bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation of the Covalent Bond Structure of Peptide‐Matrix Systems by Edman Degradation of Support‐Fixed Peptides
Authors: MECH Carla , JESCHKEIT Hans , SCHELLENBERGER Alfred 
Published: March 2005

Two model peptides Ala‐Ser‐Asp‐Tyr‐Leu and Ala‐Ser‐Glu‐Tyr‐Leu have been synthesized and coupled with an without protecting groups to the amino groups of polystyrene resins. The yield in the binding operation was 93–97% by using water‐soluble carbodiimides as condensing reagents. Edman degradation of the products and quantitative estimation of the amino acid phenylthiohydantoins, as well as amino acid analysis of the partially and completely degraded resins, showed about 7–10% of the fixed peptides not to be involved in the degradation procedure.That part of the material which was not involved in the Edman degradation during the first step also remained inactive in the following degradation operations.The analytical data presented in the paper explain not only the course of the Edman degradation of peptides linked via the side‐chain carboxylic groups to the resin, but they also describe the chemical reactivity of the various carboxylic groups in the binding operation. 22% of the peptides are bound to the resin by side‐chain carboxylic groups in the case of the aspartate‐containing peptide, 26–27% in the case of the glutamate‐containing peptide. 75–77% and 72–75% respectively of the peptides are fixed to the resin via the C‐terminal carboxylic group. No more than 3% of the fixed material may be bound bifunctionally to the support.
Influence of Side‐Chain Structure of Aliphatic Amino Acids on Binding to Isoleucyl‐tRNA Synthetase from Escherichia coli MRE 600
Authors: FLOSSDORF Josef , PRÄTORIUS Hans‐Jörg , KULA Maria‐Regina 
Published: March 2005

The binding of 10 isomeric α‐amino‐heptanoic acids, of two isomeric α‐amino‐octanoic acids, of isoleucinol and valinol, and of various α‐hydroxy acids to isoleucyl‐tRNA synthetase from Escherichic coli MRE 600 has been investigated by an ultracentrifuge method. It was found that the enzyme requires a primary amino group together with a not‐too‐small side chain as prerequisites for ligand recognition. Though the enzyme is absolutely specific for the L isomers, it is fairly tolerant against replacement of the carboxyl group of the natural substrate by more or less hydrophobic substituents. These findings can be explained in terms of Ogston's three‐point‐attachment model, if it is additionally assumed that there is no further space available in the binding region normally occupied by the α‐hydrogen atom to accept other substituents which are as bulky as the carboxyl moiety.Similarly, the architecture of the binding region of the aliphatic side chain is discussed. Our measurements show that the free energy of binding strongly depends on the size and the structure of the remainder of the molecule. None of the isoleucine analogues employed is bound as tightly as the natural substrate itself, but there is a clear preference for side chains branched at the β‐carbon atom. The functioning of the side‐chain recognition site is best understood by imagining a two‐finger glove, of which one finger is tailored to a methyl and the other to an ethyl group. Both these fingers, together with the binding region for the glycine moiety and a steric barrier against a fourth substituent bulkier than hydrogen, are responsible for a high steric specificity towards the one side chain over its Cβ epimer.
Light Chains from Slow‐Twitch Muscle Myosin.
Authors: WEEDS Alan G. 
Published: March 2005

Myosin light chains have been isolated from slow‐twitch soleus muscles of rabbit and cat. Two chemically related light chains of molecular weight about 22000 have been identified from their thiol sequences in each species, and these have been further characterized by amino acid analysis and peptide mapping studies. These light chains are related to the alkali light chains of rabbit fast‐twitch muscles and to the larger cardiac light chain from bovine heart muscle. The presence of two chemically related but phenotypically distinct light chains within single muscles suggests the presence of myosin isoenzyme or that the myosin molecule has a different light chain associated with each subfragment‐1 head. Although the stoichiometry of these two light chains had not been determined, the former of these two conclusions is favoured by analogy with experiments on fasttwitch myosins. In addition to these related light chains, soleus muscle myosin, like fast‐twitch myosins, contains a third light chain of about 19000 molecular weight. Unlike the corresponding light chain of rabbit fast‐twitch myosin, this 19000‐Mr light chain contrains no cysteine residues.The distribution of thiol peptides together with the characteristic mobilities of these light chains on polyacrylamide gels in the presence of sodium dodecyl sulphate provides a ‘fingerprint’ of myosins from different muscle types. This has been used to look for the presence of fast‐twitch myosin in soleus muscle, the results showing both rabbit and cat soleus muscles are homogeneous in their myosin type within the sensitivity of detection of the methods. These techniques have also been used to confirm the reciprocal transformation of light chains in myosins isolated from crossreinnervated muscles. Finally the relationships between these criteria for different myosin types and histochemical procedures for muscle fibre typing are discussed.
Enhancement of Hydroxyindole‐O‐Methyltransferase and DNA‐Dependent RNA Polymerase Activities Induced by Oestradiol in Rat Pineals in Culture
Authors: MIZOBE Fumio , KUROKAWA Masanori 
Published: March 2005

Hydroxyindole‐O‐methyltransferase and DNA‐dependent RNA polymerase activities were determined in the pineal gland removed from the ovariectomised rat and cultured under various experimental conditions.
The transferase activity declined very slowly during 24 h of incubation. 17β‐Oestradiol significantly increased the transferase activity within 2 h after the addition, and the extent of increase was dose‐dependent within the concentration range from 0.1 to 15 nM, being increased by 80% at 15 nM. Enhancement of the transferase activity by oestradiol was abolished not only by inhibitors of protein synthesis (cycloheximide and puromycin), but also by those of RNA synthesis (actinomycin D and α‐amanitin). It was also blocked by clomiphene citrate, an agent which is known to inhibit the binding of steroid hormones to their respective receptors.
RNA polymerase activity (forms A and B) declined rapidly during the initial period of pineal culture. Oestradiol (15 nM) increased the RNA polymerase B activity by 50% within 2 h after the addition. The increase was dose‐dependent within the concentration range from 0.1 to 15 nM, and was abolished by clomiphene citrate.
The possibility is suggested that the pineal is a target organ of oestradiol, and that the steroid‐induced reaction sequence in the pineal conforms to that is known in other target organs.
Studies on Chromatin. Free DNA in Sheared Chromatin
Authors: VARSHAVSKY Alexander J. , BAKAYEV Valery V. , ILYIN Yurii V. , BAYEV Alexey A. , GEORGIEV Georgii P. 
Published: March 2005

Chromatin which has been hydrodynamically sheared in a low‐ionic‐strength buffer lacking divalent cations (I = 0.005 M) contains a heterogeneous set of deoxyribonucleoprotein particles but no molecules of free DNA. The main finding is that a transference of sheared chromatin to 1–2 mM MgCl2 or to 0.1–0.2 M NaCl results in appearance of completely free DNA molecules. A salt‐induced rearrangement of DNA‐bound histories but not a partial loss of them is responsible for the observed phenomenon. Formation of free DNA molecules is accompanied by aggregation of the majority of remaining deoxyribonucleoprotein particles.The percentage of free DNA molecules in the chromatin which was sheared to an average DNA length of about 400 base pairs is increased from zero in the initial sample to 8–9% in 1 mM MgCl2 and further to ∼ 25% of the total DNA in 0.15 M NaCl, 2 mM MgCl2. Free DNA molecules in the sheared chromatin are observed not only upon isopycnic banding of formaldehyde‐fixed deoxyribonucleoproteins in CsCl gradients but also in non‐ionic metrizamide gradients with either fixed or unfixed deoxyribonucleoprotein samples. The process of free DNA formation is a reversible one; its direction and the equilibrium state depend in particular on the ionic conditions of the medium.
The Use of Several Energy‐Coupling Reactions in Characterizing Mutants of Escherichia coli K12 Defective in Oxidative Phosphorylation
Authors: SCHAIRER Hans U. , FRIEDL Peter , SCHMID Brigitte I. , VOGEL Günter 
Published: March 2005

Oxidative phosphorylation, ATP‐32Pi exchange, ATP‐dependent quenching of acridine‐dye fluorescence, ATP‐dependent transhydrogenase and ATP‐dependent transport of thiomethyl β‐d‐galactoside are shown to be experimentally equivalent tools to study the functional state of the ATPase complex in Escherichia coli wild‐type and mutant strains defective in oxidative phosphorylation.According to these criteria ten mutants in the ATPase complex were classified having lesions in the unc A,B region of the chromosome. The first mutant type lacks ATPase activity, but the membrane‐integrated part of the complex remains functional (class I). The second mutant type lacks a functional membrane‐integrated part, but retains ATPase activity (class II). The third mutant type is shown to be defective in both parts of the ATPase complex (class III).
NADH‐Dependent Aryl Hydrocarbon Hydroxylase in Rat Liver Mitochondrial Outer Membrane
Authors: UEMURA Tomihiko , CHIESARA Enzo 
Published: March 2005

NADH‐dependent 3,4‐benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3‐methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH‐dependent 3,4‐benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH‐ to NADH‐dependent 3,4‐benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5.Moreover, it was found that NADH‐dependent 3,4‐benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide‐insensitive, while that in microsomes was cyanide‐sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide‐insensitive cytochrome P‐450 and is inducible markedly by 3‐methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P‐450 oxidase system are discussed.
The Specificity Requirements of Bacteriophage T4 Lysozyme
Authors: JENSEN Harald B. , KLEPPE Gunnar , SCHINDLER Melvin , MIRELMAN David 
Published: March 2005

A series of bacterial cell wall glycopeptides of low molecular weight and cell wall nucleotide precursors have been tested for their inhibitory action on the digestion by T4 lysozyme of a radioactively labeled linear uncrosslinked peptidoglycan. The disaccharide‐peptides GlcNAc‐MurNAc‐l‐Ala‐d‐Glu(A2pm) (C5) and GlcNAc‐MurNAc‐l‐Ala‐d‐Glu(A2pm‐d‐Ala) (C6) as well as the monosaccharide‐peptide MurNAc‐l‐Ala‐d‐Glu(A2pm) were found to be good competitive inhibitors (with similar K1 values) whereas the disaccharide‐pentapeptide GIcNAcMurNAc‐l‐Ala‐d Glu‐Gly l‐Lys‐d‐Ala was a poor inhibitor. T4 lysozyme did not catalyse transglycosylation reactions from Escherichia coli B peptidoglycan to the disaccharide‐peptide C6. No changes were seen in the circular dichroism spectra (200–250 nm) or fluorescence emission spectra upon binding of the good inhibitors. The results obtained indicate that T4 lysozyme has a small active site capable of recognizing a unit consisting of MurNAc‐l‐Ala‐d‐Glu(A2pm).
Limited Proteolysis of Cytoplasmic and Nuclear Uterine Estradiol Receptors Yields Identical Estradiol‐Binding Fragments
Authors: VALLET‐STROUVE Claude , RAT Luce , SALA‐TREPAT José M. 
Published: March 2005

Limited tryptic hydrolysis of the estradiol cytoplasmic receptor from calf uterus has been demonstrated to yield in a high‐salt buffer a stable estradiol‐binding molecule with the following characteristics: sedimentation coefficient 4,0 ± 0.1 S; Stokes radius 3.5 ± 0.05 nm; molecular weight 60000 (for an assumed v̄ value of 0.73 ml g−1) and frictional ratio 1.36.Nuclear KCI extracts, prepared from uteri preincubated at 37°C with labeled estradiol, were analysed by Sephadex G‐200 chromatography and sucrose density gradient centrifugation. The following molecular parameters were found for the estradiol receptor complex: sedimentation coefficient 4.4 ± 0.1 S; Stokes radius 4.12 ± 0.02 nm; molecular weight 77000 and frictional ratio 1.47 (v̄= 0.73 ml g−1). Limited tryptic proteolysis of this extract gave an estradiol‐binding fragment with molecular characteristics identical to the trypsin‐modified cytoplasmic receptor. In addition, mild tryptic digestion of whole labeled nuclei allowed us to solubilize almost quantitatively the nuclear [3H]estradiol in a macromolecular bound form. The molecule thus obtained showed molecular parameters very similar to the 60000‐dalton trypsin fragments obtained from high‐salt cytoplasmic and nuclear extracts. These molecules were undistinguishable by gel electrophoresis analysis at six different acrylamide concentrations. These results in conjunction with those derived from dissociation kinetics experiments and ligand specificity studies indicate the cytosolic protein is a functional part of the nuclear receptor.Based upon these and other studies we suggest that proteolytic cleavage of the estradiol receptor complex, which results in the removal of the estradiol‐binding sites from the nuclear recognition sites of the molecule, could play a role in the inactivation of the estradiol receptor in vivo.
The Molecular Basis of Skeletal Muscle Phosphorylase Kinase Deficiency
Authors: COHEN Patricia T. Wade , BURCHELL Ani. , COHEN Philip 
Published: March 2005

The molecular basis of phosphorylase kinase deficiency was investigated in ICR/IAn mice, which show < 0.2% of normal activity in skeletal muscle (Cohen, P. T. W. and Cohen, P., 1973). The genetics of the deficiency indicate it is a single gene defect on the X‐chromosome (Lyon, J. B., 1970).Phosphorylase kinase was purified from skeletal muscle of a control strain, C 3 H/He‐mg, by three different procedures. (a) Ammonium sulphate precipitation and gel filtration on Sepharose 4B. (b) Hydrophobic chromatography and affinity chromatography on Sepharose 4B to which antibody to rabbit muscle phosphorylase kinase has been linked covalently. (c) Precipitation from muscle extracts with anti‐phosphorylase kinase antibody.All three procedures showed C 3 H/He‐mg phosphorylase kinases were similar to the rabbit muscle enzymes, the structures of the two isoenzyrnes being (αβ?)4 and (α'βγ)4 respectively. The proportion of the (α′βγ)4 isoenzyme relative to the (αβγ)4 isoenzyme was however about 1:1 in murine muscle compared to about 1:10 in rabbit muscle. Since the α and α′ subunits appear to be distinct gene products, the defect in ICR/IAn mice cannot be caused by a mutation in the genes coding for either the α or α′ chains, or 50% of normal activity would be observed.All three procedures for C3H/He‐mg mice failed to detect any of the four subunits α, α′, β and γ in ICR/IAn mice, suggesting that all four chains are absent in the deficiency.An allele for the β‐subunit was identified in rabbits, and the inheritance of the allele showed that it was determined by an autosomal gene. Assuming conservation of X‐linkage between mammals, the defect in ICR/lAn mice cannot be caused by a mutation in a β‐subunit gene.It is proposed that ICR/IAn mice are defective in a control gene located on the X‐chromosome which is required for the expression of structural genes, at least one of which, the gene for the β‐subunit, is located on an autosome. The results imply that interchromosomal transfer of information takes place during the synthesis of phosphorylase kinase.
Glycosidases of Molluscs
Authors: REGLERO Angel , CABEZAS José A. 
Published: March 2005

An α‐l‐fucosidase has been purified approximately 300‐fold from the liver (hepatopancreas) of the marine mollusc Chamelea gallina L. (=Venus gallina L.).During the different steps of the purification procedure it was difficult to remove the contaminant N‐acetylglucosaminidase activity; but, after electrofocusing, a final preprration free of this and other glycosidades present in the crude extract was obtained. The purified enzyme has a broad specificity; it hydrolyzes p‐nitrophenyl α‐l‐fucoside and natural substrates such as oligosaccharides containing fucosidic residues with α 1–2, α 1–3 and α 1–4 linkages; also it hydrolyzes fucose‐containing glycopeptides, such as thyroglobulin glycopeptide, and glycoproteins as porcine submaxillary mucin (previously rendered free of sialic acid). The enzyme has a pH optimum of 5.2 ± 0.2, with a Km of 7 × 10−5 M using p‐nitrophenyl l‐fucoside as substrate. It is inhibited by Hg2+ and some sugars, and activated by CN−, Zn2+, Ca2+ and EDTA. It shows two peaks by isoelectric focusing (at 6.3 and 6.6). The molecular weight of the α‐l‐fucosidase by gel filtration was over 200000.
Comparative Structural and Immunochemical Properties of Leghaemoglobins
Authors: HURRELL John G. R. , NICOLA Nicos A. , BROUGHTON William J. , DILWORTH Michael J. , MINASIAN Elizabeth , LEACH Sydney J. 
Published: March 2005

Circular dichroism studies on leghaemoglobins from snake bean, lupin, serradella and other plants show that, in common with soybean (reported earlier) they have a similar overall polypeptide chain conformation and haem environment and orientation. Immunochemical studies, on the other hand, suggest that the antigenic determinants on the surface of the leghaemoglobins vary considerably.Thus, firstly the α‐helix content of the leghaemoglobins as a class is very similar (60–65%) and approaches that of the myoglobins, secondly, the sign, magnitude and shape of their circular dichroism spectra in the near ultraviolet, Soret and visible regions suggest close similarities in the environment and orientation of a structurally important tryptophan residue and of the haem moiety, and thirdly, there is comparatively weak haem‐protein interaction.The extent of immuno cross‐reactivity was found to be best demonstrated using the Farr radioimmunoassay procedure. The results were (a) 5 leghaemoglobins from one plant (soybean) crossreacted completely but with varying affinities. (b) The extent of cross reactivity between leghaemoglobins from different plants was compared to that within a single plant; the reaction of antiserum to a soybean leghaemoglobin with a serradella leghaemoglobin was weak, with a snake bean leghaemoglobin still weaker (and incomplete) while lupin leghaemoglobins showed no cross reactivity at all. (c) The “rapid” attenuation of cross reactivity among different plant leghaemoglobins is explicable in terms of the extensive amino acid substitutions which have been demonstrated in the literature and in the present studies. (d) In view of this rapid divergence it is not surprising that sperm whale and horse heart myoglobins showed no cross reactivity with soybean leghaemoglobins.In summary, amino acid substitutions in the leghaemoglobin family are conformationally but not immunochemically conservative.
Preferential Stimulation of the Plant mRNA Synthesis by Gibberellic Acid
Authors: WASILEWSKA Lidia D. , KLECZKOWSKI Kazimierz 
Published: March 2005

Treatment of the etiolated maize seedlings with the plant hormone, gibberellic acid results in a significant enhancement of heavy polyribosome formation.
This is accompanied by highly increased incorporation of the labelled RNA precursors into RNA engaged in the polyribosotnal complex, as well as by an increased rate of protein synthesis in viva.
Determination of the specific radioactivity of particular RNA classes isolated from polyribosomes reveals that gibberellic acid stimulates mostly the synthesis of the rapidly labelled, nonribosomal RNA fraction.
A considerable amount of this rapidly labelled RNA fraction, whose synthesis is preferentially stimulated by exogenous gibberellic acid contains poly(A) sequences, as shown by affinity chromatography on oligo(dT)‐cellulose, indicating that phytohormone causes an increased transcription of mRNA in etiolated maize seedlings.
When [3H]adenosine served as the RNA precursor it was found that the ratio between the heteropolymeric and polyadenylic parts of the poly(A)‐RNA chain markedly changed under gibberellin treatment, suggesting that, in addition to an increased rate of mRNA synthesis, the plant hormone also affects the process of post‐transcriptional polyadenylation of the newly made mRNA precursors. Possible extension of the polyadenylate segment in the presence of gibberellin may account for a longer functional half‐life of the mRNA synthesized in plants treated with the phytohormone, and may explain significantly enhanced heavy polyribosome formation, as well as a higher efficiency of protein synthesis in plants treated with gibberellic acid.