bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA Polymerase I and recBC Enzyme Support the Covalent Closing of Hydrogen‐Bonded λDNA Circles in Extracts of Escherichia coli Cells
Authors: PYHTILÄ Mauno J. , SYVÄOJA Juhani E.
Published: March 2005

The covalent closing of hydrogen‐bonded λDNA circles in Escherichia coli extract was observed to require DNA polymerase I, recBC enzyme and ATP. This covident closing activity was lost in strain harbouring it mutation in one of the genes responsible for production of the enzymes mentioned above, and was recovered by combining these mutant extracts. ATP could be replaced with any of the other nucleoside triphosphates. High concentrations of ATP inhibited the closure. K+ or NH+14 (0.2 M) was required for optimal activity and NMN was it strong inhibitor.
Photo‐Induced Crosslinking between Phenylalanine Transfer RNA and Messenger RNA on the Escherichia coli Ribosome
Authors: MATZKE Antonius J. M. , BARTA Andrea , KUECHLER Ernst
Published: March 2005

Crosslinking between phenylalanyl transfer RNA from brewer's yeast and poly(U) can be obtained when ribosomal complexes are irradiated at > 300 nm; the ribosomal complexes were formed by enzymatic binding of the tRNA. The crosslinked product was isolated on oligo(dA)cellulose columns. The experiments suggest that the photo‐crosslinking occurs via the wybutine in the anticodon loop of the tRNA. This system can be used to study interactions between tRNA and messenger RNA on the ribosome.
Direct Enzyme Titration Curve of NADH: Cytochrome b5 Reductase by Combined Isoelectric Focusing/Electrophoresis
Authors: LOSTANLEN Danielle , GACON Gérard , KAPLAN Jean‐Claude
Published: March 2005

Methemoglobin reduction in human red cells involves successively an electron transport from NADH to a soluble form of cytochrome b5 (step 1) and from cytochrome b5 to methemoglobin (step 2). Step 1 is catalysed by an enzyme, soluble NADH:cytochrome b5 reductase (EC 1.6.2.2). Step 2 is non‐enzymatic and involves complementary electrostatic interactions between acidic residues of cytochrome b5 and basic residues of hemoglobin [Gacon et al. (1980) Proc. Natl Acad. Sci. USA, 77, 1917–1921]. Here we present data indicating a similar mode of interactions occurring in step 1 between cytochrome 65 reductase and cytochrome 65. These results have been obtained by using the combined isoelectric focusing/electrophoresis method [Righetti et al. (1978) J. Chromatogr. 166, 455–460] allowing a direct titration of both entities either separately or in a mixture. This is the first report on the obtention of a direct titration curve of an enzyme visualized after specific staining (zymogram). The pH dependence of the Michaelis constant for cytochrome b5 is also in agreement with the hypothesis that electrostatic charges, which are maximal below pH 7.0, are essential in the interaction between cytochrome b5 and its reductase.
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.
A Histone‐Specific Acetyltransferase is Associated with Simian‐Virus‐40 Chromatin
Authors: OTTO Bemd , BÖHM Joachim , KNIPPERS Rolf
Published: March 2005

[14C]Thymidine‐labeled simian virus 40 nucleoprotein. complexes were prepared from nuclei of lytically infected African green monkey kidney cells. The nucleoprotein complexes were further purified by sucrose gradient centrifugation and then incubated in the presence of [3H]acetyl‐coenzyme A. We observed a transfer of [3H]acetyl groups to the endogenous histones H2B, H3 and H4. The enzyme was solubilized in the presence of 0.5 M NaCl and showed properties described for the DNA‐binding acetyltransferase (J. Böhm, E. J. Schlaeger and R. Knippers, preceding paper in this journal).
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.
Isolation of the Cytochrome‐bc1 Complex from Rat‐Liver Mitochondria
Authors: GELLERFORS Par , JOHANSSON Tomas , NELSON B. Dean
Published: March 2005

The cytochrome bc1 complex has been isolated from rat‐liver mitochondria by two different procedures. The enzyme isolated by either procedure exhibits a specific cytochrome b and cytochrome c1 heme content of approximately 8 and 4 nmol/mg protein respectively. Both preparations contain only seven polypeptides on sodium dodecylsulfate gel electrophoresis, with the following apparent molecular weights: I, 50000: II, 46000: III, 33000; IV, 25000; V, 12500; VI, 10000; VII, 5600. The polypeptide composition is identical to that of the beef‐heart enzyme isolated by cholate/ammonium sulfate fractionation. Furthermore, with the exception of subunit II (core protein 2), the apparent molecular weights of the subunits are identical in the rat‐liver and beef‐heart enzymes.
Synthetic Substrates for Thyroid Oligosaccharide Transferase
Authors: RONIN Catherine , GRANIER Claude , CASETI Christiane , BOUCHILLOUX Simone , Van RIETSCHOTEN Jurphaas
Published: March 2005

The N‐glycosylation of proteins is initiated by transfer of preassembled oligosaccharides from lipid carriers to an asparagine residue in the sequence Asn‐Xaa‐Ser(Thr). Various synthetic peptides were previously shown to act as effective acceptors when supplied to microsomal membranes. The present work was undertaken to delineate further within such peptides the structural determinants required for the N‐glycosylation process. Several new peptides were synthesized to evaluate the effects of the chain length and of the modifications on the asparagine, Xaa or threonine residues: asparagine was replaced by glutamine, Xaa by proline or N‐methylated alanine, threonine by an homoserine residue. Furthermore, both side chains of asparagine and threonine were simultaneously replaced by thaw of glutamine and homoserine respectively to restore a putative hydrogen bonding between the carbonyl and hydroxyl groups. The assays were performed with a solubilized form of the oligosaccharide transferase and the acceptor capacities of the peptides expressed by the ratio V/Km of their apparent kinetic parameters V and Km. Results showed that N‐acetyl‐tripeptide amides are excellent substrates for the enzyme. All modifications in the‐Asn‐Xaa‐Thr‐region resulted in a loss of acceptor capacity. The importance of the conformational aspect is discussed.
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.
Arrest of Chain Growth of Replicon‐Sized Intermediates by Aphidicolin During Rat Fibroblast Cell Chromosome Replication
Authors: YAGURA Tatquo , KOZU Toinoko , SENO Takeshi
Published: March 2005

The effect of aphidicolin, a specific inhibitor of DNA polymerase α, on size maturation of nascent DNA intermediates was studied in cultured rat fibroblast cells. Results provided the first evidence of DNA synthesis associated with merging of intermediates of larger than replicon size. Aphidicolin at a concentration (1.4 μg/ml) caus‐ ing 90‐95%, inhibition of [3H]thymidine incorporation, resulted in accumulation of intermediates of nearly the same size as the replicon (2‐5 × l07 Da); although the synthesis of short nascent fragments (referred to as Okazaki fragments) continued in the presence of aphidicolin, the rate of their elongation to the replicon size was greatly decreased. On removal of aphidicolin, these accumulated intermediates merged into high‐molecular‐weight DNA. This merging of the intermediates was ociated with DNA synthesis in paps between adjacent intermediates, as revealed by photolysis of bromodeoxyuridine‐DNA leader with long‐wave ultraviolet light; when the cells had been pulse‐labeled for 5 min with bromodeoxyuridine immediately after removal of the drug, the large DNA arising from aphidicolin‐arrested intermediates was cut into fragments of the original size by long‐wave ultraviolet light irradiation. The arrest of chain elongation at the replicon‐size by aphidicolin might be due to inhibition of this DNA synthesis in gaps, because aphidicolin did not cause degradation of nascent DNAs.
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.
A Restriction Map of Xenopus laevis Mitochondrial DNA
Authors: CORDONNTER Agnès M. , VANNIER Patrick A. , BRUN Gilbert M.
Published: March 2005

The mitochondrial DNA from Xenopus laevis is a 17.4 × 103‐base‐pair circular DNA molecule. The mapping of this DNA, using 19 different restriction endonucleases is reported here. The sites are as follows: 1 for BamHI, PstI, SacI, SalI, BalI; 2 for BglII, SacII, EcoRI, ClaI, 3 for XhoI, 4 for AvaI, XbaI, PvuII, 5 for HindIII, 6 for HhaI, BclI, HpaI, 10 for AvaII and 11 for HincII. The same sites (except for one of the two ClaI sites) are observed in the molecule cloned in pBR322 DNA. The fragments corresponding to 62 cleavage sites have all been ordered and precisely located. They provide suitable conditions for further investigations connected with the study of replication and nucleotide sequence determination of this molecule.
On the Relationship between Rate of ATP Synthesis and H+ Electrochemical Gradient in Rat‐Liver Mitochondria
Authors: ZORATTI Mario , PIETROBON Daniela , AZZONE Giovanni Felice
Published: March 2005

The relationship between rate of ATP synthesis, JATP, and value of the proton electrochemical gradient, ΔH, has been analyzed in intact mitochondria.

Onset of phosphorylation causes a depression of ΔH of 1.5 kJ/mol. There is a close parallelism between inhibition of JATP and restoration of ΔH to its state‐4 value during titrations with oligomycin or atractyloside.

Titrations with ionophores display the following features: (a) ΔH can be depressed by 3–4 kJ/mol by valinomycin + K+ without affecting the rate of ATP synthesis; (b) uncouplers abolish JATP completely while depressing ΔH by 3 kJ/mol; (c) complete abolition of ATP synthesis by inhibitors of electron transport is accompanied by a depression of ΔH of only 1 kJ/mol.

The results indicate that: (a) there is a close functional relationship between redox and ATPase H+ pumps, whereby inhibition of electron transfer is accompanied by simultaneous inhibition of the ATPase H+ pumps; and (b) uncoupling of oxidative phosphorylation is not due to depression of ΔH of only 1 kJ/mol.

The consistence of the present data with either a chemiosmotic model where ΔH is the sole and obligatory intermediate for energy coupling, or models where there is a direct transfer of energy between the two pumps is discussed.
Evidence that the Ya and Yc Subunits of Glutathione Transferase B (Ligandin) are the Products of Separate Genes
Authors: BEALE Dennis , KETTERER Brian , CARNE Tom , MEYER David , TAYLOR John B.
Published: March 2005

A study of the structure of glutathione transferase B (ligandin) has been made with a view to understanding the relationship between the structures of the subunits of which it is composed. It consists of a mixture of a homodimer (Ya Ya) and a heterodimer (Ya Yc) in which the monomers are denned by their apparent molecular weights, that of Ya being 22000 and Yc 25000.

Soluble tryptic peptides from the native homodimer Ya Ya have been compared with those from an artificial homodimer Yc Yc produced by rehybridization of native Ya Yc. Approximately 10 peptides specific to Ya Ya, 12 specific to Yc Yc and 21 common to both have been detected.

Some of the above peptides are derived from variants of the monomers themselves. Ya Ya and Yc Yc have two C termini which are the same in both dimers, namely phenylalanine and lysine. Also there are four cysteinyl peptides, of which three are common to Ya Ya and Yc Yc and one specific to each.

These results suggest that Ya and Yc are derived from at least two different but related genes.
Aldoximes: Active‐Site Probes of Alcohol Dehydrogenases
Authors: SIGMAN David S. , FROLICH Marianne , ANDERSON Robert E.
Published: March 2005

The oximes of aliphatic aldehydes inhibit horse liver and yeast alcohol dehydrogenase. The pattern of inhibition of these enzymes by the oximes reflects their substrate specificity. For example, acetaldoxime is a more effective inhibitor of the yeast enzyme than butyraldoxime (K1 0.06 mM and 2 mM respectively). However, for the liver enzyme, the K1 for butyraldoxime is 6.6 nM while that for acetaldoxime is 0.68 μM. The inhibition constants of hexaldoxime, octaldoxime and decaldoxime for the liver enzyme are 2 nM, 1 nM and 0.7 nM, respectively.

The inhibition of the liver enzyme by the aliphatic oxime is attributable to the anti isomer which forms spectroscopically identifiable ternary complexes with the enzyme and NAD+. All complexes exhibit a transition with a maximal absorption from 290–305 nm and an absorption coefficient of approximately 7 mM−1 cm−1 which most likely results from the addition of the hydroxyl group of the oxime to the nicotinamide moiety of the coenzyme. An analogous complex is not formed with yeast alcohol dehydrogenase. Since the dissociation constants determined from steady‐state inhibition data and the kinetics of formation and dissociation of the spectroscopically identifiable complex are identical, the enzyme‐NAD+‐oxime complex must be exclusively responsible for the observed inhibition, even though ternary complexes with NADH also form. The ternary complex formed with the oxime of p‐dimethylaminocinnamaldehyde has the characteristic absorption at 290–305 nm, indicative of oxygen addition to the nicotinamide moiety, and in addition exhibits a spectral shift in its long‐wavelength transition from 330 nm to 425 nm, suggestive of inner‐sphere coordination of the zinc ion by the nitrogen of the oxime.

Anti‐oximes are structurally homologous to 4‐alkyl pyrazoles which are also very effective inhibitors of horse liver alcohol dehydrogenase. The relative insensitivity of the yeast enzyme to pyrazole and oximes suggests that the catalytic function of the zinc ion in these two enzymes cannot be assumed to be identical.
On the Function of the Natural ATPase Inhibitor Protein in Intact Mitochondria
Authors: SÁNCHEZ‐BUSTAMANTE Victor J. , DARSZON Alberto , GÓMEZ‐PUYOU Armando
Published: March 2005

The recently described methodology to extract the mitochondrial ATPase along with other mitochondrial proteins into organic solvents, and their subsequent incorporation into liposomes [Eur. J. Biochem. (1982) 121, 427–433] has been employed to estimate the number of ATPases that contain the natural ATPase inhibitor protein in its inhibitory site in intact mitochondria incubated in various metabolic states. It was found that in the presence of electrochemical gradients about 50% of the ATPases are without inhibitor protein in its inhibitory site (active ATPases). In the transition from state 4 to state 3 the percentage of active ATPases diminishes from about 50% to approximately 20%. This indicates that during steady‐state phosphorylation only a limited number of ATPases are in the active catalytic state, and that not only during active hydrolysis does the inhibitor protein interact with its inhibiting site; rather the inhibitor seems to interact with an intermediate state of the enzyme that appears either during the synthetic or hydrolytic reactions. In addition it was found that ATP, with or without uncoupler, induces the interaction of the inhibitor protein in more than 80% the ATPases through an oligomycin‐sensitive process. Thus, notwithstanding other factors the interaction may account for the low hydrolytic activity of mitochondria.
Biosynthesis of Ascorbate in Yeast
Authors: BLEEG Henry S. , CHRISTENSEN Flemming
Published: March 2005

An enzyme from Saccharomyces cerevisiae which catalyzes the reaction: l‐galactonolactone + O2→l‐ascorbate + H2O2 has been purified 466‐fold from the mitochondrial fraction of a yeast homogenate. The enzyme has several properties that are different from the l‐galactonolactone oxidase described by Nishikimi et al. [Arch. Biochem. Biophys. 191, 479–486 (1978)]. By gel filtration in the presence of sodium deoxycholate an apparent Mr of 70000 was obtained for the active enzyme. Polyacrylamide‐gradient gel electrophoresis in the presence of deoxycholate gave an Mr of 74000, whereas sodium dodecylsulfate/polyacrylamide gel electrophoresis showed only one protein band corresponding to an Mr of 18000. A tetrameric structure of the enzyme is thereby suggested.

The substrate specificity is confined to the aldonoacid lactones l‐galactono‐, d‐altrono‐, l‐fucono‐, d‐arabino‐ and d‐threono‐1,4‐lactones. Competitive inhibition was demonstrated with l‐gulono‐ and d‐galactono‐1,4‐lactones. p‐Chloromercuriphenyl sulfonate, iodoacetamide, N‐ethylmaleimide, sulfite and sulfide were all inhibitory to the enzyme. No effect was seen when cyanide, azide, EDTA, α,α'‐bipyridyl or bathocuproine disulfonate was added.

An apparent Km of 0.3 mM with l‐galactonolactone as a substrate was found. The Km for oxygen was 0.18 mM. The pH/activity curve exhibited a maximum around pH 8.9 and a shoulder at pH 6.5.

Evidence of a covalently bound flavin coenzyme and involvement of an iron‐sulfur cluster was obtained from difference spectra of oxidized minus substrate‐reduced enzyme with peaks or shoulders of the oxidized enzyme at 475, 445, 410, 375 and 350 nm. In sodium dodecylsulfate/polyacrylamide gels the enzyme subunit(s) had a bright yellow fluorescence after fixation in 7% acetic acid or 5% formaldehyde.

The galactonolactone oxidase is stable with 50 % activity being lost in 6 months at + 5°C.
Partial Amino‐Acid Sequence of the Epidermal Growth‐Factor‐Binding Protein
Authors: ANUNDI Helena , RONNE Hans , PETERSON Per A. , RASK Lars
Published: March 2005

The partial amino acid sequence of the epidermal growth‐factor‐binding protein was determined. Residues in 108 unique positions, corresponding to 45% of the molecule, were identified. The protein is a serine protease, closely related to the nerve growth factor γ subunit. It is suggested that the epidermal growth‐factor‐binding protein, like other serine protease, is synthesized as a single polypeptide chain which undergoes limited endoproteolysis.

The isolated material also contained minor amounts of a second serine protease. This protease is closely related to the epidermal growth‐factor‐binding protein, differing from it in 7 out of the 45 amino acid positions available for comparison. The latter protease may be identical to the previously described protease A.
The Purification and Properties of 2,4‐Dichlorophenol Hydroxylase from a Strain of Acinetobacter Species
Authors: BEADLE Carol A. , SMITH Anthony R. W.
Published: March 2005

2,4‐Dichlorophenol hydroxylase has been purified 13‐fold from Acinetobacter grown on 2,4‐dichlorophenoxyacetic acid as sole carbon source. The enzyme was estimated to be 80–90% pure by electrophoresis.

The enzyme has a relative molecular mass of about 240000 and consists of four subunits of identical size.

The enzyme cont,ains FAD as the prosthetic group. FAD could not be replaced by riboflavin or FMN in reconstituting active enzyme from apoenzyme.

The reaction catalysed is an NADPH‐dependent hydroxylation of 2,4‐dichlorophenol with the formation of 3,5‐dichlorocatechol as product. The reaction stoichiometry is typical of a monooxygenase with an external electron donor. NADPH is the preferred reduced pyridine nucleotide substrate but the enzyme can function with NADH.

The enzyme possesses broad effector specificity. In addition to 2,4‐dichlorophenol, 4‐chlorophenol and 4‐chloro‐2‐methylphenol are true substrates for the enzyme. A number of ‘non‐substrate effectors’ has been found.

The enzyme is sensitive to thiol‐inhibiting reagents.
Phosphorylation of 40‐S Ribosomal Subunits by cAMP‐Dependent, cGMP‐Dependent and Protease‐Activated Protein Kinases
Authors: DEL GRANDE Robert W. , TRAUGH Jolinda A.
Published: March 2005

The phosphorylation of 40‐S ribosomal subunits by cyclic‐nucleotide‐dependent and protease‐activated protein kinases from rabbit reticulocytes was studied in vitro. Under optimal conditions the CAMP‐dependent protein kinases incorporated up to 2 mol phosphate/mol S6. The electrophoretic mobility of S6 following phosphorylation indicated that this value was not an average for a population of maximally phosphorylated and non‐phosphorylated S6 but represented a uniform population of diphosphorylated 40‐S ribosomal subunits. Tryptic digests of S6 were analyzed by two‐dimensional fingerprinting following phosphorylation with the cAMP‐dependent protein kinase; two phosphopeptides, A and B, were observed. When 40‐S ribosomal sub‐ units were examined with the cGMP‐dependent protein kinase, 1 mol phosphate was incorporated/mol S6. Upon analysis of the phosphopeptides obtained with the cGMP‐dependent protein kinase, only peptide A was observed. S6 was also modified by a cyclic‐nucleotide‐independent protein kinase, protease‐activated kinase II, following activation of the enzyme by limited proteolytic digestion. These findings suggest that a multiple protein kinase system may regulate the phosphorylation‐state of S6. A second ribosomal protein, S10, was phosphorylated by a different cyclic‐nucleotide‐independent protein kinase, protease‐activated kinase I, and up to 1 mol phosphate was incorporated.
The Transmembrane Electrical Potential in Rhodopseudomonas sphaeroides Determined from the Distribution of Tetraphenylphosphonium after Correction for Its Binding to Cell Components
Authors: LOLKEMA Juke S. , ABBING Arend , HELLINGWERF Klaas J. , KONINGS Wil N.
Published: March 2005

The membrane potential was determined in intact cells of Rhodopseudomonas sphaeroides from the distribution of the lipophilic cation tetraphenylphosphonium (Ph4P+) after correction for probe binding to cell components. The concentration of Ph4P+ in the external medium of the cells was recorded with a Ph4P+ ‐selective electrode. Probe concentrations up to 20 μM have no effect on the membrane potential. The binding of the probe to the cells is proportional to the free probe concentration up to 80 μM and increases with the pH of the medium. The binding characteristics were determined for the different cell components: the cytoplasm, the cytoplasmic membrane and the outer membrane (and cell wall). The results indicate that the binding sites are for 47% located at the inside of the cytoplasmic membrane and for 53% at the outside of the cytoplasmic membrane. With the equation derived previously [J. S. Lolkema, K. J. Hellingwef, W. N. Konings, Biochim. Biophys. Acta, 681 (1982) 85–94] the membrane potential can be calculated after correction for probe binding. These membrane potentials were 58 mV lower than the uncorrected values. In the dark the membrane potential is – 68 mV. Upon illumination of the cells the membrane potential increases to – 97 mV if nigericin is present. In the absence of nigericin a rapid conversion of the membrane potential into a pH gradient occurs and steady state membrane potentials are generated which are only slightly higher than the membrane potentials found in the dark.
tuf Gene Dosage Effects on the Intracellular Concentration of EF‐TuB
Authors: van der MEIDE Peter H. , KASTELEIN Rob A. , VIJGENBOOM Erik , BOSCH Leendert
Published: March 2005

In this paper we have studied the effect of raising the intracellular EF‐Tu concentration on the expression of tufB. To this aim cells were transformed with multicopy plasmids carrying either tufA or tufB. The intracellular EF‐Tu concentrations were determined by the specific immunoelectrophoresis assay described in the preceding paper in this journal.

We have cloned the tufA gene in a plasmid, containing the powerful major leftward promoter (Pl) of phage λ Transcription from Pl can be repressed at low temperature by a temperature‐sensitive repressor and acitvated by heat induction. Cloning occurred in two orientations in a single EcoRI site about 150 base pairs downstream of Pl. Cells carrying either plasmid were shown to contain an almost doubled amount of EF‐Tu at temperatures from 28°C to 37°C. This indicates that transcription of tufA can proceed from a possible binding site for RNA polymerase on these cloned fragments. The EF‐Tu level was further increased to about 30% of total cellular protein after a temperature shift from 37°C to 43°C.

The multicopy plasmid pTuB1 described by Miyajimaet al. [FEBS Lett. 102, 207–210 (1979)] and a derivative (pTuBo, compare preceding paper in this journal) were used to study the expression of both chromosomal and plasmid‐borne tufB. Transformation with either plasmid raised the intracellular EF‐Tu concentration by 30–60% depending on the nutritional conditions.

Suppression of tufB expression was observed when the intracellular level of EF‐Tu increased after transformation with all plasmids mentioned above. The results are in accord with the concept that EF‐Tu acts as an autogenous feedback inhibitor involved in the regulation of tufB.
Arginyl‐tRNA Synthetase from Brewer's Yeast
Authors: THIEBE Rainer
Published: March 2005

tRNAArg and arginyl‐tRNA synthetase have been purified to homogeneity from brewer's yeast by chromatographic methods. Arginyl‐tRNA synthetase is a monomeric enzyme with a molecular weight of 72000. Two active forms of the enzyme can be found, they are interconvertible. The more stable conformation is probably the natural one. Arginyl‐tRNA synthetase seems to recognize arginine very specifically. No evidence for any proof‐reading mechanism could be found. The steady‐state mechanism is somewhat different from the types found with arginyl‐tRNA synthetase from other sources. However, all these results are compatible with a concerted reaction. Simultaneously with the release of AMP or pyrophosphate an allosteric rearrangement occurs. This conversion seems to be determining for the reaction mechanism.
Galactose‐Specific Endocytosis in Rat Liver
Authors: QUINTART Joël , COURTOY Pierre‐Joseph , LIMET Joseph‐Nicolas , BAUDHUIN Pierre
Published: March 2005

Galactosylated bovine serum albumin (galBSA) and its peroxidase‐conjugate (galBSA‐HRP) are rapidly cleared after intravenous injection and accumulate in rat liver. Competition experiments indicate that these galBSA derivatives are taken up by the galactose‐specific receptor. By cytochemistry on liver slices, galBSA‐HRP is found almost exclusively in hepatocytes, in similar structures as those reported for asialoorosomucoid.

After differential centrifugation of liver homogenates, galactosylated ligands are concentrated in the light mitochondrial (L) and the microsomal (P) fractions. Together, these two fractions contain roughly 80% of the galBSA or of its peroxidase‐conjugate present in the homogenate 10 min after injection to the animals. By isopycnic centrifugation of L or combined LP fractions in sucrose gradients, ligand‐containing structures equilibrate at low densities (1.11–1.13 g/ml). Up to 52‐fold enrichment over the homogenate can be achieved for these structures, with a 14% yield. By isopycnic centrifugation, the distribution of ligand‐containing structures is clearly distinct from the bulk of plasma membrane (5′‐nucleotidase), endoplasmic reticulum (glucose‐6‐phosphatase) and lysosomes (cathepsin B), but considerable overlapping with galactosyltransferase, a marker for the Golgi complex, is observed.

GalBSA‐HRP‐containing structures have been identified by ultrastructural cytochemistry. In low density fractions, labelled structures are vesicles or tubules heterogenous in size and shape and surrounded by a smooth membrane. Other components of such preparations are mostly Golgi and endoplasmic reticulum elements.

Quantitative assessment of the purity in our best preparations of ligand‐containing structures leads to the inference that they are about 50% pure. It may nevertheless be concluded that they constitute a biochemically and morphologically distinct intracellular compartment. This compartment could be specialized in ligand‐receptor and ligand‐ligand sorting.
RNA Polymerase: Interaction of RNA and Rifampicin with the Subassembly α2β
Authors: Huaifeng MI , Hartmann Guido R.
Published: March 2005

We studied the inhibition of tryptic digestion of the subassembly α2β of Escherichia coli DNA‐dependent RNA polymerase to investigate its interaction with RNA and rifampicin. Both agents decreased distinctly the cleavage of subunit β in the subassembly as well as the degradaion of the transiently formed polypeptides (Mr > 80000). Short RNAs with a chain length of approximately 35 nucleotides were most protective at a concentration of 1 mg/ml while long RNAs were less effective at the same concentration. DNA did not exert any observable protective effects. The association of RNA with α2β was shown by chromatography on phosphocellulose, which separates α2β bound to RNA from free α2β. The association of α2β with RNA was inhibited by rifampicin.