bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein‐Bound Adenosine 3′:5′‐Monophosphate in Liver of Glucagon‐Treated Rats
Authors: SCHWOCH Gerhild , HILZ Helmuth 
Published: March 2005

The adenosine 3′:5′‐monophosphate (cyclic AMP) fraction bound to high‐affinity proteins of rat liver cytosol was determined by homogenization of the frozen tissue with a charcoal suspension followed by centrifugation. Protein‐bound cyclic AMP in the supernatant appears to represent mainly or exclusively cyclic AMP bound to the regulatory subunit of protein kinases as indicated by its identity with the phosphodiesterase‐resistant cyclic AMP fraction in homogenates and its close correlation with protein kinase activation in vivo.Parallel determination of total cyclic AMP, protein‐bound cyclic AMP and protein kinase activation in the livers of rats sacrificed between 2 and 120 min after glucagon injection made it possible to correlate these parameters at different levels of total cyclic AMP in vivo. In non‐stimulated tissue, total cyclic AMP was 0.7 pmol/mg tissue, about 50 % being present in the form of protein‐bound cyclic AMP. At a total cyclic AMP level of 1.1–1.2 pmol/mg, half‐maximal values for bound cyclic AMP as well as half‐maximal activation of protein kinases were found. This shows not only a close correlation between these two parameters but also indicates that rather small changes in total cyclic AMP are required in vivo to effect binding of cyclic AMP to the regulatory subunit, R, with the concomitant activation of protein kinase.The reliability of protein‐bound cyclic AMP as an indicator of protein kinase activation was further documented in the livers of stressed animals, in which total cyclic AMP was elevated nearly two‐fold while bound cyclic AMP and protein kinase remained at basal values.
Activity of Protein Kinase Dependent on Adenosine 3′:5′‐Monophosphate and of Its Thermostable Protein Inhibitor in Rat Hepatoma (HTC) Cells
Authors: ROUSSEAU Guy G. 
Published: March 2005

Normal expression of a variety of hormonal effects which depend on cyclic AMP (adenosine 3′:5′‐monophosphate) requires the presence of glucocorticoids. Our hypothesis was that glucocorticoids control directly or indirectly the activity of cyclic‐AMP‐dependent protein kinase. This has been investigated in cultured hepatoma (HTC) cells in which N6,O2′‐dibutyryladenosine 3′:5′‐monophosphate increases the activity of tyrosine transaminase only after glucocorticoid treatment.In these cells, we have determined the concentration and half‐life of protein kinase, the sensitivity of this enzyme in vitro to cyclic AMP and to its thermostable protein inhibitor, the state of dissociation of protein kinase holoenzyme in vivo and its sensitivity, in the intact cell, to dibutyryladenosine 3′:5′‐monophosphate and to the inhibitor diamide, and we have also determined the concentration of endogenous thermostable protein inhibitor of protein kinase.None of these parameters were influenced by glucocorticoids under conditions where these hormones stimulate the activity of tyrosine transaminase and restore sensitivity to dibutyryladenosine 3′:5′‐monophosphate.It is concluded that the permissive action of glucocorticoids probably results from a control of cyclic‐AMP‐dependent processes exerted at a level beyond the protein kinase system.
Inhibition of Cholesterol Synthesis in vitro and in vivo by ML‐236A and ML‐236B, Competitive Inhibitors of 3‐Hydroxy‐3‐methylglutaryl‐Coenzyme A Reductase
Authors: ENDO Akira , TSUJITA Yoshio , KURODA Masao , TANZAWA Kazuhiko 
Published: March 2005

1. ML‐236A and ML‐236B, potent inhibitors of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase, were readily absorbed into the liver, the major site of cholesterogenesis, after oral administration in mice; peak concentration of these inhibitors in the liver, which were attained within 60 min after administration, were 4% of the dose for ML‐236A and 10% for ML‐236B, respectively.2. ML‐236B reduced sterol synthesis from both [14C]acetate and [14C]octanoate in rat liver slices to 50% of control at a concentration of 0.05 μg/ml (0.12 μM) but not that from [14C]mevalonate at higher concentrations up to 1.25 μg/ml. Inhibition of sterol synthesis was also observed in liver slices obtained from rats which had previously received this compound.3. ML‐236B was also inhibitory in vivo to cholesterol synthesis in various tissues of rats in which [14C]acetate was intraperitoneally injected to the animals and labeled digitonin‐precipitable sterols synthesized were isolated and determined. Sterol synthesis in the liver was far more strongly inhibited (over 90 % at 4h after oral administration of 50 mg/kg of ML‐236B) than that in other organs tested including kidney, lung, adrenal, spleen, and testis.4. These findings strongly suggest that hypocholesterolemic activity of ML‐236A and ML‐236B is owing largely to specific inhibition of hepatic cholesterol synthesis.
The Involvement of a Complex between Formylmethionyl‐tRNA and Initiation Factor IF‐2 in Prokaryotic Initiation
Authors: VAN DER HOFSTAD Gerard A. J. M. , FOEKENS John A. , BOSCH Leendert , VOORMA Harry O. 
Published: March 2005

A complex between initiation factor IF‐2 and fMet‐tRNA can be formed under ionic conditions, which are optimal for initiation complex formation. The complex can be retained on cellulose nitrate filters after fixing with glutaraldehyde.The IF‐2 · fMet‐tRNA complex formation is not influenced by GTP and GDP. Other nucleoside di or triphosphates also have no effect. Evidence is presented that this complex acts as an intermediate in polypeptide chain initiation. The IF‐2 · fMet‐tRNA complex formation is not influenced by initiation factors IF‐1 and IF‐3.The binary complex can be bound to the 30‐S subunit in the absence of GTP, which indicates that there is no concomittant binding of the IF‐2 · fMet‐tRNA complex and the nucleotide moiety to the 30‐S subunit. The binding of the binary complex is stimulated by GTP. The influence of some inhibitors of initiation on the IF‐2 · fMet‐tRNA complex formation has been tested. Aurin tricarboxylic acid appeared to be a strong inhibitor, whereas the sulfhydryl reagents N‐ethylmaleimide and p‐chloromercuribenzoate had no effect.
Hydrolysis and Isomerization of trans,trans‐Farnesyl Diphosphate by Andrographis Tissue‐Culture Enzymes
Authors: MACKIE Hugh , OVERTON Karl H. 
Published: March 2005

1. Incubation of (3R,5S)‐[5‐3H1]mevalonate + (3RS)‐[2‐14C]mevalonate with Andrographis cell‐free extract leads to trans,trans‐farnesol and cis,trans‐farnesol which both totally retain tritium.2. This conflicts with our previous results which predict one third tritium loss in the cis,trans‐farnesol. Inversion at C‐1 during hydrolysis of trans,trans‐farnesyl diphosphate to trans,trans‐farnesol could explain this anomaly.3. (1S)‐trans,trans‐[1‐3H1]Farnesyl diphosphate and phosphate and (1R)‐trans,trans‐[1‐3H1]‐farnesyl diphosphate and phosphate, all prepared chemically, were hydrolysed with Andrographis phosphatase, and alkaline phosphatase and hydrogenolysed with lithium aluminium hydride and the product alcohols exchanged with liver alcohol hydrogenase.4. Both Andrographis phosphatase and alkaline phosphatase hydrolyse trans,trans‐farnesyl diphosphate and trans,trans‐farnesyl phosphate with retention.5. Hydrolysis of trans,trans‐[1‐18O]farnesyl diphosphate in H218O with both phosphatases supports P‐O fission.6. The C‐1 configuration in (1S)‐trans,trans‐[1‐3H1]farnesyl diphosphate and phosphate and (1R)‐trans,trans‐[1‐3H1]farnesyl diphosphate and phosphate is progressively racemised in 0.01 M NH4OH/MeOH (1/9) at –20°C.
Reduction in β‐Adrenergic Response of Cultured Glioma Cells Following Depletion of Intracellular GTP
Authors: FRANKLIN Trevor J. , TWOSE Patricia A. 
Published: March 2005

1. When C6 glioma cells were incubated with mycophenolic acid, a potent and specific inhibitor of IMP:NAD oxidoreductase (EC 1.2.1.14) there was a marked depletion of the cellular content of GTP. The viability of the cells was unaffected.2. The adenosine 3′:5′‐monophosphate (cyclic AMP) response of C6 glioma cells to the β‐adrenergic stimulant, (±)isoprenaline, was considerably reduced after treatment with mycophenolic acid. The diminished response to (±)isoprenaline was prevented by the inclusion of guanine in the culture medium along with mycophenolic acid.3. The adenylate cyclase response to (±)isoprenaline of whole homogenates from C6 cells treated with mycophenolic acid was also depressed; the response was restored to normal by the addition of GTP.4. The adenylate cyclase response to (±)isoprenaline of a membrane fraction prepared from homogenates of C6 cells was almost totally dependent on the presence of added GTP. Membrane fractions from control and mycophenolic‐acid‐treated C6 cells gave similar adenylate cyclase responses to (±)isoprenaline in the presence of GTP.5. It is concluded that mycophenolic acid may depress the β‐adrenergic sensitivity of C6 cells by depleting the cellular content of GTP.
Purification and Characterization of Vicilin Peptidohydrolase, the Major Endopeptidase in the Cotyledons of Mung‐Bean Seedlings
Authors: BAUMGARTNER Bruno , CHRISPEELS Maarten J. 
Published: March 2005

1. Vicilin peptidohydrolase, the major endopeptidase present in the cotyledons of bean seedlings, has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis and isoelectrofocusing, from the cotyledons of mung bean [Vigna radiata (L.) Wilczek] seedlings.2. The protease has an apparent molecular weight of 23 000 as estimated by gel filtration and polyacrylamide gel electrophoresis, an isoelectric point of 3.75 and belongs to the class of the sulfhydryl proteases as judged by the effect of various protease inhibitors on the activity of the enzyme.3. The purified enzyme readily hydrolyzes synthetic esters between amino acids and p‐nitrophenol coupled to N‐carbobenzoxy and N‐tert‐butoxycarbonyl residues. Esters with asparagine and glutamine residues are hydrolyzed most readily.4. The purified enzyme readily degrades vicilin, the principal reserve protein present in mung bean seeds. Degradation is optimal at pH 5.1. The various polypeptides of vicilin are gradually digested to smaller polypeptides and oligopeptides. Vicilin peptidohydrolase and carboxypeptidase cooperate in the complete digestion of vicilin with the resulting release of amino acids.
Cellulases from Chaetomium thermophile var. dissitum
Authors: ERIKSEN Jonny , GOKSØYR Jostein 
Published: March 2005

When cultivated in a liquid medium with cellulose, Chaetomium thermophile var. dissitum produced extracellular cellulolytic enzymes. By concentration of the culture filtrate, followed by ion‐exchange chromatography on DEAE‐Sephadex A‐50 and gel filtration on Biogel P‐100, three electrophoretically pure components were obtained. Of these, one was a typical Cx enzyme (endoglucanase) causing rapid decrease of the viscosity of carboxymethylcellulose solutions, while showing low effect on native cellulose.The other was active towards native cellulose, but had little effect on the viscosity of carboxymethylcellulose. It is concluded that this enzyme is an exoglucanase (C1 enzyme), possibly a cellobiohydrolase.The third component showed only cellobiase (β‐glucosidase) activity, and had no effect on cotton or carboxymethylcellulose. The three components, when mixed, showed synergistic effects on highly ordered cellulose. The endo‐ and exoglucanases were characterized with regard to molecular size and iso‐electric point (pI). Both cellulases had pI near 4.55, but their molecular weights were different: 67 000 (exoglucanase) and 41 000 (endoglucanase). The effect of temperature on the activity of the cellulases was examined with both cotton and carboxymethylcellulose as substrate. Arrhenius activation energies, Q10, and temperature optima for the different reactions were determined.
Preparation of N6‐[N‐(6‐Aminohexyl)carbamoyl]‐Adenine Nucleotides and Their Application to Coenzymically Active Immobilized ADP and ATP, and Affinity Adsorbents
Authors: YAMAZAKI Yoshimitsu , MAEDA Hidekatsu , SUZUKI Hideo 
Published: March 2005

Reaction of ADP with hexamethylene diisocyanate in hexamethylphosphoramide followed by treatment in an acidic medium afforded three new adenine nucleotide analogues, N6‐[N‐(6‐aminohexyl)carbamoyl]‐ADP, N6‐[N‐(6‐aminohexyl)carbamoyl]‐ATP, and N6‐[N‐(6‐aminohexyl)carbamoyl]‐AMP in yields of 13%, 12% and 17%, respectively. The occurrence of the ATP analogue may be interpreted in terms of the equilibrium, 2ADP = ATP + AMP.Coenzymic activities of the ADP analogue against acetate kinase and pyruvate kinase were 82% and 20%, respectively, relative to ADP and those of the ATP analogue against hexokinase and glycerokinase were 63% and 87%, respectively, relative to ATP. These analogues were bound to CNBr‐activated soluble dextran through their terminal amino group to give an immobilized ADP and an immobilized ATP, each of which was recycled in a system comprising acetate kinase and hexokinase, and when placed in a membrane reactor together with the enzymes, functioned as an immobilized coenzyme continuously yielding glucose 6‐phosphate.A series of chemically defined affinity adsorbents were obtained by coupling these analogues to CNBr‐activated Sepharose, and were used to separate the enzymes in a mixture of hexokinase, pyruvate kinase, phosphoglycerate kinase, lactate dehydrogenase, and alcohol dehydrogenase.
Metabolic Behaviour of Nonhistone Chromosomal Proteins in Proliferating and in Resting Fibroblasts
Authors: DJONDJUROV Lalju Petrov , IVANOVA Emilia Christova , TSANEV Roumen Georgiev 
Published: March 2005

The metabolism of nonhistone chromosomal proteins was studied in two lines of cells showing a different degree of contact inhibition: human diploid fibroblasts, which are easily contact‐inhibited, and Chinese hamster fibroblasts, which had been made to stop proliferating by fasting. By following the 3H/14C ratio of [3H]tryptophan‐labelled nonhistone chromosomal proteins and [14C]thymidine‐labelled DNA in chase experiments three main groups of these proteins could be detected with respect to their metabolic behaviour: (a) a metabolically stable group which is acid‐insoluble and represents the bulk of nonhistone chromosomal proteins in proliferating cells; this group is conserved when the cells enter a resting phase; (b) a metabolically labile group which is acid‐insoluble and is observed as a minor fraction in proliferating cells; (c) a metabolically labile group which is acid‐insoluble and accumulates in resting cells; this fraction is much larger in contact‐inhibited cells.Stimulation of cell proliferation by trypsinization decreases the amount of nonhistone chromosomal proteins in resting cells to the basic level observed in proliferating cells.
Thermophilic Polynucleotide Phosphorylase from Thermus thermophilus
Authors: HISHINUMA Fumio , HIRAI Keiko , SAKAGUCHI Kenji 
Published: March 2005

A thermophilic polynucleotide phosphorylase lacking polynucleotide phosphorolytic activity was purified from Thermus thermophilus HB‐8 strain.The enzyme is an altered form of the native polynucleotide phosphorylase, probably attacked by the proteinase(s) of this extreme thermophile during the purification process. This modified enzyme lacks phosphorolytic activity to poly(A) while retaining weak activity to phosphorolyse tetranucleotides or hexanucleotides. The purified enzyme was shown to be homogeneous by electrophoretic analysis in polyacrylamide gel. This enzyme had a molecular weight of 190000 as calculated both from electrophoresis on polyacrylamide gel and from the Stokes' radius derived from the gel filtration pattern and the sedimentation coefficient. The enzyme was separated into three polypeptide chains by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate; their molecular weights were calculated to be 92000, 73000 and 35000.The enzyme was thermophilic and thermotolerant, exhibiting its maximal activity at 70 °C. The four ribonucleoside diphosphates (ADP, GDP, UDP and CDP) were polymerized to the extent of 7‐S size.
Translation of Enzymically Decapped Messenger RNA
Authors: ABRAHAM K. Abraham , PIHL Alexander 
Published: March 2005

An enzymic procedure was used to remove the 7‐methylguanosine diphosphate moiety at the 5′ ends of rabbit hemoglobin mRNA and mouse immunoglobulin light‐chain mRNA. Evidence was obtained that the procedure, which involves the use of polynucleotide kinase, does not result in any further degradation of the mRNA.The enzymically decapped mRNA was as effective as untreated mRNA in supporting protein synthesis in a wheat germ system. This was the case over a wide range of mRNA concentrations and over a considerable period of time. The presence in the incubation mixture of S‐adenosylhomocysteine, an inhibitor of methylation, did not affect the results.The data indicate that the presence of a 7‐methylguanosine diphosphate residue at the 5′ end of mRNAs is not an obligatory requirement for translation in eucaryotic systems.
Primary Structure of Human Fibrinogen
Authors: GÅRDLUND Bengt , HESSEL Birgit , BLOMBÄCK Birger , MARGUERIE Gerard , MURANO Genesio 
Published: March 2005

Five disulfide‐containing fragments obtained from cyanogen‐bromide‐cleaved human fibrinogen (N‐DSK, Hi2‐DSK, Ho1‐DSK, Ho2‐DSK and Ho3‐DSK) have been characterized in this report. The complete primary structure of the ‘N‐terminal disulfide knot’, N‐DSK, has been elucidated earlier. The individual chains of the fragments composed of more than one chain have been purified following reduction and alkylation.Hi‐2‐DSK, Mr 26600, a one‐chain fragment with one intra‐chain disulfide bond is derived from the Aα chain of fibrinogen. Ho1‐DSK, Mr 43000, consists of five chains linked together by six disulfide bridges. The largest chain of Ho1‐DSK, Mr 21000, is derived from the γ chain of fibrinogen, a smaller chain, Mr 5900, from the Aα chain and the three remaining, Mr 5800, 4300 and 2200, from the Bβ chain. Ho2‐DSK, Mr 5400, is derived from the Bβ chain of fibrinogen and constitutes a single‐chain peptide with one disulfide bond. Ho3‐DSK, Mr 7400, consists of two chains connected with one disulfide bridge. Amino acid analysis of the disulfide knots and their individual chains is presented. Partial amino acid sequence of Hi2‐DSK, the four larger chains of Ho1‐DSK and Ho2‐DSK and the complete sequence of the smallest chain of Ho1‐DSK is shown.All disulfide knots except N‐DSK were found to be monomeric. One mole of N‐DSK and two moles of each of the other disulfide knots are present per mole of fibrinogen. These five disulfide knots account for all disulfides in human fibrinogen (fraction 1–4). The location of the disulfide knots in the fibrinogen molecule and the arrangement of the disulfide bonds is discussed.
Nucleolar Chromatin in Chinese Hamster Ovary Cells
Authors: BACHELLERIE Jean‐Pierre , NICOLOSO Monique , ZALTA Jean‐Pierre 
Published: March 2005

A stepwise physico‐chemical dissection of nucleoli isolated from Chinese hamster ovary cells has allowed the isolation of actively transcribed regions of chromatin, highly enriched in ribosomal genes. Nucleolar chromatin could be separated into its various topographical components. This separation should improve our knowledge of the structure/function relationships in chromatin as the fine structure of the nucleolus is very sensitive to changes in ribosomal RNA synthesis.The spatial distribution of ribosomal genes among the various chromatin areas of the nucleolus was assayed by hybridization with rRNA. In exponentially growing Chinese hamster ovary cells ribosomal genes are predominantly located in the intranucleolar stretches of nucleolus‐associated chromatin. However, ribosomal sequences represent but a very minor part (less than 1 %) of intranucleolar DNA. In an attempt to improve the analysis of the actively transcribed chromatin areas, intranucleolar chromatin was submitted to additional fractionation steps involving, among others, isopycnic separation in metrizamide gradients. In these latter conditions a small subset of intranucleolar chromatin banded with preribosomal ribonucleoprotein. In this subfraction, about one‐third of the DNA sequences corresponded to preribosomal RNA matrix units. The yield of recovery of this intranucleolar chromatin subfraction being closely related to the level of transcriptional activity of ribosomal genes, it appears likely that this separating procedure depends on the presence of ribosomal RNA transcription complexes and could, therefore, represent a general procedure for the isolation of actively transcribed areas from chromatin.
The Reversible Inhibition of Gluconeogenesis in Kidney Cortex by Diazenedicarboxylic Acid Bis(N,N‐dimethylamide)
Authors: PILLION Dennis , LEIBACH Frederick H. , ROCHA Hector , VON TERSCH Francis J. , MENDICINO Joseph 
Published: March 2005

Gluconeogenesis in rat kidney cortex slices was reversibly inhibited by diazenedicarboxylic acid bis(N,N‐dimethylamide). The rate of formation of glucose from pyruvate, malate, succinate and D‐fructose decreased at least 3‐fold in the presence of this inhibitor, commonly called diamide. The extent of inhibition of gluconeogenesis from pyruvate was dependent on the concentration of diamide and closely correlated with the degree of inhibition of protein kinase. The inhibition of gluconeogenesis and protein kinase were completely reversed after removal of the inhibitor. The effects of diamide on particulate enzymes which may be involved at rate‐limiting steps in transport processes which support gluconeogenesis in renal tissue was examined. Several enzymes present in kidney brush border and microsomal membrane preparations were inhibited by diamide. Na+, K+‐dependent ATPase and glucose‐6‐phosphatase were inhibited, but Mg2+‐dependent ATPase, alkaline phos‐phatase and y‐glutamyl transpeptidase, which are also present in these same subcellular fractions, were not inhibited by diamide. Based on the results obtained in these studies it is proposed that diamide may inhibit gluconeogenesis at rate‐limiting steps catalyzed by enzymes which are regulated by protein kinase. These enzymes, in turn, appear to play a role in the transfer of substrates across the plasma membrane.The mechanism of inhibition of protein kinase by diamide was also investigated. Protein kinase present in a wide variety of rat tissues as well as phosphorylase kinase and pyruvate dehydrogenase kinase was inhibited by diamide. Kinetic studies showed that the inhibition of protein kinase by diamide was non‐competitive with respect to Mg2+, ATP, protein substrates and cyclic AMP. Sedimentation studies in sucrose‐density gradients containing histone and cyclic AMP showed that diamide inhibited both the intact and dissociated forms of protein kinase. These results support earlier observations which showed that diamide did not bind at the active site of protein kinase, but instead formed inactive complexes by binding to hydrophobic areas on the surface of the enzyme.
Further Evidence that Elongation Factor 1 Remains Bound to Ribosomes during Peptide Chain Elongation
Authors: GRASMUK Hans , NOLAN Robert Dwyer , DREWS Jürgen 
Published: March 2005

This paper describes three types of experiments which indicate that the binding sites for elongation factor 1 (EF‐1) and elongation factor 2 (EF‐2) on ascites cell ribosomes are not identical and perhaps not even overlapping. The experimental evidence presented includes direct competitive binding of labeled elongation factors to ribosomes as well as the influence of pokeweed antiviral protein and Escherichia coli anti‐L7/L12 proteins on the binding and function of the two factors. It is further shown that EF‐1/β from Artemia salina does not function in displacing EF‐1 from mouse ascites tumor cell ribosomes.These results also support our recently proposed model that EF‐1 remains bound to the ribosome during the peptide chain elongation cycle.