bluuebunny/crossref_metadata_2025 huggingface dataset:
The active centers of adenylylsulfate reductase from Desulfovibrio gigas
Authors: LAMPREIA Jorge , MOURA Isabel , TEIXEIRA Miguel , PECK Harry D. , LEGALL Jean , HUYNH Boi H. , MOURA José J. G.
Published: March 2005

In order to utilize sulfate as the terminal electron acceptor, sulfate‐reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non‐identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (∼ 400 kDa). It is an iron‐sulfur, flavincontaining protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa.

Low‐temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g= 2.02 and only detectable below 20 K. This signal represented a minor species (0.10–0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe‐grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe‐S center I) observable below 30 K and a concomitant decrease in intensity of the g= 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase‐reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g= 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin–spin‐coupled nature (observable below 20 K), indicating the presence of at least two iron‐sulfur centers, (centers I and II).

Mössbauer studies on 57Fe‐enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin‐type [4Fe‐4S] cluster. Center I exhibits spectra with atypical Mössbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin‐type [4Fe‐4S] cluster, suggesting a different structure and/or a distinct cluster‐ligand environment.
Evidence for variable metal‐radical spin coupling in oxoferrylporphyrin cation radical complexes
Authors: BILL Eckhard , DING Xiao‐Qi , BOMINAAR Emile L. , TRAUTWEIN Alfred X. , WINKLER Heiner , MANDON Dominique , WEISS Raymond , GOLD Avram , JAYARAJ Karupiah , HATFIELD William E. , KIRK Martin L.
Published: March 2005

Oxoferrylporphyrin cation radical complexes were generated by m‐chloroperoxybenzoic acid oxidation of the chloro and trifluoromethanesulfonato complexes of tetramesitylporphyrinatoiron(III) [(TMP)Fe] and the trifluoromethanesulfonato complex of tetra(2,6‐dichlorophenyl)porphyrinatoiron(III) [TPP(2,6‐Cl)Fe]. Coupling between ferryl iron (S= 1) and porphyrin radical (S′= 1/2) spin systems was investigated by Mössbauer and EPR spectroscopy. The oxoferrylporphyrin cation radical systems generated from the TMP complexes show strong ferromagnetic coupling. Analysis of the magnetic Mössbauer spectra, using a spin Hamiltonian explicitly including a coupling tensor J̃ suggests an exchange‐coupling constant J > 80 cm−1. The EPR spectra show non‐zero rhombicity, the origin of which is discussed in terms of contributions from the usual zero‐field effects of iron and from iron‐radical spin‐dipolar interaction. A consistent estimate of zero‐field splitting parameter D∼+ 6 cm−1 was obtained by EPR and Mössbauer measurements. EPR and Mössbauer parameters are shown to be slightly dependent on solvent, but not on the axial ligand in the starting (TMP)Fe complex. In contrast to the TMP complex, the oxoferrylporphyrin cation radical system generated from [TPP(2,6‐Cl)FeOSO2CF3] exhibits Mössbauer and EPR spectra consistent with weak iron‐porphyrin radical coupling of |J|∼ 1 cm−1.
The interaction of Triton X‐100 with purple membranes
Authors: GONZÁLEZ‐MAÑAS Juan M. , VIRTO M. Dolores , GURTUBAY José‐Ignacio G. , GOÑI Félix M.
Published: March 2005

The interaction of the non‐ionic surfactant Triton X‐100 with Halobacterium purple membranes has been examined at sublytic and lytic surfactant concentrations. These membranes present a number of important peculiarities in their behaviour towards the surfactant. Although solubilization is a very slow process, with a half‐time of the order of hours, detergent binding appears to occur at the same fast rate as that found in other membranes. Lipids are solubilized more easily than proteins, so that hardly any protein is solubilized at surfactant concentrations at which about 75% of the lipid is in the form of detergent‐mixed micelles; once started, protein solubilization takes place within a narrow range of surfactant concentrations. Retinal provides a built‐in probe to monitor detergent‐induced conformational changes by spectroscopy in the visible range. No spectral variation is detected at the prelytic stage, i.e. when detergent is incorporated into the membrane in monomeric form. Membrane disruption is accompanied by a blue shift in the absorption maximum, retinal isomerization (from all‐trans to 13‐cis), and a decrease in specific absorbance (bleaching). Increasing detergent concentrations after solubilization is completed do not produce further shifts in the spectral maximum, but the specific absorbance is progressively decreased. It is shown that Triton X‐100 has a complex effect on the retinal chromophore, modifying its configuration and microenvironment (changes in maximum wavelength) and promoting hydrolysis of the retinal‐bacterioopsin Schiff's base (bleaching).
Purification, characterization and inhibition of d‐3‐aminoisobutyrate aminotransferase from the rat liver
Authors: TAMAKI Nanaya , KANEKO Masae , MIZOTA Chizuru , KIKUGAWA Mariko , FUJIMOTO Sigeko
Published: March 2005

d‐3‐Aminoisobutyrate–pyruvate aminotransferase was purified 2000‐fold from rat liver extract using heat treatment, ammonium sulfate fractionation, carboxylmethyl‐Sepharose CL‐6B, DEAE‐Sepharose CL‐6B, hydroxyapatite, Sephacryl S‐200 and electrofocusing chromatographies. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 220 kDa and the subunit molecular mass was 52 kDa. The enzyme exhibited absorption maxima at 280 nm and 412 nm with a shoulder at 330 nm at neutral pH. The pH optimum for enzyme activity was 9.5 and the Km values for β‐alanine and pyruvic acid were calculated to be 0.81 mM and 0.45 mM, respectively. The purified enzyme catalyzed the transamination of ω‐amino acids; β‐alanine and d‐3‐aminoisobutyric acid served as good amino donors, and pyruvic acid, glyoxylic acid and oxaloacetic acid were favorable amino acceptors.

6‐Azauracil and 6‐azathymine were found to be potent inhibitors of purified rat liver d‐3‐aminoisobutyrate–pyruvate aminotransferase. 6‐Azauracil acted as a competitive inhibitor with respect to β‐alanine, and was an uncompetitive inhibitor with respect to pyruvic acid with a Ki of approximately 8.9 mM.
Functional and structural analysis of photosystem II core complexes from spinach with high oxygen evolution capacity
Authors: HAAG Elisabeth , IRRGANG Klaus‐D. , BOEKEMA Egbert J. , RENGER Gernot
Published: March 2005

Oxygen‐evolving photosystem II core complexes were prepared from spinach by solubilizing photosystem II membrane fragments with dodecyl‐β‐d‐maltoside. The core complexes consist of the intrinsic 47‐kDa, 43‐kDa, D1 and D2 polypeptides, the two subunits, of cytochromeb559and the extrinsic 33‐kDa protein. In the presence of 50 mM CaCl2they exhibit a high oxygen evolution rate of 1.3 ± 0.2 mmol O2· mg chloropyll−1· h−1with either 2,6‐dichloro‐benzouinone or K3[Fe(CN)6] as acceptor. Electron micrographs of these complexes reveal an obtuse triangular structure in when viewed from the top, measuring 15.3 nm on one side and 10.6 nm on the other two sides. An average height of 7.3 nm was determined from the side view position. These data are in good agreement with previously reported dimensions for photosystem II core complexes [Irrgang, K.‐D., Boekema, E. J., Vater, J. and Renger, G. (1988)Eur. J. Biochem. 178, 209–217]. In contrast to previous reports the extrinsic 33‐kDa subunit could be resolved for the first time. It appears as a small protrusion when the complex is viewed from the side and seems to cover the lumenal side of the core complex appearing as a disk with a thickness of 1.5–3.3 nm.
The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene
Authors: THOMPSON Daphne E. , BREHM John K. , OULTRAM John D. , SWINFIELD Tracy‐Jane , SHONE Clifford C. , ATKINSON Tony , MELLING Jack , MINTON Nigel P.
Published: March 2005

A 26‐mer oligonucleotide probe was synthesized (based on the determined amino acid sequence of the N‐terminus of the Clostridium botulinum type A neurotoxin, BoNT/A) and used in Southern blot analysis to construct a restriction map of the region of the clostridial genome encompassing BoNT/A. The detailed information obtained enabled the cloning of the structural gene as three distinct fragments, none of which were capable of directing the expression of a toxic molecule. The central portion was cloned as a 2‐kb PvuII‐TaqI fragment and the remaining regions of the light chain and heavy chain as a 2.4‐kb ScaI‐TaqI fragment and a 3.4kb HpaI‐PvuII fragment, respectively. The nucleotide sequence of all three fragments was determined and an open reading frame identified, composed of 1296 codons corresponding to a polypeptide of 149 502 Da. The deduced amino acid sequence exhibited 33% similarity to tetanus toxin, with the most highly conserved regions occurring between the N‐termini of the respective heavy chains. Conservation of Cys residues flanking the position at which the toxins are cleaved to yield the heavy chain and light chain allowed the tentative identification of those residues which probably form the disulphide bridges linking the two toxin subfragments.
Isolation and characterization of a cDNA encoding porcine gastric haptocorrin
Authors: HEWITT Jane E. , SEETHARAM Bellur , LEYKAM Joseph , ALPERS David H.
Published: March 2005

A cDNA encoding the gastric haptocorrin was isolated from a porcine gastric mucosal λgt11 cDNA library using oligonucleotide probes. The 1.4‐kb cDNA contains a 1.25‐kb open reading frame and 178 nucleotides of 3′ noncoding region. Although no initiator methionine is present, primer extension analysis indicated that the transcription initiation site is only 100 bp upstream of the 5′ end of this clone. Northern blot analysis showed that a single mRNA species of 1.6 kb exists in hog gastric mucosa. There was no cross‐hybridization between this cDNA and the mRNA for haptocorrin in rat submaxillary gland or gastric RNA by Northern blot analysis. This lack of cross‐reactivity was also seen on Southern blots, where cow, sheep and dog but neither rat nor mouse genomic DNAs showed cross‐hybridizing bands. Comparison of the deduced amino acid sequence of this cDNA with that previously reported for rat intrinsic factor [Dieckgraefe, B. K. et al. (1988) Proc. Natl Acad. Sci. USA 85, 46–50] showed considerable similarity, suggesting that these cobalamin‐binding proteins may have a common evolutionary origin.
Purification and characterization of heme oxygenase from chick liver
Authors: BONKOVSKY Herbert L. , HEALEY John F. , POHL Jan
Published: March 2005

A major inducible form of heme oxygenase (EC 1.14.99.3) was purified from liver microsomes of chicks pretreated with cadmium chlride. The purification involved solubilization of microsomes with Emulgen 913 and sodium cholate, followed by DEAE‐Sephacel, carboxymethyl‐cellulose (CM‐52) and hydroxyapatite chromatography, and FPLC through Superose 6 and 12 columns operating in series. The final product gave a single band on silver‐stained SDS/polyacrylamide gels (Mr= 33000). Optimal conditions for measurement of activity of solubilized heme oxygenase were studied. In a reconstituted system containing purified heme oxygenase, NADPH‐cytochrome reductase, biliverdin reductase and NADPH, the Km for free heme was 3.8 ± 0.5 μM; for heme in the presence of bovine serum albumin (5 mol heme/3mol albumin) the Km the 5.0 ± 0.8 μM; and the kmfor NADPH was 6.1 ± 0.4 μM (all values mean ± SD,n= 3). Oxygen concentration as low as 15üM, with saturating concentrations of heme and NADPH, did not affect the reaction rate, indicating that the supply of oxygen is not involved in the physiological regulation of activity of the enzyme. The pH optimum of the reaction was 7.4; at 37°C, the apparent Vmax was 580 ± 44 nmol biliverdin. (mg protein)−1· min−1 and the molecular activity was 19.2 min−1. Biliverdin IXa was the sole biliverdin isomer formed. In the presence of purified biliverdin reductase, biliverdin was converted quantitatively to bilirubin. Addition of catalase to the reconstituted system decreased the breakdown of heme to non‐biliverdin products and led to nearly stoichiometric conversion of heme to biliverdin. Activity of the enzyme in the reconstituted system was inhibited by metalloporphyrins in the following order of decreasing potency: tin mesoporphyrin > tin protoporphyrin > zine protoporphyrin > manganese protoporphyrin > cobalt protoporhyrin. Protoporphyrin (3.3 or 6.6 μM) (and several other porphyrins) and metallic ions (100 μM) alone had little if any inhibitory effect, except for Hg2+ which inhibited by 67% at 10 μM and totally at 15 μM. Following partial cleavage, fragments of the purified enzyme were sequenced. Comparison of sequences to those derived from cDNA sequences for the major inducible rat and human heme oxygenase showed 69% and 76% similarities, respectively. The histidine residue at position 132 of rat heme oxygenase‐1 and the residues (Lys128‐Arg136) flanking His132 were conserved in all three enzymes, as well as in the corresponding portion of a fourth less highly similar rat enzyme, heme oxygenase‐2. It is suggested that this region plays a critical role in heme binding and in catalyzing the heme oxygenase reaction.
Structure determination of the major N‐ and O‐linked carbohydrate chains of the β subunit from equine chorionic gondotropin
Authors: DAMM Jan B. L. , HÅRD Karl , KAMERLING Johannis P. , van DEDEM Gijs W. K. , VLIEGENTHART Johannes F. G.
Published: March 2005

The carbohydrate moieties of equine chorionic gonadotropin α and β subunits were released from the protein backbones by successive treatments with peptide‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F and alkaline borohydride and then fractionated by FPLC and HPLC. The major N‐ and O‐linked glycans of the β subunit were characterized by 500‐MHz 1H‐NMR spectroscopy, showing a remarkable structural heterogeneity for the N‐glycosidically linked chains, comprising mono‐,di‐, tri‐ and tri′‐antennary N‐acetyllactosamine type of glycans, being partly α1‐6 fucosylated at the Asn‐bound GlcNAc residue and having α2‐6 and α2‐3 linked N‐acetyl‐ and N‐acetyl‐4‐O‐acetylneuraminic acid residues as sialic acid constituents. Significant differences in this respect were detected for the partially characterized glycans of the α subunit. The major part of the O‐linked carbohydrate chains, occurring solely in the β subunit, is formed by tri‐, tetra‐, penta‐ and hexa‐saccharides. There are indications for the presence of oligo(N‐acetyllactosamine) units in both the N‐ and O‐linked glycans of the β subunit.
Reconstitution of photophosphorylation in EDTA‐treated thylakoids by added chloroplast coupling factor 1 (ATPase) and chloroplast coupling factor 1 lacking the δ subunit
Authors: ENGELBRECHT Siegfried , ALTHOFF Gerd , JUNGE Wolfgang
Published: March 2005

Upon EDTA treatment thylakoids lose the chloroplast coupling factor 1 (CF1) part of their ATP synthase, CFoCF1, this exposes the proton channel, CFo. The previously established ability of the CF1 subunit δ to block the proton leak through CFo prompted us to study (a) the ability of complete CF1 and, for comparison, CF1 lacking the δ subunit to block proton leakage and thereby to reconstitute structurally some photophosphorylation activity of the remaining CFoCF1 molecules and (b) their ability to form functional enzymes (functional reconstitution). In order to discriminate between activities caused by added CF1 or CF1 (−δ) and remaining CFoCF1, the former were inhibited by chemical modification of subunit β by N,N′‐dicyclohexyl carbodiimide (DCCD) and the latter by tentoxin. We found that added CF1 acted both structurally and functionally while added DCCD‐treated CF1 (DCCD‐CF1) acted only structurally. In contrast to previous observations, CF1(−δ) and DCCD‐CF1(−δ) also acted structurally although the reduction of proton leakage was smaller than with DCCD‐CF1. Hence there was no functional reconstitution without subunit δ present. Previous studies indicated that only a small fraction of exposed CFo is highly conducting and that this small fraction is distinguished by its high affinity for added CF1. The results of this study point rather to a wider distribution of CFo conductance states and binding affinities.
The conformation of single‐stranded nucleic acids
Authors: PAQUETTE Jean , NICOGHOSIAN Krikor , QI Guo‐rong , BEAUCHEMIN Nicole , CEDERGREN Robert
Published: March 2005

Conformational analyses using the single‐strand‐specific nuclease from mung bean and restriction endonucleases have been performed on a series of DNA fragments related to the sequence of the yeast initiator tRNAMet. Mung bean nuclease cleaves DNA fragments exclusively in some, but not all, single‐stranded regions as predicted by RNA secondary rules. Comparison of cleavage patterns of yeast initiator tRNAMet, tDNAMet (a DNA oligomer having the sequence of tRNAMet) and the anti‐tDNAMet (the complement of tDNAMet) suggests that the conformation of the three molecules is very similar. Furthermore, both tDNA and anti‐tDNA are cleaved by HhaI and CfoI restriction endonucleases at two GCG/C sites which would be in double‐ stranded regions (the acceptor and dihydrouridine stem), if the two molecules adopt the tRNA cloverleaf structure. On the other hand, minor cleavage products show that the core region, i.e. the extra loop area, is slightly more exposed in tDNA and in anti‐tDNA than in tRNA. Therefore, we submit that the global conformation of nucleic acids is primarily dictated by the interaction of purine and pyrimidine bases with atoms and functional groups common to both RNA and DNA. In this view the 2′‐hydroxyl group, in tRNA at least, is an auxiliary structural feature whose role is limited to fostering local interactions, which increase the stability of a given conformation.
Escherichia coli DNA helicase I
Authors: BENZ Inga , MÜLLER Holger
Published: March 2005

Gene traI of the Escherichia coli F sex factor which encodes DNA/helicase I was subcloned in a λ pL‐based plasmid vector and expressed in a background of pL non‐repressing cells. Neither the non‐repressed pL promoter nor the production of a high level of functional helicase I are toxic. Enzyme purified from this source was studied in the electron microscope. The results show that helicase I binds cooperatively to single‐stranded DNA. DNA covered with the helicase appears in fixed, negatively stained specimens as a smooth‐contoured filament with a diameter of 12.5 ± 0.4 nm and an axial periodicity of 7.0 ± 0.2 nm. In unfixed specimens, discrete particles with axes of 12.7 ± 0.5 nm and 7.2 ± 0.5 nm are visible. They are consistent in size with helicase I monomers (Mr 180000) suggesting that the molecule is almost isometric, despite a frictional ratio of 1.71 calculated from its diffusion coefficient. Helicase I free of DNA appears as aggregates.

For comparison, a truncated traI. lacking coding for the amino‐terminus of the product, was cloned by fusing it to an MS2 replicase gene fragment. The chimeric gene product (named helicase I del29) retains strand‐separating activity although it fails to show cooperative DNA binding behavior. Judged from the length of the helicase‐I‐specific sequence of this polypeptide. tral is located 1.3 kb nearer to the distal end of the F transfer operon compared to the position proposed in a previous genetic map. The revised location of traI has implications for understanding distal functions of the transfer operon.
DNA sequence of a citrate carrier of Klebsiella pneumoniae
Authors: van der REST Michel E. , SCHWARZ Elisabeth , OESTERHELT Dieter , KONINGS Wil N.
Published: March 2005

The citrate transport determinant of plasmid pES1 from Klebsiella pneumoniae [Schwarz E. and D. Oesterhelt (1985) EMBO J. 4, 1599–1603] has been subcloned in Escherichia coli DH1. The DNA sequence of a 1723‐base fragment that codes for the citrate carrier has been determined and the gene product has been characterized with the T7 promoter system. The DNA fragment contains an open reading frame of 1332 base pairs and codes for a protein of 444 amino acids. The hydropathy profile suggests that the protein is very hydrophobic and contains 12 membrane‐spanning segments centered around a hydrophilic core. The gene for the citrate carrier has 66% similarity with a citrate carrier determinant from a naturally occurring plasmid responsible for secondary transport of citrate across the cytoplasmic membrane of E. coli.
Selective purification of sex‐influenced esterase from rat serum by immunoaffinity chromatographies
Authors: HIROSE Tohru , MORITA Atsushi , YAMADA Makoto , NIKAIDO Hiroko , HAYAKAWA Jun‐ichiro , NIKAIDO Osamu
Published: March 2005

Sex‐influenced esterase (ES‐SI) is a female‐specific serum esterase of rats. There are two alleles at the Es‐Si locus, Es‐Sia coding for ES‐SI and Es‐Sib, a silent allele. ES‐SI was purified to electrophoretic homogeneity from the serum of adult female SI3 rats by a four‐step purification. First, albumin was removed from the serum, by Blue‐Sepharose CL‐6B affinity chromatography. Sera of rats carrying Es‐Sia and Es‐Sib were utilized to make two novel immunoaffinity columns. Rat anti‐(ES‐SI) alloantibody was obtained after the immunization of rats carrying Es‐Sib with rat ES‐SI‐positive serum. ES‐SI bound to the gel in this antibody‐conjugated affinity chromatography. Simultaneously, rabbit anti‐(rat serum protein lacking ES‐SI) antibody was raised against ES‐SI‐negative rat serum and used to exclude other serum proteins. ES‐SI was recovered in the flow‐through fractions in this antibody‐conjugated immunoaffinity chromatography, whereas the other serum proteins bound to the gel. ES‐SI was selectively purified about 240‐fold by combining these immunoaffinity chromatographies. This combination is a very powerful technique for purification of the protein. Finally, ES‐SI was purified by preparative plyacrylamide gel electrophoresis after which it appeared as a single protein band (using silver staining) on SDS/polycrylamide gel electrophoresis with a molecular mass of 72 kDa. In the ordinary estimation of enzyme purification, the purification of ES‐SI was 30‐fold at most, because the specific activity of the enzyme was calculated from the bulky esterase activity. However, ES‐SI was selectively purified 2280‐fold comparing its recovery to that of total protein. ES‐SI exhibited a pI of 4.6 and optimum pH of 6.7. The activity of ES‐SI was completely inhibited by diisopropyl fluorophosphate at 10 nM but not at all by eserine.
A point mutation in the tyrosinase gene of BALB/c albino mouse causing the cysteine → serine substitution at position 85
Authors: SHIBAHARA Shigeki , OKINAGA Shoji , TOMITA Yasushi , TAKEDA Atsushi , YAMAMOTO Hiroaki , SATO Michihiko , TAKEUCHI Takuji
Published: March 2005

Murine albinism is characterized by complete lack of melanin pigments in skin and retina. In order to study the molecular basis of albinism, we have cloned and characterized the tyrosinase gene of BALB/c mice (c/c). Sequence analysis of this gene reveals a point mutation at nucleotide residue 387 (G → C transversion) causing a Cys→Ser substitution at position 85 in one of the cysteine‐rich domains of the tyrosinase molecule. Since this G→C transversion creates an additional DdeI site, we were able to confirm that this mutation is actually present in BALB/c genomic DNA using DNA amplification techniques. In contrast, both C57BL/6 (C/C) and DBA/2 (C C) mouse strains carry the G residue at the same position, suggesting that this point mutation is specific for the albino mutation at the c locus. Moreover, we were able to show that the tyrosinase containing Ser‐85 is not functional in transient expression of its cDNA. We therefore suggest that a G→C transversion at nucleotide residue 387 of the tyrosinase gene could lead to the albino phenotype of BALB/c mouse.
Interaction of hemin with placental glutathione transferase
Authors: CACCURI Anna Maria , ACETO Antonio , PIEMONTE Fiorella , DI ILIO Carmine , ROSATO Nicola , FEDERICI Giorgio
Published: March 2005

To verify a possible involvement of glutathione transferase π in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high‐affinity site) and 400 nM (low‐affinity site). In the presence of glutathione or S‐methylglutathione the high‐affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase π was studied using two different glutathione concentrations. With 1 mM glutathione a non‐linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 μM and the inhibition appeared to be non‐competitive with respect to 1‐chloro‐2,4‐dinitrobenzene. CD spectra of the bilirubin–glutathione‐transferase complex (350–600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high‐affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase π in the hemin transport.
Purification and properties of carnitine acetyltransferase from human liver
Authors: BLOISI Wilma , COLOMBO Irma , GARAVAGLIA Barbara , GIARDINI Roberto , FINOCCHIARO Gaetano , DIDONATO Stefano
Published: March 2005

Carnitine acetyltransferase was purified from the supernatant obtained after centrifugation of human liver homogenate to a final specific activity of 78.75 unit · mg−1 with acetyl‐CoA as a substrate. Human carnitine acetyltransferase is a monomer of 60.5 kDa with maximum activity in the presence of propionyl‐CoA and a pH optimum of 8.7. Apparent Km values for acetyl‐CoA are three times lower than for decanoyl‐CoA. Km values for L‐carnitine in the presence of acetyl‐CoA are six times lower than in the presence of decanoyl‐CoA. Km values for acetylcarnitine are three times lower than for octanoylcarnitine.

The polyclonal antibodies against human carnitine acetyltransferase recognize a 60.5‐kDa peptide in the purified preparation of human liver and brain homogenates and in immunoblots of mitochondrial and peroxisomal fractions from human liver.

Immunoprecipitation and SDS/PAGE analysis of 35S‐labelled proteins produced by human fibroblasts indicate that mitochondrial carnitine acetyltransferase is synthesized as a precursor of 65 kDa.

We also purified carnitine acetyltransferase from the pellet obtained after centrifugation of liver homogenate. The pellet was extracted by sonication in the presence of 0.5% Tween 20. The chromatographic procedures for the purification and the kinetic, physical and immunological properties of pellet‐extracted carnitine acetyltransferase are similar to those of carnitine acetyltransferase purified from the supernatant of human liver homogenate.
Hydrogen peroxide activates immediate binding of a Drosophila factor to DNA heat‐shock regulatory element in vivo and in vitro
Authors: BECKER Jacqueline , MEZGER Vallérie , COURGEON Anne‐Marie , BEST‐BELPOMME Martin
Published: March 2005

The synthesis of heat‐shock proteins via activation of heat‐shock genes occurs in response to heat and various physical or chemical stressing agents. Transcriptional activation of heat‐shock genes requires a heat‐shock regulatory element in their promoter, to which a heat‐shock specific transcription factor binds. In Drosophila cells, the heat‐shock factor already exists in unstressed cells in an inactive form and acquires the capacity to bind to the heat‐shock element following stress. The mechanism of this activation is not known; neither is it known whether the different stressing agents induce the heat‐shock response through a common mechanism. We previously proposed that many agents known to induce the heat‐shock response (substances interfering with respiratory metabolism, agents reacting with sulphydryl groups, metals, recovery from anaerobiosis and ischemia) might act via accumulation of reactive oxygen species, i. e. superoxide ion or H2O2. We show here that H2O2, introduced either in Drosophila cell cultures or in cell extracts, was able to activate heat‐shock‐element binding. Activation was rapid and H2O2 concentration dependent, with a threshold of 1 μM. These results were confirmed with mouse fibroblast cells. This very rapid activation, in vivo or in vitro, suggests a direct effect of H2O2 either on the heat‐shock factor itself or on its activator.
1H‐NMR study of heme propanoate mobility in the active site of myoglobin from Galeorhinus japonicus
Authors: YAMAMOTO Yasuhiko , INOUE Yoshio , CHÛJÔ Riichirô , SUZUKI Tomohiko
Published: March 2005

Time‐dependent NOE studies of the C131 and C171 methylene proton resonances of the heme peripheral propanoate groups have elucidated their mobility in the active site of the ferric high‐spin form of Galeorhinus japonicus myoglobin. A large difference in the chemical shift due to the non‐equivalence of the heme C131 and C171 methylene proton resonances allows selective irradiation of a given proton resonance by a high‐power selective decoupler pulse in spite of their fast relaxation rates. NOE accumulation of the resonance of one methylene proton after saturation of the resonance of the other proton essentially follows the theoretical prediction derived using the two‐spin approximation, and the cross‐relaxation retes for the heme C131 and C171 methylene proton spin systems were quantitatively determined. The correlation time for the mobility of the internuclear vector connecting the heme C131 or C171 methylene protons was then calculated from the cross‐relaxation rate and values of approximately 11 ns were obtained for both C131 and C171 methylene groups in 2 mM Galeorhinus japonicus myoglobin at 35°C. The immobile C131 and C171 methylenes of the heme propanoate groups, together with a large difference in chemical shift between the methylene proton resonances, dictate their fixed orientation with respect to the protein moiety as well as the heme plane. and are therefore consistent with the immobile heme in the active site of myoglobin.
Assignment of tyrosine resonances in the 1H‐NMR spectrum of tryptophan synthase α‐subunit
Authors: SAWADA Shintaro , AKUTSU Hideo , OGASAHARA Kyoko , YUTANI Katsuhide
Published: March 2005

In order to monitor the conformational changes of tryptophan synthase α‐subunit from Escherichia coli in solution resulting from amino acid substitutions, we have assigned the Tyr resonances in the aromatic region of the 1H‐NMR spectrum to specific residues. In the spectrum of the α‐subunit deuterated with [2,3,4,5,6‐2H5]Phe and [3,5‐2H2]Tyr, the C2 and C6 protons of Tyr gave completely isolated signals at acidic p2H. Some of the C3 and C5 proton resonances overlapped overlapped with each other at acidic p2H. By using a series of mutant α‐subunits in which each Tyr was singly substituted with His or Phe, we can now assign each of seven Tyr resonances in the aromatic region to a specific residue. We have previously studied the conformational stability of a series of variant α‐subunits at position 49 [Yutani et al. (1987) Proc. Natl Acad. Sci. USA 84, 4441–4444]. We now compare the 1H‐NMR spectra in the aromatic region of the wild‐type α‐subunit and mutant α‐subunits substituted with Phe or Gly in place of Glu‐49. The results suggest that the major conformational effects of substitutions at position 49 are localized close to the position of substitution.
Molecular analysis of a cellular decision during embryonic development of Drosophila melanogaster: epidermogenesis or neurogenesis
Authors: CAMPOS‐ORTEGA José A. , KNUST Elisabeth
Published: March 2005

In Drosophila melanogaster, the neuroblasts (neural progenitor cells) develop from a special region of the ectoderm, called the neuroectoderm. During early embryonic development, the neuroblasts separate from the remaining cells of the neuroectoderm, which develop as epidermoblasts (epidermal progenitor cells). The separation of these two cell types is the result of cellular interactions. The available data indicate that a signal chain formed by the products of several identified genes regulates the cell's decision to enter either neurogenesis or epidermogenesis. Various kinds of data, in particular from cell transplantation studies and from genetic and molecular analyses, suggest that the proteins encoded by the genes Notch and Delta interact at the membrane of the neuroectodermal cells to provide a regulatory signal. This signal is thought to lead, on the one hand, to epidermal development through the action of the genes of the Enhancer of split complex, a gene complex that encodes several functions related to the transduction and further processing of the signal, including the genetic regulation in the receiving cell; on the other hand, the signal is thought to lead to neural development through the participation of the genes of the achaete‐scute complex and daughterless, which are members of a family of DNA‐binding regulatory proteins and of the gene vnd whose molecular nature is still unknown.
Metabolism and actions of 2‐deoxy‐2‐fluoro‐d‐galactose in vivo
Authors: GRÜN Bernhard R. , BERGER Ulrike , OBERDORFER Franz , HULL William E. , OSTERTAG Hermann , FRIEDRICH Eckhard , LEHMANN Jochen , KEPPLER Dietrich
Published: March 2005

The synthetic d‐galactose analog 2‐deoxy‐2‐fluoro‐d‐galactose (dGalF) offers unique advantages for studies of the d‐galactose pathway by non‐invasive techniques using 19F‐NMR spectroscopy or positron emission from the 18F‐labeled compound. The metabolism of 2‐deoxy‐2‐fluoro‐d‐galactose was studied in rodents using the unlabeled, the 18F‐labeled, and the 14C‐labeled d‐galactose analog. Analyses for the metabolites of 2‐deoxy‐2‐fluoro‐d‐galactose were performed by HPLC, enzymatic methods, and 19F‐NMR spectroscopy in vivo and in vitro. The metabolism of 2‐deoxy‐2‐fluoro‐d‐galactose was most active in the liver which took up the major part of the administered dose of the 14C‐labeled d‐galactose analog, but renal excretion was also pronounced. This was confirmed by in vivo scanning of the rat using the 18F‐labeled sugar (1.5 μCi/g; 25 nmol/g) and examination by positron‐emission tomography and gamma camera.

The dose dependence of the levels of the hepatic metabolites of 2‐deoxy‐2‐fluoro‐d‐galactose was investigated for doses between 25 nmol/g body mass and 1 μmol/g body mass. After 1 h, the major part of the acid‐soluble uracil nucleotides consisted of UDP‐2‐deoxy‐2‐fluoro‐d‐hexoses when the dose was at least 0.1 μmol/g. With higher doses, 2‐deoxy‐2‐fluoro‐d‐galactose 1‐phosphate became the predominant initial metabolite. After a dose of 1 μmol/g 2‐deoxy‐2‐fluoro‐d‐galactose 1‐phosphate accumulated rapidly (5.3±0.4 μmol/g liver after 30 min) followed by the formation of UDP‐2‐deoxy‐2‐fluoro‐d‐galactose and UDP‐2‐deoxy‐2‐fluoro‐d‐glucose (0.7±0.1 μmol/g and 1.8±0.1 μmol/g, respectively, after 5 h).

The diversion of uridylate, due to the accumulation of UDP‐2‐deoxy‐2‐fluoro‐d‐hexoses, was associated with a rapid depletion of hepatic UTP, UDP‐glucose, and UDP‐galactose. The UTP content was decreased to 11±6% of normal within 15 min after administration of 2‐deoxy‐2‐fluoro‐d‐galactose at a dose of 1 μmol/g. The UTP‐depleting action was minimal, however, at a dose of 25 nmol/g or less, indicating that interference in uridylate metabolism would be negligible at the doses required for positron‐emission tomography of the liver using the 18F‐labeled compound. At higher doses, the UTP deficiency induced by 2‐deoxy‐2‐fluoro‐d‐galactose could be useful in the chemotherapy of d‐galactose‐metabolizing tumors such as hepatocellular carcinoma. At moderate doses of this d‐galactose analog, 19F‐NMR spectroscopy in vivo or in vitro could pinpoint defects of enzymes in galactosemia, e.g. of galactokinase, uridylyltransferase, or UDP‐glucose 4‐epimerase.
Organization and complete nucleotide sequence of the core‐histone‐gene cluster of the annelid Platynereis dumerilii
Authors: SELLOS Daniel , KRAWETZ Stephen A. , DIXON Gordon H.
Published: March 2005

The arrangement of the core‐histone genes, their transcriptional polarity and their nucleotide sequences have been determined for the polychaete annelid Platynereis dumerili.

A clone containing the core‐histone genes was isolated from a annelid genomic library constructed in the EMBL‐4 phage vector, using a trout H3 genomic probe. This clone was found to contain two and a half repeats of a 6‐kbp EcoRV fragment that contained one copy of each of the core‐histone genes.

The clusters are tandemly arrayed in the genome and the gene order within the core‐histone cluster does not vary. Absolutely no differences were found in the nucleotide sequences comprising the same part of two adjacent clusters (bases −225 to 2776 and bases 5821 to 8825).

The number of copies of the cluster appeared to be high: approximately 660 copies/diploid cell, as also observed in sea urchins and amphibians. There are also some additional subtypes of histone gene organization: multimers of tandemly arrayed genes and isolated genes; these are present at a much lower copy number (an average of 40–50 copies/diploid genome). Two mRNAs (for H2B and H3) are transcribed from one DNA strand and the two other histone mRNAs (for H2A and H4) from the other strand as is the case for some insects and certain vertebrates.

No H1‐coding sequence has been found in the completely sequenced four‐membered cluster. The organization of histone genes in P. dumerilii is similar to the clustering found in Caenorhabditis elegans but in this nematode worm several different types of organization are observed with a low copy number for each.
GAP1, the general amino acid permease gene of Saccharomyces cerevisiae
Authors: JAUNIAUX Jean‐Claude , GRENSON Marcelle
Published: March 2005

In Saccharomyces cerevisiae, mutations at the GAP1 locus selectively abolish the activity of the general amino acid transport system. This permease catalyses active transport of apparently all biological amino acids across the plasma membrane. We have determined the nucleotide sequence of the GAP1 gene. The sequence contains an open reading frame of 601 codons corresponding to a polypeptide of Mr 65578. This polypeptide is strongly hydrophobic; it exhibits three potential glycosylation sites. Hydropathy analysis suggests 12 membrane‐spanning regions. The N‐terminal domain is charged, it does not resemble hydrophobic signal sequences found in secreted proteins. Hence the GAP1 gene encodes a protein with characteristics typical of integral membrane proteins translocating ligants across cellular membranes. The deduced amino acid sequence of GAP1 protein presents strong similarities to those of the yeast arginine, histidine and proline permeases, suggesting a common evolutionary origin for these amino acid permeases.

Nitrogen‐source regulation of the GAP1 permease is believed to occur at two distinct levels, i.e. permease synthesis and permease activity [Grenson (1983) Eur. J. Biochem. 133, 135–139]. Northern analysis of GAP1‐specific transcripts in wild‐type and in mutant strains is in agreement with these views and indicates that nitrogen catabolite repression of GAP1 synthesis occurs at the RNA level.
Monoclonal antibodies against different epitopes of peptide hormones
Authors: JURZAK Mirek , BOER Rainer , FRITZSCH Günter , KOJRO Elzbieta , FAHRENHOLZ Falk
Published: March 2005

In order to produce monoclonal antibodies directed against different epitopes of the neurohypophyseal hormone vasopressin, the hormone was coupled to carrier proteins via photoreactive groups at different positions in the vasopressin sequence: [2‐(4‐azidophenylalanine), 8‐arginine]vasopressin (peptide P1, photoreactive group at position 2) and desamino‐[8‐N6‐(4‐azidophenylamidino)lysine]vasopressin (peptide P2, photoreactive group at position 8) were conjugated to thyroglobulin by flash photolysis. Monoclonal antibodies against these conjugates bound {[3H]8‐arginine}vasopressin with dissociation constants ranging over 40–400 nM. Epitope analysis by means of competitive ELISA showed that the monoclonal antibody obtained with peptide P1 as hapten was directed against the C‐terminal acyclic tripeptide when its conformation was stabilized by interaction with the disulphide‐linked cyclic hexapeptide. In contrast, the epitope analysis of three monoclonal anti‐(peptide P2) antibodies demonstrated that they recognized antigenic determinants in the cyclic hexapeptide ring, mainly the hydrophobic surface formed by Tyr2 and Phe3.

Our results suggest that monoclonal antibodies against different epitopes in small peptide hormones can be generated selectively by using photoreactive peptides in such a way that different antigenic sites are exposed in the hapten‐carrier conjugate.
Fluorescence spectroscopic analysis of ligand binding to kringle 1 + 2 + 3 and kringle 1 fragments from human plasminogen
Authors: MATSUKA Yury V. , NOVOKHATNY Valery V. , KUDINOV Stanislav A.
Published: March 2005

The ligand binding of kringle 1 + 2 + 3 and kringle 1 from human plasminogen has been investigated by fluorescence spectroscopy. Analysis of fluorescence titration of kringle 1 + 2 + 3 with 6‐aminohexanoic acid shows that this fragment, besides the high‐affinity lysine‐binding site with Kd= 2.9 μM, contains two additional lysine‐binding sites which differ in binding strength (Kd= 28 μM and Kd= 220 μM). This strongly suggests the existence of a lysine‐binding site in each of the first three kringles.

6‐Aminohexanoic acid, pentylamine, pentanoic acid and arginine were used for investigation of the ligand specificity of isolated kringle 1 prepared by pepsin hydrolysis of kringle 1 + 2 + 3. It has been established that kringle 1 has high affinity to 6‐aminohexanoic acid, pentylamine and arginine (Kd values are 3.2 μM, 4.8 μM and 4.3 μM, respectively). At the same time pentanoic acid did not bind with kringle 1. These facts indicate, firstly, a broad ligand specificity of kringle 1 and, secondly, the paramount importance of the positively charged group of the ligand for its interaction with lysine‐binding site of this kringle.
Phospholipid specificity of bovine heart bc1 complex
Authors: SCHÄGGER Hermann , HAGEN Thomas , ROTH Brigitte , BRANDT Ulrich , LINK Thomas Andreas , von JAGOW Gebhard
Published: March 2005

Bovine heart bc1 complex was reversibly inactivated by a new simple and effective chromatographic delipidation method. Upon phospholipid replenishment, catalytic activity increased from values near zero to values 2–6‐times higher than those of the original preparation. Compared to original preparations maximally activated by additional phospholipid, the degree of reactivation was up to 100%. By this delipidation method, the 6.4‐kDa protein subunit was removed with the phospholipid. The loss of this protein neither diminished electron transport activity nor abolished proton translocation.

Two requirements were necessary to obtain quantitative data: (a) only bc1 complexes, homogeneously dissolved before and after relipidation had to be used and (b) the phospholipid bound to the complex had to be determined. The correlation of catalytic activity to bound phospholipid was studied in the range of low phospholipid/protein ratios, which had previously been insufficiently resolved. Catalytic activity increased linearly with added phospholipid up to a molar ratio of 80–100 lipid molecules/dimeric complex. This corresponds to the number of phospholipid molecules that complete a single bilayer annulus.

The activating effect of phospholipid is not merely due to a hydrophobic phase effect, since it strongly depends on the nature of the polar head group of the added phospholipid. Of the three major phospholipids bound to the bc1 complex, only phosphatidylethanolamine and phosphatidylcholine activated when added as sole phospholipid. Tightly bound diphosphatidylglycerol was needed for preservation of the native complex structure.
Renin is sorted to the regulated secretory pathway in transfected PC12 cells by a mechanism which does not require expression of the pro‐peptide
Authors: CHIDGEY Martyn A. J. , HARRISON Timothy M.
Published: March 2005

The rat pheochromocytoma cell line PC12 targets secretory proteins into two distinct pathways. When DNA encoding human prorenin was transfected into PC12 cells, the protein was sorted into the regulated secretory pathway and released with similar kinetics to noradrenaline upon carbachol stimulation. To determine whether information for targeting prorenin lies within the pro‐peptide we have transfected PC12 cells with a construct lacking the pro‐peptide coding sequence. The transformed line secretes an apparently fully active enzyme and responds to carbachol stimulation with a rapid release of renin activity. We conclude that the pro‐peptide of renin is not essential for targeting the protein to the regulated pathway in PC12 cells.
Analysis of hepatic copper, zinc, metallothionein and metallothionein‐Ia mRNA in developing sheep
Authors: PAYNTER Jennifer A. , CAMAKARIS James , MERCER Julian F. B.
Published: March 2005

The concentrations of zinc, copper, metallothionein and metallothionein‐Ia mRNA in sheep livers during development was determined. It was found that early sheep foetuses (30–40 days gestation) had very high concentrations of hepatic zinc (2305±814 μg/g dry mass), and that these levels declined steadily to 644±304 μg/g near to term. The copper concentrations in the foetal livers were not higher than those in the adult. The concentrations of metallothionein and metallothionein‐Ia mRNA were also very high in the foetal livers and declined steadily during gestation from 261±94 molecules/pg RNA to 71±18 molecules/pg near to term. Metallothionein‐Ia mRNA concentrations were closely correlated with hepatic zinc concentrations but not with copper. Metallothionein concentrations also decreased during gestation: e.g. 3044 μg/g (wet mass) in one foetus on day 34 of gestation to 862 μg/g on day 125. After birth, however, the concentrations of metallothionein declined to less than 100 μg/g and this decline occurred despite the presence of significant quantities of mRNA. The ratio of metallothionein/metallothionein‐Ia mRNA decreased from 1.3 to 3.2 × 105 molecules metallothionein/molecule of metallothionein‐Ia mRNA during gestation to between 0.28–0.64 × 105 molecules/molecule in the postnatal animals. We conclude that the major function of metallothioneins in the foetal liver is protection of the liver against the potentially toxic accumulation of zinc. In the postnatal sheep there appears to be a decreased synthesis or increased degradation of metallothionein.