or Con A‐mediated hemadsorption does not respond to these changes in membrane lipid composition. Though the amount of Con A bound to the cell surface is a determinant of Con A‐mediated agglutinability, the major components of the decreases in Con A‐mediated hemadsorption which occur at both these critical temperatures do not have their origin in the decreases in Con A binding which occur over these same temperature ranges – that is 5–7°C and 15–19°C.

Con A‐mediated hemadsorption measured at 22°C was dramatically inhibited when LM cells were first incubated at 7°C or less. Reversal of this inhibition required 20–30 min of subsequent incubation at 22°C, indicating that factors other than membrane lipid “fluidity” are determinants of agglutinability. LM cells treated with the microtubule‐disrupting alkaloids colchicine, colcemid, or vinblastine at concentrations as low as 10−6 M were as much as fourfold more agglutinable with Con A. By contrast, lumicolchicine, an inactive derivative of colchicine, had a slight inhibitory effect on Con A‐mediated hemadsorption. Colchicine, vinblastine, or lumicolchicine treatment of LM cells did not alter the quantitative binding of labeled lectin. The results suggest that membrane lipid “fluidity” and the cell cytoskeleton (microtubule/microfilament system) are important determinants of lectin interactions with cell surfaces. The results are interpreted in terms of a model of cell–cell and cell–lectin interactions which assigns a central role to the Con A receptor.
Oxidation of lipids and membranes II: Effects of oxidants on cerebral white matter homogenates
Authors: Bubis J. J. , Wolman M.
Published: May 2005

Bovine cerebral white matter homogenates were oxidized by various oxidizing solutions of equal molarity and subsequently extracted with water. Most of the oxidants tested (K‐dichromate, FeCl3, H2O2, O2, and chloroperbenzoic, ascorbic, performic, and periodic acids) rendered the various myelin constituents less extractable than the constituents of unoxidized control homogenates. KMnO4, and to a lesser degree hemoglobin, rendered myelin constituents more extractable with water than those of the control homogenates. The findings indicate that most of the oxidants produced stabilization of the lamellar pattern, probably through cross‐linking and polymerization. KMnO4 and hemoglobin caused labilization and breakdown of the membranous structure. Proof that stabilization of membranes is caused by some oxidants and that fragmentation occurs by the action of KMnO4 and hemoglobin was obtained by the light‐scattering technique and by electron microscopy of the oxidized homogenates. Evidence obtained indicated that formation of hydrophobic end groups during oxidation favors polymerization, while prevalence of hydrophilic groups is associated with fragmentation.
Electron paramagnetic resonance and nanosecond fluorescence depolarization studies on creatine‐phosphokinase interaction with myosin and its fragments
Authors: Botts Jean , Stone Deborah B. , Wang Alice T. L. , Mendelson Robert A.
Published: May 2005

Recent reports in the literature have indicated a physical association of creatinephosphokinase (CPK) with the tail portion of the myosin molecule. The present paper describes further studies on the interaction of CPK with myosin and myosin fragments, using the techniques of electron paramagnetic resonance (EPR) and nanosecond fluorescence depolarization. From EPR work, spin‐labeled CPK appears to interact with myosin, tail‐less myosin (heavy meromyosin [HMM]), and myosin heads (subfragment‐1 [S1]), the extent of interaction being proportional to the S1 content of myosin or its fragments. Spin‐labeled CPK did not evidence interaction with the headless myosin “rods”, with myosin tails (light meromyosin [LMM]), with S2 necks (which connect S1 to the rest of the myosin molecule), or with actin. When a fluorescent dye is directed to the essential ϵ‐amino group of CPK, nanosecond fluorescence depolarization studies indicate a substantial interaction with myosin, HMM, and S1, but very little with F‐actin. When the “fast‐reacting” thiol of the S1 moiety or the “essential thiol” of CPK was labeled with either a fluorescent dye or a spin label, no interaction between CPK and myosin (or S1) was detected.
Heat production and metabolism during the contraction of mammalian skeletal muscle
Authors: Kretzschmar K. M.
Published: May 2005

Methods are described whereby initial processes of muscular contraction may be investigated in a mammalian preparation, the soleus muscle of the rat. Conditions are chosen so that recovery is avoided. An isometric tetanus is investigated and an energy balance sheet is drawn up. It is found that there is more heat evolved than can be accounted for in terms of measured chemical reaction. This discrepancy is discussed with reference to the similar results that have been obtained using frog muscle.
Energy conservation and uncoupling in mitochondria
Authors: Hatefi Youssef
Published: May 2005

Energy conservation and uncoupling in mitochondria are examined in the light of three important new findings: (a) Studies with the photoaffinity‐labeling uncoupler 2‐azido‐4‐nitrophenol have shown that mitochondria contain a specific uncoupler binding site (apparently a polypeptide of Mr = 30,000 ± 10%). (b) This site fractionates into an enzyme complex (complex V), which is capable of oligomycin‐ and uncoupler‐sensitive ATP‐Pi exchange. It is absent from electron transfer complexes I, III, and IV, which represent segments of the respiratory chain containing coupling sites 1, 2, and 3, respectively. (c) Trinitrophenol is a membrane‐impermeable uncoupler (uncouples submitochondrial particles, but not mitochondria) and a poor protonophore. There is an excellent correlation between the uncoupling potencies and the affinities of uncouplers for the mitochondrial uncoupler‐binding site. There is no correlation between uncoupling potency and protonophoric activity of uncouplers when a membrane‐permeable uncoupler is compared with a membrane‐impermeable one.
Exploring sites on mitochondrial ATPase for catalysis, regulation, and inhibition
Authors: Lardy Henry A. , Schuster Sheldon M. , Ebel Richard E.
Published: May 2005

Evidence is presented that mitochondrial ATPase has two types of sites that bind adenine nucleotides. The catalytic site, C, binds the substrates ATP, GTP, or ITP and the inhibitor guanylyl imidodiphosphate (GMP‐PNP). A second type of site, R, binds ATP, ADP, adenylyl imidodiphosphate (AMP‐PNP), and the chromium complexes of ATP or ADP. All of these substances binding to the R site inhibit the hydrolysis of ATP in a competitive manner; their inhibition of hydrolysis of ITP and GTP is noncompetitive. GMP‐PNP inhibits oxidative phosphorylation in submitochondrial particles but AMP‐PNP does not. The localization on mitochondrial membranes of sites for the binding of various antibiotics that inhibit oxidative phosphorylation is discussed.
Studies of the oligomycin‐sensitive ATPase from yeast mitochondria. Reconstitution of ATP‐32Pi exchange in the presence of phospholipids
Authors: Ryrie Ivan J.
Published: May 2005

A purified preparation of the oligomycin‐sensitive ATPase from yeast mitochondria has been shown to elicit an oligomycin‐ and uncoupler‐sensitive ATP‐32Pi exchange in the presence of phospholipids. Reconstitution was normally achieved by dialysis of an ATPase‐phospholipid‐cholate mixture. Following this procedure, vesicles with diameters between 200 and 1,500 Å were seen by electron microscopy. As in mitochondria, ATPase activity in the reconstituted system was stimulated by a range of uncouplers which inhibited ATP‐32Pi exchange. These and other findings suggest that the coupling mechanism may still be intact within the ATPase complex.
The effects of ADP on reverse electron flow and the oxygen exchange reactions catalyzed by bovine heart muscle submitochondrial particles
Authors: Mitchell Robert A. , Russo John A. , Lamos Candace M.
Published: May 2005

1,N6‐Ethenoadenosine diphosphate (ϵ‐ADP) inhibits reverse electron flow (succinate → NAD+ driven by ATP) by competing with ATP, in contrast to ADP which we have shown previously to be a noncompetitive inhibitor. From these and other data it is concluded that the noncompetitive inhibition noted with ADP results from a combination of competitive inhibition plus non‐ or uncompetitive inhibition, the former occuring at a relatively nonspecific catalytic site and the latter at an extracatalytic site apparently quite specific for ADP.

ADP, which stimulates ATP ⇌ H2O and Pi ⇌ H2O exchanges appears to be necessary for inhibition by arsenate of these exchanges. It is suggested that the ATP‐supported Pi ⇌ H2O exchange may be predominantly of the medium or intermediate type, depending on the concentrations of the Mg2+ complexes of ADP and Pi. Thus only exchanges involving medium ADP and Pi would be expected to show arsenate sensitivity.
Intermediate complex of ATP hydrolysis and synthesis by muscle proteins
Authors: Hotta K.
Published: May 2005

Myosin catalyzed exchange between 32Pi and ATP in reaction medium during its enzymatic hydrolysis of ATP only by a very small amount. Addition of actin increased to a great extent the rate of incorporation of 32Pi in the presence of Mg. Glycerinated smooth muscle fibers also exhibited the ability to exchange 32Pi and ATP upon the application of external force (repeated stretching and releasing). A schematic mechanism of the action of actin and external force on acceleration of 32Pi incorporation is proposed and the importance of the M*‐ADP complex for force generation is suggested.
Myosin structure. Proximity measurements by fluorescence energy transfer
Authors: Haugland Richard P.
Published: May 2005

The results of energy transfer experiments on the proximity of six sites on the globular head region of myosin are discussed. A large hydrophobic crevice has been detected on each myosin head which is sufficiently large to accommodate six aromatic rings simultaneously. In the crevice is located a thiol residue not involved in activation of myosin Ca2+ ATPase and a lysine residue which is specifically trinitrophenylated with 2, 4, 6‐trinitrobenzenesulfonic acid. A second sulfhydryl whose modification activates the Ca2+ ATPase is located near the hydrophobic thiol site. The tryptophan whose fluorescence is enhanced by ATP binding is sufficiently close to the thiols and lysine residue to quantitatively transfer its energy to probes at these sites. The site of myosin ATPase has been tentatively located as being near the other five sites by energy transfer to or from synthetic chromophoric substrates. Implications of these results on the possibility of determining the location of the myosin light chain and actin binding sites are discussed.
On the enthalpy of binding of ADP to heavy meromyosin
Authors: Goodno Charles C. , Swenson Charles A.
Published: May 2005

The binding of ADP to heavy meromyosin has been studied by microcalorimetry. Minute amounts of myokinase interfere with binding measurements, but by selection of appropriate conditions, we can estimate that the value of the apparent ΔHbinding lies between −1.0 and −3.0 kcal per mole of ADP bound (0.3 M KCl, 2 mM MgCl2, 20 mM Tris, pH 8.00, 20°C). Values of ΔHbinding reported to date are an order of magnitude larger, and we suggest that these values are artifactual results due to myokinase contamination.
Calcium regulation of cardiac myofibrillar activation: Effects of MgATP
Authors: Solaro R. John
Published: May 2005

In 2 mM MgATP, 0.08 ionic strength and 1 mM free Mg++ cardiac myofibrils bound 3.5 nmoles Ca/mg protein at maximal ATPase activation. Significant amounts of Ca were also bound to cardiac myosin with these same conditions. By subtraction of this myosin‐bound Ca we obtained an estimate of 4 moles Ca bound per mole of myofibrillar troponin at maximal ATPase. We found, however, that Ca activation of myofibrillar ATPase could be estimated assuming that only two of troponin's Ca‐binding sites are engaged in regulation of crossbridge activity. Increase in MgATP from 0.3 to 5.0 mM raised the free Ca, giving half‐maximal isometric tension or ATPase. Although part of this shift is most probably due to changes in the number of rigor (nucleotidefree) actin‐myosin linkages, the rightward shift of the free Ca++‐activation relation with increase in MgATP from 2 to 5 mM appears to be due to effects of active (nucleotide‐containing) actin‐myosin linkages.
The use of aryl azido ATP analogs as photoaffinity labels for myosin ATPase
Authors: Jeng Stella J. , Guillory Richard J.
Published: May 2005

The synthesis of ATP analogs containing a photoactive aryl azido grouping coupled to the 3′ hydroxyl of ATP is described. The potential effectiveness of these analogs in the investigation of nucleotide‐binding regions is outlined and this effectiveness demonstrated by their photodependent inhibition of subfragment 1 ATPase. The use of 14C‐labeled azido ATP demonstrates an almost stoichiometric covalent binding of the analog.

Because of their potential application to other systems, a number of reactions describing the reactivity of the 3′ hydroxyl of the nucleotide ribose are outlined in an Appendix.
Synthesis of adenosine triphosphate by way of potassium‐sensitive phosphoenzyme of sodium, potassium adenosine triphosphatase
Authors: Post Robert L. , Toda Gotaro , Kume Shoji , Taniguchi Kazuya
Published: May 2005

The sodium and potassium ion pump is an intrinsic enzyme of plasma membranes. In these experiments it was driven backward in a transient two‐step operation involving, first, phosphorylation of the enzyme from inorganic phosphate, and second, transfer of the phosphate group from the enzyme to ADP upon addition of a high concentration of Na+. There was no evidence of a significant concentration gradient across the membranes. Na+ presumably reached the solutions on both faces of the membrane simultaneously and provided the energy for synthesis simply as a consequence of ligand binding. An interaction free energy between the free energy of the binding of Na+ presumably reached the solutions on both faces of the membrane simultaneously and provided the energy for synthesis simply as a consequence of ligand binding. An interaction free energy between the free energy of the binding of Na+ and the free energy of hydrolysis of the phosphate group on the enzyme was estimated. The experiments also suggested a feature of the transport mechanism. This is control by phosphorylation of access pathways from the solutions in contact with the faces of the membrane to an active center for cation binding. In the dephosphoenzyme access would be to the intracellular solution and in the phosphoenzyme access would be to the extracellular solution.
Nonrandom distribution of receptors for melanocyte‐stimulating hormone on the surface of mouse melanoma cells
Authors: Varga Janos M. , Saper Mark A. , Lerner Aaron B. , Fritsch Peter
Published: May 2005

An improved bubble method was developed for applying an ultrathin layer of nuclear track emulsion on the surface of cells labeled with I125‐MSH. The autoradiographs of I125‐MSH binding indicate a nonrandom distribution of receptors on the surface of mouse melanoma cells. It is suggested that MSH receptors are displayed in clusters previous to an independently of their exposure to the hormone.
Concanavalin a perturbation of membrane enzymes of mammary gland
Authors: Carraway Coralie A. , Carraway Kermit L.
Published: May 2005

The plant lectin concanavalin A (Con A) specifically inactivates the 5′ ‐nucleotidase of a plasma membrane‐enriched fraction from lactating mammary gland. The lectin also causes an activation of the membrane Mg++ ‐ATPase, but does not affect galactosyltransferase or alkaline phosphatase. The enzyme perturbations are prevented by α‐methylmannoside, an inhibitor of Con A binding, indicating that specific binding to carbohydrate structures rather than nonspecific protein‐protein interaction is involved. Solubilization of the 5′ ‐nucleotidase in detergents (0.2% Triton X‐100 or 1% deoxycholate) does not prevent Con A inactivation, indicating that incorporation into the membrane structure is not a requirement for the Con A effect. The results suggest that Con A inactivates the 5′ ‐nucleotidase by a direct interaction with the enzyme and that this enzyme is a Con A receptor site on the surface of mammary cells.
Selective modification of cell surface proteins and thymidine transport in hamster cells exposed to cholera toxin
Authors: Rieber M. , Bacalao J.
Published: May 2005

The increased adherence and morphological response which occurs in Chinese hamster ovary cells as a result of exposure to cholera toxin is paralleled by modification in the relative exposure of outer proteins. Mild proteolysis treatment of the cells prelabeled with [3H] glucosamine reveals a markedly different kinetics of release of external glycopeptides as a result of exposure to cholera toxin. Selective alterations in external tyrosyl‐rich proteins can also be detected by lactoperoxidase‐catalyzed radioiodination. The above modifications are accompanied by a decrease in the rate of thymidine uptake by toxintreated cells.
Proteins of the hepatoma tissue culture cell plasma membrane
Authors: Tweto John , Friedman Else , Doyle Darrell
Published: May 2005

The specificity of lactoperoxidase‐catalyzed iodination for the proteins of the hepatoma tissue culture cell plasma membrane was examined by histochemical, biochemical, and cell fractionation techniques. Light microscope autoradiography of sectioned cells shows the incorporated label to be localized primarily at the periphery of the cell. Most of this label can be released from the cell by trypsin but not by collagenase or hyaluronidase. The label is recovered from the cells as either monoiodotyrosine or diiodotyrosine after hydrolysis of cell extracts with a mixture of proteolytic enzymes. The label co‐purifies during cell fractionation with an authentic liver cell plasma membrane marker enzyme, 5′‐nucleotidase. Thus, the incorporated iodide is itself a valid marker for those membrane polypeptides having tyrosine residues accessible to the lactoperoxidase. The polypeptide complexity of the purified plasma membrane was examined by high resolution dodecyl sulfate‐polyacrylamide gel electrophoresis. At least 50 polypeptides in the membrane are accessible to iodination. These polypeptides probably represent the bulk of the protein mass of the membrane and iodinating them does not affect cell viability, growth rate, or cell function. Labeling experiments with fucose and glucosamine show that at least nine of the iodinated peptides may be glycoproteins.
Heterogeneity in the conformation of different protein fractions from the human erythrocyte membrane
Authors: Holzwarth G. , Yu John , Steck Theodore L.
Published: May 2005

We have isolated 5 families of proteins from human red blood cell membranes and characterized their secondary structure by ultraviolet circular dichroism measurements. The protein families were prepared by selective solubilization from ghosts under nondenaturing conditions. We find that the intact ghost has a mean α‐helix fraction of 0.37, whereas a low‐ionic‐strength extract (bands 1, 2, 5, “spectrin”) has a substantially higher helix fraction, 0.55. Further extraction of the ghosts with para‐chloromercuribenzoate yields bands 2.1, 4.1, 4.2, and 6; their helix content is only 0.17. Finally, the major intrinsic protein, band 3, was solubilized by a nonionic detergent. Its helix fraction is 0.38.
DNA synthesis in cultured human fibroblasts: Regulation by 3′ : 5′‐cyclic amp
Authors: Rechler Matthew M. , Bruni Carmelo B. , Podskalny Judith M. , Carchman Richard A.
Published: May 2005

The addition of serum to density‐inhibited human fibroblast cultures induced a wave of DNA synthesis, measured as [3H] thymidine incorporation into acid‐precipitable material, beginning after 8–12 hr and reaching maximum levels at 16–24 hr. Addition of dibutyryl‐3′ : 5′‐cyclic AMP (DBcAMP) together with serum inhibited [3H] thymidine incorporation by 75–95%. When DBcAMP was added for the first 4 hr of serum stimulation and then removed, the wave of DNA synthesis was not delayed. This suggested that serum could induce DNA synthesis even though cyclic AMP concentrations were maintained at high levels by DBcAMP during this initial period. These results are inconsistent with the hypothesis that it is the immediate transient reduction in 3′ : 5′‐cyclic AMP concentration following the addition of serum that triggers DNA synthesis. By contrast, DBcAMP added 8 hr after serum inhibited [3H] thymidine incorporation to the same extent as DBcAMP added at the same time as serum. This indicated that a step essential for DNA synthesis and occurring late in G1 was inhibited by high concentrations of 3′ : 5′‐cyclic AMP.
Some kinetic and chromatographic properties of detergent‐dispersed adenylate cyclase
Authors: Johnson Roger A. , Garbers David L. , Pilkis Simon J.
Published: May 2005

Studies on the reaction kinetics and chromatographic properties of detergent‐dispersed adenylate cyclase are described. Detergent‐dispersed enzyme was prepared from whole rat cerebellum and from partially purified plasma membranes from rat liver.

Data were simulated to fit kinetic models for which an inhibitor is added in constant proportion to the variable substrate. Models were chosen to distinguish whether the adenylate cyclase reaction may be controlled by an inhibitory action of free ATP−4 (or HATP−3) or by a stimulatory action of free divalent cations. The various kinetic models were then tested with the dispersed brain adenylate cyclase with both Mg++ and Mn++ and in two different buffer systems. The experimental data indicate that this enzyme has a distinct cation binding site, but exhibits no significant inhibition by HATP−3 or ATP−4.

The detergent‐dispersed adenylate cyclase both from liver plasma membranes and from brain have been chromatographed on anion exchange material and have been chromatographed on anion exchange material and have been subjected to gel filtration. The presence of detergent was required for elution of cyclase activity from DEAE‐Sephadex but was not required when DEAE‐agarose was used. Dispersed brain cyclase was also chromatographed on agarose‐NH(CH2)3 NH(CH2)3‐NH2 which exhibits both ionic and hydrophobic properties. Fifty percent of the applied activity was recovered with a fivefold increase in specific activity. The data suggest that the relative effectiveness of a given chromatographic procedure for detergent‐dispersed adenylate cyclase may reflect the in fluence of both hydrophobic and ionic factors.
Studies of bacterial chemotaxis in defined concentration gradients. A model for chemotaxis toward L‐serine
Authors: Dahlquist F. W. , Elwell R. A. , Lovely Peter S.
Published: May 2005

The details of the chemotactic response of Salmonella typhimurium to gradients of L‐serine have been examined in some detail. Two relatively macroscopic techniques have been employed to measure the bacterial response. These include measurements of the average velocity as the bacterial population moves toward attractants, and measurement of the upward‐to‐downward flux ratio, R, in the stable preformed attractant gradients. The dependence of the average velocity on gradient appears to be hyperbolic in nature, while the flux ratio depends linearly on the gradient. These data suggest a microscopic model for the dependence of bacterial behavior on the serine gradient. The model involves a linear dependence of the mean lifetime of a bacterial trajectory on the gradient for those bacteria moving toward higher attractant concentration. Those moving toward low concentrations of attractant do not change the mean duration of their trajectories, or the speed at which a given bacterium swims through the solution. This model generates the observed dependences of the average velocity and flux ratio on gradient. Interpretation of the experimental data suggests that a gradient which increases serine concentration by a factor of 2 in 10 mm is sufficient to double the average duration of a trajectory for a bacterium moving directly up the gradient. The concentration dependence of the chemotactic response to serine is more complicated. It suggests that more than one receptor of serine may be involved in determining chemotactic behavior to this attractant.
Immunological studies of acetylcholine receptors
Authors: Lindstrom Jon
Published: May 2005

Immunochemical techniques for the study of acetylcholine receptors are described. Immunization of rabbits, rats, guinea pigs, and goats with acetylcholine receptor protein purified from Electrophorus electric organ tissue results in muscular weakness and death due to impaired neuromuscular transmission. Serum from immunized animals contains high concentrations of antibodies directed at receptors from the electric organ and low concentrations of antibodies directed at receptors from skeletal muscle. The detailed similarities between the disease of receptor‐immunized animals, “experimental autoimmune myasthenia gravis” (EAMG), and myasthenia gravis are compared. Reactions of antisera from animal with EAMG with receptor from Electrophorus and Torpedo are studied. Antireceptor antibodies in these antisera are directed predominantly at determinants other than the acetylcholine‐binding site.
Reconstitution of a purified acetylcholine receptor
Authors: Michaelson D. M. , Duguid J. R. , Miller D. L. , Raftery M. A.
Published: May 2005

Dialysis of the purified acetylcholine receptor from Torpedo californica electroplax with lipids from the same organ results in a vesicular membrane system in which the receptor is embedded in the bilayer and oriented so that most of the neurotoxin‐binding sites appear to be on the outer surface. The constituted vesicles are chemically excitable by acetylcholine and carbamylcholine, as measured by 22Na+ efflux. The excitability is specifically blocked by the antagonist α‐bungarotoxin. These results demonstrate that the purified reconstituted receptor system not only can specifically bind neurotransmitter but also can trigger ion translocation. It therefore has the properties necessary to effect postsynaptic depolarization in vivo.
The role of cell membrane in the antiviral effect of interferon
Authors: Pitha Paula M. , Vengris Vitolis E. , Reynolds Fred H.
Published: May 2005

The mechanism of interferon action in human fibroblasts has been studied by use of both antisera to human fibroblast interferon and the antisera to the surface of human fibroblast cell. The anti‐interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Interferon antiserum prevented the intracellular antiviral state from developing when added to the medium of the cells in which interferon synthesis had already been induced by poly (I · C). This suggests that development of the antiviral state involves interferon interaction with the external part of the producing cell. Treatment with the serum directed against the surface of human fibroblast cells failed to inhibit the antiviral activity of human interferon in these cells.

In addition, the effect of gangliosides on the antiviral activity of human interferon was studied and it was found that human interferon binds to gangliosides and that this interaction leads to inactivation of the antiviral effect of interferon. Pretreatment of human fibroblasts with gangliosides had no effect on the sensitivity of these cells to exogenous interferon.
Effect of polyamines on the uptake of poly (2′‐fluoro‐2′‐deoxyuridylic acid) by a mammalian cell line
Authors: Janik Borek , Sommer Ronald G.
Published: May 2005

VERO cells can take up poly(dUfl)1 from the medium. The uptake involves surface adsorption and, most probably, intracellular penetration. Part of the poly(dUfl) is hydrolyzed during incubation with the cells but the hydrolysis products are not incorporated into de novo synthesized nucleic acids. The uptake is reduced by serum and stimulated by polycationic ionenes. The magnitude of stimulation depends on the structure of the ionene and the treatment regimen.
Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat
Authors: Louis S. L. , Brivio‐Haugland R. P. , Williams M. A.
Published: May 2005

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid‐supplemented diet (+EFA) or from rats fed an essential fatty acid‐deficient diet (‐EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the −EFA preparations were one‐half or less than the values for the +EFA preparations. Basal, F−, or glucagon‐stimulated adenylate cyclase activities were significantly lower in EFA‐deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon‐stimulated adenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA‐deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in −EFA livers. When glucagon was used in vitro at 1–1,000 nM, stimulation of adenylate cyclase remained lower in EFA‐deficient membranes, but extent of stimulation above basal activity was larger in −EFA membranes than in +EFA. Total Na+, K+ (Mg2+)‐ATPase from EFA‐depleted LPM exhibited significantly higher values of apparent Km and Vmax. 5′‐Nucleotidase activity, in contrast, was considerably decreased in EFA‐deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane‐bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.
Drug‐resistant mutants of chinese hamster ovary cells possess an altered cell surface carbohydrate component
Authors: Juliano Rudy , Ling Victor , Graves James
Published: May 2005

Surface label experiments using the galactose oxidase‐[3 H] ‐borohydride technique reveal that cells from drug‐resistant Chinese hamster ovary clones possess a surface carbohydrate component of apparent molecular weight 165,000 which is absent from wild‐type cells. The component may also be demonstrated by [14C] glucosamine incorporation but not by [3 H] leucine incorporation or by the lactoperoxidase surface labeling reaction.
A scanning electron microscope study of concanavalin a receptors on retinal rod cells labeled with latex microspheres
Authors: Molday Robert S.
Published: May 2005

Con A‐methacrylate microsphere conjugates prepared by a two‐step glutaraldehyde reaction were used to label Con A‐binding sites on bovine rod photoreceptor cells for visualization by scanning electron microscopy. A dense distribution of markers was observed on the surface of the rod outer segment, the inner segment, and the synaptic region. Disk membranes also appear to be heavily labeled with the Con A‐microsphere conjugates. The Con A inhibitor, α‐methyl mannoside, inhibited the binding of the conjugate to the surface of these visual cells.
Some changes in the properties of dynein ATPase in situ and after extraction following heat treatment of cilia
Authors: Blum J. J. , Hayes Alveron
Published: May 2005

Glycerol‐extracted cilia from Tetrahymena pyriformis were demembranated by treatment with Triton X‐100 and then heated for up to 30 min at temperatures between 34–38°C. Heat treatment carsed an uncoupling of the ATPase from motility as indicated by an increase in ATPase activity and a loss of pellet height response. After heat treatment, the ATPase activity of the dynein in situ differed from that in unheated cilia as shown by an increased sensitivity to a lower temperature of assay (0°C) and by a loss of the activation normally observed upon reaction with N‐ethylmaleimide or p‐phenylenedimaleimide. Upon extraction of the heat‐treated cilia by Tris‐EDTA, there was a large loss in ATPase activity so that the heat‐treated cilia yielded a crude dynein fraction with a lower specific activity compared with that obtained from unheated controls. The difference was not due to a change in the amount of protein recovered or in the amount of ATPase activity which remained unextracted. Resolution of the crude dynein by sucrose density sedimentation indicated that activity was lost from both the 14S and 30S peaks but more so from the latter than from the former. Thus dynein in situ in cilia in which the ATPase has been uncoupled from motility by gentle heat treatment differs in several important respects from dynein inside unheated cilia.
Isolated cortical granules: A model system for studying membrane fusion and calcium‐mediated exocytosis
Authors: Vacquier Victor D.
Published: May 2005

Cortical granules are secretory vesicles bound to the inner surface of the plasma membrane of sea urchin eggs. Intact granules can be isolated by shearing away the cytoplasm of eggs which have been bonded to a protamine‐coated surface. When Ca2+ is added to preparations of isolated granules the granules fuse with each other and release their contents. It is believed that isolated cortical granules may be an excellent model system for the biochemical study of exocytosis.
Control of membrane morphogenesis in bacteriophage PM2
Authors: Brewer Gregory J.
Published: May 2005

The regulation of membrane formation in bacteriophage PM2 serves as a simple model for changes in membrane structure in eukaryotic cells. Prior to Pseudomonas host lysis, wild‐type virions mature to an icosahedral morphology at the inner face of the cytoplasmic membrane. The proliminary charcterization of two temperature‐sensitive mutants of PM2 is described.

In cells infected at the restrictive temperature with ts 1, an abundance of “empty” virus‐size membrane vesicles are seen. Synthesis of DNA is also reduced in ts 1 infected cells. The preponderance of vesicles is not sen in cells infected with wil‐type virus or with ts 1 at the permissive temperature. The “empty” appearance of the viral membranes suggests that viral DNA is not encapsulated.

The major viral capsid protein (MW 26,000) is located just out side the viral membrane and normallyl sediments with host and virus membranes; insted, large amounts of capsid protein can be precipitated from the supernatant with TCA. Compared to cells infected with wild type virus, cells infected with is 5 at th restrictive temperature produce inside the cell an aboundance of virus‐soze membrane vesicles.

Taken Together, These results with viral mutants suggest that formation of a viral membrane of the proper size does not require a DNA core around which to form, or an outer scaffolding of coat protein against which to form a spherical bilayer.
Immunospecific labeling of mouse lymphocytes in the scanning electron microscope
Authors: Carter David P. , Wofsy Leon
Published: May 2005

Bone marrow‐derived (B) and thymus‐derived (T) Balb/c mouse lymphocytes were identified in the scanning electron microscope (SEM) by the immunospecific attachment of one of several kinds of large‐molecular‐weight markers distinguishable in SEM. These markers (tobacco mosaic virus, keyhole limpet hemocyanin, bushy stunt virus, and bacteriophage T4) could be modified with hapten groups and linked with anti‐hapten antibody, in an indirect (sandwich) scheme, to hapten‐modified anti‐cell‐surface antibody bound to the cell surface.

Hapten‐modified antibodies to B cell antigens (goat anti‐mouse‐immunoglobulin) or to T cell antigens (rabbit anti‐mouse brain) were employed to identify these two lymphoid cell types in unfractionated spleen, mesenteric lymph node, bone marrow, and thymus cell populations. The topography of B cells was always indistinguishable from that of T cells. No surface features were found to be unique to either cell type. In suspension, the majority of B and T cells had one or no microvilli regardless of the tissue source of the labeled cells. Cells in suspension that had microvilli (usually 10% of the total cell population) were always unlabeled. However, after cell contact with a glass surface, approximately half of both the B and T cell populations had a villous topography.
Cyclic nucleotides, thioldisulfide status of proteins, and cellular control processes
Authors: Rebhun Lionel I. , Miller Marcia , Schnaitman Terry C. , Nath Jayasree , Mellon Margaret
Published: May 2005

It is shown that cyclic nucleotides can have a variety of effects on cell division, cell shape, cell adhesion, and cell movement, depending on the cells selected and the conditions under which they are used. For example, while CHO cells elongate under the influence of exogenous dibutyryl CAMP, Y‐1 adrenal tumor cells round up and polyoma‐transformed 3T3 cells show no change in shape. The totality of experience with cyclic nucleotides suggests that where they have been used by cells as control elements involving the four processes listed above, they are superimposed on basic cellular processes that progress in their absence – that is, they must be acting indirectly. In attempting to understand the inhibitory action of methyl xanthines on egg development, we were forced to abandon the idea that they acted through cyclic nucleotides. We found that methyl xanthines inhibited the activation of glutathione reductase and that glutathione oxidizing agents act as mitotic inhibitors. Further, we found that tubulin polymerizability, NAD‐kinase activity, and a mitotic apparatus associated Ca+2‐ATP‐ase were all inhibited by oxidation of some of their sulfhydryls and were activated by reduction of the resulting disulfides. These results are discussed in terms of reported cycles and activations of glutathione reductase (GR) in cells and reports that mixed disulfides of glutathione and proteins can act as substrates for GR. Using the fact that a CAMP‐dependent protein kinase has been reported to be activated by glutathione, we have suggested potential sites where sulfhydryl control processes and cyclic nucleotide control processes may interact in certain restricted cases.
Cell shape and hexose transport in normal and virus‐transformed cells in culture
Authors: Bissell Mina J. , Farson Deborah , Tung Agatha S. C.
Published: May 2005

The rate of hexose transport was compared in normal and virus‐transformed cells on a monolayer and in suspension. It was shown that: (1) Both trypsin‐removed cells and those suspended for an additional day in methyl cellulose had decreased rates of transport and lower available water space when compared with cells on a monolayer. Thus, cell shape affects the overall rate of hexose transport, especially at higher sugar concentrations. (2) Even in suspension, the initial transport rates remained higher in transformed cells with reference to normal cells. Scanning electron micrographs of normal and transformed chick cells revealed morphological differences only in the flat state. This indicates that the increased rate of hexose transport after transformation is not due to a difference in the shape of these cells on a monolayer.