bluuebunny/crossref_metadata_2025 huggingface dataset:
Reaction of bovine‐liver copper‐zinc superoxide dismutase with hydrogen peroxide
Authors: JEWETT Sandra L. , CUSHING Susan , GILLESPIE Frieda , SMITH Dave , SPARKS Shawn
Published: March 2005

The reaction of hydrogen peroxide with the copper‐zinc bovine‐liver superoxide dismutase at low molar ratios (0.2–20.0) of H2O2/active site between pH 7.3–10.0 leads to the loss of native enzyme as a distinct form monitored by electrophoresis. The pH dependence of the loss of native enzyme between 7.3 and 9.0 indicates the involvement of a conjugate base on the enzyme of pka of 8.7 ± 0.1. The rate of loss of the native enzyme is first order with respect to the concentration of both enzyme and hydrogen peroxide between pH 7.3 and 9.0 with no evidence for binding of peroxide. A second‐order rate constant of 3.0 ± 1.0 M−1 s−1 is obtained from these data. At pH 10.0 the reaction is first order with respect to enzyme concentration but saturable in H2O2. All data are consistent with the interpretation that H2O2 reacts with the enzyme at the lower pH where the reaction is dependent upon the conjugate base of a functional group on the enzyme. At the higher pH, the data are consistent with the reaction of HO2− and H2O2 with the dismutase. The dissociation constant for HO2− calculated from the kinetic data at pH 10.0 is between 25–50 μM and the rate constant for the breakdown of the HO2−· dismutase complex is 1.10 ± 0.05 × 10−2 s−1. The change in the electrophoretic pattern at all pH values is accompanied by the loss of the ability of the enzyme to bind copper. Weakly bound or free copper can be detected using bathocuproine disulfonate. Furthermore copper‐defficient forms of the enzyme can be detected by staining gels of the peroxide‐treated dismutase with diethyldithiocarbamate.
The brain‐specific gene for rat aldolase C possesses an unusual housekeeping‐type promoter
Authors: VIBERT Magdeleine , HENRY Joëlle , KAHN Axel , SKALA Henriette
Published: March 2005

A DNA fragment encompassing the first exon and about 750 bp of the 5′‐flanking sequence has been isolated and sequenced. The gene has multiple start sites of transcription which are dispersed over about 200 bp. The promoter lacks TATA and CAAT boxes and is very G + C‐rich, with putative binding sites for the transcriptional factors Sp1 and AP2. Similar features are shared with two other brain‐specific genes encoding thy‐1 antigen and γ‐enolase. The existence of a conserved block of similarity upstream of the human and rat aldolase C genes suggests that this region could be involved in tissue‐specific expression whose mechanism seem to be, at least in part, transcriptional.
1H‐NMR studies of high‐potential iron‐sulfur protein from the purple phototrophic bacterium, Rhodospirillum tenue
Authors: KRISHNAMOORTHI Ramaswamy , CUSANOVICH Michael A. , MEYER Terrence E. , PRZYSIECKI Craig T.
Published: March 2005

The high‐potential iron‐sulfur protein (HiPIP) from Rhodospirillum tenue (strain 3761) shows only a weak (20–25%) sequence similarity to HiPIPs from Chromatium vinosum, Ectothiorhodospira halophila and Ectothiorhodospira vacuolata, including the strict conservation of only two of the twelve residues assumed to be in the 4Fe‐4S cluster packing region [Tedro, S. M., Meyer, T. E. and Kamen, M. D. (1979) J. Biol. Chem. 254, 1495–1500]. In spite of these differences, the general range and distribution of hyperfine‐shifted 1H‐NMR peaks of oxidized and reduced R. tenue HiPIP resemble those of E. halophila HiPIP I [Krishnamoorthi, R., Markley, J. L., Cusanovich, M. A., Pryzycieki, C. T. and Meyer, T. E. (1986) Biochemistry 25, 60–67]. Temperature‐ and pH‐dependence and longitudinal relaxation behavior were determined for hyperfine‐shifted peaks of the oxidized protein. Tentative assignments of peaks to ligands and aromatic residues suggest the presence of common apoprotein—active‐site interactions in these proteins. Differences occur in the pattern of paramagnetically shifted peaks attributed to hydrogens bonded to the 4Fe‐4S cluster. Hyperfine‐shifted peaks of R. tenue HiPIP are not perturbed by pH changes in the range 5–9. In contrast, those of the C. vinosum protein exhibit a pH‐dependence of chemical shifts that has been attributed to the titration of His42 [Nettesheim, D. G., Meyer, T. E., Feinberg, B. A. and Otvos, J. D. (1983) J. Biol. Chem. 258, 8235–8239]. Since R. tenue HiPIP contains no histidine, the present observation confirms the above hypothesis.
The G · C base‐pair preference of 2‐N‐methyl 9‐hydroxyellipticinium
Authors: SCHWALLER Marc‐Antoine , AUBARD Jean , AUCLAIR Christian , PAOLETTI Claude , DODIN Guy
Published: March 2005

Among the DNA‐intercalating drugs in the ellipticinium series, 9‐hydroxy derivatives elicit the highest antitumoral properties. In water these drugs display a very low fluorescence quantum yield. Replacement of H2O by D2O partially restores the fluorescence of the ellipticinium chromophore. The possibility that such a proton‐exchange mechanism could be involved in a base‐recognizing process at the DNA level (and therefore be responsible for some base preference) was examined by direct fluorescence titration in deuterated buffer and DNA/drug fluorescence energy transfer. These experimental approaches provide mutually consistent results showing that the 9‐hydroxylated drug recognizes specific DNA sites that are not recognized by the non‐hydroxylated drug.

When compared to 2‐N‐methyl ellipticinium, the 2‐N‐methyl 9‐hydroxyellipticinium presents: (1) higher binding constants for each DNA studied; (2) a base dependence of the fluorescence properties of the bound form (fluorescence increment upon DNA binding varying over 5–11); (3) a base dependence of its DNA affinity constants (1.1–3.3 × 106 M−1) and of its site size (exclusion parameters varying over 3.0‐4.4); (4) a base dependence of its energy transfer from DNA bases.

Analysis of the binding data suggests that the 9‐hydroxyl group of 2‐N‐methyl ellipticinium is responsible for a G · C base‐pair preference, the preferred binding site being a doublet sequence of two adjacent G · C which could be flanked either by a additional G · C base pair or by an A · T base pair.
Molecular organization of RNP complexes containing P11 antigen in heat‐shocked and non‐heat‐shocked Drosophila cells
Authors: SCHULDT Claus , KLOETZEL Peter M. , BAUTZ Ekkehard K. F.
Published: March 2005

Immunofluorescence analysis of polytene chromosomes of Drosophila melanogaster using the monoclonal antibody P11 has shown that after heat‐shock the 38‐kDa P11 antigen almost exclusively localizes at heat‐shock puff 93D where it is part of giant puff‐specific RNP granules. The biochemical experiments reported here show that, independent of growth temperature, the P11 antigen is a component of nuclear 10S RNP particles. The P11‐containing 10S snRNPs can be stabilized in CsCl with 20mM Mg2+ and possess a buoyant density of ρ= 1.4 g/cm3. Sucrose gradient analysis of nuclear RNP extracts of heat‐shocked Schneider's S‐3 tissue culture cells shows that, after a 37 °C heat‐shock, the 10S RNPs associate with large RNP complexes sedimenting at 170–220S. The change in distribution is a temperature‐dependent process with intermediate forms at 29 °C and 33 °C. In thermotolerant cells this observed change in distribution is strongly reduced. DEAE‐Sephacel column chromatography and sucrose gradient analysis of nuclear RNP, followed by Northern blot analysis using 93D‐specific probes of the Taq I repeat and immunoblotting experiments, show that the P11‐containing 10S snRNPs are distinct from the RNP complexes formed by the 93D transcripts, suggesting an indirect association after heat‐shock. Our experiments demonstrate that, despite the fact that a 37 °C heat‐shock does not affect the overall integrity of nuclear RNP, it imposes changes on the general organization and interaction of the nuclear RNP population, resulting in the formation of large nuclear RNP aggregates and complexes. Such changes may be important for the survival strategy of the cell and for hnRNA processing and storage events which are effected by heat‐shock.
Energy requirement and kineties of transport of poly(A)‐free histone mRNA compared to poly(A)‐rich mRNA from isolated L‐cell nuclei
Authors: SCHRÖDER Heinz C. , FRIESE Ursula , BACHMANN Michael , ZAUBITZER Thomas , MÜLLER Werner E. G.
Published: March 2005

ATP‐promoted efflux of poly(A)‐rich RNA from isolated nuclei of prelabeled mouse lymphoma L5178y cells has an activation energy of 51.5 kJ/mol, similar to that found for the nuclear envelope nucleoside triphosphatase (48.1 kJ/mol) assumed to be involved in mediating nucleocytoplasmic transport of at least some RNA. Here we show that efflux of two specific poly(A)‐rich mRNAs (actin and β‐tubulin) from isolated L‐cell nuclei is almost totally dependent on the presence of ATP, while efflux of poly(A)‐free histone mRNA (H4, H2B, and H1) also occurs to a marked extent in the absence of this nucleotide. Measurements of temperature dependence of transport rate revealed an activation energy of 56.1 kJ/mol for actin mRNA, while the activation energy for histone‐H4‐mRNA efflux was in the same range as that found for ATP‐induced release of RNA from demembranated nuclei (about 15–20 kJ/mol). Addition of nonhydrolyzable nucleotide analogs of ATP to the in vitro system used for measurement of RNA transport did not result in release of nonhistone mRNA (actin), but enhanced the efflux of H4 mRNA to approximately the same extent as ATP. Although not absolutely required, addition of ATP stimulated the rate of export of histone mRNA about twofold. Only the poly(A)‐rich RNA, but not the poly(A)‐free RNA, released from isolated nuclei was found to compete with poly(A) for the nuclear envelope mRNA‐binding site, indicating the mechanism of transport for both RNA classes to be distinct. Export of both nonhistone and histone mRNA was found to be inhibited by a monoclonal antibody against a p60 nuclear‐pore‐complex antigen. This antibody had no effect on the nucleoside triphosphatase, mediating transport of poly(A)‐rich mRNA.
Covalent structure of collagen
Authors: SEYER Jerome M. , HASTY Karen A. , KANG Andrew H.
Published: March 2005

Bovine articular type II collagen was prepared by limited pepsin digestion, differential salt fractionation and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type II collagen α chains yielded twelve distinct peptides designated CB1–12. The peptide α1(II)‐CB11 was isolated by carboxymethylcellulose chromatography and Sephadex G‐75S gel filtration. Automated Edman degradation together with chymotrypsin, thermolysin and trypsin digestion enabled identification of its complete amino acid sequence. Compared with type I and type III collagen, the data show similarity with α1(I)‐CB8 and α1(III)‐CB6‐1‐8‐10‐2 peptides, respectively. The peptide is located within residues 124–402 of the α1(II) collagen chain and with its identification, now extends the known amino acid sequence of bovine type II cartilage collagen to 660 amino acid residues including α1(II)‐CB1‐2‐6‐12‐11‐8‐10 (partial). This corresponds to α1(I)‐CB0‐1‐2‐4‐5‐8‐3‐7 (partial; 1–660) and α1(III)‐CB3A‐3B‐3C‐7‐6‐1‐8‐10‐2‐4‐5 (partial; 1–660) of bovine α1(I) and α1(III) collagen chains.
Isolation and characterization of the rainbow trout erythrocyte band‐3 protein
Authors: MICHEL Frank , RUDLOFF Victor
Published: March 2005

Rainbow trout (Salmo gairdneri) band‐3 protein was isolated from trout erythrocyte plasma membranes by a combination of preparative SDS/PAGE and electroelution. High purity and recovery of the plasma membranes were achieved by a new method. This was demonstrated using 4,4′ diiso‐thiocyano[3H2]dihydro‐stilbene 2,2′disulfonic acid (3H2DIDS) which specifically labels band‐3 protein. On SDS/PAGE, band‐3 protein yields a similarly diffuse pattern, as does mammalian band‐3 protein, with an apparent Mr of 116000. In situ chymotryptic cleavage/cross‐linking experiments with 3H2DIDS reveal that the fragments cross‐link as in human and mouse band‐3 proteins but that there are minor differences. Treatment of trout erythrocytes with trypsin results in cleavage of the band‐3 protein. Purified polyclonal antibodies raised against trout band‐3 protein react with trout band‐3 protein and do not crossreact with mouse or human band‐3 protein. They react specifically with only one chymotryptic fragment of trout band‐3 protein.
Substrate‐modulated reactions of putidamonooxin
Authors: WENDE Peter , BERNHARDT Frithjof‐Hans , PFLEGER Karl
Published: March 2005

4‐Methoxybenzoate monooxygenase is fairly nonspecific. The enzyme system with putidamonooxin as its oxygen‐activating component catalyses:(a) O‐, S‐ and N‐demethylation; (b) the oxygenation of 4‐methylbenzoate and 4‐methylmercaptobenzoate, with formation of 4‐carboxybenzyl alcohol and 4‐carboxyphenylmethyl sulfoxide, respectively, and (c) attack of the aromatic ring of 4‐and 3‐hydroxybenzoate and 4‐aminobenzoate, yielding 3,4‐dihydroxybenzoate and 4‐amino‐3‐hydroxybenzoate, respectively.

Compounds which are bound by the active sites of putidamonooxin have two essential features in common: a planar aromatic ring system, and a free carboxyl group attached to it.

By a substrate‐modulated reaction putidamonooxin can be induced to function not only as a monoxygenase but also as a peroxotransferase, i.e. it incorporates both atoms of the activated oxygen molecule into a substrate molecule. This finding supports the hypothesis that a mesomeric state of the iron · peroxo complex, [FeO2]+, is indeed the active oxygenating species of putidamonooxin.

The lifetime of the ternary complex consisting of enzyme · iron‐peroxo‐complex · substrate is significantly prolonged by uncoupling and partially uncoupling substrates, except when it is inactivated by protonation of the iron · peroxo complex by a proton transported into the active sites by a special kind of substrate (i.e. isomers of monoaminobenzoate), with the direct formation of H2O2.

The lifetime of the active oxygen species is determined by (a) the rate of the oxygenation reaction in the presence of tight‐coupling substrates and (b) the rate of the oxygenation reaction as well as detoxification by the availability of a dissociable proton in the presence of partial uncoupling (and uncoupling) substrate analogues.

The rate of the oxygenation reaction depends on the lifetime of the active oxygen species, [FeO2]+, in the presence of partial uncoupling substrates.

The iron · peroxo complex attacks an aromatic ring system according to the empiric rules of electrophilic substitution, whereas the attack of aliphatic substituents at the aromatic ring is controlled by steric criteria.
4‐Chloroacetylpyridine adenine dinucleotide
Authors: TRITSCH Denis , EILER‐SAMAMA Brigitte , SVIRCEVIC Jasmina , ALBRECHT Anne‐Marie , BRANLANT Guy , BIELLMANN Jean‐François
Published: March 2005

The analogue of NAD+, 4‐chloroacetylpyridine‐adenine dinucleotide (clac4PdAD+), invactivated the glyceraldehyde‐3‐phosphate dehydrogenase from sturgeon at a high rate. An affinity labeling was shown to occur with clac4PdAD+. The mononucleotide 4‐chloroacetylpyridine 1‐β‐d‐ribose 5′‐phosphate (clac4PdMN+) reacted with the enzyme in a second‐order reaction whose rate was much smaller than that calculated for clac4PdAD+ taken as a second‐order rate reagent. The rate of the reaction of clac4PdAD+ with the enzyme was determined by stopped flow, using as a probe the long‐wavelength absorption maximum (430 nm) formed concomitantly with inactivation of the enzyme. Computer‐assisted graphic simulation showed that the clac4PdAD+ analogue could bind to the active site of the enzyme from Bacillus stearothermophilus in a similar manner to that of NAD+, and that the reactive carbon and the reactive thiolate of Cys‐149 were within bonding distance. The absorption at 430 nm was linearly proportional to the substoichiometric concentration of clac4PdAD+/mole subunit. Thiol titration suggested the modification of one thiol residue per subunit. The modified thiol was identified by degradation as Cys‐149. In contrast to the absorption band generated during the reaction of the 3‐chloroacetylpyridine‐adenine dinucieotide (clac3PdAD+) with the same enzyme [Eur. J. Biochem (1982) 127, 519–524; 129, 437–446], enzyme inactivated with clac4PdAD+) and clac4PdMN+ exhibited an absorption maximum at long wavelength which was still present after denaturation. The chromophore is proposed to be the enol form of the α‐thioether ketone produced by alkylation of the thiolate of Cys‐149 by the chloroacetyl group.
Release of phospholipase A2 activity from rat vascular smooth muscle cells mediated by cAMP
Authors: PFEILSCHIFTER Josef , PIGNAT Werner , MÄRKI Fritz , WIESENBERG Irmgard
Published: March 2005

Primary cultures of smooth muscle cells (SMC) derived from rat aorta release a phospholipase A2 activity into the culture medium. Phospholipase A2 activity was determined with [1‐14C]oleate‐labelled Escherichia coli as substrate. The enzyme has a neutral pH optimum and the activity is critically dependent on the free calcium concentration, with significant activity in the micromolar range of free calcium. Treatment of SMC with the β agonist salbutamol, forskolin or cholera toxin, which all activate adenylate cyclase and increase intracellular cAMP concentration, increase the release of phospholipase A2 activity in a dose‐dependent manner. Likewise, the addition of the membrane‐permeable cAMP analogues, (Sp)‐adenosine 3′,5′‐[thio]phosphate and N6,O‐2′ ‐dibutyryladenosine 3′,5′‐phosphate, enhance the release of phospholipase A2 activity from SMC in a dosedependent manner. There is a lag period of about 4 h before a significant secretion of phospholipase A2 can be detected under basal, as well as under stimulated conditions. The forskolin analogue 1,9‐dideoxyforskolin, which is inactive as a stimulator of adenylate cyclase, has no effect on phospholipase A2 secretion. Likewise, the potent vasoconstrictive peptide angiotensin II activates inositol phospholipid turnover in SMC, but has no effect on phospholipase A2 release. Pretreatment of SMC with actinomycin D or cycloheximide completely suppresses basal and cAMP‐stimulated secretion of phospholipase A2 activity, thus demonstrating that transcription and protein synthesis are necessary for enzyme release.
Galanin receptor and its ligands in the rat hippocampus
Authors: FISONE Gilberto , LANGEL ülo , CARLQUIST Mats , BERGMAN Tomas , CONSOLO Silvana , HÖKFELT Tomas , UNDÉN Anders , ANDELL Siv , BARTFAI Tamas
Published: March 2005

Receptors for the 29‐amino‐acid peptide, galanin, in membranes from the rat ventral hippocampus were examined using chloramine‐T‐iodinated porcine galanin as ligand. The equilibrium binding of125I‐galanin showed the presence of a high‐affinity binding site (Kd=1.91 ± 40 nM). The concentration of the high‐affinity‐binding sites was 107 ± 15 fmol/mg membrane protein. The on rate constant was estimated to be 2.6 ± 0.1 M−1 min−1 at 37°C.

The affinity of rat galanin (differing in three amino acid residues from the porcine protein) was equal to that of porcine galanin. The125I‐galanin‐binding site is a trypsin‐sensitive membrane protein, which is heat‐denaturated at 60°C within 5 min. The effect of GTP and its analogs and of pertussis‐toxin‐catalyzed ADP‐ribosylation on the binding of125I‐galanin suggest that the galanin receptor is coupled to an inhibitory G protein (Gi protein).

127I‐galanin was shown to be a ligand with affinity equal to that of galanin in displacing 125I‐galanin. The 125I‐galanin‐binding site in the ventral hippocampus recognizes as a ligand the tryptic fragments 1–20 and 21–29 of rat galanin and the synthetic fragments 12–29, 18–29 and 21–29 of porcine galanin. None of these afforded full inhibition of the binding of fragment 1–29 of 125I‐galanin at a concentration of 1μM.
Hepatic processing of insulin
Authors: SMITH Geoffrey D. , CHRISTENSEN Jillian R. , RIDEOUT James M. , PETERS Timothy J.
Published: March 2005

The effect of selected weak bases on the subcellular distribution and processing of internalized insulin by the liver has been studied. The effect of these bases on both the degradation products formed and on the kinetics of degradation have also been studied.

Methylamine, ammonium chloride and dansyl cadaverine but not chloroquine reduce the total amount of insulin endocytosed.

Ammonium chloride, dansyl cadaverine and chloroquine but not methylamine inhibit subsequent degradation and/or translocation of degradation products.

None of the weak bases changed the species of the degradation products found within the endocytic vesicles.

Kinetic analysis of intravesicular degradation indicates that dissociation from the receptor is the ratelimiting process in degradation.

Chloroquine and dansyl cadaverine but not methylamine or ammonium chloride showed specific inhibition of insulin degradation in isolated endocytic vesicles.

The effect of chloroquine and dansyl cadaverine on the kinetics of degradation suggest that they are acting by switching the receptor into a tight‐binding conformation thereby slowing dissociation.
On the role of phospholipids in the reconstituted cytochrome P‐450 system
Authors: RIETJENS Ivonne M. C. M. , ANCHER Leo J. , VEEGER Cees
Published: March 2005

The difference in pentoxyresorufin O‐dealkylating activity observed in a reconstituted system containing dilauroylglycerophosphocholine (Lau2GroPCho) or distearoylglycerophosphocholine (Ste2GroPCho) was used as a model to study the role of phospholipids in the reconstituted cytochrome P‐450b (IIB1) system.

The hypotheses proposed in the literature for the role of phospholipids in the reconstituted cytochrome P‐450 system, mainly based on the comparison of systems without phospholipid and with Lau2GroPCho, were either validated or shown to be unlikely when tested by comparing reconstituted systems with different phosphatidylcholines. The higher activity in the Lau2GroPCho system as compared to the Ste2GroPCho system cannot be ascribed to (a) an increased affinity of cytochrome P‐450 for the NADPH‐cytochrome reductase in the Lau2GroPCho system, also not to (b) a Lau2GroPCho‐dependent dissociation of protein multimers, nor to (c) a change in the spin state of the heme.

We found a different apparent Km for pentoxyresorufin in the Lau2GroPCho system compared with the Ste2GroPCho system. Furthermore, we found a difference between the cytochrome P‐450b tryptophan fluorescence polarization of the Lau2GroPCho system and the Ste2GroPCho system as well as with a system without phosphatidylcholine. From these observations it is concluded that the higher activity of the Lau2GroPCho system compared with the Ste2GroPCho system or with a system without additional phosphatidylcholine may at least in part be caused by a difference in the conformation of the cytochrome P‐450 molecules in these systems.

Furthermore, the different effects of both phosphatidylcholines on the Km and V for pentoxyresorufin not only suggest a role of phospholipids in the binding of the substrate to the active site of the cytochrome P‐450 molecule, but also on the efficiency of electron transfer from NADPH‐cytochrome reductase to cytochrome P‐450.
In vitro studies on the translocation of acid phosphatase into the endoplasmic reticulum of the yeast Saccharomyces cerevisiae
Authors: KREBS Harald O. , HOFFSCHULTE Hedda K. , MÜLLER Matthias
Published: March 2005

We demonstrate here the in vitro translocation of yeast acid phosphatase into rough endoplasmic reticulum. The precursor of the repressible acid phosphatase from Saccharomyces cerevisiae encoded by the PHO5 gene, was synthesized in a yeast lysate programmed with in vitro transcribed PHO5 mRNA. In the presence of yeast rough microsomes up to 16% of the acid phosphatase synthesized was found to be translocated into the microsomes, as judged by proteinase resistance, and fully core‐glycosylated. The translocation efficiency however, decreased to 3% if yeast rough microsomes were added after synthesis of acid phosphatase had been terminated. When a wheat‐germ extract was used for in vitro synthesis, the precursor of acid phosphatase was translocated into canine pancreatic rough microsomes and thereby core‐glycosylated in a signal‐recognition‐particle‐dependent manner. Replacing canine with yeast rough microsomes in the wheat‐germ translation system, however, resulted in a significant decrease in the ability to translocate and glycosylate the precursor. Translocation and glycosylation were partially restored by a high‐salt extract prepared from yeast ribosomes. The results presented here suggest that yeast‐specific factors are needed to translocate and glycosylate acid phosphatase efficiently in vitro.
cDNA‐directed expression of rat testosterone 7α‐hydroxylase using the modified vaccinia virus, T7‐RNA‐polymerase system and evidence for 6α‐hydroxylation and Δ6‐testosterone formation
Authors: AOYAMA Toshifumi , KORZEKWA Kenneth , NAGATA Kiyoshi , GILLETTE James , GELBOIN Harry V. , GONZALES Frank J.
Published: March 2005

The modified vaccinia virus, T7‐RNA‐polymerase cDNA‐expression system was used to express rat cytochrome P‐450a. Various parameters such as host‐cell type and density, and duration of infection were tested to optimize the level of expression of cytochrome P450a enzyme activity. Cytochrome P‐450a expressed from the cDNA sequence was exclusively incorporated into the membrane‐containing portions of the cell lysates, as expected from its normal association in the liver endoplasmic reticulum. The enzyme displayed a carbon‐monoxide‐reduced‐cytochrome‐P‐450a difference spectrum with a Soret maximum of 450 nm. Activity measurements revealed that cytochrome P‐450a produced three metabolites of testosterone; 7α‐hydroxytestosterone and 6α‐hydroxytestosterone and Δ6‐testosterone at a ratio of about 38:1:1. Under the appropriate conditions, the vaccinia‐virus, T7‐RNA‐polymerase system produces high levels of a single form of cytochrome P450 in cells that are virtually devoid of endogenous cytochrome P‐450. Analysis of the cytochrome P‐450 in its natural membrane‐bound state, as opposed to artificial‐lipid reconstitution studies of purified enzymes, allows accurate and confident measurements of substrate specificities.
A phosphodiester bridge between two arabinose residues as a structural element of an extracellular glycöprotein of Volvox carteri
Authors: HOLST Otto , CHRISTOFFEL Volker , FRÜND Rüdiger , MOLL Hermann , SUMPER Manfred
Published: March 2005

The sulphated glycoprotein SSG 185 is the monomeric precursor of a highly aggregated structural element in the extracellular matrix of the multicellular green alga Volvox carteri. A phosphodiester of arabinose was isolated from a saccharide fragment of SSG 185. The structure of this phosphodiester was investigated by methylation analysis, 13C‐NMR, photometric methods and enzymatic assays and identified as d‐Araf‐5‐phospho‐5‐d‐Araf. The function of this phosphodiester bridge as a crosslink of different carbohydrate chains in SSG 185 is discussed.
Bioluminescence emission of bacterial luciferase with 1‐deaza‐FMN
Authors: KURFÜRST Manfred , MACHEROUX Peter , GHISLA Sandro , HASTINGS J. Woodland
Published: March 2005

The reaction of reduced 1‐d‐FMN with oxygen and decanal results in bioluminescence with kinetic and spectral properties similar to those of the reaction with FMNH2, even though the spectral (absorbance, fluorescence) and chemical properties of the oxidized forms differ greatly. This emission, which is about 10–15% as efficient as with FMNH2, is postulated to involve the intermediacy of the corresponding 4a‐hydroperoxide, the fluorescence of which occurred transiently. The N(1) protonated species had been proposed as the emitter in the reaction with FMNH2, but the 1‐deaza analog cannot be protonated at the corresponding position, thus excluding this possibility.
Binding Ca2+ to intracellular or to extracellular sites of dihydropyridine receptor of rabbit skeletal muscle discriminates between in vitro binding of Ca2+‐channel agonist and antagonist
Authors: KANNGIESSER Uwe , PONGS Olaf
Published: March 2005

Transverse tubule membrane vesicles contain dihydropyridine receptor of rabbit skeletal muscle in an inside‐out orientation. Digitonin‐solubilized, purified dihydropyridine receptor is embedded in digitonin vesicles in an outside‐out orientation. Ca2+ selectively stimulates binding of the Ca2+‐channel antagonist [3H]PN200‐110 to dihydropyridine receptor in the outside‐out but not the inside‐out orientation. The dissociation constant for binding Ca2+ to the extracellular Ca2+‐specific binding site of dihydropyridine receptor is 2–3μM. The data demonstrate that binding Ca2+ to the extracellular high‐affinity Ca2+‐binding site is required for binding dihydropyridines to dihydropyridine recepptor. This binding is inhibited, however, by 1–10 mM concentrations of any divalent cation tested (Ba2+, Mn2+, Mg2+). Also, Ca2+ selectively stimulates binding of the Ca2+‐channel agonist [3H]BayK8644 to dihydropyridine receptor in the inside‐out orientation. The titration of this Ca2+ dependence indicates that the dissociation constant for binding Ca2+ to the intracellular Ca2+‐specific binding site of dihydropyridine receptor is in the millimolar range. Thus, binding Ca2+‐channel agonist or antagonist to dihydropyridine receptor is modulated by binding Ca2+ to different sites of the receptor. Measurements of dissociation rate constants for binding [3H]PN200‐110 to dihydropyridine receptor in the presence of diltiazem, verapamil and/or Ca2+ indicate that Ca2+, like diltiazem or verapamil, is an allosteric effector of this receptor.
Solubilization and reconstitution of the sodium‐and‐chloride‐coupled glycine transporter from rat spinal cord
Authors: LOPEZ‐CORCUERA Beatriz , ARAGON Carmen
Published: March 2005

Synaptic membranes from rat spinal cord were solubilized in the presence of 2% sodium cholate, phospholipids and 15% ammonium sulphate. The soluble extract was incorporated into liposomes consisting of asolectin and crude rat brain lipids. Reconstitution of the functional transporter protein was achieved by removal of detergent by gel filtration. Several parameters proved to be important for optimal reconstitution efficiency: (a) the lipid composition of the liposomes, (b) the type of detergent, and (c) the phospholipid/protein and detergent/protein ratio during reconstitution. In the reconstituted system, the transport of glycine showed a specific activity about twice that of native vesicles. The ionic dependence of the transport, the inhibitory effect of nigericin in the presence of external sodium and the stimulatory effect of valinomycin in the presence of internal potassium on glycine transport were preserved and more clearly observed in the reconstituted system. These results indicate that, in this preparation, the glycine transporter protein retains the same features displayed in the synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogenicity and inhibitor sensitivity.
Requirement of DNA topoisomerases for in vitro chromatin assembly by 3T6 mouse cell extracts
Authors: CÁCERES Javier F. , BLANGY Daniel , GLIKIN Gerardo C.
Published: March 2005

Nuclear extracts of 3T6 mouse cells were able to assemble in vitro minichromosomes which displayed a 150‐bp periodicity. Activities of both DNA topoisomerases I and II were detected in these extracts. When a supercoiled pUC DNA was added, it was first relaxed in less than 3 min, then slowly supercoiled again in 1–4 h. Both reactions occurred either in the absence or the presence of added Mg2+ and/or ATP, they were not blocked by DNA topoisomerase II inhibitors and they were inhibited by an antiserum against DNA topoisomerase I and by camptothecin. These findings led us to propose that, under our in vitro assay conditions, chromatin assembly is mainly carried out by a DNA topoisomerase I.
Structure and expression of an α‐tubulin gene of Physarum polycephalum
Authors: WALDEN Paul D. , MONTEIRO Mervyn J. , GULL Keith , COX Robert A.
Published: March 2005

Fragments of Physarum polycephalum DNA generated by partial digestion with Sau3A wer cloned into phage‐λ EMBL4. A recombinant (phage‐λ EαTu) containing an α‐tubulin (Eα‐tubulin) gene was isolated. The Eα‐tubulin gene is part of the altB locus. The gene was sequenced and was found to contain seven intervening sequences. the α‐tubulin isotype (Eα‐tubulin) encoded by the gene has a methionine residue at the C‐terminus. The Eα‐tubulin gene has much in common with the Nα‐tubulin gene cloned into phage‐λ NM1149 [M. J. Monteiro & R. A. Cox (1987) J. Mol. Biol. 193, 427–438]. However, the gene products Eα‐tubulin and Nα‐tubulin differ in amino acid sequence near to the C‐terminus. E‐peptide (corresponding to amino acids 440–448 of Eα‐tubulin) and N‐peptide (corresponding to amino acids 437–445 of Nα‐tubulin) were synthesised and used to raise antibodies (E‐antibodies and N‐antibodies). The antibodies were used to show that on two‐dimensional gel elctrophoresis Nα‐tubulin travels to the α1 position and Eα‐tubulin moves to the α2 position. Gene‐specific DNA probes were used to show that transcripts of the Eα‐tubulin gene were present in the plasmodial but not in the amoebal phase of the life cycle. The E‐ and N‐antibodies detected Eα‐ and Nα‐tubulins in plasmodia but not in amoebae, confirming that the expression of the Eα‐ and Nα‐tubulin genes is regulated during development. Eα‐ and Nα‐tubulin were shown to be components of spindle microtubules by indirect immunofluorescence microscopy.
The amino terminus regulates binding to and activation of cGMP‐dependent protein kinase
Authors: LANDGRAF Wolfgang , HOFMANN Franz
Published: March 2005

The allosteric regulation of binding to and the activation of cGMP‐dependent protein kinase (cGMP kinase) was studied under identical conditions at 30°C using three forms of cGMP‐kinase which differed in the aminoterminal segment, e.g. native cGMP kinase, phosphorylated cGMP kinase which contained 1.4±0.4 mol phosphate/subunit and constitutively active cGMP kinase which lacked the amino‐terminal dimerization domain. These three enzyme forms have identical kinetic constants, e.g. number of cGMP‐binding sites, Km values for MgATP and the heptapeptide Kemptide, and Vmax values. In the native enzyme, MgATP decreases the affinity for binding site 1. This effect is abolished by 1 M NaCl. In contrast, high concentrtions of Kemptide increase the affinity of binding site 2 about fivefold. Under the latter conditions, identical Kd values of 0.2 μM were obtained for sites 1 and 2. Salt, MgATP and Kemptide do not affect the binding kinetics of the phosphorylated or the constitutively active enzyme, suggesting that allosteric regulation depends solely on the presence of a native amino‐terminal segment. Cyclic GMP activates the native enzyme at Ka values which are identical with the Kd values for both binding sites. The activation of cGMP‐dependent protein kinase is noncooperative but the Ka value depends on the substrate peptide concentration. These results show that the activity of cGMP kinase is primarily regulated by conformational changes within the amino‐terminal domain.
A further insight into the binding of blood clotting factors to membranes
Authors: PRIGENT‐DACHARY Jeanne , LINDHOUT Théo , BOISSEAU Michel René , DUFOURCQ Jean
Published: March 2005

The active site of factor Xa, labelled with dansylglutamylglycylarginine (DnsEGR) is sensitive to association with Ca2+, factor Va and phospholipids. When bound to factor Va, DnsEGR — factor‐Xa does not change the composition of the binding site of factor Va, as shown by fluorescence energy‐transfer experiments between the Trp residues of factor Va and pyrene‐labelled phospholipids.

Prothrombin was cleaved by α‐chymotrypsin into two parts: N‐terminal residues 1–41 (peptide 1–41) containing the γ‐carboxyglutamic acid residues (Gla), and des‐(1–41)‐prothrombin; their membrane association was investigated. Peptide 1–41 contains the aromatic residues Tyr and Trp in positions 24 and 41, respectively, and is suitable for fluorescence spectroscopy. The absence of fluorescence energy transfer between these residues suggests that they are more than 2.8 nm apart. Binding of Ca2+ and of phospholipids involves essentially the Tyr residue, while the C‐terminal characteristics of the Trp residue remain unchanged. The conformational change which takes place on binding does not shorten the distance between Tyr and Trp beyond 2.8 nm. Our conclusion is that peptide 1–41 has an extended conformation. This result is compatible with the disordered character of the Gla region found in the crystalline structure of fragment 1 of prothrombin.

Ca2+ induces a greater fluorescence energy transfer between prothrombin and membranes labelled with pyrene but has no influence on the binding of des‐(1–41)‐prothrombin. Moreover, the binding curves of des(1–41)‐prothrombin are similar to those of prothrombin in the absence of Ca2+. It is concluded that the Ca2+‐independent association of prothrombin with membranes involves essentially that part of the prothrombin molecule deleted in the Gla region.
The C‐terminal half of the 11‐kDa subunit VIII is not necessary for the enzymic activity of yeast ubiquinol: cytochrome‐c oxidoreductase
Authors: SCHOPPINK Peter J. , DE JONG Marianne , BERDEN Jan A. , GRIVELL Leslie A.
Published: March 2005

Inactivation of the gene encoding the 11‐kDa subunit VIII of yeast ubiquinol: cytochrome c oxidoreductase leads to an inactive complex, which lacks detectable cytochrome b [Maarse, A. C., De Haan, M., Schoppink, P. J., Berden, J. A. and Grivell, L. A. (1988) Eur. J. Biochem. 172, 179–184] and in which the steady‐state levels of the Fe‐S protein and the 14‐kDa subunit VII are severely reduced. When the 11‐kDa° mutant is transformed with a gene encoding a protein consisting of the 11‐kDa protein minus its last 11 amino acids and fused to a 7‐aminoacid sequence encoded by a stop oligonucleotide, the complex is assembled normally. Enzyme activity is similar to that of the wild type, as is also the sensitivity of the complex to antimycin and myxothiazol. Transformation of the mutant with a gene encoding a protein consisting of the 11‐kDa protein lacking the last 43 amino acids (i.e. almost half the protein) and fused to the same 7‐amino‐acid sequence as above, gives partial restoration of the complex. The Fe‐S protein and the 14‐kDa subunit VII still exhibit low steady‐state levels, but cytochrome b is present again, albeit at a strongly reduced level. Electron transport activity is also partially restored and correlates with the level of cytochrome b indicating that the turnover number of the complex is similar to that of wild‐type complex III. These findings demonstrate the important role played by the 11‐kDa protein in the stabilization of cytochrome b. They also imply that at least the C‐terminal half of the 11‐kDa protein is not part of an ubiquinol‐binding site. Moreover, since the deletion has no effect on the sensitivity of the complex to myxothiazol and antimycin, at least this part of the protein is probably not involved in binding of these inhibitors.
A combination of intracellular leucine with either glutamate or aspartate inhibits autophagic proteolysis in isolated rat hepatocytes
Authors: CARO L. Heleen P. , PLOMP Peter J. A. M. , LEVERVE Xavier M. , MEIJER Alfred J.
Published: March 2005

It has been shown previously that the inhibition of autophagic proteolysis in liver by a physiological mixture of amino acids can be mimicked completely by addition of leucine in combination with alanine [Leverve, X. M., Caro, L. H. P., Plomp, P. J. A. M. and Meijer, A. J. (1987) FEBS Lett. 219, 455–458]. We have now further defined conditions which lead to this inhibition.

Isolated rat hepatocytes were incubated in the perifusion system in which the cells can be maintained at a steady state in the presence of low amino acid concentrations.

Combinations of leucine (0.5 mM) with either alanine, glutamine, asparagine or proline (2 mM) inhibited proteolysis by 40–50%. Under these conditions, both in the absence and presence of the transaminase inhibitor, aminooxyacetate, a correlation was found between the extent of inhibition of proteolysis and the sum of the total intracellular amounts of asparate and glutamate.

Inhibition of proteolysis by leucine and leucine analogues did not correlate with their ability to activate glutamate dehydrogenase.
Glycosylation of human leukocyte locus A molecules is dependent on the cell type
Authors: MERLU Benoît , NEEL Dominique , GOUSSAULT Yves , CHARRON Dominique J.
Published: March 2005

Peripheral blood monocytes and B cells were isolated from a normal donor, and a portion of the B cells was transformed by the Epstein‐Barr virus (EBV). Human leukocyte locus A (HLA) class‐I and class‐II molecules were immunoprecipitated by specific monoclonal antibodies after cell labeling with [3H]mannose. Glycopeptides of HLA molecules were obtained by pronase digestion and were analysed by lectin‐affinity chromatography. Complex structures were hydrazinolysed, and their sialic acid content was analysed by ion‐exchange chromatography, whereas the high‐mannose structures were separated by HPLC. In normal cells, class‐I antigens bear principally fucosylated biantennary structures while HLA‐DR class‐II antigens bear bi‐, tri‐ and tetraantennary structures and high‐mannose structures. HLA antigens are more sialylated on normal B cells than on normal monocytes. An EBV cell line had a very different pattern of HLA‐antigen glycosylation when compared with the original B cells. In the transformed cells, the fractions containing biantennary structures are largely decreased. In contrast, an increase of the tri‐ and tetra‐antennary structure fractions is noticed, particulary in class‐II molecules, while both triantennary and high‐mannose structures are increased in class‐I molecules. Moreover, when compared to normal B cells, the complex structures of class‐I antigens in the EBV‐transformed B‐cell line are undersialylated while they are oversialylated in the case of the class‐II molecules.
Serum albumin binding of palmitate and stearate
Authors: BRODERSEN Rolf , VORUM Henrik , SKRIVER Elisabeth , PEDERSEN Anders Overgaard
Published: March 2005

In usual studies of ligand binding to a carrier, free and bound ligand concentrations are measured in equilibrium mixtures with varying carrier and ligand concentrations. The observed data are then analyzed by a binding equation such as Scatchard's or the general binding equation. With palmitic, stearic and oleic acids as ligands we found that the aqueous solubility is too low to allow this procedure. We have consequently transformed the general binding equation so that it does not contain parameters related to aqueous solutions of the ligand. While the classical binding equations describe affinities of transfer of a ligand from an aqueous solution to the carrier, the new equation is valid for transfer of a ligand from one bound state to another, i.e. for relative binding description. The relative binding constants, L1, L2, L3…Li, in the new equation thus define the transfer affinity for the ligand from a 1:1 complex with a standard carrier to an i:1 complex of the ligand with the carrier investigated.

Binding of palmitate and stearate to human serum albumin was studied by determination of dialytic exchange rates between identical fatty acid/albumin solutions. The results were analyzed by the new equation without reference to ligands in aqueous solution.
Circular dichroic spectroscopy of membrane haemoproteins
Authors: DEGLI ESPOSTI Mauro , PALMER Graham , LENAZ Giorgio
Published: March 2005

The circular dichroism (CD) of dihaem cytochrome b from mitochondrial and bacterial ubiquinol:cytochrome‐c reductase (bc1 complex) has been characterized. The dichroic properties of the yeast purified cyt b are very similar to those of the native cyt b within the mitochondrial bc1 complex. The CD spectra in the Soret region of the native cytochrome b present in all species studied show an intense bisignate Cotton effect having a zero‐crossing wavelength close to the absorbance maximum.

In preparations partially or completely depleted of the low‐potential b haem (b1) the CD spectra exhibit a single positive Cotton effect resembling the corresponding absorption spectrum. This is particularly evident in the purified cytochrome b‐562 from Rhodobacter sphaeroides R26, which contains only the high‐potential b haem (bh). These spectral features together with the reconstitution of the cytochrome b1 haem have been used to resolve the CD contribution of each haem to the CD spectra of cytochrome b.

The mechanisms which might be responsible for the optical activity have been examined. It appears that the CD spectra of cytochrome b derive from both the mutual interaction of its two haems (giving rise to exciton coupling) and to the interaction of each haem with nearby aromatic residues, other than the pairs of histidines which coordinate the iron. The dipole coupling between haem and aromatic residues appears to be more important than exciton coupling in the CD spectra of oxidized b cytochromes and correlations have been made between the CD features and the proposed structure of cytochrome b.
Méthionyl‐tRNA synthétase du germe de blé
Authors: ARCHAMBAULT de VENÇAY Jacques
Published: March 2005

Methionyl‐tRNA synthetase from wheat germ. – Effect of an endogenous protease and correlations between structural characteristics and catalytic properties. Methionyl‐tRNA synthetase (MetRS) has been described as a free monomeric or oligomeric enzyme, or included in a multienzyme complex. Moreover, on limited tryptic digestion, it can generate shorter forms.

So, when purified from wheat‐germ lysate, the possible presence of proteases able to hydrolyse this enzyme was investigated.

When extraction was performed with sulfhydryl‐blocking reagents, an active monomeric MetRS of Mr 105000 was purified. This enzyme form was identical to the structure exhibiting methionyl‐tRNA synthetase activity in multisynthetic complexes. Without this inhibitor, MetRS was purified as an active dimeric form of Mr 165000 with identical subunits of Mr 82000.

A protease inhibited by sulfhydryl‐blocking reagents and included in a complex of Mr 2 · 106 was isolated from this wheat‐germ lysate. This protease was able to hydrolyse different proteins (albumin, casein), but was without activity for a trypsin substrate, such as N‐α‐benzoyl‐dl‐arginine p‐nitroanilide. When added to a solution of Mr‐105000 MetRS, it yielded an inactive peptide of Mr 20000, containing numerous charged amino acids and a protein of Mr 82000, able to give an active dimeric enzyme of Mr 165000. Amino acid analysis of this last form, indicated an identical structure with the active dimeric MetRS of Mr 165000, purified in the absence of protease inhibitors. Moreover, the affinity for methionine was the same for the monomeric enzyme of Mr 105000 and the dimeric form of Mr 165000, probably because proteolysis did not affect the catalytic domain.

When enzymic activity of the proteolysed form (Mr 2×82000) was studied versus enzyme concentration, a decrease in specific activity, at low concentrations, was seen. This phenomenon was analysed on the basis of the existence of an equilibrium between an active dimer and two inactive monomers. With the active monomeric form of Mr 105000, no change in specific activity with decreasing enzyme concentration occurred.