bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparison of the effects of gentle heating, acetone, and the sulfhydryl reagent bis (4‐fluoro‐3‐nitrophenyl) sulfone on the ATPase activity and pellet height response of tetrahymena cilia
Authors: Blum J. J. , Hayes A.
Published: May 2005

Incubation of glycerol‐extracted, Triton X‐100 demembranated Tetrahymena cilia with 2–10 vol % acetone caused an enhancement of ATPase activity by 2‐ to 3‐ fold, depending on concentration and time of incubation. Axonemal ATPase activity was also increased upon incubation with bis (4‐fluoro‐3‐nitrophenyl) sulfone (FNS). Acetone and FNS enhanced the activity of solubilized 30S dynein, but slightly inhibited that of 14S dynein. Heating at 38°C, incubation with FNS, and incubation with acetone activated axonemal ATPase to the same extent. Subsequent studies of (1) the effect of time of preincubation with a spin‐labeled maleimide (SLM) at 25°C as a function of pH on the ATPase activity, (2) the concentration dependence of the inhibition of ATPase activity by N‐ethylmaleimide or SLM, (3) the ratio of ATPase activity assayed at 25°C to that assayed at 0°C, and (4) the ratio of ATPase activity at pH 8.6 to that at pH 6.9 did not reveal any difference in the properties of the axonemal ATPase after near maximal enhancement by the heat, acetone, or FNS treatments. It was concluded that enhancement of ATPase activity by gentle heat treatment, by incubation with acetone (or other organic solvents), or by FNS results from a conformation change of 30S dynein.

The effect of acetone and of FNS on the pellet height response (a measure of the increase in height of the pellet of cilia precipitated by brief centrifugation in the presence of ATP as compared to the absence of ATP) was also determined. Enhancement of ATPase by these reagents did not lead to a decrease in pellet height response. This observation, in conjunction with other data, indicates that there are at least 3 states of the cross‐bridge cycle of dynein arms in cilia.
Conformation and intermolecular interactions of carbohydrate chains
Authors: Morris Edwin R. , Rees David A. , Thom David , Welsh E. Jane
Published: May 2005

For consideration of their conformations and interactions, carbohydrate chains can conveniently be divided into 3 classes on the basis of their covalent structure; namely periodic (a), interrupted periodic (b), and aperiodic (c) types. In aqueous solution carbohydrate chains often exist as highly disordered random coils. Under appropriate conditions, however, polysaccharides of types (a) and (b) can adopt a variety of ordered conformations. Physical methods, and in particular optical rotation, circular dichroism, and nuclear magnetic resonance, provide sensitive probes for the study of the mechanism and specificity of these disorder‐order transitions in aqueous solution.

Intermolecular interactions between such polysaccharide chains arise from co‐operative associations of long structurally regular regions which adopt the ordered conformations. For acidic polysaccharides these cooperative associations may involve alignment of extended ribbons with cations sandwhiched between them. In other systems the interactions involve double belices which may then aggregate further, and geometric “matching” of different polysaccharide chains can also occur. These ordered, associated regions are generally terminated by deviations from structural regularity or by “kinks” which prevent complete aggregation of the molecules.

The complex carbohydrate chains which occur at the periphery of animal cells have very different, aperiodic structures and although their conformations are as yet poorly understood, preliminary indications are considered.
The sialoglycoprotein subunits of human placental brush border membranes characterized by two‐dimensional electrophoresis
Authors: Wada H. Garrett , Góarnicki Stella Z. , Sussman Howard H.
Published: May 2005

A brush border membrane enriched fraction was isolated from human, full‐term placenta. This membrane fraction exhibited large membrane fragments with microvilli projecting from the basal membrane in electron micrographs and was enriched tenfold in alkaline phosphatase, a brush border enzyme marker. The sialoglycoproteins associated with this membrane fraction were tritiated by mild periodate oxidation of sialic acid and reduction with tritiated NaBH4. The membranes were solubilized in 8 M urea, 2% Triton X‐100, and the tritiated glycoprotein subunits were reduced with β‐mercaptoethanol and characterized by 2‐dimensional polyacrylamide gel electrophoresis using a method similar to that described by O'Farrell and Bhakdi, Knüferman, and Wallach. The tritiated subunits were detected in the gels by autofluorography. The 2‐dimensional subunit “maps” resolved at least 17 major sialoglycoprotein subunits whereas only 10 major periodate‐Schiff reagent staining components were resolved by 1‐dimensional SDS polyacrylamide gel electrophoresis. Placental alkaline phosphatase (PAP) was identified on the subunit maps by inclusion of 32P‐labeled PAP in the tritiated membrane sample. The 32P‐labeled PAP corresponded to a major tritiated sialoglycoprotein subunit, which was heterogeneous with respect to charge as demonstrated by 3 closely running spots of the same molecular weight.
Hexose transport regulation in cultured hamster cells
Authors: Christopher C. William
Published: May 2005

Hamster (nil) cells maintained overnight in culture medium containing cycloheximide and either glucose or fructose exhibit strikingly different rates of hexose transport and metabolism (i.e., uptake). Pretreatment of cultures with sulfhydryl reagents makes it possible to determine initial transport rates for a physiological sugar such as galactose which is a catabolite in hamster cells. Using galactose transport as a model, hexose uptake enhancements can now be shown to be due almost entirely to increase in the rate of the transport step. The transport regulation can best be accounted for by a model comprised of 2 antagonizing mechanisms. This model involves turnover of transport carriers as well as inhibitory units (“regulators”). The experimental as well as the theoretical model may also apply to the well‐known uptake enhancements observed in oncogenically transformed cells.
High‐affinity phlorizin binding to brush border membranes from small intestine: Identity with (a part of) the glucose transport system, dependence on the Na+‐gradient, partial purification
Authors: Tannenbaum Carl , Toggenburger Gerhard , Kessler Markus , Rothstein Aser , Semenza Giorgio
Published: May 2005

In the presence of an NaSCN gradient phlorizin binds with a high affinity (Kd ⋍ 4.7 μM) to vesicles derived from brush border membranes of intestinal cells of rabbits. The value for Kd corresponds closely to that of Ki determined from phlorizin inhibition of sugar transport. The apparent affinity for phlorizin is decreased if NaCl is substituted for NaSCN and decreased substantially if the gradient of NaSCN is allowed to dissipate prior to the phlorizin binding. The number of high affinity binding sites is about 11 pmol/mg protein. Additional binding to low affinity sites can amount to as much as 600 pmol/mg protein after prolonged exposure to phlorizin (5 min). The high affinity sites are related to glucose transport based on the similarity of the Kd and Ki values under a variety of conditions and on the inhibition of the binding by D‐glucose but not by D‐fructose. The transport system and the high affinity phlorizin binding sites can be enriched by a factor of 2–3 by treatment of vesicles with papain, which does not affect the transport system, but considerably hydrolyzes nonrelevant protein.
The amino‐ and carboxyl‐terminal sequence of bovine rhodopsin
Authors: Hargrave Paul A. , Fong Shao‐Ling
Published: May 2005

The amino terminus of bovine rhodopsin is blocked and has the sequence x‐Met‐Asn(CHO)‐Gly‐Thr‐Glu‐Gly‐Pro‐Asn‐Phe‐Tyr‐Val‐Pro‐Phe‐Ser‐Asn(CHO)‐Lys‐Thr‐Gly‐Val‐Val‐Arg, where CHO represents sites of carbohydrate attachment. The carboxyl‐terminal sequence of rhodopsin is Val‐Ser‐Lys‐Thr‐Glu‐Thr‐Ser‐Gln‐Val‐Ala‐Pro‐Ala. Upon short‐term digestion of rod outer segment (ROS) membranes with thermolysin, opsin (∼ 35,000 daltons) is converted to a membrane‐bound fragment O′ (∼ 30,500 daltons) and 2 peptides containing 12 amino acids are released from the carboxyl terminus of rhodopsin into the supernatant. Upon long‐term digestion of ROS with thermolysin, opsin and O′ are replaced by the membrane‐bound fragments F1 (∼25,000 daltons), and F2 (∼9,500 daltons). When 32P‐ROS are digested, F2 carries the 32P. Both O′ and F1 contain the amino‐terminal glycopeptide.
Kinetics of Na+‐dependent D‐glucose transport
Authors: Hopfer Ulrich
Published: May 2005

The kinetic parameters of the Na+‐dependent glucose transport system have been determined in isolated membrane vesicles for D‐glucose, Na+, and phlorhizin. The D‐glucose flux measurements were carried out by the equilibrium exchange procedure at constant external and internal Na+ concentrations and zero potential. Equations were developed to extract information about Km and Vmax from uptake measurements into a vesicle population that is heterogeneous with respect to size (surface to volume ratio). The Km for D‐glucose was 14 mM and independent of the Na+‐concentration, while the Vmax was strongly Na+‐dependent and increased 15‐fold between 1 and 100 mM Na+. The Km of Na+ for activation of the Vmax was 18 mM. The calculated KI values for phlorhizin were 2.7 and 1.9 μM when determined under active and equilibrating D‐glucose flux conditions, respectively.
Orientation of the protonmotive force in membrane vesicles of escherichia coli
Authors: Adler Lawrence W. , Ichikawa Tomio , Hasan Syed M. , Tsuchiya Tomofusa , Rosen Barry P.
Published: May 2005

Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D‐lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside.

The latter type of vesicle, on the other hand, exhibits D‐lactate‐dependent proline transport but does not accumulate calcium with D‐lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D‐lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy‐linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).
Involvement of membrane sulfhydryls in the activation and maintenance of nutrient transport in chick embryo fibroblasts
Authors: Smith‐Johannsen H. , Perdue J. F. , Ramjeesingh M. , Kahlenberg A.
Published: May 2005

At 5 μg/ml, insulin stimulates hexose, A‐system amino acid, and nucleoside transport by serum‐starved chick embryo fibroblasts (CEF). This stimulation, although variable, is comparable to that induced by 4% serum. The sulfhydryl oxidants diamide (1–20 μM). hydrogen peroxide (500 μM), and methylene blue (50 μM) mimic the effect of insulin in CEF.

PCMB‐S,1 a sulfhydryl‐reacting compound which penetrates the membrane slowly, has a complex effect on nutrient transport in serum‐ and glucose‐starved CEF. Hexose uptake is inhibited by 0.1–1 mM PCMB‐S in a time‐ and concentration‐dependent manner, whereas A‐system amino acid transport is inhibited maximally within 10 min of incubation and approaches control rates after 60 min. A differential sensitivity of CEF transport systems is also seen in cells exposed to membrane‐impermeant glutathione‐maleimide I, designated GS‐Mal. At 2 mM GS‐Mal reduces the rate of hexose uptake 80–100% in serum‐ and glucose‐starved CEF; in contrast A‐system amino acid uptake is unaffected. D‐glucose, but not L‐glucose or cytochalasin B, protects against GS‐Mal inhibition. These results are consistent with the hypothesis that sulfhydryl groups are involved in nutrient transport and that those sulfhydryls associated with the hexose transport system and essential for its function are located near the exofacial surface of the membrane in CEF.
The inhibitory effect of the artificial electron donor system, phenazine methosulfate‐ascorbate, on bacterial transport mechanisms
Authors: Eagon R. G. , Gitter B. D. , Rowe J. J.
Published: May 2005

The artificial electron donor system, phenazine methosulfate (PMS)‐ascorbate, inhibited active transort of solutes in Pseudomonas aeruginosa irrespective of whether the active transport systems were shock sensitive or shock resistant. N,N,N′,N′‐tetramethylphenylenediamine could be substituted for PMS but a higher concentration was required. PMS‐ascorbate also inhibited active transport in several other bacterial species with the exception of Escherichia coli and of a nonpigmented strain of Serratia marcescens. PMS‐ascorbate previously has been shown to energize active transport in isolated membrane vesicles, even those prepared from the same bacterial species in whose intact cells active transport was inhibited. The apparent Km of glucose active transport in untreated cells of P. aeruginosa was 40 μM while the Km of glucose transport in cells incubated with PMS‐ascorbate was 25 mM, and PMS‐ascorbate had no effect on efflux of accumulated glucose. These results strongly suggested that facilitated diffusion resulted upon exposure of the cells to PMS‐ascorbate. Thus, PMS‐ascorbate appeared to have an uncoupler‐like effect on cells of P. aeruginosa. The experimental data also pointed out that there are fundamental differences between the response of intact cells and membrane vesicles to exogenous electron donors.
Localization of some glycolipid glycosylating enzymes in the golgi apparatus of rat kidney
Authors: Fleischer Becca
Published: May 2005

Cell fractions from rat kidney were isolated and studied for their ability to synthesize several possible intermediates in the biosynthesis of sulfatides and gangliosides. The enzymes studied include UDP‐Gal:ceramide galactosyltransferase, UDP‐Gal:glucosylceramide galactosyltransferase, UDP‐Gal:galactosylceramide galactosyltransferase, and CMP‐NAN:lactosylceramide sialyltransferase activities. The initial glycosylation of ceramide was found to be present in all of the kidney cell fractions studied. The remaining glycosylating enzymes were largely localized in the Golgi apparatus of kidney. Thus, in addition to modifying glycoproteins for secretion, the Golgi apparatus in kidney is involved in the modification of a number of glycolipids which are destined to form cell membrane components.
Structural analysis of a membrane glycoprotein: Glycophorin A
Authors: Furthmayr Heinz
Published: May 2005

Glycophorin A is the major sialoglycoprotein of the human erythrocyte membrane. Structural studies indicate that this molecule is made up of 3 domains composed of 2 hydrophilic segments which are separated by a region of 22 nonpolar amino acids. The N‐terminal half of the molecule contains all the carbohydrate associated with this protein.

Glycophorin A forms high‐molecular‐weight complexes which can be dissociated only under certain conditions. The site of subunit interaction is located within the hydrophobic segment, which serves both to mediate protein‐protein and protein‐lipid interactions within the bilayer membrane. Glycophorin A spans the membrane presumably as a demeric complex with the carboxyterminal ends extending into the cytoplasm of the red cell. The transmembrane nature of the polypeptide chains finds strong support from the use of specific antibody‐ferritin conjugates applied to thin sections of fixed and frozen intact cells.

Preliminary information on the analysis of human red cell variants which may lack some or all of the sialoglycopeptides are consistent with the presence in normal cells of a second sialoglycoprotein, provisionally labeled glycophorin B.
Prediction for secondary structures of ten proteins from the 50S subunit of the escherichia coli ribosome
Authors: Dzionara Michael , Robinson Stephen Michael Lewis , Wittmann‐Liebold Brigitte
Published: May 2005

Predictions of the secondary structures of the following 10 proteins from the large subunit of the E. coli ribosome were made using their known amino acid sequences: L6, L16, L19, L27, L28, L30, L31, L32, L33, and L34. The predictions were made according to 4 different methods and the results for each protein are presented as diagrams indicating the conformational states, helix, extended structure, turn, and random coil, of each residue. From these diagrams, regions of highly probable secondary structure for the proteins are calculated. Estimates are made of the maximum possible lengths of the proteins in order to correlate these with the results obtained from antibody binding sites in the 50S subunit as determined by electron microscopy.
Cell surface carbohydrates of preimplantation embryos as assessed by lectin binding
Authors: Brownell Anna G.
Published: May 2005

Preimplantation embryos were obtained from the uteri and oviducts of 2 strains of mice, Swiss CD‐1 and B6 CBA. After removal of the zona pellucida by treatment with pronase, FITC‐lectins were bound to the embryonic cell surfaces at either 4°C or 37°C. Both morula and blastocyst stage embryos bound the following lectins, FITC‐ConA, FITC‐WGA, FITC‐RCAII and FITC‐RCAI. No difference in binding was observed between the morula stage and the blastocyst stage within each mouse strain for each specific lectin. However B6 CBA embryos bound less FITC‐ConA and FITC‐WGA than the corresponding Swiss CD‐1 embryos. The topographical arrangement of the lectin receptors was observed to differ between 4°C and 37°C for FITC‐Con A, FITC‐RCAII, and FITC‐RCAI. While lectins bound at 4°C showed a pattern of continuous labeling, the same lectin at 37°C showed aggregation of lectin receptors into patches indicating lateral mobility of these receptors within the embryonic cell membranes. In contrast FITC‐WGA bound at 4°C and 37°C demonstrated continuous labeling of embryos at both temperatures. FITC‐fucose binding protein did not bind to Swiss CD‐1 embryos.

The invasiveness of trophoblastic cells of mouse blastocysts was studied by culturing isolated embryos without prior enzyme treatment on reconstituted collagen gels. After 4 days in BME containing only glutamine and bovine serum albumin as supplements, the embryos shed their zona pellucida and implanted into the collagen gel as indicated by zones of lysis in proximity to the embryonic cells when analyzed by scanning electron microscopy.
Interaction between cytoplasmic (Ca2+—Mg2+) ATPase activator and the erythrocyte membrane
Authors: Vincenzi Frank F. , Farrance Martha L.
Published: May 2005

Human red blood cells (RBC) contain a cytoplasmic, nonhemoglobin protein which activates the (Ca2+‐Mg2+) ATPase of isolated RBC membranes. Results presented in this paper confirm that activation of (Ca2+‐Mg2+)ATPase is associated with binding of the cytoplasmic activator to the membrane. Binding of the cytoplasmic activator is reversible and dependent on ionic strength and Ca2+. Cytoplasmic activator is sensitive to trypsin but is not degraded when intact RBC are exposed to trypsin. Cytoplasmic activator does not modify the (Ca2+‐Mg2+)‐ATPase of membranes from RBC exposed to activator prior to hemolysis. Thus, the activator is located in the cell and appears to act by binding to the inner membrane surface.
Organization and polysaccharides of sponge aggregation factor
Authors: Humphreys Susie , Humphreys Tom , Sano James
Published: May 2005

Aggregation factor, the macromolecular complex which mediates species‐specific aggreagation of dissociated sponge cells, was isolated from several species, partially characterized, and visualized by electron microscopy. All factors were large fibrous complexes with a backbone and side chains or arms. In some factors, the backbone is linear. In others it is circular and the complex appears as a sunburst with arms extending like rays from the circle. The size and location of the polysaccharide chains have been studied using purified preparations of Microciona prolifera. “Sunbursts” treated with ethylenediaminetraacetate (EDTA) for 4 weeks at 0°C dissociate into 3 protein‐ and polysaccharide‐containing components. Sodium dodecyl sulfate does not cause the sunburst to dissociate nor does it inhibit dissociation in the presence of EDTA suggesting that dissociation is not due to hydrolytic enzymes. The dissociation products were tractionated on a 977‐Å pore size micropore glass column. Fifteen percent of the material is excluded and appears in the electron microscope as the central circle of the sunburst. Digestion of the circles with 10−3 M dithiothreitol (DTT) and 0.5 mg/ml proteinase K for 72 h at 37°C produces 2 polysaccharide chanis of 65,000 and 6,000 daltons as fractionated and sized on a 233‐Å pore size micropore glass column using Pharmacia dextrans as standards. The included fractions of the EDTA‐treated material are subunits of the arms which contain 70% of the polysaccharide. A single polysaccharide of 6,000 daltons as measured on 233‐Å size glass beads and Sephadex G‐75 is released from these subunits by proteinase digestion. Pharmacia dextrans are used as standard on both columns. We calculate that there would be four 65,000‐dalton chains and one hundred 6,000‐dalton chains per circle and fifty 6,000‐dalton chains per arm. The third component of the EDTA‐treated preparation is partially included on the column. It appears as linear fibrils in the electron microscope and contains polydisperse polysaccharides of several‐hundred‐thousand daltons. It may be an impurity since there is apparently less than 1 of the large polysaccharide chains per sunburst.
The biosynthesis of mannolipids and mannose‐containing complex glycans by the retina
Authors: Kean Edward L.
Published: May 2005

Large‐scale incubations were carried out with homogenates of the retinas of the 15–16‐day‐old chick embryo in the presence of GDP[U‐14C] mannose, from which there were isolated a mannolipid (Lipid I), oligosaccharide‐lipids (Lipid II), and glycoprotein (residue). These incubations were performed in the presence of endogenous acceptors as well as dolichyl phosphate. [14C] Mannolipid I was subjected to chromatography on DEAE cellulose and silicic acid. The response to these, as well as TLC, enzymatic, and chemical treatments, were consistent with the product being dolichyl phosphomannose. [14C] Lipid II was purified by DEAE cellulose chromatography and gel filtration on LH‐20. Responses to these treatments, as well as TLC and paper chromatography, were consistent with this product being of the class of the oligosaccharide‐pyrophosphate‐lipids. The residue remaining after removal of the lipids was shown to contain glycoproteins by conversion of high‐molecular‐weight radioactive material to low‐molecular‐weight [14C] mannose‐containing glycopeptides by the action of pronase. These reactions and their products are consistent with there being in the retina, the pathway for glycoprotein synthesis involving the participation of the lipid‐activated carbohydrates.

When the incubations were performed in the presence of ATP or ADP there was a decrease in the labeling of Lipid I, accompanied by an increase in the labeling of Lipid II and glycoprotein. When incubated in the presence of dolichyl phosphate and deergent, however, the stimulatory effect of ATP did not occur. The effect on these activities of a variety of other nucleotide phosphates was also examined.
The turnover of a tissue specific cell surface ligand which inhibits lectin induced capping
Authors: McDonough James , Lilien Jack
Published: May 2005

Ten‐day‐old embryonic chick neural retina release into the environment glycoprotein ligands which bind to homologous cells, inhibiting the lectin‐induced redistribution of cell surface receptors. Material with identical activity is released from trypsin‐dissociated neural retina cells that are allowed to repair in culture for 2 h and are then transferred to fresh medium. Release of ligand is inhibited by cytosine arabinoside, hydroxyurea, UDP, and EDTA, and is potentiated by MnCl2. These data suggest that a glycosyltransferase reaction plays a critical role in the turnover of the cell surface ligand. Reactivation of enzymatically deglycosylated ligand solutions by intact cells provides further support for this hypothesis.

Release of ligand is also accompanied by a loss of the agglutinability of the cells by a tissue‐specific component which accumulates in monolayer conditioned medium. Conditions which inhibit release maintain maximal agglutinability suggesting similar mechanisms mediate both processes.
The structure of intrinsic membrane proteins
Authors: Guidotti Guido
Published: May 2005

Intrinsic membrane proteins are embedded in the lipid bilayer so that the polypeptides come in contact with the non‐polar region of the bilayer. There are two major types of intrinsic proteins: those with most of their mass outside the cytoplasm (Type I) and those with most of their mass inside the cytoplasm (Type II). In the latter group are the membrane transport systems. The anion exchange system of the human erythrocyte is a dimer of band 3 polypeptides. These polypeptides span the bilayer, have most of their mass in the cytoplasm, and are glycosylated. About 20–25% of the polypeptide, however, is in the bilayer. Arguments are presented to support the view that the intramembrane segments of the protein are α‐helical and that the major protein‐protein interactions between the subunits are in the cytoplasmic portion of the protein.
Regulation of glucose carriers in chick fibroblasts
Authors: Amos Harold , Musliner Thomas A. , Asdourian Hovan
Published: May 2005

The derepression of glucose transport initiated by removing glucose from the incubation medium requires both protein and RNA synthesis. The synthesis and accumulation of putative mRNA for the carrier protein(s) can be demonstrated by inhibiting protein synthesis with cycloheximide (2 μg/ml). Release from inhibition with simulataneous addition of actionmycin D (1–5 μg/ml) results in a burst of carrier synthesis that achieves virtually maximal derepression in 4–6 h. An external energy source provided by a “nonrepressive” sugar (D‐fructose, D‐xylose) or by pyruvate is required to accomplish carrier synthesis. Previous failure to demonstrate mRNA accumulation was due to the depletion of energy in the starved cells. Glucose acts as a repressor at a posttranscriptional step, probably at the level of turnover of formed carrier.

The protection of formed carrier in the absence of glucose and by inhibitors of protein synthesis even in the presence of glucose has encouraged conjecture that a protease is activated by a metabolic product of glucose that is analogous to a co‐repressor. The glucose metabolite either activates the protease by direct interaction with it or alters the conformation of the carrier to expose a critical region to protease attack. Indeed the regulation of carrier density in the membrane of chick fibroblasts may be achieved entirely by carrier inactivation, the rate of which is a function of glucose concentration in the culture medium.
Cell surface glycosyltransferases—do they exist?
Authors: Deppert Wolfgang , Walter Gernot
Published: May 2005

The presence of glycosyltransferases on surfaces of mammalian cells has been reported by many investigators and a biological role for these enzymes in cell adhesion and cell recognition has been postulated. Critical analysis, however, showed 2 major complications regarding the assay for cell surface glycosyltransferases: (1) hydrolysis of the nucleotide sugar by cell surface enzymes and subsequent intracellular use of the free sugar and (2) loss of cell integrity if trypsinized or EDTA‐treated cells were used in suspension asays. We have assayed intact, viable cells in monolayer for cell surface glycosyltransferases using conditions under which intracellular utilization of free sugars generated by hydrolysis of the nucleotide sugar was prevented. Our data demonstrate that the presence of galactosyltransferases on the surface of a variety of cells, including established (normal and virally transformed) as well as nonestablished cells, is unlikely. No evidence for the existence of cell surface fucosyl‐and sialyltransferases could be obtained, but our data do not exclude the possibility that low levels of these enzymes are present.
Direct modification of the glycocalyx of a cultured muscle cell line by incorporation of foreign gangliosides and an integral membrane glycoprotein
Authors: Barratt Dwight G. , Rogers Jackilynn D. , Sharom Frances J. , Grant Chris W. M.
Published: May 2005

As part of a program to better understand the cause‐or‐effect nature of the relationship between cell surface carbohydrate and cell properties and behaviour, experiments have been carried out on direct modification of the glycocalyx of cultured cells. Modification was by incorporation of gangliosides and an integral membrane glycoprotein chosen to be dissimilar to species occurring naturally in the cell line. Two methods of incorporation were investigated: simple addition of the new components to the culture medium for various times, or assembly of the components into the walls of lipid vesicles which were subsequently fused with cells. Gangliosides from beef brain and glycophorin, the major human erythrocyte sialoglycoprotein, were successfully added to the surface of myoblasts in quantities sufficient to represent a significant perturbation. Changes in cell adhesion, morphology, and viability were observed which seem to be a direct result of glycocalyx modification.
Temperature‐dependent morphological changes in membranes of bacillus stearothermophilus
Authors: Halverson Craig A. , Esser Alfred F. , Souza Kenneth A.
Published: May 2005

Bacillus stearothermophilus cells vary the lipid fatty acid composition of cytoplasmic membranes with growth temperature. Spin label studies of such membranes have been interpreted to indicate lateral lipid phase separations at the growth temperature. We have now used freeze‐fracture electron microscopy to confirm the spin label studies. Freeze‐fracture faces of protoplasts indicate slight but distinct protein aggregation at the growth temperature. Aggregation increases rapidly with decreasing quench temperature in wild‐type cells. In contrast we were unable to demonstrate extended protein segregation in membranes of a temperature‐sensitive mutant that contains more than 58% branched fatty acids.

Storage of protoplasts for prolonged times below the lipid phase transition results in the appearance of corrugated fracture faces with 300‐ to 500‐Å repeat patterns, although this organism does not synthesize lecithins.
Biochemical and ultrastructural studies of ectoglycosyltransferase systems of murine L1210 leukemic cells
Authors: Bernacki Ralph J. , Porter Carl W.
Published: May 2005

Intact murine L1210 leukemic cells incorporated significant quantities of [3H]‐N‐acetylneuraminic acid directly from CMP‐N‐acetylneuraminic acid. When pretreated with Vibrio cholerae neuraminidase, incorporation increased sixfold to tenfold. Biochemical studies comparing incorporation of N‐acetyl‐neuraminic acid from the nucleotide sugar with that from free sugar demonstrated that the relatively high levels of incorporation from CMP‐N‐acetyl‐neuraminic acid could not be due to the incorporation of free sugar generated by extracellular degradation of the nucleotide sugar. Very little N‐acetylneuraminic acid was taken up or incorporated by L 1210 cells from free sugar and this incorporation was not increased by neuraminidase pretreatment. Moreover, extracellular breakdown of CMP‐N‐acetylneuraminic acid during incubations with L 1210 cells was rather insignificant.

Electron microscope autoradiography of cells incubated with CMP‐N‐acetylneuraminic acid demonstrated that greater than 84% of the incorporated radioactivity was associated with the plasma membrane and less than 1% with the Golgi apparatus. These findings are consistent with the conclusion that incroporation of N‐acetylneuraminic acid from CMP‐N‐acetylneuraminic acid is the consequence of a cell surface sialytransferase system. Pretreatment of cells with the nonpenetrating reagent, diazonium salt of sulfonilic acid, significantly inhibited this ectoenzyme system while only marginally affecting galactose uptake and incorporation at the Golgi apparatus. Interestingly, incorporation from CMP‐N‐acetylneuraminic acid declined as the viability of the cell population declined. When taken together, the above evidence develops a rigorous argument for the presence of a sialyltransferase enzyme system at the cell surface of L 1210 cells.

Studies directed towards the detection of a similar ectogalactosyltransferase system were also undertaken. Cells incubated in the presence of UDP‐[3H]‐galactose incorporated radioactivity into a macromolecular fraction. The presence of excess unlabeled galactose in the incubation medium significantly reduced this incorporation. Electron microscope autoradiographs of cells incubated with UDP‐[3H]‐galactose, demonstrated that incorporation occurred primarily at the Golgi apparatus. The grain distribution in these autoradiographs was similar to that for free galactose. Thus, the incorporation observed for L‐1210 cells incubated in UDP‐[3H]‐galactose was due primarily to the intracellular utilization of free galactose generated by extracellular degradation of the nucleotide sugar. Inability t o demonstrate an ectogalacto‐syltransferase system on L1210 cells does not rule out the possibility that the enzyme is present but undetectable due t o the absence of appropriate cell surface acceptor molecules.
Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins
Authors: Fowler Velia , Bennett Vann
Published: May 2005

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water‐soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate‐labeled integral membrane proteins (> 70% of label in band 3 and PAS‐1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus‐fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside‐out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000‐dalton fragment binds to spectrin in solution, competitively inhibits association of 32P‐spectrin with inside‐out vesicles with a Ki of 10−7M, and causes rapid dissociation of 32P‐spectrin from vesicles. Both acid‐treated 72,000‐dalton fragment and the 45,000 dalton‐cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.
Retention of insulin‐stimulated D‐glucose transport activity by adipocyte plasma membranes following extraction of extrinsic proteins
Authors: Pillion Dennis J. , Shanahan Michael F. , Czech Michael P.
Published: May 2005

Plasma membrane vesicles prepared from adipocytes incubated with insulin exhibited accelerated D‐glucose transport activity characteristic of insulin action on intact fat cells. Both control and insulin‐stimulated D‐glucose transport activities were inhibited by cytochalasin B and thiol reagents. Extraction of plasma membranes with dimethylmaleic anhydride eluted 80% of the protein from plasma membrane vesicles. The two major glycoprotein bands (94,000 and 78,000 daltons) and small amounts of a 56,000‐dalton band were retained in dodecyl sulfate gels of the extracted membranes. Both control and insulin‐activated D‐glucose transport activities were retained by plasma membrane vesicles extracted with dimethylmaleic anhydride. Cytochalasin B binding activity was also retained by extracted membrane vescles and D‐glucose uptake into extracted vescles derived from untreated or insulin‐treated fat cells was inhibited by cytochalasin B. These results suggest that the modification of the adipocyte hexose transport system elicited by insulin action is not altered by a major purification step which involves quantitative extraction of extrinsic membrane proteins.
Characterization of the ribosomal binding site in rat liver rough microsomes: Ribophorins I and II, two integral membrane proteins related to ribosome binding
Authors: Kreibich Gert , Czakó‐Graham Maria , Grebenau Ruth , Mok Winnie , Rodriguez‐Boulan Enrique , Sabatini David D.
Published: May 2005

Rat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: (i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; (ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detegent Kyro EOB; (iii) in intact rough microsomes ribophorins can be crosslinked chemically to the ribosomes and therefore are in close proximity to them.

Treatment of rough microsomes with a low Triton X‐100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton‐treated microsomes containing invaginations led to the recovery of smooth and “rough‐inverted” vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosome‐binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents sugest that ribophorins are necessary for in vitro ribosome binding. Ribophorin‐like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross‐react with antisera against them.
Membrane permeability of bifunctional, amino site‐specific, cross‐linking reagents
Authors: Miyakawa Tokichi , Takemoto Larry J. , Fox C. Fred
Published: May 2005

The membrane permeability of a series of reversible cross‐linking reagents which are diazide tartarate derivatives has been compared with that of dimethyl‐3,3′‐dithiobispropionimidate (DTBP). The diazide tartarate derivatives tested include tartryl‐diazide (TDA), tartryl‐di(glycylazide) (TDGA), tartryl‐di(β‐alanylazide) (TDAA), tartryl‐di‐(γ‐aminobutyrylazide) (TDBA), tartryl‐di (ϵ‐aminocaproylazide) (TDCA). TDA, which has the shortest chain length of the diazide tartarate derivatives tested, proved to be readily permeable through the erythrocyte membrane. When added at equal concentration to unsealed ghosts, TDGA was at least as reactive as DTBP in its ability to cross link the internally displayed proteins 1, 2, 4.1, 4.2, and 6. Treatment of resealed ghosts by DTBP produced oligomeric complexes of these proteins plus apparent homooligomeric complexes of hemoglobin. TDGA at the same concentrations did not cross‐link any of these components, indicating its membrane‐impermeable nature. As the chain length of the homologous series increased from TDGA to TDCA, the cross‐linkers became increasingly permeable through the erythrocyte membrane.
Vesicles isolated from ATP‐depleted erythrocytes and out of thrombocyte‐rich plasma
Authors: Lutz Hans U.
Published: May 2005

When human erythrocytes are depleted of endogenous ATP they release spectrin‐free vesicles as a light vesicle fraction [Lutz et al: J Cell Biol 73:548, 1977] and chains of rounded vesicles as well as flattened myelin forms in a heavy vesicle fraction. The heavy fraction retains some spectrin, and glycophorin is partially degraded. The release of both types of fragments is not dependent upon added Ca+2, and 50 μM EGTA does not prevent the vesicle release. Concomitant with vesicle release, a large fraction of the major protein components of the cell is found in disulfide‐bonded aggregates.

A protocol is outlined to recover erythrocyte‐specific fragments from thrombocyte‐rich plasma. It allows detection of spectrin‐free vesicles in whole blood stored under blood bank conditions for 12 days. In freshly drawn blood no such vesicles are observed, but particles are obtained that are different from thrombocyte fragments and that show a prominent glycoprotein running slightly faster than glycophorin.
Spectrin binding and the control of membrane protein mobility
Authors: Goodman Steven R. , Branton Daniel
Published: May 2005

Transmembrane proteins of the human erythrocyte show restricted in‐plane mobility. Many of the restrictions on mobility are attributable to the molecules of spectrin which are located on the protoplasmic surface of the erythrocyte membrane. These molecules are elongate, form end‐to‐end heterodimer associations, and bind selectively to protein (or proteins) accessible on inside‐out, but not right‐side out, membrane vesicles.
Isolation and partial characterization of human erythrocyte membrane NADH: (Acceptor) oxidoreductase
Authors: Wang Chi‐Sun , Alaupovic Petar
Published: May 2005

The NADH: (acceptor) oxidoreductase (EC 1.6.99.3) was isolated from human erythrocyte ghosts by a procedure including Triton X‐100 solubilization, affinity chromatography on an NAD+‐Sepharose 4B column, ammonium sulfate precipitation, and isoelectric focusing. This enzyme preparation was characterized by a single band on the urea‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and by a single precipitin line with its corresponding antiserum on double diffusion and immunoelectrophoresis. A 103‐fold purification indicates that the oxidoreductase represents approximately 1% of the ghost protein mass. The specific activity of the purified enzyme was 112 units/mg protein. The pH optimum was 6.8 and the isoelectric point, pI, was 6.6. The oxidoreductase has a specificity for NADH as a cofactor. The NADPH was ineffective as a reducing agent. The enzyme activity was strongly temperature‐dependent, displaying maximal activity between 35 and 40°C. The energy of activation was 4.9 kcal. The enzyme activity was inhibited by sulfhydryl reagents, anionic detergents, and divalent ions. The amino acid composition of the purified enzyme is characterized by the presence of all common amino acids including half‐cystine and tryptophan. The results of carbohydrate and lipid analyses indicated that the oxidoreductase is a glycolipoprotein with fucose, galactose, mannose, and glucosamine as the sugar components and cholesterol and sphingomyelin as the lipid constituents. The apparent subunit molecular weight estimated by urea‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the absence and presence of 2‐mercaptoethanol was 40,000. The antiserum completely inhibited the enzymic activity at the equivalence point. We suggest that the membrane‐bound NADH: (acceptor) oxidoreductase might be a transmembrane protein.
Mitogenic response to epidermal growth factor: Relationship to number, affinity, and down‐regulation of EGF receptors in three murine embyro cell lines
Authors: Aharonov Aharon , Passovoy Dennis S. , Herschman Harvey R.
Published: May 2005

Swiss 3T3 and C3H‐M2 cells have a greater mitogenic response to epidermal growth factor (EGF) than do C3H‐10T1/2 cells. The latter cell line, however, has a number of EGF receptors per cell intermediate between the two cell lines that have a more vigorous response to EGF. Scatchard analysis of binding data indicate that all three cell lines have one class of EGF receptor, with indistinguishable affinity for the ligand. When exposed to 10‐nM EGF all three cell lines “down‐regulate” their EGF receptors with the same time course, and to the same precentage of initial receptors.
Studies of the diphtheria toxin receptor on chinese hamster cells
Authors: Draper Rockford K. , Chin Daniel , Stubbs Lisa , Simon Melvin I.
Published: May 2005

Concanavalin A, wheat germ agglutinin and the ovalbumin glycopeptide are all inhibitors of the cytotoxic effect of diphtheria toxin on Chinese hamster cells. Ovalbumin glycopeptide loses its inhibitory property after treatment with β‐N‐acetylglucosaminidase. This demonstrates the importance of the glycopeptide structure for the mechanism of inhibition. The glycopeptide may be a toxin cell‐surface receptor analogue.

Diphtheria toxin‐resistant mutants were isolated in order to search for cells that might have an altered toxin receptor. One mutant was 10‐to 15‐fold more resistant to diphtheria toxin than wild‐type cells when protein synthesis was measured as a function of toxin concentration. However, when protein synthesis was measured as a function of time at a high toxin concentration, the time before onset of inhibition was identical in the mutant and wild‐type cells. We present evidence indicating that the resistance of this mutant can be accounted for by a decreased affinity of toxin for a cell‐surface receptor.
Erythrocyte membrane alterations in Huntington disease: Effects of γ‐aminobutyric acid
Authors: Butterfield D. A. , Braden M. L. , Markesbery W. R.
Published: May 2005

The interaction of the inhibitory neurotransmitter γ‐aminobutyric acid (GABA) with erythrocyte membranes from patients with Huntington disease and normal controls has been studied by electron spin resonance. GABA affects the physical state of erythrocyte membrane proteins in control and Huntington disease differently. In addition, after exposure of spin‐labeled Huntington disease erythrocyte membranes to 0.1 mM GABA, the relevant electron spin resonance parameters reflecting the physical state of membrane proteins are indistinguishable from those of untreated control membranes. These findings support the concept that this disease is associated with a generalized membrane defect.
Independence of normal phenotypic properties and cell surface receptor mobility in variant cell lines
Authors: Williams Robert E. , Linsley Peter S. , Rittenhouse Harry G. , Fox C. Fred , Boone Charles W.
Published: May 2005

We report the use of three classes of variants from the long‐established malignantly transformed LM cell line to demonstrate that the apparent mobility of cell surface receptors need not be dependent on the expression of the transformed phenotype in vitro.