bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell‐Free Translation of Messenger RNAs from Adult and Fetal Human Muscle
Authors: KAHN Axel , COTTREAU Dominique , DAEGELEN Dominique , DREYFUS Jean‐Claude 
Published: March 2005

Using a procedure of ethanol precipitation in concentrated guanidine · HCl solutions followed by chloroform/isoamylic alcohol extraction and washing in 3 M sodium acetate, we isolated high‐molecular‐weight cellular RNA from human fetal and adult skeletal muscle. About 500 μ RNA were obtained/g of fetal muscle and 50 μ RNA g of adult muscle. Both RNA preparations were efficiently translated in a cell‐free reticulocyte lysate system and directed synthesis of various polypeptides, one of them of Mr 200000 probably corresponding to myosin heavy chains. Dodecylsulphate/polyacrylamide gel electrophoretic pattern of polypeptides neosynthesized using either fetal or adult RNA exhibited several differences.Three neosynthesized cytosolic muscle enzymes were purified from the translation mixtures using a micromethod of immunoaffinity chromatography; specificity of the neosynthesized polypeptides purified according to this procedure was checked by immunological competition with the corresponding unlabeled pure muscle enzymes.Successful cell‐free translation of RNA from adult skeletal muscle and purification of neosynthesized human enzymes are reported for the first time. These methods, indispensable for further studies on human adult muscle gene expression, could also shed light on the mechanism of some inherited molecular diseases and tumoral or dystrophic processes.
Turnover of Cellular Glutathione in Isolated Rat‐Kidney Cells
Authors: MOLDÉUS Peter , ORMSTAD Kari , REED Donald J. 
Published: March 2005

Turnover of cellular glutathione in isolated rat kidney cells was studied using cystine or methionine as sulfur donor. In the absence of any sulfur donor a continuous decrease of intracellular reduced glutathione (GSH) during incubation of the cells was observed. This decrease was abolished in the presence of cystine and, as indicated by incorporation of 35S, there was also a rapid synthesis of GSH. In the presence of a γ‐glutamyltransferase inhibitor, the synthesis of intracellular GSH was accompanied by an accumulation of extracellular cysteineglutathione mixed disulfide whereas only minor amounts of GSH and glutathione disulfide could be detected. The intracellular levels of both the cysteine‐glutathione and blutathione disulfides were at all time points very low.Even though the uptake of cystine was rapid and not rate‐limiting for GSH synthesis, almost no cystine could be detected intracellularly. An increasing intracellular cysteine concentration was however observed, indicating a rapid reduction of cystine.In contrast to cystine, methionine did not protect from the loss of intracellular GSH and only a low rate of incorporation of 35S into GSH was observed. Methionine was rapidly taken up into the cells but was apparently converted to cysteine only to a very limited extent. This is most likely due to a low activity of the enzyme cystathionase since neither homocysteine nor cystathionine was very effective in supporting GSH synthesis.
On the Molecular and Submolecular Structure of the Semiquinone Cations of Alloxazines and Isoalloxazines as Revealed by Electron‐Paramagnetic‐Resonance Spectroscopy
Authors: MÜLLER Franz , GRANDE Hans J. , HARDING Leonard J. , DUNHAM William R. , VISSER Antonie J. W. G. , REINDERS Jan H. , HEMMERICH Peter , EHRENBERG Anders 
Published: March 2005

The thermodynamically stable and, therefore, analytically most important alloxazine and isoalloxazine radical cations have been studied in detail by electron paramagnetic resonance (EPR) spectroscopy. Isotopic and chemical substitutions have been made as in earlier studies with the less stable neutral and anionic species. The experimental spectra have been calculated with the aid of a more sophisticated computer‐simulation program than previously used. Excellent fits were obtained only when all of the following atoms were taken into account in the hyperfine coupling scheme: N‐5 H. N‐10 H or CH3. C‐6 H, C‐7 H, C‐8 H or CH3 and C‐9 H. An additional but small coupling constant was required for the fit. This latter coupling constant is assigned to the nitrogen atom(s) of the pyrimidine subnucleus of (iso)alloxazine radical cations. The EPR‐active proton is attached to N‐5 as we also found for the neutral flavosemiquinone.The alloxazine and isoalloxazine radical cations exhibit an identical hyperfine coupling scheme but differ especially in the pyrazine nucleus with respect to the spin density distribution. This suggests that the geometrical structure of the two kinds of radicals is somewhat different. The highest spin density is, however, located at N‐5 of (iso)alloxazine as has been found for the other flavosemiquinone species.The hyperfine coupling constants are interpreted in terms of spin densities and comparison is made with the most recently available quantum chemical calculations. All monomeric flavosemiquinone species are compared with each other and their differences in the submolecular structure are discussed briefly.
Asymmetric and Symmetric Membrane Reconstitution by Detergent Elimination
Authors: HELENIUS Ari , SARVAS Matti , SIMONS Kai 
Published: March 2005

The dissociation and reconstitution of the Semliki Forest virus membrane using the nonionic detergent octyl β‐d‐glucoside was studied by sucrose density gradient centrifugation. The dissociation occurred in three stages: lysis at a free equilibrium octyl glucoside concentration of 14–18 mM, solubilization at 18–20 mM, and delipidation of the spike glycoproteins at the critical micellar concentration (22 mM) or higher. After solubilization the spike glycoproteins were present as soluble complexes with sedimentation coefficients of 19 S and 6 S. The 6‐S form probably corresponded to a glycoprotein monomer complexed to detergent and the 19‐S form consisted of oligomeric detergent‐protein complexes. The two forms were in slow equilibrium with each other.When the soluble spike protein complexes and egg lecithin solubilized with octyl glucoside were mixed and the octyl glucoside concentration lowered either by dialysis or by dilution, reconstitution occurred. Three types of products were obtained: vesicles with 30% of the spike protein facing inwards and 70% facing outwards, vesicles with virtually all (95%) of the spike proteins pointing outwards, and small protein‐rich soluble aggregates [Helenius et al. (1977) J. Cell Biol. 75, 866]. It was demnnorated that during reconstitution the symmetric vesicles were formed at 19 mM free equilibrium octyl glucoside by the association of the 6‐S protein complexes with the phospholipids and the asymmetric vesicles were formed at 10–16 mM octyl glucoside when the 19‐S complexes associated with the lipids. Asymmetric membrane vesicles were also obtained when membrane penicillinase from Bacillus licheniformis was reconstituted with egg lecithin using octyl glucoside. It could be shown that the penicillinase was oligomeric at the octyl glucoside concentration where the reconstitution occurred.The results demonstrate that different mechanisms of reconstitution give rise to the symmetric and the asymmetric vesicles. The critical factor in determining the mechanism is the state of aggregation of the proteins at the octyl glucoside concentration where membranes begin to form from the solubilized lipids.
Isolation and Characterization of Dipeptidyl Peptidase IV from Human Placenta
Authors: PÜSCHEL Gerd , MENTLEIN Rolf , HEYMANN Eberhard 
Published: March 2005

Human placenta is surprisingly rich in post‐proline dipeptidyl peptidase activity. Among various cell fractions, microsomes have the highest specific activity. A homogeneous enzyme preparation is obtained in a six‐step purification procedure. The final preparation appears homogeneous upon dodecyl sulfate electrophoresis, but analytical isoelectric focussing reveals various active bands with isoelectric points in the range of pH 3–4. The enzyme is a glycoprotein containing about 30% carbohydrate. Treatment with neuraminidase lowers the isoelectric points but does not reduce the heterogeneity of the band pattern. The subunit molecular weight is 120000 as estimated by dodecyl sulfate electrophoresis, whereas Mr of the native enzyme is > 200000, as can be concluded from gel filtration experiments.The purified dipeptidyl peptidase cleaves various synthetic and natural peptides, including substance P, kentsin, casomorphin and a synthetic renin inhibitor. In general, the specificity of the placenta peptidase is similar to that of post‐proline dipeptidyl peptidase from other sources. Phenylalanylprolyl‐β‐naphthylamide (Km= 0.02 mM, V= 92 U/mg) is the best substrate among various synthetic peptide derivatives. Only peptides with a free N‐terminal amino group and proline, hydroxyproline, or alanine in position 2 of the N‐terminal sequence are cleaved. However, X‐Pro‐Pro‐… structures, e.g. as in bradykinin, are not attacked.1 mM bis‐(4‐nitrophenyl)phosphate or 1 mM diisopropylfluorophosphate completely inactivate the peptidase within 30 min at 30°C (pH 8). The peptidase is also completely inhibited by 1 mM Zn2+ and by other heavy metals.
Intracellular Localization of Rat‐Liver Uracil‐DNA Glycosylase
Authors: COLSON Pierre , VERLY Walter G. 
Published: March 2005

Most of the uracil‐DNA glycosylase of the rat liver cell is located in chromatin; there is, however, some activity in the nuclear sap and in the cytoplasm. The chromatin uracil‐DNA glycosylase has been purified; the preparation is devoid of endonuclease and exonuclease activities; the enzyme does not need divalent cations, has a broad optimum pH around 8, is strongly inhibited by increasing ionic strength and free uracil.The apparent Km is independent of the strandeness of the DNA substrate containing uracil, but V is slightly higher with the single‐stranded substrate. The frequency of uracil substitution in the double‐stranded DNA influences the kinetic parameters: a higher frequency increases both Km and V.The inhibitory effects of NaCl and free uracil are greater when the greater when the substrate is double‐stranded rather than single‐stranded. It is speculated that, acting either on the DNA or on the enzyme, both oppose the opening of the double helix necessary for the formation of the enzyme‐substrate complex.The increased reaction rate with a higher frequency of uracil residues in double‐stranded DNA is interpreted as a tendency for the repair enzyme to work in a processive way. It is supposed that processivity also occurs with single‐stranded DNA and that it is opposed by both NaCl and free uracil, explaining a greater inhibition when the single‐stranded substrate has a higher uracil content.
The Ribosomal Elongation Cycle: tRNA Binding, Translocation and tRNA Release
Authors: RHEINBERGER Hans‐Jörg , SCHILLING Susanne , NIERHAUS Knud H. 
Published: March 2005

Experiments concerning the ribosomal elongation cycle have been performed; the following results were obtained.

A measurements of the percentage of tightly coupled ribosomes participating in a tRNA binding reaction is obtained by determining both the stoichiometry of binding and the site location. In the case of AcPhe‐tRNA it is sufficient to determine the maximal binding since at moderate Mg2+ concentration (≤ 15 mM) one ribosome cannot bind more than one AcPhe‐tRNA molecule, which can be present at either the A or P site (exclusion principle).
tRNA binding to poly(U)‐programmed ribosomes does not follow the predictions of simple models with rigid tRNA binding sites as postulated by the classical A/P site model. For example, 2.7 molecules of tRNAPhe are found per ribosome at a ribosome concentration of 0.33 μM and a [14C]tRNAPhe excess over ribosomes of about 40‐fold, in agreement with our previous report. At a 70S ribosome concentration of 3.3 μM and a sevenfold excess of tRNAPhe a binding of 2.5 tRNA molecules/ribosome would be expected according to the binding constants reported by us, but in fact a binding of only 1.8 molecules/70S ribosome is found. These findings explain the current discrepancies concerning the number of tRNA sites on the ribosome and confirm previous reports that three tRNA molecules can be bound simultaneously to one ribosome.
The ternary complex EF‐Tu · GTP · Phe‐tRNAPhe binds to the A site at 0°C even if the P site is not occupied. Only if the P site is free does the addition of EF‐G provoke a translocation to the P site at 0°C, whereas at 37°C the Phe‐tRNAPhe‘slides’ from the A to the P site without EF‐G. This EF‐G‐independent sliding is not observed if the P site is filled by a deacylated tRNAPhe. Thus, EF‐G is directly involved in the translocation reaction, and its role is not restricted to tRNA release.
Kinetics of the puromycin reaction with Phe‐tRNA and AcPhe‐tRNA, respectively, were compared. The initial rate of AcPhe‐puromycin formation exceeds that of Phe‐puromycin formation by a factor of 3.6 (0°C, 15 mM Mg2+).
Poly(U)‐programmed ribosomes carrying only [14C]tRNA release tRNA upon addition of EF‐G. tRNA release is observed only if at least two tRNAPhe molecules are present per ribosome. The tRNA bound first is released with a higher probability than that bound second. Such a complex is not related to a classical pretrans‐location complex carrying a peptidyl‐tRNA at the A site. Therefore, when tRNA release during the course of an EF‐G‐induced translocation reaction is studied, care must be taken that no significant amounts of the complex 70S · mRNA · (deacylated tRNA) are present.
Induction and Characterization of a Microsomal Flavonoid 3′‐Hydroxylase from Parsley Cell Cultures
Authors: HAGMANN Marie‐Luise , HELLER Werner , GRISEBACH Hans 
Published: March 2005

A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen‐dependent hydroxylation of (S)‐naringenin [(S)‐5, 7, 4′‐trihydroxyflavanone] to eriodictyol (5, 7, 3′, 4′‐tetrahydroxyflavanone). Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3′‐hydroxylase reaction. In contrast prunin (naringenin 7‐O‐β‐glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4‐hydroxylase, did not catalyse hydroxylation of 4‐coumaric acid to caffeic acid.3′‐Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P‐450‐dependent flavonoid 3′‐monooxygenase. Pronounced differences in the inhibition of flavonoid 3′‐hydroxylase and cinnamate 4‐hydroxylase were found with EDTA, potassium cyanide and N‐ethylmaleimide.Irradiation of the cell cultures led to increase of flavonoid 3′‐hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease. This is similar to light‐induction of phenylalanine ammonialyase and cinnamate 4‐hydroxylase.In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3′‐hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia‐lyase, cinnamate 4‐hydroxylase, and NADPH‐cytochrome reductase. The results prove that flavonoid 3′‐hydroxylase and cinnamate 4‐hydroxylase are two different microsomal monooxygenases.
Monoclonal Antibodies against Human β‐Glucocerebrosidase
Authors: BARNEVELD Ruud A. , TEGELAERS Frans P. W. , GINNS Edward I. , VISSER Pim , LAANEN Elly A. , BRADY Roscoe O. , GALJAARD Hans , BARRANGER John A. , REUSER Arnold J. J. , TAGER Joseph M. 
Published: March 2005

Monoclonal antibodies were obtained against the membrane‐bound lysosomal enzyme β‐glucocerebrosidase (acid β‐glucosidase), which is deficient in Gaucher's disease.BALB/c mice were immunized with homogeneous enzyme protein extracted from a sodium dodecyl sulphate/polyacrylamide gel. The mice were subsequently hyperimmunized with partially purified enzyme prior to fusion of spleen cells with myeloma cells. After fusion, 32 primary hybrid cell populations were obtained which continued to produce antibodies against β‐glucocerebrosidase after prolonged time of culture. All antibodies reacted with both native and denatured enzyme.Four primary cell populations were subcloned and the antibodies produced were characterized. The antibodies were all four of the IgG1 subclass. Three of these antibodies bind to protein A whereas one does not. The results of binding assays indicated that three of the antibodies react with the same antigenic domain (epitope 1), but the fourth with a different one (epitope 2). Probably two antigenic determinants are present in epitope 1 since one of the antibodies with specificity for epitope 1 is inactivated after iodination by the chloramine‐T procedure whereas a second one is not.
Microsomal Glutathione Transferase
Authors: MORGENSTERN Ralf , DePIERRE Joseph W. 
Published: March 2005

The procedure developed for purification of the N‐ethylmaleimide‐activated microsomal glutathione transferase was applied successfully to isolation of this same enzyme in unactivated form. The microsomal glutathione transferases, the unactivated and activated forms, were shown to be identical in terms of molecular weight, immunochemical properties, and amino acid composition.In addition the microsomal glutathione transferase purified in unactivated form could be activated 15‐fold with N‐ethylmaleimide to give the same specific activity with 1‐chloro‐2,4‐dinitrobenzene as that observed for the enzyme isolated in activated form. This activation involved the binding of one molecule N‐ethylmaleimide to the single cysteine residue present in each polypeptide chain of the enzyme, as shown by amino acid analysis, determination of sulfhydryl groups by 2,2'‐dithiopyridyl and binding of radioactive N‐ethylmaleimide.Except for the presence of only a single cysteine residue and the total absence of tryptophan, the amino acid composition of the microsomal glutathione transferase is not remarkable. The contents of aspartic acid/asparagine + glutamic acid/glutamine, of basic amino acids, and of hydrophobic amino acids are 15%, 12% and 54% respectively. The isoelectric point of the enzyme is 10.1.Microsomal glutathione transferase conjugates a wide range of substrates with glutathione and also demonstrates glutathione peroxidase activity with cumene hydroperoxide, suggesting that it may be involved in preventing lipid peroxidation. Of the nine substrates identified here, the enzymatic activity towards only two, 1‐chloro‐2,4‐dinitrobenzene and cumene hydroperoxide, could be increased by treatment with N‐ethylmaleimide. This treatment results in increases in both the apparent Km values and V values for 1‐chloro‐2,4‐dinitrobenzene and cumene hydroperoxide.Thus, although clearly distinct from the cytosolic glutathione transferases, the microsomal enzyme shares certain properties with these soluble enzymes, including a relative abundance, a high isoelectric point and a broad substrate specificity. The exact role of the microsomal glutathione transferase in drug metabolism, as well as other possible functions, remains to be established.
Phosphorylation of purified bovine cardiac sarcolemma and potassium‐stimulated calcium uptake
Authors: FLOCKERZI Veit , MEWES Rainer , RUTH Peter , HOFMANN Franz 
Published: March 2005

Sarcolemmal vesicles were prepared from bovine cardiac muscle by differential and discontinuous sucrose density gradient centrifugation. Na+/K+‐ATPase was purified 33‐fold to a specific activity of 53 ± 0.5 (12) μmol Piμmg±1×h−1, binding sites for strophantin 20‐fold to a density of 56.3 ± 5.3 (14) pmol/mg and that for the calcium antagonist nitredipine 5.5‐fold to a density of 0.72 ± 0.07 (6) pmol/mg. The specific activity of the Na+/Ca2+ exchanger was 61.1 ± 3.7 (6) nmol/mg. The vesicles had an intravesicular volume of 20 ± 4 (4) μl/mg and 56.9 ± 6 (4)% of the vesicles were right‐side‐out oriented.Several peptides of the purified membranes were phosphorylated in the presence of Mg · ATP and EGTA. Most of the radioactive phosphate was incorporated into a peptide with an apparent molecular mass of 22 kDa. Denaturation of the membranes at 100°C changed the mobility of this peptide to 15 kDa and 11 kDa. This peptide could not be distinguished from a sarcoplasmic reticulum peptide of similar molecular mass. The phosphorylation of the sarcolemmal peptide was stimulated by Ca2+/calmodulin, cAMP and the catalytic subunit of cAMP‐dependent protein kinase. A comparison of the phosphorylation of sarcolemmal membranes with that of sarcoplasmic reticulum showed that Ca2+/calmodulin stimulated in each membrane, the phosphorylation of the 22‐kDa peptide and a 44‐kDa peptide, and in the sarcoplasmic reticulum the phosphorylation of an additional peptide of 55‐kDa. Ca2+/calmodulin‐dependent phosphorylation of a 55‐kDa peptide could not be demonstrated in sarcolemma, regardless if sarcolemmal membranes were incubated together with sarcoplasmic reticulum or if the phosphorylation was carried out in the presence of purified cardiac myosin light chain kinase or phosphorylase kinase.‘Depolarization’ induced Ca2+ uptake which was measured according to Bartschat, D. K., Cyr, D. L. and Lindenmayer, G. E. [(1980) J. Biol. Chem. 255, 10044–10047] was 5 nmol/mg protein. This uptake was not enhanced after preincubation of the vesicles with Mg · ATP or Mg · ATP and cAMP‐dependent protein kinase. The value of 5 nmol/mg protein is in agreement with the theoretical amount of Ca2+ which can be accumulated by the bovine cardiac sarcolemma in the absence of a driving force other than the Ca2+ gradient. The potassiumstimulated Ca2+ uptake was not blocked by the organic Ca2+ channel blockers. Prolonged incubation of Mg · ATP with sarcolemmal vesicles in the presence of various ATPase inhibitors led to the hydrolysis of ATP. The liberated phosphate precipitated with Ca2+ in the presence of LaCl3. These precipitates amounted to an apparent Ca2+ uptake ranging from 50 to over 1000 nmol/mg. The results suggest that potassium‐stimulated Ca2+ uptake of bovine cardiac sarcolemmal vesicles is not enhanced in the presence of ATP or by phosphorylation of a 22‐kDa peptide.
Biosynthesis of the ubiquinol‐cytochrome c reductase complex in yeast
Authors: VAN LOON Adolphus P. G. M. , KREIKE Jan , DE RONDE Antony , VAN DER HORST Gijsbertus T. J. , GASSER Susan M. , GRIVELL Leslie A. 
Published: March 2005

The mitochondrial ubiquinol – cytochrome c reductase complex (complex III or cytochrome bc1 complex) is thought to consist of eight subunits, seven of which are specified by nuclear genes and synthesized in the cytoplasm. We have studied the synthesis of five of the nuclear‐encoded subunits both in vivo and in vitro and show that of these the 44‐kDa, 40‐kDa and 17‐kDa subunits are synthesized with cleavable extensions, while the 14‐kDa and 11‐kDa proteins are synthesized without detectable extra sequences. The sizes of the pre‐sequences, as determined by the relative mobility of the precursor proteins in sodium dodecyl sulphate/polyacrylamide gels, range from 0.5‐kDa for the 44‐kDa and 40‐kDa subunits to 9‐kDa for the 17‐kDa subunit. The existence in vivo of precursor forms to the 44‐kDa, 40‐kDa and 17‐kDa subunits implies that import is at least partially a post‐translational process. The precursor of the 44‐kDa subunit can be processed post‐translationally in vitro by isolated mitochondria.The messenger RNAs for subunits of the complex have been studied. Those coding for the 44‐kDa, 40‐kDa, 14‐kDa and 11‐kDa proteins and cytochrome c1 are of different sizes, indicating that each of these subunits is synthesized as a separate protein, rather than as part of a polyprotein precursor.