bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fluorescence Polarization and Energy‐Transfer Studies on the Pyruvate Dehydrogenase Complex of Escherichia coli
Authors: SCOUTEN William H. , VISSER Antonie J. W. G. , GRANDE Hans J. , DE KOK Arie , DE GRAAF‐HESS Adriana C. , VEEGER Cees 
Published: March 2005

We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrcgenase complex isolated from Escherichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested that the flavin moiety is at a substantial distance (over 4.5 nm) from the labeled lipoyl group. Since the lipoyl group must supply electrons to the FAD during the catalytic decarboxylation of pyruvate, we have investigated several potential mechanisms whereby this could happen. Movement within the camplex, possibly triggered by the presence of substrate, seemed to be a strong possibility. Complex labeled with fluorophores on the accessible sulfhydryls, or on the lipoyl functions, did not give evidence of such triggering upon addition of substrate as judged by both static and dynamic fluorescence depolarization.The mobility of the subunits of labeled lipoamide dehydrogenase exceeded that expected for the total complex. Pyrene maleimide bound to the lipoyl functions also exhibited considerably faster rotations than the predicted one of the whole complex (τc > 3 μs). This suggests that a constant movement within the complex, coupled with the rotation of the lipoyl group, may bring the active sites of the complex transiently close enough together to interact on a time scale much faster than enzyme turnover. At the same time, the lipoyl group and the active sites of the complex can spend most of their time at points which are rather distant from each other.
Purification and Chemical Characterization of the Vitamin‐B12‐Dependent 5‐Methyltetrahydrofolate: Homocysteine Methyltransferase from Escherichia coli B
Authors: PAESSENS Arnold , RÜDIGER Harold 
Published: March 2005

The transferase was isolated by means of hydrophobic chromatography and combination of ionexchange and gel filtration at different pH values and ionic strengths. As judged by disc electrophoresis, the enzyme is homogeneous. Electrophoresis in the presence of sodium dodecylsulfate reveals only one band with Mr= 49500 ± 10%. In gel filtration the native enzyme has a Mr of 200000. The subunits can be crosslinked by iminothiolane followed by hydrogen peroxide oxidation. In sodium dodecylsulfate electrophoresis this results in a band pattern of integer multiples of 50000 up to 200000 but not higher. The high‐Mr aggregates disappear on splitting the crosslinks by reduction. Thus the enzyme appears to be composed of four subunits identical or nearly identical in Mr.By the dansyl method, only phenylalanine and methionine were found as the amino‐terminal residues, suggesting the existence of two different types of subunits.
Inhibition Studies in situ of Yeast Catalases
Authors: SELS André A. , BRYGIER Jeanne 
Published: March 2005

The catalase activity of the intact yeast cells towards external substrate is generally lower than the ‘cryptic’ activity which is revealed after cell lysis. The physiological basis for the reduced catalytic activity of the intact cell (‘patent’ activity) has been investigated by establishing the inhibition profiles of catalases in situ using selected probes; to this end we utilized either nonpenetrating acids and/or catalase poisons able to cross the plasmic membrane.Owing to the peculiar features of the reaction mechanism, competitive inhibitors, which are known to interact with the prosthetic group of catalases, show an efficiency that is unlinked to the hydrogen peroxide concentration under the usual assay conditions ([H2O2] ·Km). This mode of interaction, which also characterizes the action of the penetrating probes HCOOH and HCN, is particularly well adapted to the study of the behaviour of the cytoplasmic catalases in situ.By this experimental approach, it has been shown that the catalase of the inner cellular region contributes, together with an isoenzyme present at the cell surface, to the patent activity.The mathematical processing of the data, which takes into account a rate‐limiting diffusion of external substrate into the intact yeast cell, has allowed us to predict accurately the resulting apparent efficiency of inhibitors as a function of the physiological variations of the intracellular enzyme concentration.
The Fidelity of Gramicidin S Synthetase
Authors: AARSTAD Kjell , ZIMMER Trine‐Lise , LALAND Søren G. 
Published: March 2005

The amino acid analog l‐cyclohexylalanine, which may be considered an analog of the hydrophobic amino acids leucine and valine, was found to thioester‐bind to the heavy enzyme of gramicidin S synthetase. The results indicate that it preferably binds to the thiol site of leucine, although binding to the valine site also occurs. In the synthesis of the cyclic decapeptide by gramicidin S synthetase, the results suggest that l‐cyclohexylalanine can replace l‐leucine and l‐valine. We have also found that in the synthesis of the cyclic decapeptidase l‐leucine can replace l‐valine and vice versa.The results further indicate that the previously reported thioester binding of d and l‐phenylalanine to the heavy enzyme takes place at the thiol site of leucine.
The Complete Primary Structure of the Phosphatidylcholine‐Transfer Protein from Bovine Liver
Authors: AKEROYD Robert , MOONEN Peter , WESTERMAN Jan , PUYK Wouter C. , WIRTZ Karel W. A. 
Published: March 2005

The complete primary structure of the phosphatidylcholine transfer protein from bovine liver consists of: Ac‐Met‐Asp‐Pro‐Gly‐Ala‐Gly‐Ala‐Phe‐Ser‐Glu10‐Glu‐Gln‐Phe‐Arg‐Glu‐Ala‐Cys‐Ala‐Glu‐Leu20‐Gln‐Arg‐Pro‐Ala‐Leu‐Ser‐Gly‐Ala‐Ala‐Trp30‐Glu‐Leu‐Leu‐Val‐Glu‐Thr‐Gln‐Gly‐Ile‐Ser40‐Val‐Tyr‐Arg‐Leu‐Leu‐Asp‐Gln‐Gln‐Thr‐Gly50‐Leu‐Tyr‐Ala‐Tyr‐Lys‐Val‐Phe‐Gly‐Val‐Leu60‐Glu ‐Asp‐Cys‐Leu‐Pro‐Asp‐Leu‐Leu‐Ala‐Asp70‐Val‐Tyr‐Met‐Asp‐Leu‐Ala‐Tyr‐Arg‐Lys‐Gln80‐Trp‐Asp‐Gln‐Tyr‐Val‐Lys‐Glu‐Leu‐Tyr‐Glu90‐Lys‐Glu‐Cys‐Ser‐Gly‐Glu‐Thr‐Val‐Val‐Tyr100‐Trp‐Gin‐Val‐Lys‐Tyr‐Pro‐Phe‐Pro‐Met‐Ser110‐Asn‐Arg‐Asp‐Tyr‐Val‐Tyr‐Val‐Arg‐Gln‐Arg130‐Gln‐Glu‐Leu‐Asp‐Phe‐Glu‐Gly‐Gln‐Lys‐Val130‐His‐Val‐Ile‐Leu‐Ala‐Gln‐Ser‐Thr‐Ser‐Glu140‐ Pro‐Gln‐Phe‐Pro‐Glu‐Lys‐Ser‐Gly‐Val‐Ile150‐Arg‐Val‐Lys‐His‐Tyr‐Lys‐Gln‐Arg‐Leu‐Ala160‐Ile‐Gin‐Ser‐Asp‐Gly‐Lys‐Arg‐Gly‐Ser‐Lys170‐Val‐Phe‐Met‐Tyr‐Tyr‐Phe‐Asp‐Asn‐Pro‐Gly180‐Gly‐Gln‐Ile‐Pro‐Ser‐Trp‐Val‐Ile‐Asn‐Trp190‐Ala‐Ala‐Lys‐Asn‐Gly‐Val‐Pro‐Asn‐Phe‐Leu200‐Lys‐Asp‐Met‐Val‐Lys‐Ala‐Cys‐Gln‐Asn‐Tyr210‐Lys‐Lys‐Thr.Elucidation of the sequence was based on digestion of the protein by cyanogen bromide, trypsin, protease from Staphylococcus aureus, protease from mouse submaxillaris gland and clostripain, and the manual and automated Edman degradation of the peptides. The sequence of the N‐terminal half (residues 1–122) has been previously published [Moonen, P., Akeroyd, R., Westerman, J., Puyk, W. C., Smils, P., and Wirtz, K. W. A. (1980) Eur. J. Biochem. 106, 279–290]. The protein contains two disulfide bridges at Cys17‐Cys63 and Cys93‐Cys=207. It has a calculated molecular weight of 24681 and a molar absorbance at 280 nm of 60365 M−1 cm−1.
Primary Structure of Porcine Plasminogen
Authors: BRUNISHOLZ René A. , RICKLI Egon E. 
Published: March 2005

The single polypeptide chain of native porcine plasminogen (molecular weight approx. 90000 after CNBr‐cleavage and geiltration (Sephadex G‐75) yielded a high molecular weight core fraction of fragments linked by disulfide bridges and three fragments of lower molecular weight (N‐terminal and C‐terminal CNBr‐fragments and dodecapeptide). From the reduced and S‐carboxamidomethylated core fraction an additional seven fragments with molecula5r weights between 2000 and 38000 were obtained. The CNBr‐fragments were aligned in the porcine plasminogen polypeptide chain according to sequence homologies with the known primary structure of human plasminogen. Due to the lack of two methionine residues in kringle 1 and in the N‐terminal part of the light chain region and to an additional methonine residue in kringle 2 the CNBr‐fragment pattern differs from that of human plasminogen.Affinity chromatography of elastase‐digested, native plasminogen yielded three fragments with intact lysine binding sites, originating from the heavy chain region and a non‐adsorbable fragment, corresponding to human ‘mini’‐plasminogen. This fragment was converted by urokinase into a proteolytically active protein which served for the isolation of the porcine plasmin light chain.With the aid of the fragments produced by the CNBr and elastase cleavage approx. 350 residues were sequenced, of which about 80% showed identity with the sequence of human plasminogen. This percentage varied depending on the region of the molecule, with the highest extent of identity (80 ‐ 90%) found in the analyzed kringles 2 and 4.
The Size and Distribution of Cytochrome c Oxidase Subunit IV mRNA between Free and Membrane‐Bound Polyribosomes
Authors: NORTHEMANN Wolfgang , SCHMELZER Elmon , HEINRICH Peter C. 
Published: March 2005

A length of 990 ± 80 nucleotides was estimated for the poly(A)‐rich mRNA of the higher‐molecular‐weight precursor of rat liver cytochrome c oxidase subunit IV. Assuming a molecular weight of 19500 for the cytochrome c oxidase subunit IV precursor, a non‐coding region of about 350 necleotides must exist.In experiments in the site of synthesis it is shown that the poly(A)‐rich RNA for the higher‐molecular‐weight precursor of cytochrome c oxidase subunit IV is found in free, loosely and tightly membrane‐bound polyribosomes. This finding does not agree with the present picture of free polysomes as the exclusive site of synthesis for mitochondrial proteins.
Modification of Trytophanase with Tetranitromethane
Authors: NIHIRA Takuya , TORAYA Tetsuo , FUKUI Saburo 
Published: March 2005

Modification of apotryptophanase with tetranitromethane [C(NO2)4] resulted in a loss of enzymatic activity, whereas holotryptophanase was highly resistant against C(NO2)4‐inativation. The essential importance of the active‐site‐bound pyridoxal 5′‐phosphate (pyridoxalP) for the protection was confirmed by the agreement of K1/2 (protection) (1.2 μM) for pyridoxal‐P with Km (1.5μM) in enzyme catalysis. Amino acid analyses and inactivation stoichimetry showed that modification of 1‐2 tyrosyl residues per monomer caused completee inactivation. The appearance of 430‐nm species upon incubation of C(NO2)4‐inactivated apoenzyme with pyridoxal‐P indicated that the (NO2)4‐inactivated apoenzyme could still bind the coenzyme, although an affinity of the enzyme for pyridoxal‐P(Kd= 51μM) was much lower than that of the native enzyme (Kd= 0.7μM). A close relationship was observed between the cofactor activity of monovalent cations and their effecctiveness in the protection yb pyridoxal‐P: in the presence of active monovalent cations (K+, NH4+ and Rb+) pyridoxal‐P could provide the protection but not in the presence of inactive cations (Li+, Na+ and Cs+) as well as in the absence of inorganic nonovalent ations.From teh experimental results obtained it was suggested strongly that tryptophanase has essential tyrosyl residues near the active site. The tryosyl residues were prevented from the attack of C(NO2)4 by the active‐site‐bound pyridoxal‐P only in the catalytically active holoenzyme.
Dioxygen Activation by Putidamonooxin
Authors: BERNHARDT Frithjof‐Hans , KUTHAN Hartmut 
Published: March 2005

In the presence of substrates not favourable for hydroxylation, more than 80% of the dioxygen consumed by purified, reconstituted 4‐methoxybenzoate monooxygenase appears in the reaction mixture as hydrogen peroxide. We have investigated whether under these conditions (a) reduced putidamonooxin, the oxygenase of this enzyme system, either autoxidizes in the presence of dioxygen, with liberation of superoxide anion radicals which then disproportionate to H2O2 and O2, or (b) dioxygen is reduced by two sequential single‐electron steps leading to the active oxygen species that forms hydrogen peroxide directly when inactivated by protonation.Quantitative estimation of O−2 radicals, with either succinylated ferricytochrome c or epinephrine used as O−2 scavengers, revealed that only about 6% of the total electron flux channeled via putidamonooxin to dioxygen led to the monovalent reduction of dioxygen. This means that not more than 3% of the hydrogen peroxide found under uncoupling conditions arises from the rapid bimolecular disproportionation of initially formed O−2 radicals.Inconsistent results were obtained when lactoperoxidase was used as an O−2 trap. Our measurements indicate that the conversion of lactoperoxidase into compound III is an inappropriate method of detecting any O−2 radicals that may be formed by the uncouplex 4‐methoxybenzoate monooxygenase. The stoichiometry of about 1:1 for O2 uptake: H2O2 formation indicates that under uncoupling conditions H2O is virtually not formed.The role of [FeO2]+ as the active oxygenating species of putidamonooxin is discussed.
Altered Calmodulin Activity in Fluphenazine‐Resistant Mutant Strains
Authors: KURN Nurith , SELA Ben‐Ami 
Published: March 2005

Genetically altered calmodulin activity in spontaneously derived mutant strains, which were selected for resistance to the toxic effect of a specific inhibitor, the phenothiazine drug fluphenazine, is demonstrated. Partially purified calmodulin preparations from wild‐type and fluphenazine‐resistant strains of the multicellular alga Volvox carieri, were tested for the ability to activate Ca2+‐ATPase of the erythrocyte membranes, and the inhibition of this stimulatory activity by fluphenazine. Unlike the preparation obtained from wild‐type cells, mutant calmodulin is shown to be insensitive to fluphenazine inhibition, in one case, and calmodulin from another strain was found to be inactive in vitro, i.e. it did not activate Ca2+‐ATPase.The pleiotropic phenotype of the spontaneously derived mutant strains involved aberrant multicellular or‐ ganization and hormone‐independent committment of the multipotent asexual reproductive cells, gonodia, to sexual development. These results clearly implicate calmodulin in the control of development and morphogenesis in this simple multicellular eukaryote. In addition, intracellular inhibition of calmodulin in wild‐type cells is shown to block the morphogenic process of embryo inversion and to arrest motility.The availability of mutant calmodulin will facilitate further investigation of the role of this ubiquitous re‐ gulatory protein in the control of development and differentiation in multicellular eukarytes, as well as the fine structure/function relationship with regard to calmodulin modulation of a wide variety of cellular processes.
Multisite Phosphorylation of Glycogen Synthase from Rabbit Skeletal Muscle
Authors: PICTON Colin , AITKEN Alastair , BILHAM Terry , COHEN Philip 
Published: March 2005

Glycogen synthase is a substrate for five distinct protein kinases in skeletal muscle which phosphorylate seven different serine residues on the enzyme. Cyclic‐AMP‐dependent protein kinase phosphorylates sites 1a, 1b and 2, phosphorylase kinase, site 2, glycogen synthase kinase 3, sites 3a, 3b and 3c, glycogen synthase kinase 4, site 2 and glycogen synthase kinase 5 site 5. Site 2 is seven residues from the N‐terminus of glycogen synthase and is located in a cyanogen bromide peptide termed CB1 (apparent Mr= 9000). The other six phosphorylation sites are located in a cyanogen bromide peptide termed CB 2 (apparent Mr= 24000) at the C‐terminal end of the molecule.The sequence of the N‐terminal 123 residues of peptide CB2, has been completed. Sites 3a, 3b, 3c, 5. la and 1b are located at residues 30, 34, 38, 46, 87 and 100 from the N‐terminus of CB2 respectively. Sitc l a is the next serine residue after site 5. The region surrounding sites 3a, 3b and 3c is very rich in proline residues while that surrounding sites la and 1b contains many serine and threonine residues. The 23 residues following site 5 contain 15 aspartic acid and glutamic acid residues, while the region immediately N‐terminal to site 1a is very basic. The whole region is remarkably hydrophilic and is the region at which the native enzyme is attacked by proteinases. The sites at which glycogen synthase is cleaved by trypsin, chymotrypsin and thermolysin have been identified. The finding that trypsin cleaves the enzyme C‐terminal to site 3c while chymotrypsin cleaves N‐terminal to site 3a has formed the basis of a simple procedure for determining the state of phos‐ phorylation of the seven serine residues in vivo [Parker, P. J., Embi, N., Caudwell, F. B., and Cohen, P. (1982) Eur.j. Biochem. 124, 47–551.
Substrate Specificity of Cerebral GDP‐fucose: Glycoprotein Fucosyltransferase
Authors: BROQUET Pierre , LEON Monique , LOUISOT Pierre 
Published: March 2005

Solubilized sheep brain fucosyltransferase was shown to transfer fucose from GDP‐fucose onto glycoprotein and glycopeptide acceptors, such as asialofetuin, asialotransferrin, their glycopeptides and glycopeptides from ovalbumin, but not on to monosaccharides and disaccharides such as galactose, N‐acetylglucosainine and lactose. Competition studies between asialofetuin and glycopeptide V from ovalbumin provided evidence that both substrates compete for a common enzyme active site.The position of the fucosyl linkage was then investigated. Endo‐β‐N‐glucosaminidase D digestion of fucosylated and acetylated glycopeptide V showed that fucose is not linked to asparagine‐linked N‐acetylglucosamine. Hydrazinolysis and nitrous acid deamination performed on asialofetuin and glycopeptide V proved that fucose is not linked to external galactose or N‐acetylglucosamine either. Thus we assume that fucose is linkcd to the oligomannochitobiosyl core of the glycan, and probably to the second N‐acetylglucosamine.
Binding and Screening by Cations and the Effect on Exogenous NAD(P)H Oxidation in Neurospora crassa Mitochondria
Authors: MOLLER Ian M. , SCHWITZGUEBEL Jean‐Paul , PALMER John M 
Published: March 2005

The uncoupled oxidation of exogenous NADH by mitochondria from Neurospora crassu has a pH optimum at 7.0. In the presence of EDTA (1 mM) the optimum is at pH 6.5; maximal inhibition (65%) occurs at pH 7.2. This is comparable to the results with higher plant mitochondria.
The corresponding pH optima for NADPH oxidation are 7.75 (control), 7.0 (+ EDTA) and 8.0 (effect of EDTA), respectively. NADPH oxidation is completely inhibited by EDTA at pH 8.0. These pH optima are all about 1 higher than observed in mitochondria from higher plants.
The inhibition of NADH oxidation by EDTA is shown to be due to the removal of Mg2+ bound to the mitochondria1 membranes.
It is shown that 9‐aminoacridine can be used to monitor the surface potential of the membranes of Neurospora mitochondria.
Cations stimulate NADH oxidation by Neurospora mitochondria in a manner consistent with the theory of the diffuse layer. Quantitatively, the rcsults suggest that Neurospora mitochondria contain fewer charges per mass of protein than Jerusalem artichoke (Heliantthus tuberosus) mitochondria but more than mitochondria from Arum maculatumspadices.
A good correlation is found between the effect of La3+ on the fluorescence of 9‐aminoacridine in the presence of mitochondria and on the oxidation of NADH by the mitochondria. La−3 has different effects on mitochondria from Neurospora, Jerusalem artichoke tubers and Arum spadices. The results indicate that the fluorescence of 9‐aminoacridine can be used to monitor binding sites on biological membranes.
The Yield of Reactivation of Lactic Dehydrogenase after Guanidine · HCl Denaturation is Not Determined by Proline cis⇋trans Isomerization
Authors: ZETTLMEISSL Gerd , RUDOLPH Rainer , JAENICKE Rainer 
Published: March 2005

Reconstitution of tetrameric lactic dehydrogenase from pig muscle after maximum unfolding in 6M guanidine · HCI produces about equal amounts of native enzyme and inactive aggregates. The decrease in the recon‐ stitution capacity is not determined by chemical modification or aggregation in the denatured state. Instead, a kinetic competition between refolding and aggregation during reconstitution must be assumed, similar to the mechanism proposed earlier for the reconstitution after acid dissociation; under this condition conformational rearrangements within residual structures of the acid‐dissociated state were found to be decisive for the decrease in rate and yield of reactivation [G. Zettlmeißl et al. (1979) Biochemistry, 18, 5567‐5571; (1981) Eur. J. Binchem. 121, 169‐1751.To determine whether the yield of reactivation after dissociation and denaturation by guanidine · HCl is influenced by proline cis⇋trans isomerization in the denatured state [cf. J. F. Brandts et al. (1975) Biochemistry, 14, 4953‐49631, time‐dependent measurements were performed. Unfolding in 6 M guanidine · HCI as determined by the decrease in protein fluorescence is found to be very fast (τ= 18.7 ms at 20 °C). Aggregation occurs even after very short times of denaturation where all X‐proline peptide bonds of the denatured chains are expected still to be in their native isomeric conformation. Therefore, the ratio of aggregation and reactivation is not likely to be determined by proline cis trans isomerism. As indicated by the independence of the rate of reactivation on the isomerization state of proline, the slow folding reaction on the correct first‐order/second‐order pathway of reactivation must be determined by rate‐limiting steps other than proline isomerism.
Source of Rapidly Labeled ATP Tightly Bound to Non‐catalytic Sites on the Chloroplast ATP Synthetase
Authors: AFLALO Claude , SHAVIT Noun 
Published: March 2005

Bound [32P]ATP is found on deenergized, washed chloroplast thylakoids which were illuminated in the presence of ADP and [32P]Pi. Tight binding of [32P]ATP occurred both during and after energization. Different classes of bound [32P]ATP were distinguished on the basis of their rates of formation, susceptibility to hexokinase and displacement by unlabeled ATP.

The rates of formation and discharge of the rapidly labeled tighly bound ATP class were much lower than that of ATP formation. The level of this bound ATP saturates at lower concentrations of substrates than does the rate of phosphorylation. Unlabeled ATP, present in the reaction medium, displaces the rapidly labeled tightly bound ATP without affecting the rate of phosphorylation.
We therefore conclude that the rapidly labeled bound ATP class does not fulfill the requirements expected for a catalytic intermediate and that the nucleotide tight binding site(s) on the ATP synthetase differ from the catalytic site(s) for ATP formation.
Since the rapidly labeled tightly bound [32P]ATP is not abolished by high concentrations of hexokinase, but is nevertheless displaced by exogenous ATP, we propose that tight binding of ATP to non‐catalytic sites occurs via a free species of newly synthesized ATP which diffuses slowly to the medium from a space accessible to ATP but not to hexokinase.