bluuebunny/crossref_metadata_2025_split huggingface dataset:
Demonstration and Characterisation of a Testicular Receptor for 1,25‐Dihydroxycholecalciferol in the Rat
Authors: MERKE Jürgen , KREUSSER Wilhelm , BIER Bert , RITZ Eberhard 
Published: March 2005

Defective luteinizing‐hormone‐mediated cAMP generation in rat testis was previously demonstrated by us in acute and chronic uremia. This defect was abolished by administration of 1,25‐dihydroxycholecalciferol (1,25‐dihydroxycalciol). In the present study, we furnish evidence for a cytosolic 1,25‐dihydroxycalciol receptor in the rat testis.Testes of non‐rachitic, rachitic or acutely uremic male Sprague‐Dawley rats were homogenized in 0.3 M KCl, 10 mM Tris/HCl, 1.5 mM EDTA, 1 mM dithiothreitol, pH 7.4. Fresh purified cytosol was incubated with 1,25‐[3H]‐dihydroxycalciol (with or without 200‐fold molar excess of unlabeled 1,25‐dihydroxycalciol, subjected to 5–20% sucrose density‐gradient separation (centrifugation with 255000xg, 21 h, 4°C) and analysed with the hydroxyapatite assay. The data document the presence of a macromolecule in testicular cytosol with the properties of the intestinal 1,25‐dihydroxycalciol receptor. The binding macromolecule migrated at 3.5 S and could clearly be distinguished from the 6‐S tissue‐binding site for 25‐hydroxycholecalciferol. The 1,25‐dihydroxycalciol receptor was present in non‐rachitic, rachitic and acutely uremic rats.Scatchard analysis of the receptor demonstrates high affinity and selectivity (1,25‐dihydroxycalciol ≫ 25‐hydroxycholecalciferol > 1 α‐hydroxycholecalciferol > 24(R),25‐dihydroxycholecalciferol), low capacity (Nmax= 82 fmol/mg protein) and an equilibrium constant of Kd= 2.6 × 10−10 M. The receptor is a protein according to enzyme degradation studies (trypsin, pronase, RNAase, DNAase). Competition studies with and without the alkylating reagent N‐ethylmaleimide demonstrate a cysteine residue in or close to the binding site for 1,25‐dihydroxycalciol. Binding of 1,25‐dihydroxycalciol protects the receptor against inactivation with N‐ethylmaleimide.
NAD‐Linked l(+)‐Lactate Dehydrogenase from the Strict Aerobe Alcaligenes eutrophus
Authors: STEINBÜCHEL Alexander , SCHLEGEL Hans G. 
Published: March 2005

The l(+)‐lactate dehydrogenase (EC 1.1.1.27) of Alcaligenes eutrophus catalyzes the NADH‐dependent reduction of pyruvate and a few other 2‐oxoacids. The Km values for NADH, NAD, pyruvate and l(+)‐lactate are 0.075 mM, 0.130 mM, 0.820 mM and 7.10 mM, respectively. The reaction follows a rapid equilibrium ordered bi‐bi mechanism and involves the formation of a dead‐end EBQ complex. The competitive inhibition of pyruvate reduction caused by NAD (with respect to NADH) is regarded to be of physiological importance.The enzyme is strongly inhibited by oxaloacetate, oxalate and to a less extent by oxamate. Oxaloacetate was found to be the most powerful inhibitor of the enzyme and exerts an almost complete inhibition of the reduction of pyruvate and some 2‐oxoacids at concentrations of 1 μM and less. At 0.1 μM oxaloacetate the inhibition of pyruvate reduction is about 90%.The kinetics of pyruvate reduction in the presence of oxaloacetate is characterized by a burst phase followed by a decreased steady‐state velocity. During the burst phase, which lasts from several seconds to some minutes, the enzyme undergoes transition to a less active enzyme form. The inhibition studies revealed the lactate dehydrogenase to be a hysteretic enzyme, due to its slow response to the ligand. The characteristics of the transient were examined.The inhibition of lactate dehydrogenase from A. eutrophus by oxaloacetate is considered to be of great physiological importance, allowing its function only at a low oxaloacetate concentration and consequently at high NADH/NAD ratios.
Amino‐Acid Sequences of the Active‐Site Sulfhydryl Peptide and Other Thiol Peptides from the Cysteine Proteinase α‐Clostripain
Authors: GILLES Anne‐Marie , DE WOLF André , KEIL Borivoj 
Published: March 2005

One free‐SH group in the heavy chain of α‐clostripain reacts rapidly with N‐tosyllysine chloromethyl ketone which inactivates the enzyme. Iodoacetic acid also reacts with the thiol group required for enzyme activity but more slowly. A tryptic peptide containing the reactive sulfhydryl group labelled with iodo[1‐14C]acetic acid was isolated and determined to be Gln‐Ser‐Val‐Asp‐Leu‐Leu‐Ala‐Phe‐Asp‐Ala‐Cys‐Met. All other cysteine peptides were isolated from the trypsin hydrolysate of the [14C]carboxymethylated enzyme. Moreover N‐terminal and C‐terminal sequences of both chains of α‐clostripain were determined.The sequences representing 20% of the primary structure of α‐clostripain are not homologus with either other cysteine proteinases or with any other protein structure known to date.
Activation of the Enzymic Activity of Hepatic Lipase by Apolipoprotein A‐II
Authors: JAHN Claus E. , OSBORNE James C. , SCHAEFER Ernst J. , BREWER H. Bryan 
Published: March 2005

Human post‐heparin plasma contains two major lipases, lipoprotein lipase and hepatic lipase. Lipoprotein lipase has been investigated extensively and in the presence of a specific activator, apolipoprotein C‐II, is of major importance in modulating the hydrolysis of triacylglycerol rich lipoprotein particles in plasma. The metabolic function and regulation of hepatic lipase is less well established. We have reported previously that apolipoprotein A‐II(apoA‐II)in vitro increased the activity of hepatic lipase by 3.6‐fold [Jahn et al. (1981) FEBS Lett. 131, 366–368]. We have extended these studies and report here the effects of normal plasma, very low density, low density and high density lipoproteins (VLDL, LDL and HDL) and apolipoproteins A‐I, A‐II, C‐I, C‐II and C‐III on hepatic lipase enzymic activity. Normal plasma and HDL activated hepatic lipase by 255 ± 53% (n = 9) and 288 ± 55% (n = 7) respectively, whereas VLDL inhibited hepatic lipase to 25% and LDL did not affect hepatic lipase enzymic activity (n = 7). Isolated apoA‐I, apoC‐I, apoC‐II, and apoC‐III inhibited hepatic lipase enzymic activity to 25 ± 8% (n = 5), 25 ± 15% (n = 5), 23 ± 7% (n = 5) and 13 ± 10% (n = 5) of the basal level.ApoA‐II recombined with HDL lipids activated by 273 ± 42% (n = 5) whereas apoC‐III recombined with HDL lipids inhibited hepatic lipase to 30% of basal activity. Activation of hepatic lipase by apoA‐II was dependent on temperature and pH, with maximal activation (5.75‐fold over basal levels) occurring at pH 7.5 and 37°C. The addition of lipid‐free apoA‐II 10 min after the hydrolysis of triacylglycerol by hepatic lipase had begun resulted in an increase in rate from 1.9 nmol min−1 ml−1 to 7.0 nmol min−1 ml−1. The rate observed by the addition of apoA‐II before the start of the reaction was 9.5 nmol min−1 ml−1. These results are consistent with the concept that apoA‐II modulates the enzymic activity of hepatic lipase, and may play a role in vivo in the regulation of hepatic lipase activity and HDL lipoprotein metabolism.
Maturatio of 5‐S rRNA: Ribonuclease E Cleavages and Their Dependence on Precursor Sequences
Authors: Roy Monoj K. , SINGH Balraj , RAY Bimal K. , APIRION David 
Published: March 2005

9‐S RNA is a processing intermediate that accumulates in an RNase E− strain of Escherichia coli. It spans from the RNase III cleavage site, after 23‐S rRNA, to the 3′ end of the transcript and is derived from rRNA genes which do not contain tRNAs distal to 5‐S rRNA. Here, we have studied the processing of 9‐S RNA with ribonuclease E. RNase E cleaves 9‐S RNA in two sites: one of these is three nucleotides upstream from the 5′ end of 5‐S rRNA, the other downstream from its 3′ end. Both cleavages are probably introduced by the same enzyme, since both cleavages are thermolabile when an extract of a temperature‐sensitive RNase E mutant was used for processing in vitro.In order to asses the role of 5′ and 3′ end precursor‐specific sequences in the RNase E reaction, we isolated the molecules lacking nucleotides at the 5′ or 3′ end. Molecules having the 5′ end of 9‐S RNA but missing nucleotides from the 3′ end (called 8‐S RNA) were as good a substrate for RNase E as 9‐S, RNA itself. However, molecules having the 3′ end of 9‐S RNA but the 5′ end of p5 (called 7‐S RNA), were less efficient substrates for RNase E. Finally, the removal of as little as seven nucleotides from the 5′ end of 8‐S RNA rendered it almost completely unstuitable as a substrate for RNase E.
Immunological Evidence for Apoproteins of the Light‐Harvesting Chlorophyll‐Protein Complex in a Mutant of Barley Lacking Chlorophyllb
Authors: RYRIE Ivan J. 
Published: March 2005

A barley mutant lacking chlorohyllband the pigmented light‐harvesting chlorophyll‐protein of photo‐system 2 is shown by several criteria to contain functional apoproteins of the light‐harvesting complex.Electrophoretic comparison of thylakoid polypeptide Patterns, and the effects of trypsin treatment on these, seuggests that the mutant contains several polypeptides equivalent in mobility to those of the wild‐type complex.An antibody monospecific for the light‐harvesting complex agglutinated both wild‐type and mutant thylakoids.‘Western blot’ immunoelectrophoretic analysis indicated that of four distinct subunits of the light‐harvesting complex in the wild‐type thylakoids, three are detectable in the mutant.As in wild‐type lamellae at least one of the light‐harvesting complex polypeptides is phosphorylated by the endogenous protein kinase. The results are considered in terms of a general role for the light‐harvesting complex polypeptides in membrane appression and the regulation of excitation energy distribution within thylakoids.
Experimental Evidence and Theoretical Discussion for Long‐Term Oscillations of Phosphofructokinase in a Compartmentalized System
Authors: HERVAGAULT Jean‐François , THOMAS Daniel 
Published: March 2005

The activity of rabbit muscle phosphofructokinase (EC 2.7.1.11) has been followed as a function of time under conditions where the enzyme is separated from the bulk solution by an inert membrane.An enzymatic coupling assay allows continuous measurement of the variations in NADH concentration, which is directly related to the enzyme catalytic activity. For given concentrations of substrates (ATP and Fru6P) in the outside reservoir and a given ratio between diffusion coefficients of both substrates, the activity of phosphofructo‐kinase exhibits an oscillatory behavior during a period of about 5 h. The phenomenon is explained in terms of coupling between diffusion of metabolites and non‐linear enzyme reaction.
Faithful Transcription of Ribosomal 5‐S RNA in vitro Depends on the Presence of Several Factors
Authors: SHI Xiao‐Ping , WINGENDER Edgar , BÖTTRICH Johannes , SEIFART Klaus H. 
Published: March 2005

Cytoplasmic extracts from HeLa cells, capable of transcribing the cloned genes for ribosomal 5‐S RNA, were employed to study the factors involved in this process. Two factors can be isolated, by gel filtration through Sephadex G‐100, which are devoid of RNA polymerase activity. They significantly enhance the extent and specificity of the transcription of 5‐S rRNA. Both proteins can jointly be purified by affinity chromatography on immobilized DNA containing the genes for ribosomal 5‐S RNA from Xenopus borealis. Besides a protein of approximately 45 kDa, possibly corresponding to TF IIIA isolated from Xenopus oocytes, a second protein with a molecular mass of 22 ± 1 kDa stimulates the formation of 5‐S RNA. This protein is contained in the breakthrough of DEAE‐cellulose; it binds to and is eluted from phosphocellulose with 0.6 M KCl.In addition, it was found that the exclusion volume obtained after gel filtration on Sephadex G‐100 contains functional complexes, which are capable of transcribing the cloned 5‐S genes and hence contain all the required factors. Direct evidence is presented that the protein of 22 kDa described above is contained in and can be isolated from such complexes. It is postulated from indirect evidence that an additional factor with a molecular mass in excess of 100 kDa is required which can be removed from functional polymerase complexes by gel filtration through Bio‐Gel A5m.
Two Homologous Cytochromes b5 in a Single Cell
Authors: LEDERER Florence , GHRIR Rachid , GUIARD Bernard , CORTRIAL Sylvie , ITO Akio 
Published: March 2005

The amino acid sequence of the heme‐binding domains of rat liver cytochromes b5 from outer mitochondrial membranes and from microsomes has been determined by a combination of autotmatic and manual degradation of fragments generated bvy trypsin digestion and by cleavage at tryptophan. Tryptic peptides were separated by high‐pressure liquid chromatography.The sequence fo microsomal cytochrome b5 is identical with the one published by Ozols and Heinemannn after completion of this study [Biochim. Biophys. Acta (1982) 704, 163–173.] The sequesnce of outer membrane cytochrome b5 differs from the microsomal one at 38 positions out of 91. There are 40 positions invariant between this sequence and the eight microsomal sequences published thus far. The non‐conservatie substitutions are located at the surface of the know three‐dimensional structure of the microsomal cytochrome b5 except of for the substitution of histidine‐15 by arginine.This paper brings the final proof that two iso‐cytchromes b5 exist in the same cell. Their high degree of similarity as well as their differential cellular localization raise some questions which are briefly discussed.
Proteins of the 30‐S Subunit of Escherichia coli Ribosomes which Interact Directly with Natural mRNA
Authors: BROUDE Natalia E. , KUSSOVA Klaudia S. , MECVEDEVA Natalia I. , BUDOWSKY Edward I. 
Published: March 2005

Ultraviolet irradiation (254 nm) of the complexes of MS2 phage RNA (mRNA) and the 30‐S aubunits of Escherichia coli ribosomes prepared at 0°C and 37°C in the presence and absence of intiation factor 3 (IF‐3) causes cross‐linking of mRNA with proteins S3, S4, S5, S7, S9, S18 and IF‐3. Hence, these proteins interact directly with mRNA within the complex 30‐S‐subunit. mRNA.Additin of IF‐3 results in an increase o f the rate of complex formation and decrease of its dissociation rate. The addition of IF‐3 changes the relative amounts of cross‐linked proteins (mainly S3, S4 and S18).Decreasing the temperature from 37°C to 0°C not only decelerates the complex formation rate but also changes the relative amount of cross‐linkes proteins, indicating the influence of the conditins on the structure of the complex.
Amino‐Acid Sequence of Feather Keratin from Fowl
Authors: ARAI Kunio Murayama , TAKAHASHI Rieko , YOKOTE Yoshiko , AKAHANE Kiso 
Published: March 2005

The amino acid sequence of a single polypeptide chain, B‐4, from fowl feather barbs has been determined. The B‐4 chain was found to consist of 96 amino acid residues and t o have a molecular weight of 10206 in the S‐carboxymethylated form. The kN terminus of this protein was an N‐acetylsrine residue. The B‐4 protein contained seven S‐carboxymethylcysteine residues, six of which are located in the N‐terminal region (residues 1–26), and other one in C terminus. The central region of teh peptide chain was rich in hydrophobic residues. There were homolohous amino acids at 66 positions in the sequences of the sequences of the feather keratins of fowl, emu and silver gull. The variation (substitution, deletion and insertion) in sequence was found to be localized in both terminal sections of the polypeptide chain.The B‐4 protein structure was predicted to contain β‐Sheet structure is mostly located in the central region (residues 22–70). On the othere hand, both terminal regions are almost devoid of secondary structure.
Characterisation of Phosphoenolpyruvate Transport across the Erythrocyte Membrane
Authors: HAMASAKI Naotaka , MATSUYAMA Hiroyuki , HIROTA‐CHIGITA Chiyo 
Published: March 2005

Phosphoenolpyruvate was found to be transported across the erythrocyte membrane by a carrier‐mediated transport system. The transport of phosphoenolpyruvate was competitively inhibited by inorganic phosphate (Ki= 24mM) and pyridoxal 5‐phosphate (Ki= 0.2 mM), whereas the transport was noncompetitively inhibited by L(+)‐lactate (Ki= 37 mM). Specific inhibitors for the inorganic anion transport system such as 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid and 4,4′‐dinitrostilbene‐2,2′‐disulfonic acid strongly inhibited the phosphoenolpyruvate transport. The transport was irreversibly inhibited by treating erythrocytes with pyridoxal 5‐phosphate and NaBH4. Transport activities of Phosphenolpyruvate and inorganic phosphate in the treated cells were similary inhibited by pyridoxal 5‐phosphate depending on its concetrations. 4,4′‐Dinitrostilbene‐2,2′‐disulfonic acid protected both transport activities against the pyridoxal 5‐phosphate/NaBH4 treatment.The major integral membrane protein, band 3, was preferentially labelled by treating erythrocytes with pyridoxal 5‐phosphate and NaB[3H]H4. the radioactive incorporation into band 3 was confirmed by two‐dimensional gel electrophoresis combining isoelectric focusing in the first dimension and sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the second dimension.Taken together, these results suggest that band 3 mediates the transport of phosphoenolpyruvate as well as inorganic phosphate.
Photoaffinity Leabeling of Chloroplastic and Cytoplasimic Leycly‐rRNA Synthetases of Euglena gracilis, by the γ‐(p‐Azidoanilidae) of ATP
Authors: KRAUSPE Rainald , LAVRIK Olga I. 
Published: March 2005

The photoreactive‐δ(p‐azidoanilidate) analog of ATP, AzAn ATP, was used to affinity‐label the chlorophlastic an cytoplasmic leucyl‐tRNA synthetases of Euglena gracilis. The analog is abvle to replacxe the substrate ATP is in the tRNA leucylation reaction catalyzed by both enzymes. In the presence of ATP, it is competitive inhibitor against ATP as well a sleucine for the two isoenzymes, as is also shown for the photoiactive‐ δ‐anilidate analog of ATP, AnATP, which does not serve as subtrate in the enzyme reaction.During ultraviolet irradiaiton, the enzymes are irrevesibly inactivated by AzAnATP in a concenytration‐dependent and time‐dependent manner indicative of phtoaffinity labelling. Both ATP and leucine, but not tRNA, protect the enzymes against ultraviolet‐induced inactivation by AzAn ATP. Comparative kinetic characterization of the inactivation process differences in the active centers of two intracelluar isoenzymes.
Identification of Monkey Lung Procathepsin d‐II as a Pepsinogen‐C‐Like Acid Protease Zymogen
Authors: MORIYAMA Akihiko , KAGEYAMA Takashi , TAKAHASHI Kenji 
Published: March 2005

Procathepsin d‐II (Mr=37500) was purifieed from Japanese monkey lung at pH 7.0, and was shown to be converted to the active form, cathepsin d‐II (Mr=33000) via an intermediate (Mr=35500) upon treatment at pH 3.0 and 14°C. Procathepsin d‐II was shown to be the inactive precursor of cathepsin d‐II based on the following results; the former was inactive toward heat‐denaturated casein at pH 5.4 whereas the latter was active; the former was not inactivated by diazoacetyl‐Dl‐norleucine methyl ester in the presence of Cu2+ ion at pH 6.0 whereas the latter was inactivattede rapidly under the same conditions; and the former had no affinity to pepstein‐Sepharose between pH5 and 7 whereas the latter was adsorbed to it.With a rabbit antiserum against procathepsin d‐II, cathepsin d‐II, pepsinogen C and pepsinC of Japanese monkey were each found to give a single precipitin line which fused completely with each other on agarose plate. On the other hand, cathepsin d‐I purified from the monkey lung, and pepsinogens A (I, II, III‐1, III‐2 and III‐3) obtained from the monkey gastric mucosa failed to precipitate with the antiserum. With the antiserum against the monkey pepsinogen C, the same results were obtained. Further, procathepsin d‐II and pepsinogen C were shown to have the same amino‐terminal amino acid sequence, Ala‐Val‐Val‐Lys‐Val‐Pro‐Leu‐Lys‐Lys‐Phe‐Lys‐. All these results indicate a strong similarly of procathepsin d‐II abd cathepsin d‐II to pepsinogen C and pepsin C, respectively.
Inactivation‐Reactivation Process and Repression of Permease Formation Regulate Several Ammonia‐Sensitive Permeases in the Yeast Saccharomyces cerevisiae
Authors: GRENSON Marcelle 
Published: March 2005

Two distinct regulatory mechanisms are responsible for the absence of general amino‐acid permease activity in cells of the wild‐type strain Σ 1278b of Saccharomyces cerevisiae grown in the presence of ammonium ions.One is a reversible inactivation process which progressively develops upon addition of ammonium ions to a proline‐grown culture, and completely suppresses the permease activity within one hour. This inactivation process is absent in mutants altered at the MUT2, MUT4, or PGR genetic loci.In these mutants, a repression of the formation of active permease may clearly be observed in the presence of ammonium ions. This second regulatory mechanism is absent in mutants affected at the GDHCR locus, which might code for a repressor molecule. It is also relieved in the presence of a glnts mutation (which makes the glutamine synthetase thermosensitive) suggesting glutamine as an effector.Two other ammonia‐sensitive permeases, namely the proline permease and the ureidosuccinic‐acid permease, seem to be subject to the same double regulation.Mutations affecting the structural gene of the anabolic NADP‐linked glutamate dehydrogenase (gdhA) seem to completely prevent repression of the general amino‐acid permease, while they partially suppress its inactivation in the presence of ammonium ions.
Translational Barrier in Central Region of Encephalomyocarditis Virus Genome
Authors: SVITKIN Yuri V. , AGOL Vadim I. 
Published: March 2005

A fractionated cell‐free system from Krebs‐2 cells was prepared which contained ribosomes and a high‐speed supernatant. When this system was programmed with encephalomyocarditis virus RNA, the synthesis of a precursor of capsid proteins, polypeptide preA, proceeded at a rate not very different from that observed in unfractionated extracts, whereas the synthesis of more distally encoded proteins, in particular polypeptide F, was greatly retarded, if not abolished. A protein was purified from the cytoplasmic extracts of Krebs‐2 cells which greatly enhanced production of polypeptide F as well as other noncapsid proteins in the fractionated system. By several criteria, this protein was identified as eukaryotic elongation factor 2 (eEF‐2). By using the ADP‐ribosylation assay, it was found that the fractionated system contained about 15% of the amount of eEF‐2 present in the unfractionated extracts.The results suggest that changes in the eEF‐2 content may affect the elongation rate differently at different regions of the RNA template.
The Pyruvate Dehydrogenase Complex of Escherichia coli K12
Authors: STEPHENS Paul E. , DARLISON Mark G. , LEWIS Hilary M. , GUEST John, R. 
Published: March 2005

The nucleotide sequence of a 3780‐base pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain‐termination method. The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites. Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post‐infection labelling studies. The relative molecular mass (99474), composition (885 amino acids), amino‐terminal residue and carboxy‐terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1).The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene. It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr= 27049) of unknown function.