bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Trypsin Treatment of Rat Adipocytes on Biological Effects and Binding of Insulin and Insulin‐Like Growth Factors
Authors: ZAPF Jürgen , SCHOENLE Eugen , WALDVOGEL Margaretha , SAND Inger , FROESCH E. Rudolf 
Published: March 2005

Trypsin‐treatment of isolated rat adipocytes abolishes the metabolic effects not only of insulin, but also of the insulin‐like growth factors: in trypsin‐treated cells, concentrations of these hormones that are otherwise maximally effective no longer stimulate 3‐O‐methylglucose transport and lipogenesis or inhibit epinephrine‐induced lipolysis. Concomitantly, the trypsin‐treated adipocytes no longer display specific insulin binding. In contrast, the characteristics of the binding of the insulin‐like growth factors are not grossly affected by prior trypsinization of the adipocytes.These findings add further support to the concept that the insulin‐like growth factors act on glucose metabolism and antilipolysis via the insulin receptor of the adipocyte.
Purification and Physicochemical Properties of ATP Citrate (pro‐3S) Lyase from Lactating Rat Mammary Gland and Studies of Its Reversible Phosphorylation
Authors: GUY Paul S. , COHEN Philip , HARDIE D. Grahame 
Published: March 2005

A simple procedure is described for the isolation of ATP‐citrate lyase, acetyl‐CoA carboxylase and fatty acid synthase from lactating rat mammary gland. ATP‐citrate lyase was purified 48‐fold by poly(ethyleneglycol) precipitation, ammonium sulphate precipitation and chromatography on DEAE‐Sepharose at pH 7.5. The preparation was completed in 2–3 days and 30 mg of enzyme was isolated from 10 animals, which represented a yield of 38%. The preparation was of a high degree of purity as judged by polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S20,w was 13.6 ± 0.2 S, the absorption coefficient, A1%280nm measured refractometrically was 12.2 ± 0.7 and the amino acid composition was determined.The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 116000 and the molecular weight of the native enzyme measured by sedimentation equilibrium was 473000. These experiments indicate that mammary ATP‐citrate lyase is a tetramer. The purified enzyme contained 0.5 mol of alkali‐labile phosphate covalently bound/subunit.The phosphorylation of ATP‐citrate lyase by cyclic‐AMP‐dependent protein kinase reached a plateau when 0.7 ± 0.2 mol of phosphate were bound to a serine residue in a single tryptic peptide. The same tryptic peptide was phosphorylated by cyclic‐GMP‐dependent protein kinase, although at a much slower rate. ATP‐citrate lyase was not phosphorylated by phosphorylase kinase or by glycogen synthase kinase 3.The phosphate introduced by cyclic‐AMP‐dependent protein kinase could be completely removed by incubation with protein phosphatase 1 or protein phosphatase 2. However the alkali‐labile phosphate present in ATP‐citrate lyase as isolated was not removed by these protein phosphatases, indicating that it is located at a distinct site(s). Phosphorylation of purified ATP‐citrate lyase by cyclic‐AMP‐dependent protein kinase did not alter the V or the apparent Km for citrate, ATP or CoA.The properties of mammary ATP‐citrate lyase are compared to those of the rat liver enzyme and the possible role of phosphorylation in the regulation of its activity is discussed.
Factors Modulating Transcription and Translation in vitro of Ribosomal Protein S20 and Isoleucyl‐tRNA Synthetase from Escherichia coli
Authors: WIRTH Reinhard , KOHLES Veronika , BÖCK August 
Published: March 2005

The DNA‐dependent protein‐synthesizing system developed by Zubay [Zubay, G. (1973) Annu. Rev. Genet. 7, 267–287] was optimized for the transcription and translation of genes from the 0.5‐min region of the Escherichia coli chromosome carried by transducing λ phages. The E. coli gene products synthesized were isoleucyl‐tRNA synthetase, ribosomal protein S20, dihydrodipicolinic acid reductase and (possibly) the two subunits of carbamoyl‐phosphate synthetase.Formation of ribosomal protein S20 is specifically stimulated by the addition of 16‐S rRNA and not by 5‐S or 23‐S rRNA. 16‐S rRNA increases the rate of S20 synthesis, the final yield of product depends on the duration of persistence of the RNA added. Addition of 16‐S rRNA to the separate transcription and translation systems showed that it is the translation of the S20 mRNA which is enhanced. Furthermore, S20 synthesis is stimulated more than fourfold when concomitant synthesis of rRNA occurs from a plasmid carrying an rrn transcriptional unit. The results described are explained in terms of a model which suggests that ribosomal protein S20 feedback inhibits its synthesis at the translational level and that removal of S20 into ribosomal assembly (i.e. binding to 16‐S rRNA) releases inhibition. The model postulates a direct link between synthesis of ribosomal RNA and ribosomal protein and between the rates of ribosomal assembly and ribosomal protein synthesis.The stimulatory effect of guanosine 3′‐diphosphate 5′‐diphosphate on isoleucyl‐tRNA synthetase formation and its inhibition of the synthesis of ribosomal protein S20 in vitro occurs at the level of transcription. Its relevance in vivo, however, remains to be demonstrated.Formation of isoleucyl‐tRNA synthetase in vitro is not influenced either by the addition of a surplus of purified enzyme nor by the limitation of protein synthesis by the addition of anti‐(isoleucyl‐tRNA synthetase) serum. There is no evidence, therefore, that isoleucyl‐tRNA synthetase is autogenously regulated.
Characterization of a Membrane‐Bound Biotin‐Containing Enzyme: Oxaloacetate Decarboxylase from Klebsiella aerogenes
Authors: DIMROTH Peter 
Published: March 2005

Oxaloacetate decarboxylase from Klebsiella aerogenes is firmly bound to the cytoplasmic membrane, from which it can be solubilized with nonionic detergents. The solubilized enzyme behaved like‐the membrane‐bound enzyme with respect to its inhibition by avidin and to the requirement of sodium ions for catalytic activity. The decarboxylase was purified 4.5‐fold over the solubilized membrane extract by conventional means. Dodecylsulfate disc‐gel electrophoretic analysis indicated that the enzyme consists of polypeptides of a single size. The molecular weight of these polypeptides is 68000. Radioactive biotin was incorporated specifically into these polypeptide chains upon growth of the bacteria in the presence of the radioactive vitamin. Biotin as the prosthetic group of oxaloacetate decarboxylase is now firmly established. The enzyme in the absence of detergent occurs in a highly aggregated form which elutes in the exclusion volume of a Biogel A 1.5 m column.The reported inhibition of oxaloacetate decarboxylase by citrate could not be repeated. On the other hand oxalate, 2‐oxomalonate and glyoxylate proved to be very potent inhibitors of the decarboxylase.The stereochemical course of the oxaloacetate decarboxylation reaction was determined starting from stereospecifically labelled malates, which by malate dehydrogenase and oxaloacetate decarboxylase were converted to chiral pyruvates. The chirality of these pyruvates was analysed via their conversion to acetates and malates by determining the extent of tritium retention upon incubation of the latter with fumarase. It was found that oxaloacetate decarboxylation occurs stereospecifically with retention of configuration.
The Regulation of Glycolysis in Human Erythrocytes
Authors: ATAULLAKHANOV Fazoil I. , VITVITSKY Victor M. , ZHABOTINSKY Anatoly M. , PICHUGIN Aleksei V. , PLATONOVA Olga V. , KHOLODENKO Boris N. , EHRLICH Lev I. 
Published: March 2005

According to the mathematical model, the dependence of the rate of glycolysis on the ATP concentration can be represented by the bell‐shaped curve with the descending part at the concentration of ATP close to physiological values. The existence of the descending part is a result of the strong inhibition of phosphofructokinase by ATP and hexokinase by glucose 6‐phosphate and provides stabilisation of the intracellular concentration of ATP.
Using arsenate as an uncoupler of the oxidation of glucose and ATP synthesis, the dependence of the rate of glycolysis on the ATP concentration in erythrocytes was measured. This dependence (glycolysis characteristic) is represented by a bell‐shaped curve. The normalized glycolysis characteristics are the same for all the donors investigated.
The increase of permeability of the erythrocyte membrane to monovalent cations by the polyene antibiotic levorin leads to an increase of glycolytic rate in erythrocytes (40–70%) and to a decrease in the ATP concentration (15%) and glucose 6‐phosphate (25–60%). The data obtained with levorin, and also other results on Na, K‐ATPase activation are in a good agreement with the characteristic obtained using arsenate.
In all cases, inhibition of Na+, K+‐ATPase of erythrocytes by ouabain leads to the decrease of the rate of lactate production (10–20%) and increase of the concentration of glucose 6‐phosphate (14–40%). The ATP concentrations remains almost unchanged or is only slightly increased. The rate of glucose utilisation in erythrocytes from different donors show two types of behaviour. In the first type of erythrocyte it drops proportionally to the decrease in the lactate production rate; in the second type of erythrocyte it remains unchanged.
The experimental results presented are in qualitative agreement with the predictions of the mathematical model.
Binding of the Chemoattractant Folic Acid by Dictyostelium discoideum Cells
Authors: VAN DRIEL Roel 
Published: March 2005

The binding of the chemoattractant folic acid to intact Dictyostelium discoideum cells has been studied. A few hundred thousand ligand molecules can be bound per cell with a dissociation constant in the order of 3 × 10−7 M. Intact cells equilibrate rapidly with extracellular folic acid, suggesting that the binding sites are located on the outer cell surface. The folic‐acid‐binding properties do not change during early cell development induced by starvation for nutrients.A number of putative folate‐binding proteins have been identified in a detergent‐solubilized plasma membrane fraction by affinity chromatography on folate‐Sepharose and specific elution with free folate. One of these proteins appeared to be an integral membrane protein as was deduced from its amphiphilic behaviour. It may be that among these proteins is the chemotactic receptor for folate.
Cytotoxicity Acquired by Conjugation of an Anti‐Thy1.1 Monoclonal Antibody and the Ribosome‐Inactivating Protein, Gelonin
Authors: THORPE Philip E. , BROWN Alexander N. F. , ROSS Walter C. J. , CUMBER Alan J. , DETRE Simone I. , EDWARDS D. Caird , DAVIES Anthony J. S. , STIRPE Fiorenzo 
Published: March 2005

Gelonin, a plant protein which can powerfully reduce the protein‐synthetic capacity of ribosome preparations, was covalently coupled to anti‐Thy1.1 antibody. The conjugate was prepared using N‐succinimidyl‐3‐(2‐pyridyldithio)propionate which generates a disulphide linkage between the component molecules. Two conjugate fractions were obtained with Mr of 180000 and > 200000.After its linkage to the antibody, gelonin suppressed those Thy1.1‐bearing T lymphocytes from AKR mice which will respond to phytohaemagglutinin and concanavalin A in tissue culture. The [3H]leucine incorporation with the T‐cell mitogens was inhibited by 50% with the 180000‐Mr fraction at a concentration of 0.4 nM and with the > 200000‐Mr fraction of pM. Unconjugated gelonin induced comparable reductions in T‐cell responsiveness but at concentrations of 30 nM. The conjugates exerted little or no effect upon B lymphocytes or T lymphocytes from CBA mice (Thy1.2+ve). Two Thy1.1‐expressing AKR lymphoma cell lines, AKR‐A and BW5147, were found to be sensitive to the conjugates, albeit much less so than the normal T lymphocytes. The conjugates injected in vivo significantly prolonged the life of CBA mice bearing an AKR‐A lymphoma allograft.It is concluded that gelonin can, by its linkage to an antibody, be rendered cytotoxic with a potency to match or exceed those of the toxins abrin and ricin.
Mechanism of Uptake of l‐Arginine by Sugar‐Cane Cells
Authors: KOMOR Ewald , THOM Margaret , MARETZKI Andrew 
Published: March 2005

Suspension cells of sugar cane were used as a model system for cells of higher plants to study the mechanism of l‐arginine uptake.The uptake system is specific for the l‐arginine molecule in the fully ionized state, i.e. δ‐guanidine group and α‐amino group positively charged and carboxyl group negatively charged. This was concluded because the Km value for uptake increased strongly for: (a) l‐arginine analogues which lack the charged carboxyl group (l‐arginine methyl ester, agmatin); (b) l‐arginine analogues, which lack the charged α‐amino group (l‐arginine acid, γ‐guanidinobutyric acid); (c) l‐arginine analogues which lack the charged δ‐guanidine group or γ‐guanidinoxy group (l‐citrulline, l‐canavanine at neutral and alkaline pH‐values).The importance of the positive charge at the δ‐guanidine group or γ‐guanidinoxy group was further documented by Km values for l‐arginine and l‐canavanine uptake at different pH values. Only at pH values where the γ‐guanidinoxy group is protonated, was there an effective uptake of l‐canavanine and effective competition of l‐canavanine with l‐arginine.The length of the l‐arginine molecule was less important: slightly larger l‐homoarginine) or shorter analogues (l‐lysine) were taken up rather well. A spatial rearrangement at the α‐carbon (d‐arginine) was, however, not tolerated.The uptake of l‐arginine proceeds by electrogenic uniport, there is no evidence for symport or antiport of another molecule (though l‐canavanine uptake at neutral pH value causes a transient alkalinization of the suspension medium). Charge equilibration is brought about by efflux of protons and potassium ions.
Effect of Chloroquine and Methylamine on Endocytosis of Fluorescein‐Labelled Control IgG and of Anti‐(Plasma Membrane) IgG by Cultured Fibroblasts
Authors: SCHNEIDER Yves‐Jacques , TROUET André 
Published: March 2005

We report here the effect of chloroquine and methylamine two lysosomotropic drugs, on the binding, uptake and subcellular localization of fluorescein‐labelled control immunoglobulm G (control IgG) a marker for non‐specific adsorptive endocytosis and of anti‐(plasma membrane) IgG (specific IgG), a specific ligand of cell‐surface antigens.At 4 °C, methylamine and chloroquine inhibit the binding of control IgG to the cell surface, probably by a reversible competition. These two drugs, methylamine more than chloroquine, considerably slow down the rate at which control IgG is transferred from its binding sites on the phagosomal membrane to the lysosomal compartment; both drugs block almost completely the intralysosomal digestion of this IgG as well as the release of degradation products into the culture‐medium. They do not aftect the binding and uptake of the specific IgG. In addition, methylamine seems to inhibit partially the return to the cell surface of membrane antigens and of membrane fragments bearing 5′‐nucleotidase or binding sites for control IgG.We conclude that important steps (binding to cell surface, delivery to lysosomes, digestion and recycling of plasma membrane) involved in the uptake and the processing of IgG by fibroblasts are inhibited by these two substances. The effects of lysosomotropic agents on the regulation and function of the endocytic pathway and of lysosomes could have many pharmacological and therapeutic implications.
Biosynthesis of the Light‐Harvesting Chlorophyll a/b Protein
Authors: BENNETT John 
Published: March 2005

When etiolated pea seedlings were exposed to continuous light for 24 h and then returned to darkness, 38% of the chlorophyll a, 74% of the chlorophyll b and 84% of the light‐harvesting chlorophyll a/b protein that had accumulated under illumination proved to be unstable in darkness. The unstable chlorophyll displayed a half‐life of abdüt 90 min. In contrast, the α and β subunits of the chloroplast coupling factor and the large and small subunits of ribulose 1,5‐bisphosphate carboxylase continued to accumulate in darkness, although at a slower rate than in plants maintained under light.
Short‐term labelling in vivo with L‐[35S]methionine showed that leaves continued to synthesize the light‐harvesting protein and the small subunit of ribulose 1,5‐bisphosphate carboxylase for up to 48 h after transfer of plants from light to darkness. However, after long‐term labelling (16 h), the light‐harvesting chlorophyll a/b protein was found to be labelled to high specific activity only in illuminated leaves.
I conclude that the light‐harvesting chlorophyll a/b protein is subject to turnover after transfer of plants from light to darkness. The site of breakdown appears to be the photosynthetic membrane. I suggest that turnover of the protein is part of the normal physiological mechanism for co‐ordinating the accumulation of the pigment and protein components of the light‐harvesting chlorophyll a/b complex.
Vicia graminea Anti‐N Lectin: Partial Characterization of the Purified Lectin and Its Binding to Erythrocytes.
Authors: DUK Maria , LISOWSKA Elwira 
Published: March 2005

Vicia graminea, lectin, purified by affinity chromatography, was homogeneous in sodium dodecylsulphate/ polyacrylamide gel electrophoresis and migrated with a velocity corresponding to a molecular weight of 125000. Heating in 0.1% sodium dodecylsulphate at 100°C caused dissociation of the lectin into subunits with an apparent molecular weight of 31000. The results of isoelectric focusing suggested non‐identity of the lectin subunits and existence of several molecular forms of the lectin. The lectin was neither dissociated nor inactivated by succinyl ation, but was irreversibly inactivated by 8 M urea. The lectin was totally bound to concanavalin‐A—Sepharose and could be eluted with α‐d‐mannopyranoside.Binding of the 125I‐labeled Vicia gramninea lectin to untreated and desialylated human erythrocytes of blood groups M and N, horse and bovine erythrocytes was characterized. The lectin was bound specifically to sites specific for blood group N on untreated human erythrocytes with an uniform affinity, and association constant Ka= 1.5 × 108 M−1. Desialylated human NN and MM erythrocytes bound more lectin, with a distinctly higher, but non‐uniform affinity. Vicia graminea lectin bound weakly to horse erythrocytes, and the effect of their desialylation was similar to that obtained with human erythrocytes. The lectin was not bound either to untreated or to desialylated bovine erythrocytes. Binding of the labeled lectin to human NN erythrocytes was inhibited by desialylated glycoproteins of the M and N blood groups, by untreated N glycoprotein, and weakly by untreated M glycoprotein.
Synthesis and Posttranslational Segregation of Glyoxysomal Isocitrate Lyase from Castor bean Endosperm
Authors: ROBERTS Lynne M. , LORD J. Michael 
Published: March 2005

Total Polyadenylated RNA isolated from 3‐day‐old germinating caster bean endosperm was translated in the rabbit reticulocyte lysate cell‐free protein‐synthesizing system in either the presence or absence of canine pancreatic microsomal vesicles. Isocitrate lyase synthesized in vivo had the same apparent molecular weight as the authentic glyoxysomal enzyme irrespective of whether synthesis occurred in the presence or absence of microsomes. Furthermore, in contrast to several protein encoded by mRNA isolated from maturing seed endosperm tissue, isocitrate lyase was not contranslationally segregated into the lumen of the microsomal vesicles.Intact 3‐day‐old endosperm tissue was labeled in vivo by adding [35S]methionine and at various times the tissue was homogenized and the cellular organelles fractionated. Separated fractions were assayed for newly synthesized isocitrate lyase. Immunoreactive product was initially detected in the cytosolic fraction and subsequently began to accumulate in glyoxysomes.The results indicate that glyoxysomal isocitrate lyase is synthesized on free polysomes and released into the cytosol. Transfer to the matrix of the glyoxysome involves a posttranslational membrane translocation step which is not accompanied by proteolytic cleavage of the polypeptide chain.
Binding of tRNA to Escherichia coli Ribosomes as Measured by Velocity Sedimentation
Authors: SCHMITT Marion , MÖLLER Achim , RIESNER Detlev , GASSEN Hans Günter 
Published: March 2005

We followed the binding of initiator and elongator tRNA to 70‐S ribosomes and its subunits by velocity sedimentation in the analytical ultracentrifuge. This technique shows the advantage over the previously used methods (adsorption of the complexes to nitrocellulose filters or fluorescence titrations) in that no kinetic effects obscure the equilibrium data and that none of the components has to be chemically modified. The concentrations of the macromolecular compounds are kept constant and the binding equilibria are shifted by varying the Mg2+ concentration in a range which is accessible to experimental analysis.Free 30‐S ribosomes bind no tRNA, whereas one tRNA molecule is bound to 50‐S ribosomal subunits. In the presence of the cognate condon one tRNA can be associated with the small subunit. Free, programmed, or misprogrammed 70‐S ribosomes bind exactly two elongator tRNAs. Only the initiator tRNA does discriminate significantly between the two ribosomal sites when bound to a ribosome. A‐U‐G complex.
Isoleucyl‐tRNA Synthetase from Baker's Yeast
Authors: FREIST Wolfgang , VON DER HAHR Frsiedrich , CRAMER Friedrich 
Published: March 2005

The order of substrate addition to isoleucyl‐tRNA synthetase from baker's yeast has been investigated by steady‐state kinetics with inhibition by four different inhibiting ATP analogs acting competitively, uncompetitively and noncopetitively with respect to ATP, namely purineriboside (=nbularin), 3′‐deoxy‐adenosine (=cordycepin), 8‐amino‐adenosine and 8‐azido‐adenosine 5′‐triphosphates. The inhibition studies were done in the amino‐acylation and in the pyrophosphate exchange reaction, the aminoacylation eas investihated in the absence and presence of inorganic pyrophosphatase. Additionally bisubstrate kinetics and product inhibition studies were carried out.The inhibition patterns indicate a multisite system with a minimum number of two sites for each of the substrates. Yhe results of the pyrophosphate exchange studies are consistent with formation of E. Ile‐AMP‐ATP.Ile complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, subsequent release of pyrophosphate and random addition of a second molecule of ATP and iso‐leucine. For the aminoacylation in the absence of pyrophosphatase an ordered ter‐ter mechanism is postulated; in the presence of pyrophosphatase the mechanism is random bi‐uni uni‐bi ping‐pong. Both the pyrophosphate and the analogs of this compound such as imidodiphosphate or methylenediphosphonate can induce the enzyme to act in the ter‐ter mechanism.