bluuebunny/crossref_metadata_2025 huggingface dataset:
The natural occurrence of insulin receptors in groups on adipocyte plasma membranes as demonstrated with monomeric ferritin‐insulin
Authors: Jarett Leonard , Smith Robert M.
Published: May 2005

This study was designed to document whether the reported distribution of insulin receptors in small groups of receptor sites randomly distributed in the glycocalyx of adipocytes and isolated adipocyte plasma membranes was a naturally occurring phenomena or due to artifacts. Possible artifacts include: (1) oligomeric forms of ferritin in the ferritin‐insulin preparation, (2) an uneven distribution of the glycocalyx on the plasma membrane, or (3) ligand‐induced aggregation of occupied receptor complexes. Biogel A 1.5m chromatography of the ferritin‐insulin conjugate revealed the ferritin in the ferritin‐insulin complex to consist of 55% monomers, 15% dimers, and 30% oligomers. The monomer peak was purified (> 95%) for use in these studies. Cationic ferritin, a glycocalyx marker, when incubated with paraformaldehyde‐fixed plasma membranes, was found to be uniformly distributed on the surface of the plasma membrane indicative of uniformly distributed glycocalyx. The ability to demonstrate and inhibit ligand‐induced aggregation on the isolated plasma membrane was established with a multivalent ligand, ferritin‐concanavalin A. More than 66% of the ferritin‐concanavalin A receptors were found in large clusters of 5 or more and 34% as singletons or clusters of up to 4 when incubated at 24°C with fresh membranes. Only 38% of the ferritin‐concanavalin A receptors were in large clusters; 62% were singletons or clusters up to 4 on membranes prefixed with paraformaldehyde before incubation. The distribution of the monomeric ferritin‐insulin was similar on both adipocytes and purified adipocyte plasma membranes and was consistent with earlier reports with ferritin‐insulin. The quantitative distribution of the monomeric ferritin‐insulin as singletons or in groups of 2–6 was comparable between the intact cells and isolated membranes incubated at 24°C. The binding of 500 μUnits monomeric ferritin‐insulin per ml to the isolated plasma membranes was studied under incubation conditions similar to those used with ferritin‐concanavalin A. Under all three conditions, fresh membranes at 24°C and 0–4°C and prefixed membranes at 24°C, the pattern of distribution of the monomeric ferritin‐insulin as singletons or groups of 2–6 was identical, indicating that the ligand was not causing aggregation into clusters as did the concanavalin A. Thus, the occurrence of insulin receptors in small groups appears to be a natural phenomenon in the plasma membrane structure of adipocytes.
Mutations affecting the binding, internalization, and lysosomal hydrolysis of low density lipoprotein in cultured human fibroblasts, lymphocytes, and aortic smooth muscle cells
Authors: Brown Michael S. , Anderson Richard G. W. , Goldstein Joseph L.
Published: May 2005

Studies comparing the metabolism of low density lipoprotein (LDL) in normal cells and in cells cultured from patients with homozygous familial hypercholesterolemia have disclosed the existence of a receptor for plasma LDL. This receptor has been identified on the surface of human fibroblasts, lymphocytes, and aortic smooth muscle cells. An extension of these studies to cell strains derived from patients with other single gene defects in cholesterol metabolism has provided additional insight into the normal mechanisms by which cells regulate their cholesterol content and how alterations in these genetic control mechanisms may predispose to atherosclerosis in man.
Transport in halobacterium halobium: Light‐induced cation‐gradients, amino acid transport kinetics, and properties of transport carriers
Authors: Lanyi Janos K.
Published: May 2005

Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na+. Measurements of 22Na flux, exterior pH change, and membrane potential, ΔΨ (with the dye 3,3′‐dipentyloxadicarbocyanine) indicate that the means of Na+ transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H+/Na+ > 1). The resulting large chemical gradient for Na+ (outside > inside), as well as the membrane potential, will drive the transport of 18 amino acids. The 19th, glutamate, is unique in that its accumulation is indifferent to ΔΨ: this amino acid is transported only when a chemical gradient for Na+ is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H+ collapses within 1 min, while the large Na+ gradient and glutamate transporting activity persists for 10–15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na+, arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with Vmax and Km comparable to light‐induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na+, in an electrically neutral fashion, so that only the chemical component of the Na+ gradient is a driving force.

The transport of all amino acids but glutamate is bidirectional. Actively driven efflux can be obtained with reversed Na+ gradients (inside > outside), and passive efflux is considerably enhanced by intravesicle Na+. These results suggest that the transport carriers are functionally symmetrical. On the other hand, noncompetitive inhibition of transport by cysteine (a specific inhibitor of several of the carriers) is only obtained from the vesicle exterior and only for influx: these results suggest that in some respects the carriers are asymmetrical.

A protein fraction which binds glutamate has been found in cholate‐solubilized H. halobium membranes, with an apparent molecular weight of 50,000. When this fraction (but not the others eluted from an Agarose column) is reconstituted with soybean lipids to yield lipoprotein vesicles, facilitated transport activity is regained. Neither binding nor reconstituted transport depend on the presence of Na+. The kinetics of the transport and of the competitive inhibition by glutamate analogs suggest that the protein fraction responsible is derived from the intact transport system.
Amino acid transport systems in animal cells: Interrelations and energization
Authors: Christensen Halvor N.
Published: May 2005

After summarizing the discrimination of the several transport systems of neutral amino acids in the cell of the higher animal, I discuss here the ways in which 2 dissimilar transport systems interact, so that one tends to run forward for net entry and the other backwards for net exodus. An evaluation of the proposals for energization shows that uphill transport continues when neither alkali‐ion gradients nor ATP levels are favorable. Evidence is presented that under these conditions a major contribution is made by another mode of energization, which may depend on the fueling of an oxidoreductase in the plasma membrane. This fueling may involve the export by the mitochondrion of the reducing equivalents of NADH by one of the known shuttles, e.g., the malate‐aspartate shuttle. After depletion of the energy reseves in the Ehrilich cell by treating it with dinitrophenol plus iodoacetate concentrative uptake of test amino acids is restoration by pyruvate but in poor correlation with the restoration of alkali‐ion gradients and ATP levels. This restoration by pyruvate but not by glucose is highly senstitive to rotenone. A combination of phenazine methosulfate and ascorbate will also produce transport restoration, before either the alkali‐ion gradients or ATP levels have begun to rise. The restoration of transport applies to a model amino acid entering by the Na+‐independent system, as well as to one entering by the principal Na+‐dependent system, restoration being blocked by ouabain, despite the weak effect of ouabain on the alkali‐ion gradients in the Ehrlich cell. Quinacrine terminates very quickly the uptake of model amino acids, before the alkali‐ion gradients have begun to fall and before the ATP level has been halved. Quinacrine is also effective in blocking restoration of uphill transport by either pyruvate or the phenazine reagent. Preliminary results show that vesicles prepared from the plasma membrane of the Ehrlich cell quickly reduce cytochrome c or ferricyanide in the presence of NADH, and that the distribution of a test amino acid between the vesicle and its environment is influenced by NADH, quinacrine, and an uncoupling agent in ways consistent with the above proposal, assuming that a majority of the vesicles are everted.
Glycosphingolipids in membrane architecture
Authors: Sharom Frances J. , Grant Chris W. M.
Published: May 2005

As part of a program to investigate the behavior and interactions of glycolipids in biological membranes we have synthesized spin‐labeled derivatives of 2 families of carbohydrate‐bearing ceramides (glycosphingolipids): simple neutral glycolipids and gangliosides. Galactosyl ceramide has been synthesized with the spin label at 3 different positions on the fatty acid chain. It has been studied in bilayers of various different lipids and lipid mixtures and compared to the corresponding phospholipid spin labels. Considerable similarity has been found between the behavior of galactosyl ceramide and phosphatidylcholine. These similarities include a negligible flip‐flop rate, a flexibility gradient in the acyl chains, and exclusion from phosphatidylserine domains in the face of a Ca2+‐induced lateral phase separation. Evidence for dramatic clustering of simple neutral glycolipids has not been found. Glycosphingolipids do seem to have the capacity to increase rigidity in fluid lipid bilayers. A general procedure has been developed for covalent attachment of a nitroxide spin label to the headgroup region of complex glycolipids such as gangliosides. Studies of beef brain gangliosides labeled in this manner and incorporated into bilayers of phosphatidylcholine indicate that the headgroup oligosaccharides are in rapid, random motion as opposed to being in any way immobilized. This headgroup mobility depends very little on the fluidity or rigidity of the bilayer. However, headgroup mobility decreases, perhaps as a result of cooperative headgroup interactions, with increasing bilayer concentration of unlabeled ganglioside.
Carbon‐13 as a tool for the study of carbohydrate structures, conformations and interactions
Authors: Nunez H. A. , Walker T. E. , Fuentes R. , O'Connor J. , Serianni A. , Barker R.
Published: May 2005

The application of 13C‐NMR spectroscopy to problems involving the structures and interactions of carbohydrates is described. Both 13C‐enriched and natural abundance compounds were used and some advantages of the use of the stable isotope are described. Carbon‐carbon and carbon‐proton coupling constants obtained from 1‐13 C enriched carbohydrates were employed in the assignment of their chemical shifts and to establish solution conformation. In all cases studied thus far, C‐3 couples to C‐1 only in the β‐anomers while C‐5 couples to C‐1 only in the α‐anomers. C‐6 and C‐2 always couple to C‐1 in both anomeric species. The alkaline degradation of glucose [1‐13 C] to saccharinic acids was followed by 13C‐NMR. The conversion of glucose [1‐13 C] to fructose‐1,6‐bisphosphate [1,6‐13 C] by enzymes of the glycolytic pathway was shown as an example of the use of 13C‐enriched carbohydrates to elucidate biochemical pathways. In a large number of glycosyl phosphates the 31P to H‐1 and 31P to C‐2 coupling constants demonstrate that in the preferred conformation the phosphate group lies between the O‐5 and the H‐1 of the pyranose ring. The influence of paramagnetic Mn2+ ions on the proton decoupled 13C‐NMR spectra of uridine diphosphate N‐acetylglucosamine indicates that the Mn2+ interacts strongly with the pyrophosphate moiety and with the carbonyl groups of the uracil and N‐acetyl groups.
On the rate limiting step in downhill transport via the LacY permease of Escherichia coli
Authors: Rotman Boris
Published: May 2005

Strains of Escherichia coli K12 were constructed for the specific purpose of evaluating the inducibility of the influx mechanism controlled by the lacY gene. These strains are heteromerodiploids characterized by a high and relatively constant level of β‐D‐galactosidase which is not affected significantly by induction of the Lac operon. These properties were obtained by introducing episomal lacI+,Oc,Z+,Y− genes into the cells. In these merodiploids the rate of o‐nitrophenyl‐β‐D‐galactopyranoside (ONPG) hydrolysis of extracted cells is 50‐times that of intact cells. This difference indicates that the rate limiting step in the ONPG hydrolysis by intact cells is influx.

Using a set of merodiploids with and without the LacY transport system, we were able to demonstrate a specific induction of ONPG influx. However, the increase in influx due to induction was only 3.5‐fold as compared to the 40‐fold increase observed when the LacY permease was measured by intracellular accumulation of [14C] TMG.
An electron microscopic and enzymic study of rat liver peroxisomal nucleoid core and its association with urate oxidase
Authors: Lata Gene F. , Mamrak Frank , Bloch Phillip , Baker Barbara
Published: May 2005

The appearance of the characteristic crystalloid core of rat liver peroxisomes is emulated by the electron microscopic (EM) appearance of highly purified urate oxidase prepared from the same tissue. The purity of the enzyme preparation was established by gel electrophoresis under various conditions and the specific enzyme activity was at least as high as any previously reported. The amino acid composition of urate oxidase was determined. As additional evidence for close association of the peroxisomal core with urate oxidase, it was demonstrated that the biphasic changes in rat liver urate oxidase activity in response to prolonged starvation were paralleled by changes in the EM appearance of peroxisomes. Under comparable conditions catalase, another peroxisomal enzyme, did not show the same changes in activity as did urate oxidase. Evidence for the possible identity of urate oxidase with the peroxisomal crystalloid of rat liver has been presented, all materials having been obtained from, and experiments performed with, the rat.
Small‐angle X‐ray scattering study of human serum low‐density lipoproteins with differential reactivity for an arterial proteoglycan
Authors: Mateu L. , Kirchhausen T. , Padrón R. , Camejo G.
Published: May 2005

The structure and thermal behavior of human serum low‐density lipoproteins showing either a high or a low reactivity against a proteoglycan isolated from human arteries have been found to be different from each other. It is suggested that modifcations in the lipoprotein surface structure induced by the physical state of the neutral lipids could modulate the affinity of the macromolecule for the arterial component.
Ganglioside structures and distribution: Are they localized at the nerve ending?
Authors: Ledeen R. W.
Published: May 2005

Gengliosides generally provide a small portion of the complex carbohydrate content of cell surfaces. An exception is the central nervous system where they comprise up to 5–10% of the total lipid of some membranes. This tissue is unique in that the quantity of lipid‐bound sialic acid exceeds that of the protein‐bound fraction. Over 30 different molecular species have been characterized to date. These range in complexity from sialosylgalactosyl ceramide with 2 sugars to the pentasialoganglioside of fish brain with 9 carbohydrate units. Virtually all cellular and subcellular fractions of brain that have been carefully examined contain gangliosides to one degree or another, but the majority of brain ganglioside is located in the neurons. Their mode of distribution within the neuron has not been entirely clarified by subcellular studies. Calculations based on reported values for axon terminal density and synaptosomal ganglioside concentration in the rat reveal that nerve endings contribute less than 12% of total cerebral cortical ganglioside. It is concluded that the plasma membranes of neuronal processes contain most of the neuronal ganglioside. These and other considerations suggest the possibility that gangliosides may be distributed over the entire neuronal surface.
Immunochemical purification of probe‐labeled plasma membrane proteins: An approach to the molecular anatomy of the cell surface
Authors: Comoglio Paolo M. , Tarone Guido , Bertini Marilena
Published: May 2005

The probe 2,4,6‐trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labelling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate‐polyacrylamide gels of probe‐labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus‐transformed cells. Moreover, the transformed cells display a nonvirion component of 80–100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37°C but not at 41°C, the expression of this component is related to the expression of the transformed phenotype.
Glycosaminoglycans of the fat globule membrane of cow milk
Authors: Lis Dora , Monis Benito
Published: May 2005

Membranes of fat globules of cow milk contained 163 μg/100 mg (dry weight) of glycosaminoglycans (expressed as uronic acid); 62.5% of the uronic acids corresponded to hyaluronic acid, the remaining consisted of sulfated glycosaminoglycans (chondroitin‐4‐(‐6) sulfates, and dermatan and heparan sulfates) with different degrees of sulfation.
Distribution of glycophorin on the surface of human erythrocyte membranes and its association with intramembrane particles: An immunochemical and freeze‐fracture study of normal and En(a−) erythrocytes
Authors: Gahmberg Carl G. , Taurén Georg , Virtanen Ismo , Wartiovaara Jorma
Published: May 2005

Human erythrocyte membranes of the En(a–) blood group lack the major sialoglycoprotein (glycophorin). By absorption of a crude antiglycophorin antiserum with En(a–) membranes a specific antiglycophorin antiserum was obtained. By immune electron microscopy we showed that glycophorin is randomly distributed on the surface of normal erythrocytes. When polycationized ferritin, which mainly binds to glycophorin, was used as a marker a similar even labeling of normal erythrocyte membranes was seen. En(a–) membranes bound much less of this marker. In freeze‐fracturing the intramembrane particles of both membrane types had a similar distribution and appeared in equal amounts. However, partial removal of spectrin from these membranes, followed by incubation at pH 6 resulted in more extensive aggregation of the particles in En(a–) membranes than in normal membranes. The results may be interpreted as glycophorin contributing by electrostatic repulsion to the random distribution of the intramembrane particles in normal cells. This repulsion is weakened in En(a–) cells by the lack of glycophorin.
The spectrin‐actin complex and erythrocyte shape
Authors: Pinder Jennifer C. , Ungewickell E. , Bray D. , Gratzer W. B.
Published: May 2005

The effects of phosphorylation of spectrin on the properties of the cytoskeletal network of the human erythrocyte have been studied. A suspension of the cytoskeletal residues obtained after extraction of the ghosts with the nonionic detergent Triton X‐100 forms a gel on addition of membrane kinase and ATP. Phosphorylation has no effect on the association state of purified spectrin. No species higher than a tetramer of polypeptide chains is formed in vitro; in the absence of divalent cations, this tetramer is an entity liberated from and evidently present in the membrane. It has not so far proved possible to detect any F‐actin in the cytoskeleton before or after phosphorylation. It is suggested that the consequence of phosphorylation is formation of additional interactions between spectrin and monomeric actin molecules. This view is supported by the formation, after phosphorylation of the Triton‐extracted cytoskeleton, of an insoluble mass of protein on treatment with a cross‐linking reagent. In the absence of divalent cations, a series of oligomeric species is progressively liberated from the cytoskeleton on extraction with solutions of low ionic strength. These oligomers contain actin as well as spectrin, and are thought to result from disruption of the network by random denaturation of the mono meric actin in the absence of divalent metal ions. A schematic view of the effects of phosphorylation on the structure of the cytoskeleton is presented.
Regulation of hemopoietic cell differentiation and proliferation
Authors: Burgess Antony W. , Metcalf Donald , Watt Suzanne M.
Published: May 2005

Differentiation and proliferation of almost all hemopoietic cell lines can now be studied in vitro. Cloning techniques and suspension cultures allow the study of proliferation of the multipotential hemopoietic progenitor cell and the committed progenitors for granulocytes, macrophages, eosinophils, megakryocytes, and erythrocytes. The proliferation of each of the committed progenitor cells is controlled by specific glycoproteins and two of these have recently been purified: granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and erythropoietin. The rate of proliferation of the GM‐progenitor cells and their pattern of differentiation depends on the concentration of the hormone. At low concentrations of GM‐CSF (10−11 M) fewer progenitor cells are stimulated and macrophage colonies rather than granulocyte colonies develop. The change in the direction of granulocyte‐macrophage differentiation appears to be related to (a) the concentration of GM‐ CSF and (b) the different sensitivity of a subpopulation of monocyte colony‐forming cells which are responsive to GM‐CSF even at low concentrations of the regulator. Analysis of the rate of RNA synthesis by bone marrow cells has shown that GM‐CSF stimulates the mature nondividing end cells of differentiation (ie, polymorphs) as well as the progenitor cells. Although GM‐CSF and erythropoietin have been radiolabeled, binding studies have been hampered by the loss of biologic activity during the labeling procedure and the heterogeneity of the target cells to which the regulators bind. Surface proteins and receptors for erythrocytes have been well characterized but the relationships between these proteins and the cell surface proteins of nucleated blood cells is not well understood. It appears that some proteins are lost from the cell surface during the development of granulocytes, which are retained on the surface of the B lymphocyte. Other proteins such as chemotactic receptors and complement receptors only appear on the mature cells. External radiolabeling of the granulocyte surface using iodogen yielded a simple profile of 125I‐labeled proteins when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Elucidation of the receptor‐bound conformation of the enkephalins
Authors: Gorin Fredric A. , Balasubramanian T. M. , Barry C. David , Marshall Garland R.
Published: May 2005

The biologically relevant conformers of enkephalin predicted by solid state, solution state, and theoretical energy studies have been compared with the published structure‐activity data on these compounds. No conformational technique proposes a model consistent with all the pharmacological data; the shortcomings of each approach are evaluated. An alternative approach, which correlates the structure‐activity data of opiate compounds with that of the enkephalins, is described and shown to produce a model consistent with the available structure‐activity data.
Neural uptake of catecholamines and their molecular structures: A histopharmacologic study
Authors: Uno H. , Fellman J. H.
Published: May 2005

Using ultrastructural and histofluorescence methods, we investigated the uptake mechanism of catecholamines by the nerve terminals in the cutaneous smooth muscles of stump‐tailed macaques (Macaca arctoides). This in vivo approach ultilized the observed cytotoxic effects of 6‐hydroxydopamine on these catecholamine‐containing terminals and the protective effects of simultaneous treatment with catecholamines (dopamine, norepinephrine, and epinephrine), their 3–0‐methylated derivatives (metanephrine and normetanephrine), and catechol acids (3,4‐dihydroxymandelic acid and 2, 4, 5‐trihy‐droxymandelic acid). Both catecholamines and 3–0‐methylated derivatives protected these nerve terminals from destruction by 6‐hydroxydopamine, but catechol acids did not. However, the 3–0‐methylated derivatives were less effective than the catecholamines. The degree of protection afforded by these amines depended largely on their concentration. Only catecholamines intensified the electron density of the intravesicular mass or the fluorescence in the nerve terminals; therefore, 3–0‐methylated derivatives may inhibit 6‐hydroxydopamine uptake at axoplasmic membrane sites, but not inside the axon.

These observations led to the discovery that there are two sites for the catecholamine uptake process. One site is the axoplasmic membrane. The terminals are protected by catecholamines and their 3–0‐methylated derivatives from 6‐hydroxydopamine uptake and thus destruction. The other site is the intra‐axonal compartments. Here competitive binding between the vesicular protein and both 6‐hydroxydopamine and the catecholamines plays a main role.
Effects of PTH and Ca2+ on renal adenyl cyclase
Authors: Nielsen Susan T. , Neuman William F.
Published: May 2005

The effects of calcium ion on the adenylate cyclase system was studied in isolated, renal basal‐lateral plasma membranes of the rat. Bovine parathyroid hormone (bPTH) and a guanyl triphosphate analogue, Gpp(NH)p were used to stimulate cyclase activity.

Under conditions of maximal stimulation, calcium ions inhibited cyclic adenosine monophosphate (cAMP) formation, the formation rate falling exponentially with the calcium concentration. Fifty percent inhibition of either bPTH‐ or Gpp(NH)p‐stimulated activity was given by approximately 50 μM Ca++. Also the Hill coefficient for the inhibition was close to unity in both cases. The concentration of bPTH giving half‐maximal stimulation of cAMP formation (1.8 × 10−8 M) was unchanged by the presence of calcium.

These data suggest that calcium acts at some point other than the initial hormone‐receptor interaction, presumably decreasing the catalytic efficiency of the enzymic moiety of the membrane complex.
Inhibition by protease inhibitors of binding of adrenal and sex steroid hormones
Authors: Baker Michael E. , Vaughn Duke A. , Fanestil Darrell D.
Published: May 2005

Binding of steroid hormones is inhibited by protease inhibitors and substrates. The protease inhibitors phenylmethyl sulphonylfluoride, tosyl‐lysine chloromethyl ketone, and tosylamide‐phenylethyl‐chloromethyl ketone and the protease substrates tosyl arginine methyl ester and tryptophan methyl ester eliminate specific binding of aldosterone, dexamethasone, dihydrotestosterone, estrogen, and progesterone to their respective receptors. These protease inhibitors and substrates also inhibit binding of progesterone to the 20,000 molecular weight mero‐receptor formed from the progesterone receptor in chick oviduct. The binding of estradiol to rat alpha‐fetoprotein is inhibited by the protease inhibitors and substrates but not by tryptophan or tryptophan amide, indicating the importance of an ester structure in the inhibition of steroid binding. Our results suggest that all steroid hormone receptors have a site with both common structural features and a role in the regulation of steroid hormone binding.
Activation of cyclic AMP‐dependent protein kinase and stimulation of protein phosphorylation in response to adenosine in C‐1300 murine neuroblastoma
Authors: Green Richard D. , Noland Thomas A.
Published: May 2005

DEAE‐cellulose chromatography of the 20,000g supernatant fraction of homogenates of C‐1300 murine neuroblastoma (clone N2a) yields one major and two minor peaks of cyclic AMP‐dependent protein kinase activity. Assessment of the endogenous activation state of the enzyme(s) reveals that the enzyme is fully activated by the treatment of whole cells with adenosine (10 μM) in the presence of the phosphodiesterase inhibitor Ro 20 1724 (0.7 mM). This treatment produces a large elevation in the cyclic AMP content of the cells. The treatment of whole cells with adenosine alone (1–100 μM) or Ro 20 1724 alone (0.1–0.7 mM) produces minimal elevations in cyclic AMP but nevertheless causes significant activations of cyclic AMP‐dependent protein kinase. The autophosphorylation of whole homogenates of treated and untreated cells was studied using [γ‐32P] ATP, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography. Treatments which activate cyclic AMP‐dependent protein kinase selectively stimulate the incorporation of 32P into several proteins. This stimulation is most prominent in the 15,000‐dalton protein band. The addition of cyclic AMP to phosphorylation reactions containing homogenate of untreated cells stimulates the phosphorylation of the same protein bands. These results indicate that adenosine may have regulatory functions through its effect on the cyclic AMP: cyclic AMP‐dependent protein kinase system.
Cholera toxin‐catalysed ADP‐ribosylation of erythrocyte proteins: General properties
Authors: Gill D. Michael
Published: May 2005

Upon incubation of lysed pigeon erythrocytes with NAD, adenosine diphosphateribose (ADP‐ribose) is incorporated into nuclear poly ADP‐ribose and into an unidentified acid‐insoluble product of the cytosol. The properties of these incorporations have been examined and a method developed for reducing their amount whilst retaining the sensitivity of the lysate to cholera toxin. This method has allowed the detection and description of a set of cholera toxin‐specific ADP‐ribose transfers to membrane‐bound and soluble proteins under conditions that lead to adenylate cyclase activation.
Lipid‐protein interactions in membranes: Effect of lipid composition on mobility of spin‐labeled cysteine residues in yeast plasma membrane
Authors: Esfahani Mojtaba , Solomon Daniel J. , Mele Lisa , Teter M. Noel
Published: May 2005

In order to gain direct evidence for lipid‐dependent protein conformation in membrane, effects of modification of lipid composition on mobility of spin‐labeled cysteine residues were investigated in the plasma membrane of the yeast Saccharomyces cerevisiae. Conversion of the bulk of phospholipids to diglycerides by treatment of the membrane with phospholipase C substantially enhanced spectral anisotropy. However, alteration of the viscosity of the lipid‐bilayer by enriching the membrane with palmitelaidic or oleic acid had no effect on mobility of spin‐labeled cysteine residues. These observations indicate that while the spin‐labeled residues are not in direct contact with the lipid core of the membrane, there are lipid‐protein interactions to the extent that removal of polar portion of the bulk of phospholipids induces conformational changes in proteins, which in turn restrict mobility of these residues. It is concluded that conformation of membrane proteins depends on lipid structure and that phospholipids have a role in preserving the native conformation of proteins.
The role of temperature in the crystallization of ribosomes in chick embryos
Authors: Barbieri Marcello
Published: May 2005

The relationship between ribosome crystallization and cell degeneration has been studied in chick embryos at various temperatures, and new methods of inducing ribosome microcrystals are described. A model is discussed that reinterprets the role of low temperatures in these phenomena and provides a unitary explanation of the various cases in which the occurrence of ribosome crystallization in chick embryos has been reported.
Phosphoprotein intermediate in the ca2+‐dependent atpase reaction of macrophage plasma membrane
Authors: Schneider C. , Mottola C. , Romeo D.
Published: May 2005

ATPase activity and phosphorylation by [γ‐32P] ATP of isolated plasma membrane of alveolar macorphages are stimulated in a parallel fashion by physiologic concentrations of Ca2+, with half‐maximal activating effect of this ion at (3–7) × 10−7 M. For various membrane preparations, a direct proportionality exists between Ca2+‐dependent ATPase activity and amount of 32P incorporated. Labeling of membrane attains the steady‐state level by 10 sec at 0°C, and is rapidly reversed by adenosine diphosphate (ADP). K+ decreases the amount of membrane‐bound 32P, mainly by enhancing the rate of dephosphorylation of the 32P‐intermediate. Hydroxylamine causes a release of about 90% of 32P bound to the membrane, thus indicating that the 32P‐intermediate contains an acyl‐phosphate bond. When the labeled plasma membrane is solubilized and electrophoresed on acrylamide gels in the presence of sodium dodecyl sulphate, the radioactivity appears to be largely associated with a single protein fraction of 132,500 ± 2,000 apparent molecular weight. These features of the macrophage Ca2+‐ATPase suggest that the enzyme activity might be part of a surface‐localized Ca2+‐extrusion system, participating in the regulation of Ca2+‐dependent activities of the macrophage.
Structure of the dna binding cleft of the gene 5 protein from bacteriophage fd
Authors: McPherson Alexander , Jurnak Frances , Wang Andrew , Rich Alexander , Molineux Ian
Published: May 2005

The structure of the gene 5 DNA unwinding protein from bacteriophage fd has been solved to 2.3‐Å resolution by X‐ray diffraction techniques. The molecule contains an extensive cleft region that we have identified as the DNA binding site on the basis of the residues that comprise its surface. The interior of the groove has a rather large number of basic amino acid residues that serve to draw the polynucleotide backbone into the cleft. Arrayed along the external edges of the groove are a number of aromatic amino acid side groups that are in position to stack upon the bases of the DNA and fix it in place. The structure and binding mechanism as we visualize it appear to be fully consistent with evidence provided by physical‐chemical studies of the protein in solution.
Nuclear control of tumorigenicity in cells reconstructed by PEG‐induced fusion of cell fragments
Authors: Shay Jerry W. , Clark Mike A.
Published: May 2005

The techniques of somatic cell hybridization have provided a valuable means of studying mechanisms of regulation of mammalian cell differentiation and transformation. Most previous studies have indicated that fusions between tumorigenic and nontumorigenic cells result in hybrid cells that are usually tumorigenic. In recent years it has been demonstrated that the phenotypic expression of tumorigenicity is at least partially due to the extensive chromosome loss that occurs in most interspecific and some intraspecific hybrid cells. In the present study we have utilized enucleation techniques that permit cells to be divided into nuclear (karyoplast) and cytoplasmic (cytoplast) cell fragments. Even though these nuclear and cytoplasmic fragments are metabolically stable for short periods of time, in our hands they ultimately degenerate. Viable cells can be reconstructed by PEG‐induced fusion of karyoplasts to cytoplasts. Since reconstructed cells apparently do not segregate chromosomes, they may provide a clearer understanding of the interactions between the nucleus and the cytoplasm in the control of the expression of tumorigenicity. We have reconstructed cells using karyoplasts from the tumorigenic Y‐1 cell line and cytoplasts from a nontumorigenic cell line, A‐MT‐BU‐A1. In addition we have reconstructed cells containing Y‐1 cytoplasts and A‐MT‐BU‐A1 karyoplasts. The reconstructed cells porduced were assayed for tumorigenicity by their ability to grow in soft agar and in nude mice. The results of these experiments indicate that the reconstructed cells containing a tumorigenic nucleus and a nontumorigenic cytoplasm ultimately are tumorigenic and conversely the reconstructed cells containing a nontumorigenic nucleus and a tumorigenic cytoplasm are nontumorigenic. These experiments support the concept that with these cell lines the nucleus (karyoplast) is sufficient to control the phenotypic expression of tumorigenicity.
The assembly of lipid‐linked oligosaccharides in plant and animal membranes
Authors: Bailey David S. , Dürr Mathias , Burke John , Maclachlan Gordon
Published: May 2005

Membrane preparations from growing regions of pea stems and activelydividing mouse L‐cells form lipid‐linked saccharides from GDP‐mannose and UDP‐N‐acetylglucosamine. These lipids have properties which are consistent with those of mono‐and di‐phosphoryl polyisoprenyl derivatives.

In experiments using plant membranes, the monophosphoryl derivative labeled with GDP‐(14C) mannose contains mannose only, while the diphosphoryl derivative labeled with the same nucleotide sugar is heterogeneous, containing oligosaccharides corresponding to mannosaccharides of 5, 7, and 9‐12 residues. Only the diphosphoryl polyisoprenyl derivatives are labeled with UDP‐(14C)glucosamine and these contain predominantly chitobiose and N‐acetylglucosamine itself. Unlabeled GDP‐mannose added after UDP‐N‐acetyl (14C)glucosamine results in the formation of higher lipid‐linked oligosaccharides which are apparently the same as those which are labeled with GDP‐(14C)mannose alone. Incubation of the membranes with GDP‐(14C)mannose in the presence of Mn2+, unlabeled UDP‐glucose or unlabeled UDP‐N‐acetylglucosamine results in marked changes in the accumulation of both the polyisoprenyl monophosphoryl mannose and polyisoprenyl diphosphoryl oligosaccharides.

Animal cell membranes synthesise lipid‐linked oligosaccharides when incubated with UDP‐N‐acetylglucosamine and GDP‐mannose. These oligosaccharides are similar in size to those synthesised by the plant membranes but their formation is more efficient. The potential roles of these compounds in glycoprotein biosynthesis in both plant and animal tissues is discussed.
Mitotic factors from mammalian cells: A preliminary characterization
Authors: Sunkara Prasad S. , Wright David A. , Rao Potu N.
Published: May 2005

The objective of this study was the preliminary characterization of the factors from mitotic HeLa cells that can induce meiotic maturation in Xenopus laevis oocytes. We found that this factor is a heat‐labile, Ca2+‐sensitive, nondialyzable protein with a sedimentation value of 4‐5S. Furthermore, no new protein synthesis was found to be required for this mitotic factor to induce maturation in the amphibian oocytes. These data suggest that the factors involved in the breakdown of nuclear membrane and the condensation of chromosomes that are associated with three different phenomena, mitosis, meiosis, and premature chromosome condensation, are very similar in different animal species.
Growth of kidney epithelial cells in hormone‐supplemented, serum‐free medium
Authors: Taub Mary , Sato Gordon H.
Published: May 2005

Madin Darby canine kidney cells can grow in synthetic medium supplemented with 5 factors – insulin, transferrin, prostaglandin E1, hydrocortisone and triiodothyronine – as a serum substitute. These 5 factors permit growth for one month in the absence of serum, and a growth rate equivalent to that observed in serum‐supplemented medium. Dibutyryl cAMP substitutes for prostaglandin E1 in the medium, suggesting that increased growth of Maden Darby canine kidney cells results from increased intracellular cAMP. Potential applications of the serum‐free medium are discussed. The medium permits the selective growth of primary epithelial cell cultures in the absence of fibroblast over‐growth, and a defined analysis of the mechanisms by which hormones regulate hemicyst formation.
Inhibition of blood coagulation factor XIIIa‐mediated cross‐linking between fibronectin and collagen by polyamines
Authors: Mosher Deane F. , Schad Peter E. , Kleinman Hynda K.
Published: May 2005

Soluble fibronectin is found in body fluids and media of cultured adherent cells. Insoluble fibronectin is found in tissue stroma and in extracellular matrices of cultured cells. Fibronectin is a substrate for factor XIIIa (plasma transglutaminase) and can be cross‐linked to collagen and to the α chain of fibrin. We have used sodium dodecyl sulfate‐polyacrylamide gel electrophoresis to investigate the possibility that factor XIIIa ‐mediated cross‐linking is influenced by polyamines. Spermidine inhibited cross‐linking between fibronectin and type I collagen, isolated α1 (I) collagen chains, or iodinated cyanogen bromide fragment 7 of α1 (I) chains (125I‐α1 (I)‐CB7). Half‐maximal inhibition of crosslinking between 125I‐α1 (I)‐CB7 and fibronectin was observed when 0.1 mM spermine or spermidine was present. Spermidine, 0.7 mM, partially inhibited cross‐linking between fibronectin and the α chain of fibrin but failed to inhibit cross‐linking between the fibrin monomers of a fibrin clot. Spermidine also failed to inhibit cross‐linking between fibronectin molecules when aggregation of fibronectin was induced with dithiothreitol. In contrast, 0.7 mM monodansylcadaverine inhibited fibronectin‐collagen, fibronectin‐fibrin, fibronectin‐fibronectin, and fibrin‐fibrin cross‐linking. Spermidine or spermine, 0.7 mM, enhanced the cross‐linking between molecules of partially amidinated fibronectin, suggesting that N1,8‐(di‐γ‐glutamyl)‐polyamine cross‐linkages were formed. Spermidine and spermine failed to enhance cross‐linking between monomers of amidinated fibrin. These results indicate that physiologic concentrations of polyamines specifically disturb transglutaminase‐catalyzed cross‐linking between fibronectin and collagen.
Influence of buffer ions and divalent cations on coated vesicle disassembly and reassembly
Authors: Woodward Michael P. , Roth Thomas F.
Published: May 2005

Disruption of the coat of coated vesicles is accompanied by the release of clathrin and other proteins in soluble form. The ability of solubilized coated vesicle proteins to reassemble into empty coats is influenced by Mg2+, Tris ion concentration, pH, and ionic strength. The proteins solubilized by 2 M urea spontaneously reassemble into empty coats following dialysis into isolation buffer (0.1 M MES–1 mM EGTA–1 mM MgCl2–0.02% NaN3, pH 6.8). Such reassembled coats have sedimentation properties similar to untreated coated vesicles. Clathrin is the predominant protein of reassembled coats; most of the other proteins present in native coated vesicles are absent. We have found that Mg2+ is important in the coat assembly reaction. At pH 8 in 0.01 M or 0.1 M Tris, coats dissociate; however, 10 mM MgCl2 prevents dissociation. If the coats are first dissociated at pH 8 and then the MgCl2 raised to 10 mM, reassembly occurs. These results suggest that Mg2+ stabilizes the coat lattice and promotes reassembly. This hypothesis is supported by our observations that increasing Mg2+ (10 μM–10 mM) increases reassembly whereas chelation of Mg2+ by (EGTA) inhibits reassembly. Coats reassembled in low‐Tris (0.01 M, pH 8) supernatants containing 10 mM MgCl2 do not sediment, but upon dialysis into isolation buffer (pH 6.8), these coats become sedimentable. Nonsedimentable coats are noted also either when partially purified clathrin (peak I from Sepharose CL4B columns) is dialyzed into low‐ionic‐strength buffer or when peaks I and II are dialyzed into isolation buffer. Such nonsedimentable coats may represent intermediates in the assembly reaction which have normal morphology but lack some of the physical properties of native coats. We present a model suggesting that tightly intertwined antiparallel clathrin dimers form the edges of the coat lattice.
Glycosaminoglycans of the plasma membranes of renal tubule and liver cells
Authors: Lis Dora , Monis Benito
Published: May 2005

Glycosaminoglycans were isolated from plasma membranes of hepatic and renal tubule cells of guinea pig. Plasmalemma of renal tubule cells contained more total glycosaminoglycans, hyaluronic acid, chondroitin‐4 sulfates and chondroitin‐6 sulfates, and less dermatan sulfates and heparin sulfates than liver plasma membranes. These glycocalyx components, owing to their polyanionic properties, may have a role in the transport of water, ions, and macromolecules across the cell membrane.
Structural comparisons of the aggregates of tobacco mosaic virus protein
Authors: Stubbs Gerald , Warren Stephen , Mandelkow Eckhard
Published: May 2005

The coat protein of tobacco mosaic virus forms numerous aggregates, including the small A‐protein, the disk, and two helical forms. The structures of the disk, the helical protein forms, and the virus are compared. Most of the differences are in the conformation of the chain between residues 89 and 113, which lies in the region of protein at the center of the virus, inside the RNA. It is disordered in the disk, but has a fixed conformation in the virus and the protein helices. The differences between the virus and the two helical protein forms are largely in the conformations of arginines and carboxylic acids in this region.
Isolation of cholera toxin receptors from a mouse fibroblast and lymphoid cell line by immune precipitation
Authors: Critchley D. R. , Ansell S. , Perkins R. , Dilks S. , Ingram J.
Published: May 2005

Cholera toxin receptors have been isolated from both a mouse fibroblast (Balbc/3T3) and mouse lymphoid cell line labeled by the galactose oxidase borotritiide technique. Tritiated receptor‐toxin complexes solubilized in NP40 were isolated by addition of toxin antibody followed by a protein A‐containing strain of Staphylococcus aureus. In both cell types by far the major species of toxin receptor isolated was ganglioside in nature, although galactoproteins were also present in the immune complexes. Whether the galactoproteins form part of a toxin‐receptor complex or are artifacts of the isolation procedure is presently unclear.

The relative specificity of cholera toxin for a carbohydrate sequence in a glycolipid suggests that the toxin might prove a useful tool in establishing the function and organization of glycolipids in membranes. For example, interaction of cholera toxin with the mouse lymphoid cell line was shown to result in patching and capping of bound toxin, raising the possibility that the glycolipid receptor interacts indirectly with cytoskeletal elements. Cholera toxin might also be used to select for mutant fibroblasts lacking the toxin receptor and therefore having an altered glycolipid profile. Such mutants might prove useful in establishing the relationship (if any) between modified glycolipid pattern and other aspects of the transformed phenotype. Attempts to isolate mutants, based on the expectation that growth of cells containing the toxin receptor would be inhibited by the increase in cAMP levels normally induced by cholera toxin, proved unsuccessful. Cholera toxin failed to inhibit significantly the growth of either Balbc or Swiss 3T3 mouse fibroblasts although it markedly elevated cAMP levels.
Gliding mycoplasmas are inhibited by cytochalasin B and contain a polymerizable protein fraction
Authors: Maniloff Jack , Chaudhuri Utpal
Published: May 2005

Studies are presented on the effect of cytochalasin B (CB) on the growth of five Mycoplasma species, three Acholeplasma species, and one Spiroplasma species. The three gliding mycoplasma species (M) gallisepticum, M pneumoniae and M pulmonis are the only mycoplasmas inhibited by CB. These are the only prolaryotes reported to be inhibited by CB. This suggested that these three mycoplasmas might have some sort of cytoskeletal structure. A protein fraction has been isolated from M gallisepticum which polymerizes in 0.6 M KC1 and depolymerizes when KC1 is removed. This fraction contains a major 58,000‐dalton protein, a 46,000‐dalton protein, and a minor 87,000‐dalton protein.