bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA Fragment Conformations
Authors: TRAN‐DINH Son , NEUMANN Jean‐Michel , HUYNH‐DINH Tam , GENISSEL Bernadette , IGOLEN Jean , SIMONNOT Guy
Published: July 2010

The helix‐coil transition, conformations and dynamic structures of d(A‐C‐A‐T‐G‐T) we re investigated as a function of temperature by 1H‐NMR spectroscopy at various frequencies (400, 276, 250 arid 90 MHz). Imino exchangeable protons of the d(A‐C‐A‐T‐G‐T) duplex were observed between 5°C and 25°C at 276 MHz. The results suggest that the fraying process mainly consists of two stages: the extreme dA · cT base pairs open at first; the internal dG · dC and central dA · dT base pairs then open simultaneously at higher temperatures. This conclusion was supported by the midpoint temperature measurements: at1/2 value of 32 2 °C was obtained for the extreme residues; the same t1/2 value of 43± 3 °C was obtained for the internal and central base pairs. The helix and coil proportions and the dissociation constant were determined as a function of temperature from the $=f(t) curves corresponding to the non‐exchangeable protons of the internal and central residues. An enthalpy of 150 kJ/mol was found for the helix‐coil transition. At 400 MHz, all the non‐exchangeable proton resonances were assigned and most of the coupling constants were determined. The H‐1' resonance of the dC residue is particularly broad compared with that of the other H‐' protons at I> 42°C. This was attributed to the fact this proton is located in the proximity of the hydrogen bond between C= 0 of cytidine and H2 of guanine. Several sugar protons, in particular H‐2', exhibited large chemical shift variations (between 20–72°C) which decrease from the central to the extreme residues; this seems to indicate a maximum helical distorsion for the residues. Comparison between the chemical shifts and tempereture‐dependent behaviours associated with d(A‐C‐A‐T‐G‐T) and d(A‐T‐G‐C‐A‐T) protons enabled the H‐1' and thymine H‐6 resonances of d(A‐T‐G‐C‐A‐T) to be assigned. The average sugar conformations of the d(A‐C‐A‐T‐G‐T) residues are very similar for both the helix and coil forms. The large predominance of the S conformation for all d(A‐C‐A‐T‐G‐T) residues and the insignificant chemical shift variation of the guanine 1–8 proton with increasing temperature are consistent with the B‐helical conformation.

The initial relaxation time T1 of the base protons was measured as a function of tem;perature at 90 and 250 MHz. At 250 MHz, the T1 values vary slightly with temperature (T), while at 90 MHz most of the curves of log T1=f(T−1) present a break point corresponding to the midpoint temperature for the hielix‐coil transition. The T1 values of the adenine H‐8, H‐2 and thymine H‐6 protons are longer for the extreme than for the internal residues whatever the frequency and temperature. The correlation time for the coil form was determined at high temperatures from the proton TI ratios, T1 (90 Hz)/Tl (250 Hz); tc= 0.2 ns at 65°C. For the helix state, the mobility appears to be different for the purine and pyrimidine bases: the former show a greater base‐sugar flexibility and the latter a stronger anisotropic character.
Species Specificity of Promoter Recognition by RNA Polymerase and Its Transfer by the Sigma Factor
Authors: ERNST Heinrich , HARTMANN Guido R. , DOMDEY Horst
Published: July 2010

RNA polymerase holoenzyme from Micrococcus luteus synthesizes in vitro a run‐off transcript of 85 nucleotides from a DNA fragment containing part of gene E of bacteriophage 174. This RNA starts with GTP as the 5' terminus 18 nucleotides downstream from the start of gene E on the viral (+)strand. Transcription does not occur when the fragment is cleaved 36 nucleotides upstream of the initiation site. No transcript is obtained with RNA polymerase core or holoenzyme from Escscherichia coli. Other DNA fragments containing the three major E. coli promoters of øx174 are transcribed by both enzymes although much less efficiently by M. luteus RNA polymerase. When subunit sigma in E. coli RNA polymerase is replaced by sigma from M. luteus the resulting hybrid enzyme actively transcribes the DNA fragment containing the inner region of gene D with formation of the same run‐off transcript which is obtained with M. luteus holoenzyme. In the presence of sigma from E. coli this RNA is not synthesized. The hybrid enzyme also transcribes it DNA fragment containing the gene A promoter of øx174 with even higher efficiency than RNA polymerase holoenzyme from E. coli.
Substrate Specificity Studies on a Calf Thymus Nuclear Phosphoprotein Kinase
Authors: SCULLEY Thomas B. , MACKINLAY Antony G.
Published: July 2010

the protein kinase activity associated with the phosphoprotein fraction of calf thymus nuclei has been examined for its ability to phosphorylate exogenous phosphoprotein substrates. β‐Casein and phosvitin are both efficient phosphate acceptors. Phosphorylation of αs1 and β‐caseins was shown, indirectly, to occur at threonyl‐49 and threonyl‐41 respectively. Both threonyl residues occur within the sequence Thr‐Glu‐Asp.

αs1‐Casein becomes a much better substrate for the kinase when partially dephosphorylated. A similar response is shown by a phosphopeptide containing the αs1‐casein phosphate cluster and is due to a new phosphorylation site becoming available. Efficient phosphorylation of β‐casein requires that the phosphate cluster (residues 15 ‐ 19) be intact and results are presented which are consistent with there being a similar requirement for phosphorylation of the site created in αs1‐casein by partial dephosphorylation.

Comparison of genetic variants of β‐casein as phosphate acceptors for the kinase shows that the presence of lysyl residues close to the phosphorylation site markedly depresses the rate of phosphorylation. Maleylation of β‐ casein increases the rate of phosphorylation for all of the variants tested, although to varying extents. Treatment of maleylated β‐casein with trypsin to remove the N‐terminal phosphopeptide inhibits phosphotylation by the kinase.

The structural determinants of β‐casein allowing it to be efficiently phosphorylated by the kinase are concluded to be the presence of a sequence surrounding the phosphorylation site, which is rich in acidic amino acid residues and from which basic residues are absent. The acidic phosphate cluster of β‐casein also promotes phosphorylation either by interacting directly with the enzyme or through its influence on the conformation of β‐casein.
Relation of Oxidation of Long‐Chain Fatty Acids to Gluconeogenesis in the Perfused Liver of the Guinea Pig:
Authors: TUTWILER Gene F. , BRENTZEL H. James
Published: July 2010

The potent, specific inhibitor of long‐chain fatty acid oxidation, 2‐tetradecylglycitiic acid (McN‐3802), at 10μM totally abolished ketogenesis from endogenous substrates in the isolated perfused guinea pig liver. This effect was accompanied by a marked inhibition of gluconeogenesis from lactate plus pyruvate and by a shift toward a more oxidized state of the mitochondrial (3‐hydroxybutyrate/acetoacetate ratio) and cytoplasmic (lactate/pyruvate ratio) compartments.

The addition of octanoate (88–500 μM) almost completely reversed the inhibitor! etyect of 2‐tetradecyl‐ glycidic acid on gluconeogenesis. Octanoate oxidation, measured by the rate of ketogenesis, was not inhibited. This protective effect of octanoate against inhibition of gluconeogenesis by 2‐tetradecylglycidic acid was seen even though in some experiments the mitochondrial redox state remained two to three trines the magnitude observed prior to octanoate addition.

Gluconeogenesis from 4 mM glycerol was not inhibited and gluconeogenesis from 3 inM propionate was only slightly inhibited by 2‐tetradecylglycidic acid.

Thus, it would appear that fatty acid oxidation in guinea pig liver is essential for maintaining maximal rates of gluconeogenesis, especially from substrates dependent on pyruvate carboxylation for conversion to glucose.

A single dose of 2‐tetradecylglycidic acid (orally, 10–25 mg/kg) given to guinea pigs previously fasted 72 h produced a highly significant decrease of total ketones, of the 3‐hydroxybutyrate/acetoacetate ratio and of plasma glucose. A smaller hypoglycemic effect was seen when the drug was administered to animals fasted for only 24 h or 48 h.

It appears from evidence in vivo and in vitro that the guinea pig and rat respond similarly to inhibition of fatty acid oxidation. This may be important since it has been suggested that the role of fatty acid oxidation in glucose synthesis is markedly different in these two species.
Residual Analysis in Determining the Error Structure in Enzyme Kinetic Data
Authors: LITTLE David I. , POAT Peter C. , GILES Ian G.
Published: July 2010

The nature of the experimental error in the initial velocities of an enzyme‐catalysed reaction is required ifmean‐ ingful least‐squares regression is to be applied. When a rate equation more complex than that of Michaelis and Menten is to be solved least‐squares techniques are the method of choice and so determination of the error structure becomes mandatory. The use of residual analysis and Tukey'sT statistics to determinc thc weights to use are described. This method has the advantage of requiring no additional experimentation over than required for the primary investigation. Using data obtained for carcinus meanas phosphofructokinase, the variance was found to increase with velocity and was approximated by either an empirical power function. var (vi)αv1.8i or by the function, var (viα0.007 +v2i. The latter function is preferred and suggests that the data contains both a constant absolute error and a constant percentage error component.
Interaction between Monobactams and Streptomyces R61 dd‐Carboxypeptidase
Authors: GEORGOPAPADAKOU Nafsika H. , SMITH Sandra A. , CIMARUSTI Christopher M.
Published: July 2010

The monobactams are a novel family of monocyclic β‐lactam antibiotics characterized by the 2‐oxoazetidine‐1‐sulfonic acid moiety. A series of monobactams bind covalently to the streptomyces R61 DD‐carboxypeptidase in a manner similar to that for bicyclic β‐lactams, especially cephalosporins. The similarity of interaction was established by the following criteria: inhibition of binding by diisopropylfluorophosphate and α‐dicarbonyls; stoichiometry of binding; similarity of partial proteolysis products of radiolabelled enzyme: rates of release of bound β‐lactams; nature of hydrolysis and hydroxylaminolysis products.
Dolichol‐Dependent Synthesis of Chitobiosyl Proteins and Their Further Mannosylation
Authors: HOFLACK Bernard , DEBEIRE Philippe , CACAN Rene , MONTREUIL Jean , VERBERT Andre
Published: July 2010

Incubation of whole lymphocytes with UDP‐N‐acetyl [3H]glucosamine used as the only preeursor leads to the formation of dolichyl diphosphate [3H]chitobiose, DolPP‐(GlcNAc)2, and dolichyl diphosphate N‐acetyl‐ [3H]glucosamine, DolPP‐GlcNAc. Although very few dolichyl diphosphate oligosaccharidcs are formed, a high level of radioactivity is recovered with proteins and has been characterized, using hydrazinolysis procedure, as [3H]chitobiosyl and N‐acetyl[3H]glucosaminyl units. Addition of tunicamycin inhibits, to the same extent, both the synthesis of DoIPP‐(GIcNAc)1−2 and the incorporation of the N‐acetyl [3H] glucobaminyl residues onto proteins, indicating that these carbohydrate units are transferred onto proteins acceptors from their dolichol derivatives. Chase experiments have indicated that, in fact, the DolPP‐(GlcNAc)1−2 were utilized in two ways: either their transfer onto proteins or their degradation into water‐soluble saccharidic matcrial. Moreover, the transfer reaction appears to be a slow process compared to the degradation since the radioactivity chased from the DoIPP‐(GIcNAc)1−2 is not recovered on proteins. This fact allows to show that part of the [3H]chitobiose previously bound to proteins is further converted into oligomannosidic glycans in the presence of GDP‐mannose. This direct niannosylation of chitobiosyl‐proteins may represent a second route for the N‐glycosylation of proteins.
One‐Electron Photoreduction of Bacterial Cytochrome P‐450 by Ultraviolet Light
Authors: BAZIN Marc , PIERRE Jacqueline , DEBEY Pascale , SANTUS René
Published: July 2010

The intramolecular photoreduction of the bacterial cytochrome P‐450 demonstrated by the formation of the complex of reduced cytochrome m with camphor and CO has been studied by photolysis with a laser flash at 265 nm. This photoreduction of the heme follows the photoionization of a proximal aromatic amino acid residue and especially tryptophan in the nanosecond time scale as evidenced by the N2O effect on the reduction yield. However most of the hydrated electrons released in the solution do not react with the ferric porphyrin. In agreement with steady state studies, the quantum yield for formation of the complex of reduced cytochrome m with camphor and CO is proportional to the concentration of the high‐spin species. The low‐spin ferric porphyrin is thus not reducible by photoejected electrons. It is also shown that the growth kinetics of the transient absorbance at 446 nm characterizing the complex of reduced cytochrome m with camphor and CO correspond to a structural change affecting the protein and not to the formation of the complex of CO with the camphor‐bound reduced cytochrome m which occurs on a shorter time scale in the present experimental conditions.
Lactose Carrier Protein of Escherichia coli
Authors: WRIGHT J. Keith , SCHWARZ Heinz , STRAUB Edith , OVERATH Peter , BIESELER Barbara , BEYREUTHER Konrad
Published: July 2010

A procedure for the reconstitution of the lactose carrier protein, a galactoside: proton symporter in Escherrchiu coli, is described. Starting from cytoplasmic membranes derived from carrier‐overproducing strains, essentially all proteins including 89% of the carrier are solubilized by a mixture of dodecyl/tetradecyl polyoxyethylene (n = 9.5) ether and dodecyl 0‐β‐D‐maltoside. In the micellar state the carrier binds substrates with reduced affinity. Addition of E. coli phospholipids and removal of detergents by a hydrophobic column yields small vesicles (50–60‐nm diameter). In these vesicles, about 70% of the carrier is recovered and reconstituted carrier is identical to native carrier in terms of substrate binding. After fusion of the small vesicles into larger vesicles (1–5 μm), rapid countertransport of galactosides is demonstrated. Attempts to show active galactoside transport by the imposition of artificial electrical potential or pH gradients were unsuccessful, most likely because the reconstituted vesicles are in fact highly permeable to protons.
Circular‐Dichroism Study of the Interaction of Aspartate‐Aminotransferase Isoenzymes with a Coenzyme Analog
Authors: CAROTTI Daniela , RIVA Francesca , SANTUCCI Roberto , ASCOLI Franca , FASELLA Paolo
Published: July 2010

The interaction between a coenzyme derivative, 4′‐N‐(2,4‐dinitro‐5‐fluorophenyl)‐pyridoxamine 5′‐phosphate, and the apoenzyme of cytoplasmic and mitochondrial aspartate aminotransferase, was studied by circular dichroism.

The specific complexes initially formed were characterized by their circular dichroic spectra. The spectra indicate that the complex is very probably the same for the two isoenzymes. In contrast the spectra recorded during further reaction, in agreement with previous results, monitor different reaction paths and characterize the irreversible labeling at the active site of the cytoplasmic enzyme and regeneration of pyridoxal 5′‐phosphate in the mitochondrial enzyme.

By following circular dichroic changes in the mitochondrial enzyme, initial kinetic characterization of the cleavage of 4′‐N‐(2,4‐dinitro‐5‐fluorophenyl)‐pyridoxamine 5′‐phosphate to form pyridoxal 5′‐phosphate at the active site, is provided.
Tryptophan 5‐Monooxygenase from Mouse Mastocytoma P815
Authors: NAKATA Hiroyasu , FUJISAWA Hitoshi
Published: July 2010

Tryptophan 5‐monooxygenase was purified 880‐fold with a 48% yield from mouse mastocytoma cells (P815) by only a one‐step purification procedure of pteridine affinity chromatography. The specific activity of the final preparation was 5280 nmol min−1mg−1. It gave a single protein band on polyactylamide gel electrophoresis in the absence and presence of sodium dodecylsulfate. The molecular weight of the enzyme was determined to be 270000 by gel filtration and 280000 by gradient polyacrylamide gel electrophoresis. Sodium dodecylsulfate/ polyacrylamide gel electrophoresis revealed the enzyme to be composed of identical subunits with a molecular weight of 53000. Tetrameric structure of the enzyme was suggested by cross‐linking studies using dimethyl suberimidate as a bifunctional reagent. The isoelectric point of the enzyme was estimated to be 6.0. Amino acid analysis showed a residue composition similar to that reported for rat liver phenylalanine 4‐nionooxygenase. The enzyme activity was stimulated approximately fivefold by preincubation with dithiothreitol and Fe2+. The purified enzyme had an activity of phenylalanine hydroxylation and also a weak activity of tyrosine hydroxylation. The kinetic properties of the enzyme are also presented.
The Role of Lactoperoxidase‐H2O2 Compounds in the Catalysis of Thyroglobulin Iodination and Thyroid Hormone Synthesis
Authors: COURTIN Françoise , DEME Danielle , VIRION Alain , MICHOT Jean Luc , POMMIER Jacques , NUNEZ Jacques
Published: July 2010

Lactoperoxidase catalyzes successively, as thyroid peroxidase, the iodination of several tyrosyl residues of thyroglobulin and the coupling of some of them to thyroid hormones. We show the following results in this paper.

The iodination reaction is catalyzed by lactoperoxidase compound I whereas lactoperoxidase ‘compound II’ catalyzes the coupling reaction. Lactoperoxidase compound III catalyzes neither reaction.

Titration of lactoperoxidase ‘compound II’ by ferrocyanide showed that this enzyme‐H2O2 species is two oxidizing equivalents above the native enzyme, and therefore constitutes a second form of lactoperoxidase ‘compound I’. These two forms of lactoperoxidase ‘compound I’ differ probably from one another in the localization of one of the two oxidizing equivalents either on porphyrin ring: π‐compound I (compound I), or on the apoprotein: R°‐compound 1 (‘compound II’). The difference in specificity between π‐compound I and R°‐compound I in catalyzing the iodination and the coupling reaction respectively, might therefore depend differences both in the distribution of the two oxidizing equivalents and in the structure of the substrates.

In the presence of free diiodotyrosine, a halophenol which stimulates at very low concentrations the coupling reaction but has no effect on the iodination reaction, the transfer of electrons between the substrate and lactoperoxidase R°‐compound I is nearly stoichiometric: 1 mole of hormone is produced for each mole of lacto‐peroxidase R°‐compound I. Thus free diiodotyrosine seems to play the role of a very specific co‐factor of the coupling reaction.

Iodide and SCN− also stimulate the coupling reaction when the H2O2/enzyme ratio used to prepare lactoperoxidase R°‐compound I is higher than one. They do so by preventing the accumulation of the inactive derivative of lactoperoxidase, compound III, which is formed in the presence of excess H2O2. Iodide prevents the formation of compound III whereas SCN− very rapidly decomposes compound III back to the native enzyme.
Influence of Ca2+and Trifluoperazine on the Structure of Calmodulin
Authors: KREBS Joachim , CARAFOLI Ernesto
Published: July 2010

Ca2+‐induced conformational changes of calmodulin under a variety of different experimental conditions have been studied by1H‐nuclear magnetic resonance techniques. The assignment for Tyr‐99 has been corrected. Ca2+titration performed at pH 7.5 and > 9.5 apparently induces a different sequence of the protein folding process as can be monitored by the resonances of His‐107. These two structural forms cannot be interconverted. The phenylalanine residue(s) responsible for the resonances at 6.47 ppm (Ca2+‐free form) and 6.64 ppm (Ca2+‐ saturated form) respectively, are apparently located close to Ca2+‐binding sites III and IV. This can be recognized from nuclear Overhauser enhancement and Gd3+‐broadening techniques. Gd3+‐broadening experiments classify Ca2+‐binding site IV as the site with the highest Gd3+/Ca2+‐exchange rate. The antipsychotic drug trifluoperazine, which is known to bind to calmodulin in a calcium‐dependent way [Levin, R. M. and Weiss, B. (1977)Mol. Pharmacol.13, 690–697], has been found to induce a conformational change of the Ca2+‐saturated form of cal‐ modulin. The methionine and phenylalanine residues were especially affected. Possible binding site(s) for trifluoperazine are discussed.
Molecular Properties of Lipoprotein Lipase
Authors: OLIVECRONA Thomas , BENGTSSON Gunilla , OSBORNE James C.
Published: July 2010

The monomer molecular size of bovine lipoprotein lipase was evaluated by sedimentation equilibrium measurements and by gel permeation chromatography in 6 M guanidinium chloride. To establish molecular weight unequivocally we determined the partial specific volume (v̄) experimentally. This was done by analyzing equilibrium concentration profiles from analytical ultracentrifugation in 6 M guanidinium chloride using buffers made up in H2O and 2H2O. The combined results gave a v̄ of 0.71 ± 0.007 ml/g and a molecular weight of 41700 ± 1000 for monomeric bovine lipoprotein lipase. This value did not change upon mild tryptic digestion; the elution volume upon gel permeation chromatography in 6 M guanidinium chloride was also unaffected by treatment with trypsin.

Sedimentation equilibrium measurements of the trypsin‐treated material in the presence of reducing agents gave limiting molecular weights of 19000 and 23000, demonstrating that mild trypsin digestion cleaved lipoprotein lipase into two polypeptide chains of similar size held together by disulfide bonds. Mild trypsin digestion also resulted in a loss of secondary structure as determined by circular dichroic measurements. Discussion centers around the correlation between these effects of trypsin on the molecular properties of lipoprotein lipase and the previously reported effects on the kinetic properties of the enzyme.
Purification and Partial Characterization of Two Acidic Proteases from the White‐Rot Fungus Sporotrichum pulverulentum
Authors: ERIKSSON Karl‐Erik , PETTERSSON Bert
Published: July 2010

Two acidic proteases from the white‐rot fungus Sporotrichum pulverulentum have been purified and partially characterized. The enzymes were purified in four steps, protease I 152‐fold and protease II 127‐fold. The purity of the enzymes was investigated by analytical isoelectric focusing and by dodecylsulfate/polyacrylamide gel electrophoresis.

The isoelectric points of protease I and protease II were at pH 4.7 and 4.2 respectively. The molecular weights were 28000 and 26000 and the pH optima 5.0 and 5.2. Both enzymes were inhibited by heavy metal ions such as Ag+, Hg2+ and to some extent also by Cu2+. Partial inhibition was also observed with EDTA and αα′‐ dipyridyl.

The specificities of protease I and II were investigated using human fibrinopeptide A as substrate. Protease splits off the arginine in the C‐terminal position, while protease II splits at the carboxyl side of both Phe‐8 and Leu‐9 in fibrinopeptide A.

A physiological effect of the two proteases has also been demonstrated. Thus, if a culture solution from S. pulverulentum grown on cellulose was treated with the individual proteases or a mixture of the enzymes a considerable increase in endo‐l,4‐β‐glucanase activity was obtained. It may be that the endo‐glucanases are produced in a zymogenic form and activated by the proteases. However, other explanations for the phenomenon are also possible.
The FI‐Gene Product of Bacteriophage
Authors: BENCHIMOL Sam , BECKER Andrew
Published: July 2010

The FI gene product of bacteriophage lambda has been purified extensively using a biochemical assay that measures assembly of lambda phage particles in vitro. The molecular weight of the native protein was estimated to be 21700 with an s20,w of 2.1 S and a Stokes radius of 2.5 nm. The molecular weight in dodecylsulfate was estimated to be 19000. The protein is highly acidic with an isoelectric point less than 4.1.
Purification and Characterization of β‐Galactoside (α2→6)sialyltransferase from Rat Liver and Hepatomas
Authors: MIYAGI Taeko , TSUIKI Shigeru
Published: July 2010

Asialofetuin sialyltransferase from Triton X‐100 extracts of rat liver was resolved by phosphocellulose chromatography into two fractions, designated I and II in order of elution. When previously treated with Arthrobacter ureafaciens neuraminidase, fraction I eluted at about the same position as II while no alteration occurred in II. Primary rat hepatomas contained only a single asialofetuin sialyltransferase, identical to fraction I in chromatographic behavior.

Transferases I and II were purified to near homogeneity. Transferase II, as well as neuraminidase‐treated I, could be sialylated auto‐catalytically, indicating that the lack of sialic acid in II is not due to the lack of a sialic‐acid‐accepting site. Both enzymes formed an (α2→6)sialylgalactoside linkage with asialo‐glycoproteins of the glycosylamine‐type and with lactose, and were indistinguishable immunologically. Nevertheless, the transferases exhibited different molecular weights of 37000 (I) and 43000 (II). When heated at 50°C, transferase I lost half its original activity within 20 min while II was scarcely inactivated. Kinetically, transferase I showed three‐times higher affinity than II for CMP‐N‐acetylneuraminic acid and for desialylated plasma membrane.

Asialofetuin sialyltransferase was also purified from primary rat hepatoma. The purified enzyme was identical to transferase I in every respect examined. We conclude that hepatomas contain transferase I but lack transferase II.
The ppp(A2′p)nA and Protein Kinase Systems in Wild‐Type and Interferon‐Resistant Daudi Cells
Authors: SILVERMAN Robert H. , WATLING Diane , BALKWILL Frances R. , TROWSDALE John , KERR Ian M.
Published: July 2010

Daudi cells, a human lymphoblastoid line, are exceptionally sensitive to the growth inhibitory effects of interferon, 1 unit/ml being sufficient to inhibit cell growth. In addition, interferon treatment of these cells severely inhibits the incorporation of exogenous thymidine into DNA and causes cells to accumulate in the G1(G0) at the expense of the S phase of the cell cycle. The possible involvement of ppp(A2′p)nA (n= 2 to 4) in these effects has been investigated. No (< 1 nM) ppp(A2′p)nA or (A2′p)nA or alternative products of the ppp(A2′p)nA synthetase [e.g. NAD (2′pA)2] were detected in interferon‐treated cells. In addition no evidence was obtained for the occurrence of ppp(A2′p)nA‐mediated ribosomal RNA cleavage in these cells even after several days of treatment with relatively high doses of interferon.

A line of Daudi cells which is resistant to all three of the above effects of interferon was selected. The wild type and resistant lines were compared with respect to the ppp(A2′p)nA and interferon and double‐stranded RNA (dsRNA)‐mediated protein kinase systems. The resistant line was not receptor‐negative as it responded to interferon by the production of elevated levels of the ppp(A2′p)nA synthetase similar to those observed in extracts from wild‐type cells. There was no detectable difference between the lines in the levels of the (2′‐5′)phosphodiesterase responsible for the degradation of ppp(A2′p)nA. There was, however, about a twofold increase in the ppp(A2′p)nA‐dependent endoribonuclease activity in response to interferon with extracts from the wild‐type but not the resistant cells. In addition, although the dsRNA‐dependent protein kinase activity increased in both types of cell there was a striking reduction in the level of protein phosphorylation in general in response to interferon with material from the wild‐type but not the resistant cells.
An Electrophoretic Characterization of the Glucocorticoid Response of the Fu5‐5 Rat Hepatoma Cell Line
Authors: PHELPS David S. , LITWACK Gerald
Published: July 2010

In this study we have further characterized the response of liver‐derived cells to glucocorticoid treatment using the rat hepatoma, Fu5‐5. Using two‐dimensional electrophoresis we have examined changes in the synthetic rates of cytosol proteins following glucocorticoid administration by using [35S]methionine. We have also demonstrated changes in the incorporation of 32P by several cytosol proteins after hormone treatment. One of these changes occurs within 1 h of hormone treatment. We were unable to detect any changes in the nuclear protein content of Fu5‐5 cells after glucorticoid treatment. We then compared the glucocorticoid‐regulated changes in cytosol protein synthesis in Fu5‐5 cells with those of the H35 rat hepatoma, monolayer cultures of rat hepatocytes and the response of rat liver in vivo. This examination revealed that although nearly all of the eighteen hormonally responsive proteins appeared to be present in all of the cell types, only three were responsive to glucocorticoid in more than one system. Moreover, the response patterns were often reversed in different cell types, the same protein being induced in one cell type and repressed in another.
Purification and Substrate Specificity of Bovine Liver‐Ferrochelatase
Authors: TAKETANI Shigeru , TOKUNAGA Rikio
Published: July 2010

Bovine ferrochelatase from liver mitochondria was purified 1434‐fold with a 31% yield to apparent homogeneity by a procedure involving solubilization, ammonium sulfate fractionation and blue Sepharose CL‐6B chromatography. The molecular weight of the homogeneous protein was 42500 when measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 200000 was obtained by Sepharose 6B gel filtration. The specific activity for mesoheme synthesis was 413nmol × mg protein−1× min−1 at 37°C and for protoheme synthesis 88 nmol × mg−1× min−1. The optimum pH was 8.0 and Km values for the substrates were: protoporphyrin IX, 54 μM; mesoporphyrin IX, 46 μM; iron with protoporphyrin IX, 46 μM, iron with mesoporphyrin IX, 44 μM. The purified enzyme inserted iron into the following dicarboxylic porphyrins in descending order: meso‐, deutero‐, 2,4‐diacetyldeutero‐, hemato‐, and protoporphyrin IX. This did not take place in the case of 2,4‐diformyldeuteroporphyrin IX. Porphyrin c was converted to only a negligible amount of heme c, and coproporphyrin III did not act as a substrate at all. When metal specificity was examined, the highest value was obtained with zinc, decreasing in order with iron, cobalt and nickel. The enzyme failed to catalyze the insertion of copper or manganese into porphyrin. An antibody specific for the purified bovine ferrochelatase was prepared, and studies comfirmed that the synthetic activities of iron‐porphyrin, zinc‐porphyrin and cobalt‐porphyrin are ascribable to ferrochelatase.
Non‐equilibrium Thermodynamic Assessment of Redox‐Driven H+ Pumps in Mitochondria
Authors: PIETROBON Daniela , ZORATTI Mario , AZZONE Giovanni F. , STUCKI Jörg W. , WALZ Dieter
Published: July 2010

Isolated mitochondria suspended in an aerobic medium with 3‐hydroxybutyrate or succinate serving as electron donor attain a stationary state with vanishing net flow of H+ ions (state 4). Adding valinomycin to such a suspension in the presence of various concentrations of K+ ions and a weak acid system such as acetate or phosphate creates new stationary states for the mitochondria which are characterized by a constant influx of K+ ions, while the net flow of H+ ions again vanishes due to the recycling of these ions by the weak acid system.

Sufficiently low concentrations of K+ ions (less than 4 mM) cause these stationary states to last long enough for a separation of the mitochondria by centrifugation. The difference in electrochemical potential for H+ ions can then be determined by means of the partitioning of radioactively labelled markers. Suitable procedures to correct for binding of the markers are described.

It is found that, for a constant affinity of the electron in the suspending medium, electron flow and the flow of K+ ions, which indicates the flow of pumped H+ ions, are linearly dependent on the electrochemical potential difference of H+ ions. The phenomenological coefficients obtained from these correlations are discussed with respect to the contributions of additive constants in the linear relations. It is found that, under the present experimental condition, such constants most likely vanish thus yielding symmetric flow‐force relations. It is concluded that the redox‐driven H+ pumps are not tightly coupled due to molecular slipping in the pumps and that the molecular stoichiometry is 2 H+ ions/electron for coupling site I and 4 H+ ions/electron for coupling sites II and III together.
Disulfide Bridges in Tropomyosin
Authors: WILLIAMS David L. , SWENSON Charles A.
Published: July 2010

The effect of rabbit skeletal tropomyosin and tropomyosin‐troponin on the ATPase activity of actomyosin was compared for tropomyosins with the Cys‐190 disulfide bridge intact (crosslinked tropomyosin) or broken and blocked with N‐ethylmaleimide (MalNEt‐tropomyosin). Experiments were carried out at 25°C under the following solution conditions: 30 mM KCl, 5 mM MgCl2, 1 mM EGTA or 0.1 mM Ca2+, 2 mM ATP, 0.02% NaN3, 10 mM imidazole at pH 7.0 (or 10 mM Tris at pH 8.0).

At pH 7.0 both MalNEt‐tropomyosin and crosslinked tropomyosin had an inhibitory effect on the ATPase activity of actomyosin subfragment‐1, with MalNEt‐tropomyosin inhibiting more strongly than crosslinked tropomyosin.

Under the same conditions the inhibitory effect of both tropomyosins on the ATPase activity of acto‐(heavy meromyosin) was less pronounced but the inhibitory effect of MalEt‐tropomyosin was still approximately twice that of crosslinked tropomyosin at the stoichiometric ratio of tropomyosin to actin. At pH 8.0 MalNEt‐tropomyosin had an inhibitory effect on the ATPase of acto(heavy meromyosin) while crosslinked tropomyosin had a potentiating effect, demonstrating the dual effect of tropomyosin at one set of solution conditions and protein ratios.

In the presence of excess troponin and 1 mM EGTA at pH 7.0 MalNEt‐tropomyosin and crosslinked tropomyosin were indistinguishable in their ability to inhibit the ATPase of acto‐(heavy meromyosin). In the presence of excess troponin and 0.1 mM Ca2+, crosslinked tropomyosin enhanced the ATPase of acto‐(heavy meromyosin) to a slightly greater degree than MalNEt‐tropomyosin. Our results suggest that potentiation and inhibition are occurring simultaneously. We have previously shown that the presence or absence of a disulfide bridge has a profound effect on the stability of the –COOH terminal half of the molecule [Williams and Swenson (1981) Biochemistry, 20, 3856]. The results presented here indicate that the presence of the disulfide bridge in tropomyosin also affects the ATPase activity of actomyosin and suggest that MalNEt‐tropomyosin and crosslinked tropomyosin may be useful tools in studying the kinetic mechanism of skeletal muscle control.
Polymères hydrosolubles de NAD+ et d'ADP
Authors: SICSIC Sames , MAVAMBU Yakini , BRÉMONT Béatrice , LE GOFFIC François
Published: July 2010

Hydrosoluble Polymers of NAD+ and ADP. Coenzymatic Properties as a Function of their Size

Hydrosoluble polymers of NAD+ and ADP were synthesized according to a described method with some modifications. The cofactor was bound to the matrix by a spacer group of five atoms at the exocyclic adenine C‐6 amino group. Cofactor incorporations were very high. The loading of NAD+ polymers were: 420 μmol NAD+/g polymer and 330 μmol ADP ribose/g polymer arising from degradation of NAD+; the loading of ADP polymers were: 1.40 and 1.43 mmol ADP/g polymer. Each polymer obtained in a same polymerization step was fractionated in two parts by gel filtration in such a manner that these two parts had the same framework but different molecular weights. The biological properties of the fractionated polymers were compared. The small sized polymers of NAD+, assayed with four dehydrogenases, were more reduced enzymatically than the larger ones and had the higher reduction rates relative to free NAD+. In a coupled system (l‐lactate dehydrogenase, formate dehydrogenase) with coenzyme regeneration, the rate of production of l‐lactate at the steady state of the system using NAD+ polymers of small size was 80% that of the system using free NAD+. The small‐sized polymers of ADP were more phosphorylated by pyruvate kinase and creatine kinase than the larger ones. But the relative rates of transformation by pyruvate kinase of large sized polymers were higher than those of smaller ones.
Binding of tRNA in Different Functional States to Escherichia coli Ribosomes as Measured by Velocity Sedimentation
Authors: SCHMITT Marion , NEUGEBAUER Ulrike , BERGMANN Cornelia , GASSEN Hans Günther , RIESNER Detlev
Published: July 2010

The binding of initiator and elongator tRNAs to 70‐S ribosomes and the 30‐S subunits was followed by velocity sedimentation in the analytical ultracentrifuge. fMet‐tRNAfMet binds to A‐U‐G‐programmed 30‐S subunits, but not to free or misprogrammed particles. Both the formylmethione residue and the initiation factors increase the stability of the 30‐S · A‐U‐G · fMet–tRNAfMet complex. fMet–tRNAfMet is bound only to the P site of the 70‐S ribosome even in the absence of A‐U‐G. Two copies of tRNAPhe or Phe‐tRNAPhe are bound to the ribosome with similar affinity. In contrast to a recent report [Rheinberger et al. (1981) Proc. Natl Acad. Sci. USA, 78, 5310–5314], it is shown that three copies of tRNA cannot be bound simultaneously to the ribosome with binding constants higher than 2 × 104 M−1. Phe‐tRNAPhe when present as the ternary complex Phe‐tRNAPhe· EF‐Tu · guanosine 5′‐[β,γ‐methylene]triphosphate binds exclusively to the A site. The peptidyl‐tRNA analogue, acetylphenylalanine‐tRNA, can occupy both ribosomal centers, albeit with a more than tenfold higher affinity for the P site. The thermodynamic data obtained under equilibrium conditions confirm the present view of two tRNA binding sites on the ribosome. The association constants determined are discussed in relation to the mechanism of ribosomal protein synthesis.
The Effect of a Cytidine‐to‐Uridine Transition on the Stability of Escherichia coli A19 5‐S RNA
Authors: DIGWEED Martin , KUMAGAI Izumi , PIELER Tomas , ERDMANN Volker A.
Published: July 2010

We have been able to isolate several species of 5‐S ribosomal RNA from Escherichia coli A19. These molecules were separated on the basis of their differing stabilities during electrophoresis on 12% polyacrylamide gels in 7 M urea. This differing stability is shown, in one case, to be due to a different primary sequence. We have determined the sequence of the least stable of these molecules and have found only one difference to the published sequence of E. coli A19 5‐S RNA, namely a uridine in place of a cytidine at position 92. The consequent G · U base pair, formed in a normally highly stable G · C‐rich region, is responsible for a drastic reduction in the stability of the molecule. This instability leads to a less constrained, more compact molecule which thus migrates faster in electrophoresis under denaturing conditions. This species of 5‐S RNA is shown to make up 30% of the total 5‐S RNA in the 50‐S ribosomal subunits in this organism. Further structural studies were carried out using S1 nuclease digestion, sodium bisulphite modification and thermal melting analysis. All these methods indicate a 5‐S RNA drastically destabilized in parts of its secondary and tertiary structure. Finally, the ability of the variant 5‐S RNA to recognize and form a complex with its 50‐S subunit binding proteins was examined and found to be impaired.
Purification and Properties of the Coupling‐Factor ATPases F1 from Rhodopseudomonas palustris and Rhodopseudomonas sphaeroides
Authors: MÜLLER Horst , NEUFANG Hartmut , KNOBLOCH Karl
Published: July 2010

The coupling‐factor ATPases from photosynthetically grown Rhodopseudomonas palustris and Rhodopseudomonas sphaeroides were purified by the same procedure to homogeneity. Gel chromatography on Sephacryl S‐300 Superfine shortened the process of purification and improved its yield. Solubilization of the ATPase from both bacteria was found to be dependent on a specific sonication treatment of the cell suspensions, indicating a very weakly bound Fi‐ATPase in R. palustris. Depleted chromatophores could be restored in photophosphorylation and membrane‐bound ATPase activities by adding the solubilized ATPase protein. The purified enzymes did not show a markedly trypsin‐stimulated or dithiothreitol‐stimulated activity. Isoelectric focusing and chromatofocusing revealed isoelectric points of 5.0 for both F1‐ATPases. The molecular weights were determined by gel chromatography plus high‐performance liquid chromatography. Hence, we calculated a molecular weight of 350000 for both F1‐ATPases. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed five subunits for both enzymes. Kinetic parameters, regarding substrate specificity, the effect of divalent cations, Km and Ki values for the membrane‐bound and solubilized ATPases were determined.
Rise of Coenzyme A‐Glutathione Mixed Disulfide during Hydroperoxide Metabolism in Perfused Rat Liver
Authors: CRANE Denis , HÄUSSINGER Dieter , SIES Helmut
Published: July 2010

The hepatic metabolism of an externally added hydroperoxide, t‐butyl hydroperoxide, results in substantial decreases in CoASH and acetyl‐CoA and a concomitant increase in CoA‐disulfides. The principal CoA‐disulfide which is formed under these conditions was identified as CoASSG, the mixed disulfide of coenzyme A and glutathione; CoASSCoA levels were unchanged. The perturbation in the coenzyme A system was absent when the hydroperoxide was infused into livers from rats maintained on a selenium‐deficient diet. In these livers, Se‐dependent glutathione peroxidase is lowered to less than 5% of control levels; the observed effects can be attributed to flux through Se‐dependent GSH peroxidase.
α‐Adrenergic Stimulation of Glycolysis and Na+, K+‐Transport in Perfused Rat Liver
Authors: BECKER Johannes , JAKOB Andrea
Published: July 2010

Interactions between α‐adrenergic‐induced lactate production, respiration and K+ uptake of perfused rat livers were investigated. Inhibition of mitochondrial electron transport by cyanide had no effect on K+ uptake, decreased phenylephrine‐induced lactate production by 60% and suppressed extra O2 consumption by more than 90%. Ouabain did not affect α‐adrenergic‐induced respiration but inhibited K+ uptake and extra lactate production. Effects of ouabain and K+‐free medium on lactate production were additive and independent of cyanide, indicating that they were mediated by extramitochondrial mechanisms. Extra ATP utilized for K+ uptake in response to phenylephrine was apparently of glycolytic rather than mitochondrial origin. We propose that α‐adrenergic‐induced lactate production is secondary to an enhanced operation of the plasma membrane (Na+,K+)‐ATPase.
Characterization of the Secondary Structure Features of Escherichia coli, Caldariella acidophila and Mammalian Ribosomal RNA Species by Chemical Modification of Sterically Exposed Bases
Authors: CAMMARANO Piero , LONDEI Paola , BIAGINI Roberto , DE ROSA Mario , GAMBACORTA Agata
Published: July 2010

The helix content of rRNA species (Escherichia coli, Caldariella acidophila, rat liver) and the G · C content of their bihelical domains have been investigated by chemical modification of uracil and cytosine residues with probes specific for sterically exposed bases. By using radioactively labelled rRNA, G · C base pairs and the sum of A · U plus G · U base pairs have been quantified assuming that they are numerically identical with the unreactive cytosine and uracil rings, respectively. Exposed uracil bases were probed by their conversion to alkali‐labile, non‐ultraviolet‐absorbing sulphonated adducts, with 1.33 M bisulfite pH 7, at 20°C; the adducts can be separated from unreacted uracil, and quantified, by cation‐exchange chromatography of RNase T2 plus pancreatic RNase digests of bisulfite‐modified rRNA. Exposed cytosines were probed by their conversion to methoxyaminated, alkali‐stable, derivatives with 1 M methoxyamine, pH 5.5, at 37°C, and quantified by monitoring the CMP/AMP radioactivity ratio after alkaline hydrolysis of modified rRNA. Exposed uracil rings can also be estimated spectrophotometrically by the alkali‐catalyzed reversal of the non‐ultraviolet‐absorbing sulphonated adducts after separation of the latter from unreacted uracil. The cytosine deamination reaction, catalyzed by bisulfite at pH 6, has also been investigated and found to exhibit little specificity for sterically exposed bases of rRNA, the (G + C)‐richer rRNA species of C. acidophila being considerably less susceptible to non‐specific deamination than the (G + C)‐poorer rRNA of E. coli. A high degree of congruence is shown to exist between results obtained by chemical modification and melting hyperchromicity experiments.
Amino Acid Sequence around the Active Site of Two Class I Fructose‐1,6‐Bisphosphate Aldolases from Staphylococci
Authors: FISCHER Stephan , TSUGITA Akira
Published: July 2010

The amino acid sequences of a 26‐residue segment containing the active‐site lysyl residue of the class I fructose‐1,6‐bisphosphate aldolases from Staphylococcus aureus and Staphylococcus epidermidis have been determined. The sequence homology within the active sites between these enzymes and those from several eucaryotic class I aldolases showed a maximum of 21%. The similarity may indicate the origin of both procaryotic and eucaryotic class I aldolases from a common ancestor.
Kinetic Parameters of a β‐d‐Galactoside α2→6 Sialyltransferase from Embryonic Chicken Liver
Authors: BENDIAK Brad , COOK Geoffrey M. W.
Published: July 2010

A β‐d‐galactoside α2→6 sialyltransferase was purified 500‐fold in 14% yield from 14‐day embryonic chicken liver. Characterization of the product of the sialyltransferase catalysis was accomplished by separation and permethylation of double‐labelled ([14C]NeuAc, [3H]Gal) oligosaccharides following their release from the glycoprotein fetuin by hydrazinolysis. The enzyme transfers NeuAc to Gal(β1→4)GlcNAc(β1→)R‐terminated oligosaccharides; no activity was found towards Gal(β1→3)GalNAc(α1→)R structures. The trisaccharide, NeuAc(α2→6)Gal(β1→4)Glc, was shown to be a good inhibitor of the sialyltransferase. Kinetic investigations of the enzyme indicate it to have a sequential, random bi‐bi mechanism.
Crosslinking of N‐Acetyl‐phenylalanyl [s4U]tRNAPhe to Protein S10 in the Ribosomal P Site
Authors: RIEHL Nadine , REMY Pierre , EBEL Jean‐Pierre , EHRESMANN Bernard
Published: July 2010

In order to identify ribosomal components involved in the peptidyl‐tRNA binding site on the ribosome, tRNAPhe molecules were prepared in which cytidine residues had been chemically converted into 4‐thiouridine (s4U). This nucleoside is photoactive at 335 nm and able to form covalent bonds with nearby nucleophilic groups. The thiolated AcPhe‐tRNAPhe was bound to the ribosomal P site in the presence of poly(U) as verified by puromycin reactivity. Direct irradiation of the AcPhe‐[s4U]tRNAPhe/poly(U)/70‐S ribosome complex induced crosslinking of the tRNA molecule exclusively to 30‐S subunits. Analysis of the covalent complex revealed that AcPhe‐[s4U]tRNAPhe was specifically crosslinked to protein S10.