bluuebunny/crossref_metadata_2025 huggingface dataset:
Phsical Map of Phage 2C DNA: Evidence for the Existence of Large Redundant Ends
Authors: CONENE Marc , HOET Philippe , COCITO Carlo
Published: March 2005

The chromosome o0f the Bacillus Subtilkis phage 2C, a linear nmolecule of duble‐stranded DNA of about 108 Da, in which thymine is completely replaced by hydroxymethluracil, was cleaved by different endonucleases. In some cases restriction segmentsd were much fewer than expectded, suggesting a possible interference oif the unusual base with the recognition mechanism of endonucleases. The physical map of 2C DNA was established by use fo SalI and HaeIII restriction endonucleases, which yielded a limited numbver of fragments. The expected number of fragments was 240 for HaeIII and 23 for SalI; in realitym, five segmenbts were obsereved up[on clevage with HaeIII and four with SalI. The terminal fragments of the genome were first identified; the other fragments were ordered by hybridizaiton and maoleculkar wight determination of restriction fragments showed the prewsence of homologous regfions at the ends of the 2C genome. The structure fo thses direct repretitive sequences was analyzed by clevage with HaeIII and hybridization with Eco RI restriction fragments. Their size (9.2 MDa) was found to be about 1/11 of that of the whole chromnosome.
Dopamine β‐Monooxygenase
Authors: SKOTLAND Tore , FlATMARK Torgeir
Published: March 2005

The binding of 64Cu to thte water‐solube form of dopamine β‐monooxygenase from bovine adrenal medulla was studied in reconstitution and exchange experiments using high‐performance size‐exclusion gel chromatography. The reconstitution experiments provide evidencs for a evidence for a specific binding of four copper atoms/enzyme terramer using either Cu(I) or Cu(II), but some wekaer copper‐binding sites were obsered in the presence of a large excess of copper. The exchanges of both Cu(I) and Cu(II) in this protein are so rapid that exact half‐lives for the exchange reactions can not be obtained by the present method. The results indicate, howeever, that the half‐life for the exchange of the enzyme bound copper in the holoenzyme with a twofold excess of 64Cu(II) at pH 6.1 was about 1 min, wherase the exchange of Ci(I) measured at similar conditions with ascorabte present, was complete in 1 min. This is by far the most rapid exchange reported for any copper‐protein, and the results points to a unique copper‐binding site in this enzyme.
Evidence for Homologous Repeating Segments within the Elementary Polypeide Chain of Guinea Pig Thyroglobulin
Authors: PALUMBO Giuseppe , TECCE Mario F. , FORMISANO Silvestro
Published: March 2005

In this paper we report on the peptide maps of the three polypetides produced by reduction of denatured 19‐S thyroglobulin from guinea peg. These maps were obatined by both chemical cleavage (CNBr) and by limited enzymatic proteolysis (Staphylococcus aureous SV 8 protease).

Analysis of these peptides showed a strict correspindence among the electrophoretic bands produced by cleavage of the guinea pig thyroglobulin components. These results support the idea that thyroglobulin, even though it has an appearently complex molecular structure, contains polypetide sequences which are repeated in the elemetary chain.

Since simile structures should correspond to similar functions, each of these regions may work as a functional domain for the biosynthesis of the thyroid hormones.
Lipid Ddpendence of Diacylglyderol Kinase from Escherichia coli
Authors: BOHNENBERGER Elisabeth , SANDERMANN Heinrich
Published: March 2005

Diacylglycerolkinase apoprotein was purified from membranes of Encherichia coli K 12. The protein was catalytically inactive, but regained activity upon recombinatin with phospholipids, certain neutral llipids, of fatty acids. Activation proceded with positive cooperativity and was independent of the exact chemical structure, bilayer arrangement or electrical charge of the lipid. The apoprotein was activated by lysophosphatidylethanolamine but not by lysohosphatidylcholone. 1‐Monooleoylglycerol was an effective activator and substrats at the same time. The fluidity and the polatity o flipids appeared to be generally imjportent for activation. Lipid polatity was estimated by a triaclyglyderl/phosphatidylcholohn‐partitioning procedure. All lipids showing preferential association with triaclglycerol failed to activate the kinase apopretein even in the presence of detergent.

It is concluded that a defined hydrophilic/lipophilic balance of the lipid was required for the formation of a functinal lipoprotein complex.
p‐Hyroxybenzoate Hydroxylase from Pseudomonas fluorescens
Authors: HOFSTEENGE Jan , WEIJER Wicher J. , JEKEL Peter A. , BEINTEMA Jaap J.
Published: March 2005

As a final step in the elucidation of the primary structure of p‐hydroxybenzoate from Pseudomonas fluorescens, the amino acid sequences of a CNBr peptide (CB1, positions 111–276), that accounts for the middle part of the sequence, and the C‐terminal CNBr peptide (CB2, positions 277–394) from the enzyme were determined. Important sequence information was obtained from two subfragments that were formed by the cleavage with CNBr of the Met‐Thr sequence (positions 346–347) in peptide CB2.

The alignment of the two subfragments from peptide CB2 and three one‐residue overlaps between peptides from one of these subfragments were confirmed by investigation of well‐resolved parts of a 0.25‐nm electron‐density map. The sequence of residues 343–346 could not be determined with chemical methods and was assigned from the size and shape of the amino acids in the electron‐density map.

An important tool in the analysis of the amino acid sequence of peptide CB1 was the proteinase Lys‐C from Lysobacter enzymogenes, which preferentially cleaves at lysine residues.
Preparation and properties of monomercurated tRNA
Authors: ABDURASHIDOVA Gulnara G. , NARGISYAN Magi G. , BUDOWSKY Edward I.
Published: March 2005

Monomercurated tRNA has been prepared by exhaustive treatment of mercaptoethanol on polymercurated tRNA under non‐denaturing conditions. The remained mercury atom is stable toward ultraviolet (254 nm) irradiation up to absorption of 100 quanta/nucleotide, but could be easily removed by mercaptoethanol under denaturing conditions.

Monomercurated tRNA quantitatively binds to thiopropyl‐Sepharose and can be completely desorbed by mercaptoethanol‐containing solutions. Monomercurated tRNA is similar to the starting tRNA preparation in efficiency and specificity of aminoacylation and in factor‐dependent polypeptide synthesis in vitro.
Redox‐linked proton translocation in the b–c1 complex from beef‐heart mitochondria reconstituted into phospholipid vesicles
Authors: PAPA Sergio , LORUSSO Michele , BOFFOLI Domenico , BELLOMO Elisabetta
Published: March 2005

A study is presented of the characteristics of redox‐linked proton translocation in the b–c1 complex isolated from beef‐heart mitochondria and reconstituted into phospholipid vesicles.

Measurements of the H+/e− stoichiometry, with three different methods, show that four protons are released from the vesicles per 2e− flowing from quinols to cytochrome c, two of these protons formally deriving from scalar oxidation of quinols by cytochrome c. This H+/e− stoicheiometry is independent of the initial redox state of the b–c1 complex (fully reduced or oxidized) and the rate of electron flow through the complex. It does not change in the pH range 6.0–7.2, but declines to 1.5 going with pH from 7.2–8.3. This decrease is accompanied by enhancement of the rate of electron flow in the coupled state.

Collapse of ΔΨ effected by valinomycin addition to turning‐over b–c1 vesicles resulted in substantial oxidation of cytochrome b‐566 and comparable reduction of cytochrome c1, with little oxidation of cytochrome b‐562. Nigericin alone had no effect on the steady‐state redox levels of b and c cytochromes. Its addition in the presence of valinomycin caused oxidation of b cytochromes but no change in the redox state of cytochrome c1. Valinomycin alone caused a marked enhancement of the rate of electron flow through the complex. Nigericin alone was ineffective, but caused further stimulation of electron flow when added in the presence of valinomycin.

The data presented are discussed in terms of two mechanisms: the Q cycle and a model based on combination of protonmotive catalysis by special bound quinone and proton conduction along pathways in the apoproteins.
Exodeoxyribonuclease from rat brain specific for single‐stranded DNA
Authors: IVANOV Vladimir A. , GAZIEV Azhub I. , TRETYAK Tatyana M.
Published: March 2005

An exodeoxynuclease with single‐stranded DNA specificity has been isolated from rat brain and purified to electrophoretic homogeneity. Approximately 1100‐fold purification with a yield of 16% has been achieved by chromatography on DNA‐agarose, hydroxyapatite and Sephadex G‐200. The enzyme has a pH optimum at 8.4, requires Mg2+ or Mn2+ as a cofactor and has a molecular weight of about 60 000. It hydrolyzes homologous, heterologous, synthetic and depurinated substrates at the same rate liberating nucleoside 5′‐monophosphates but does not attack ultraviolet‐irradiated polydeoxyribonucleotides. This DNase is localized predominantly in neuronal cell nuclei and appears to be lacking in rat liver tissue.
Specific cell‐surface labeling of polyglycosyl chains in human erythrocytes and HL‐60 cells using endo‐β‐galactosidase and galactosyltransferase
Authors: VIITALA Juha , FINNE Jukka
Published: March 2005

In order to identify the molecule components carrying polyglycosyl chains on cell surfaces a two‐step enzymatic method was developed. In the first step, the cells were incubated with endo‐β‐galactosidase to selectively expose terminal N‐acetylglucosamine residues of the lactosamine backbone to the chains. In the second step these residues were glycosylated by incubation with galactosyltransferase and radioactive UDP‐galactose. As many as 2.5–3.0 × 106 residues per cell could be transferred to human erythrocytes. Negligible amounts of labeling occurred if either of the enzymes was omitted from the incubations. Of the label 80 % was found in glycoproteins. In accordance with previous observations, bands 3 and 4.5 were found to be the main carriers of polyglycosyl chains. In human promyelotic HL‐60 leukemia cells, a major band of apparent molecular weight of 110000–140000 was labeled. In addition, bands of lower molecular weight which appear to have escaped detection by previous methods were also labeled. The novel labeling method was found to be simple to perform, uses commercially available reagents, and leads to the efficient and highly specific labeling of cell surface molecules carrying polyglycosyl chains.
Electrostatic field effects of coenzymes on ligand binding to catalytic zinc in liver alcohol dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , OLDÉN Bertil , PETTERSSON Gösta
Published: March 2005

The synergism between coenzyme and anion binding to liver alcohol dehydrogenase has been examined by equilibrium measurements and transient‐state kinetic methods to characterize electrostatic interactions of coenzymes with ligands which are bound to the catalytic zinc ion of the enzyme subunit.

Inorganic anions typically exhibit an at least 200‐fold higher affinity for the general anion‐binding site than for catalytic zinc on complex formation with free enzyme. Acetate and SCN− interact more strongly with catalytic zinc in the enzyme‐NAD+ complex than with the general anion‐binding site in free enzyme. CN− shows no significant affinity for the general anion‐binding site, but combines to catalytic zinc in the absence as well as the presence of coenzymes.

Coordination of CN− to catalytic zinc weakens the binding of NADH by a factor of 50, and tightens the binding of NAD+ to approximately the same extent through interactions which do not include any contributions from covalent adduct formation between CN− and NAD +. These observations provide unambiguous information about the magnitude of electrostatic field effects of coenzymes on anion (e.g. hydroxyl ion) binding to catalytic zinc. They lead to the important inference that coenzyme binding must be strongly affected by ionization of zinc‐bound water irrespective of the actual acidity of the latter group.

It is concluded on such grounds that the much debated pH dependence of coenzyme binding to liver alcohol dehydrogenase must derive from ionization of zinc‐bound water. The assumption that such is not the case leads to the inference that there is no detectable effect of ionization of zinc‐bound water on coenzyme binding over the pH range 6–12, a possibility which is definitely excluded by the present results.
Modification of the cation selectivity filter and the calcium receptor of the Ca‐stimulated K channel in resealed ghosts of human red blood cells by low levels of incorporated trypsin
Authors: WOOD Phillip G. , MUELLER Hannelore
Published: March 2005

The transport properties of the Ca‐activated, K channel in the resealed human red blood cell ghost can be modified by the action of incorporated trypsin. Membranes were maximally depleted of diffusible cytoplasmic components by hemolysis on a gel filtration column at 0 °C. Subsequently, isotonicity was restarted and 0.01–1 μg/ml trypsin incorporated. Partial digestion of the membrane proteins occurred during reselling. As the degree of tryptic digestion increased, the channel became initially permeable to K and later to both K and Na; and then the channel became refractory to the action of applied Ca. The observations suggest that tryplic digestion of proteins at the inner membrane surface leads to modifications of the selectivity filter and the Ca‐receptor site of the channel. The modifications probably stem from alterations at the inner surface of a trans membrane protein which acts as a channel. Under conditions where selectivity is lost, the channel is still inhibited by externally applied TbCl3.
NMR studies of a dihaem cytochrome from Pseudomonas perfectomarinus (ATCC 14405)
Authors: MOURA Isabel , LIU Ming Cheh , LeGALL Jean , PECK Harry D. , PAYNE William J. , XAVIER António V. , MOURA José J. G.
Published: March 2005

Pseudomonas perfectomarinus (ATCC 14405) dihaem cytochrome c552 was studied by 300‐MHz proton magnetic resonance. Some of the haem resonances were assigned in the fully reduced and fully oxidized states. No evidence was found for methionine haem axial coordination. The oxidation‐reduction equilibrium was studied in detail. Due to the large difference in mid‐point redox potential between the two haems (+174mV, for haem II and ‐180mV for haem I) an intermediate oxidation state could be obtained containing reduced haem I and oxidized haem II. In this way the total paramagnetic shift at different oxidation levels could be decomposed in the intrinsic and extrinsic contributions. It was found that the two haems interact. The rate of electron exchange is slow on the NMR time scale. The redox equilibria are discussed for four possible redox species in solution.
Nucleotide sequence of the sucA gene encoding the 2‐oxoglutarate dehydrogenase of Escherichia coli K12
Authors: DARLISON Mark G. , SPENCER Margaret E. , GUEST John R.
Published: March 2005

The nucleotide sequence of a 3180‐base‐pair segment of DNA, containing the sucA gene encoding the 2‐oxoglutarate dehydrogenase component (E1o) of the 2‐oxoglutarate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain‐termination method. The sucA structural gene contains 2796 base pairs (932 codons, excluding the initiation codon AUG) and encodes a polypeptide having a glutamine residue at the amino terminus, a glutamate residue at the carboxy‐terminus and a calculated Mr= 104905. The predicted amino acid composition is in good agreement with published information obtained by hydrolysis of the purified enzyme. There is a striking lack of sequence homology between the 2‐oxoglutarate dehydrogenase (E1o) and the corresponding pyruvate dehydrogenase (E1p), which suggests that the two components are not closely related in evolutionary terms.

The location and polarity of the sucA gene, relative to the restriction map of the corresponding segment of DNA, are consistent with it being the proximal gene of the suc operon, as defined in previous genetic and post‐infection labelling studies, but it could also form part of a more complex regulatory unit. The sucA gene is preceded by a segment of DNA that contains many substantial regions of hyphenated dyad symmetry including an IS‐like sequence of the type that is thought to function as an intercistronic regulatory element. This segment also contains three putative RNA polymerase binding sites and a good ribosome binding site.
Effect of pH and proton buffer on oscillations of ion fluxes in rat erythrocytes
Authors: SADYKOV Yuri H. , HOLMUHAMEDOV Ehson L. , EVTODIENKO Yuri V.
Published: March 2005

The pH‐dependence of ionophore‐induced oscillations of transmembrane H+ and K+ fluxes in rat erythrocytes has been studied. It is stated that the oscillations are strongly depressed at pH lower than 6.9 and higher than 7.3. Proton buffers of different nature (Tris/HCl, Mops and glycylglycine) are shown to effectively inhibit the oscillatory process. The significance of H+ concentration in unstirred layers adjacent to the membrane in the mechanism of oscillations is suggested.
Structure of the oligosaccharides sensitive to endo‐β‐N‐acetylglucosaminidase H in the glycoprotein of Friend murine leukemia virus
Authors: GEYER Rudolf , GEYER Hildegard , EGGE Heinz , SCHOTT Hans Henning , STIRM Stephan
Published: March 2005

The surface glycoprotein (mixture of isoglycoproteins with Mr 69000 and 71000) was isolated from the particles of Friend murine leukemia virus, and was successively digested with protease and with endo‐β‐N‐ acetylglucosaminidase from Streptomyces griseus. Roughly 20% (w/w) of the carbohydrates in this glycoprotein were thus released, and they were fractionated by high‐performance liquid chromatography after reduction with KB3H4NaBH4. The radioactive oligosaccharitol fractions obtained were analyzed by exoglycosidase digestion, by acetolysis, and, after permethylation, by fast atom‐bombardment mass spectrometry, as well as by capillary gas‐liquid chromatography/mass spectrometry following hydrolysis, reduction and peracetylation.

Around 85% (mol/mol) of the endo‐H‐sensitive viral glycans were thus found to be oligomannosidic oligosaccharitols of size classes Man5GlcNAcOH, Man6GlcNAcOH, Man8GlcNAcOH, Man7 GlcNAcOH, and Man9GlcNAcOH (in order of prevalence), and the major structural isomers of each size class were identified. About another 15% (mol/mol) of the oligosaccharitols were shown to be of the ‘mixed type’, comprising mainly four species in which the Man(α1 → 6)‐branch of the Manα1 → 6 (Manα1 → 3) Manβ1 → 4GlcNAcOH core is substituted by one or two additional α‐mannoses, while the Man(α1 → 3)‐branch carries an N‐acetyllactosamine unit substituted by sialic acid, or by Gal(α1 → 3).
Nucleotide sequence of the gene for the immunity protein to colicin A
Authors: LLOUBES Roland P. , CHARTIER Marline J. , JOURNET Agnes M. , VARENNE Stanislas G. , LAZDUNSKI Claude J.
Published: March 2005

The nucleotide sequence of the structural gene for the immunity protein to colicin A (cai) has been established. This sequence consists of 534 base pairs. According to the predicted amino acid sequence, the polypeptide chain of this immunity protein comprises 178 amino acids and has a relative molecular mass of 20462. As expected from its localization in the inner membrane, large hydrophobic fragments are found along the polypeptide chain that also contains clusters of mostly positively charged residues. The cai like the ceiA genes encode proteins that are weakly expressed as compared to the corresponding colicins (A and E1). Codon usage reflects this difference. In contrast, the four genes for immunity to cloacin DF13 and to colicin E3 and for these bacteriocins, all of which are highly expressed and are organized in operon, display similar codon usage. These results are discussed with regards to the possible relationship between expressivity and codon usage.
Molecular cloning of hepatic 5‐aminolevulinate synthase
Authors: BORTHWICK Iain A. , SRIVASTAVA Gopesh , HOBBS Andrew A. , PIROLA Byron A. , BROOKER John D. , MAY Brian K. , ELLIOTT William H.
Published: March 2005

Hepatic 5‐aminolevulinate synthase was induced in chick embryos by administration of the porphyrinogenic drugs 2‐allyl‐2‐isopropylacetamide and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine. A cDNA library was constructed from drug‐induced liver mRNA and clones containing sequences coding for 5‐aminolevulinate synthase were identified by hybrid‐selected translation. The identity of these clones was confirmed by comparing DNA sequence data with the amino acid sequence of peptides from purified 5‐aminolevulinate synthase.

From Northern blot analysis the size of the mRNA for 5‐aminolevulinate synthase was estimated to be 2800 base pairs, approximately 600 base pairs more than that required to code for the primary translation product of relative molecular mass 74000.
Oligo(methionyl) proteins
Authors: GAERTNER Hubert , PUIGSERVER Antoine
Published: March 2005

A number of model isopeptides containing oligo(methionine) chains varying in length (2–5 residues) covalently linked to the ɛ‐amino group of lysine were synthesized by solid‐phase procedures. Hydrolysis of these peptides by pepsin, chymotrypsin, cathepsin C (dipeptidyl peptidase IV) and intestinal aminopeptidase N was investigated using high‐performance liquid chromatography to identify and quantify the hydrolysis products. Methionine oligomers grafted onto lysine were cleaved to tripeptides by pepsin. Chymotrypsin preferentially hydrolyzed the methionyl‐methionine bond preceding the isopeptide bond. Cathepsin C released dimethionyl units from the covalently attached polymers. Intestinal aminopeptidase caused efficient hydrolysis of both peptides and isopeptide bonds although free methionine decreased the cleavage of the latter bond. Hydrophobic characteristics of oligo(methionine) chains promoted enzyme‐catalyzed transpeptidations resulting probably from acyl‐transfer‐type reactions. Complementary hydrolysis of the isopeptides by these digestive enzymes suggests that covalent attachment of oligo(amino acid)s to food proteins may improve their nutritional value.
Synthesis of wheat leaf nitrite reductase de novo following induction with nitrate and light
Authors: SMALL Ian S. , GRAY John C.
Published: March 2005

Nitrite reductase has been purified almost 3000‐fold, in 35% yield, to a specific activity of 77 units (mg protein)–1 from wheat leaves using a multi‐step procedure with affinity chromatography on ferredoxin‐Sepharose as the final step. The purified enzyme, although not homogeneous, exhibited absorption maxima at 278, 390, 568 and 687 nm. Minor contaminants were removed by gel filtration in the presence of sodium dodecyl sulphate to yield a single polypeptide of Mr 60500 as judged by polyacrylamide gel electrophoresis. Antibodies raised against this polypeptide were shown to cross‐react with native nitrite reductase and were used to study the synthesis of nitrite reductase in vivo and in vitro. The increase in nitrite reductase activity following exposure of dark‐grown plants to nitrate and light was shown by immunodecoration of Western blots to be due to synthesis de novo. Poly(A)‐rich RNA isolated from plants actively synthesising nitrite reductase was shown to direct the synthesis in a rabbit reticulocyte lysate of a polypeptide of Mr 64000 which was immunoprecipitated by antibodies to nitrite reductase.
Glucose‐6‐phosphate dehydrogenase
Authors: GRONEBORN Angela M. , CLORE G. Marius , HOBBS Lynda , JEFFERY Jonathan
Published: March 2005

The conformation of NADP+ in glucose‐6‐phosphate‐dehydrogenase—NADP+ binary complexes has been investigated using proton‐proton transferred nuclear Overhauser enhancement measurements to determine inter‐proton distance ratios between bound NADP+ protons. The enzymes from Saccharomyces cerevisiae (brewer's yeast and baker's yeast) and Hansenula jadinii (Candida utilis, Torula utilis) form binary complexes with NADP+ in which the glycosidic bond of the adenine moiety is in the anti conformation whereas that of the nicotinamide moiety exists as a syn (69–70%)/anti (30–40%) mixture. The enzymes have similar subunit sizes (Mr∼58000) and it is shown that they bind NADP+ in essentially similar conformations. Inactivation of the baker's yeast enzyme with acetylsalicylic acid caused little if any alteration in the conformation of bound NADP+, and the presence of NADP+ during inactivation afforded very little protection to the enzyme. Inactivation rates were, however, lower in the presence of glucose 6‐phosphate. It is concluded that the ɛ‐amino group of the lysine residue that is acetylated during the inactivation reaction with acetylsalicylic acid is not necessary for binary complex formation between the enzyme and NADP+, but that it is situated in a part of the molecule affected by formation of the enzyme—glucose‐6‐phosphate complex. The implication of the findings for the catalytic process, and related evolutionary aspects, are discussed briefly.
The interaction of mitochondrial transhydrogenase with derivatives of coenzyme A
Authors: KOZLOV Igor A. , MILGROM Yakov M. , SABUROVA Larissa A. , SOBOLEV Alexander Yu.
Published: March 2005

The 2,4‐dinitrophenyl derivative of dephospho‐CoA and the 7‐nitrobenzofurazan‐4‐yl derivative of CoA are competitive inhibitors (Ki 3 μM and 2.6 μM respectively) of mitochondrial transhydrogenase with regard to NAD+ and NADPH respectively. The 7‐nitrobenzofurazan‐4‐yl derivative of dephospho‐CoA is a competitive inhibitor with regard to both transhydrogenase substrates with the same Ki equal to 0.3 μM. The pattern of transhydrogenase inhibition with the 7‐nitrobenzofurazan‐4‐yl derivative of dephospho‐CoA indicates that one molecule of the inhibitor binds simultaneously to both the NADP(H) and the NAD(H) binding sites of the enzyme. This result is evidence of the short distance between the NADP(H) and the NAD(H) binding sites.
Resolution by high‐pressure liquid chromatography and partial characterization of multiple forms of cytochrome P‐450 from hepatic microsomes of phenobarbital‐treated rats
Authors: BANSAL Surendra K. , LOVE John H. , GURTOO Hira L.
Published: March 2005

Seven cytochromes P‐450 (A, B, C, D, E1, E2 and F) were isolated from hepatic microsomes of phenobarbital‐induced rats by a modification of the procedure of Guengerich and Martin [Arch. Biochem. Biophys. (1980) 205, 365–379]. The modification consisted of replacing DEAE‐cellulose column by two DEAE‐Sepharose CL‐6B columns connected in tandem, changing the elution scheme and monitoring the resulting fractions by high‐pressure liquid chromatography (HPLC). Cytochrome P‐450 forms D, E1, E2, and F having molecular masses of 52.5 kDa, 52.5 kDa, 53.3 kDa and 53.2 kDa, respectively were resolved from the major form of cytochrome P‐450 ‘peak B2’ of Guengerich and Martin (above reference). These four cytochromes P‐450 were immunologically identical by Ouchterlony double‐diffusion analysis. Slight but significant differences were evident in the partial peptide digest maps of these four cytochromes P‐450 and catalytic properties of these four forms, though qualitatively similar, demonstrated distinct quantitative differences. Furthermore, HPLC retention times of these four cytochrome P‐450s were quite different. Cytochrome P‐450 forms A, B and C were distinctly different from each other and from the forms D, E1, E2 and F in the following respects: partial peptide digest maps, catalytic activities, and HPLC retention times.

The present results show that cytochromes P‐450 considered homogeneous by sodium dodecyl sulfate/polyacrylamide gel electrophoresis may be heterogeneous and contain multiple forms of cytochromes P‐450 with different net charges but similar molecular‐masses. These studies also demonstrate the capability of HPLC in providing a simple and effective tool for monitoring the separation of cytochromes P‐450 showing charge heterogeneity.
Kinetics of activation of l‐lactate dehydrogenase from Streptococcus lactis by fructose 1,6‐bisphosphate
Authors: HARDMAN Michael J. , CROW Vaughan L. , CRUICKSHANK Denise S. , PRITCHARD Graham G.
Published: March 2005

A lag is observed before the steady state during pyruvate reduction catalysed by lactate dehydrogenase from Streptococcus lactis. The lag is abolished by preincubation of enzyme with the activator fructose 1,6‐bisphosphate before mixing with the substrates. The rate constants for the lag phase showed a linear dependence on fructose‐1,6‐bisphosphate concentration, with a second‐order rate constant of 2.0 − 104 M−1 s−1, but were independent of enzyme concentration.

Binding of fructose 1,6‐bisphosphate produces a decrease in the protein fluorescence of the enzyme. The second‐order rate constant for the fluorescence change is twice that for the lag in pyruvate reduction.

The results suggest that binding of fructose 1,6‐bisphosphate induces a conformational change in the enzyme, producing a form with reduced protein fluorescence and increased activity towards pyruvate reduction.
Tryptophanamide formation by Escherichia coli tryptophanyl‐tRNA synthetase
Authors: ANDREWS David , TREZEGUET Véronique , MERLE Michel , GRAVES Pierre‐Vincent , MUENCH Karl H. , LABOUESSE Bernard
Published: March 2005

When tryptophanyl‐tRNA synthetase from Escherichia coli is allowed to react with L‐tryptophan and ATP‐Mg in the presence of inorganic pyrophosphatase, the fluorescence change of the reaction mixture reveals three or four sequential processes, depending on the buffer used. Quenched‐flow and stopped‐flow experiments show that the first two processes, which occur in the 0.001–1.0‐s time scale, can be correlated to the formation of two moles of tryptophanyl‐adenylate per mole of dimeric enzyme. These two processes are reversible by adding PPi, as seen in the fluorimeter. The third process leads to a reaction product that can no longer reform ATP after addition of PPi and that represents tryptophanyl‐ATP ester, as demonstrated by thin‐layer chromatography. This compound has been previously shown to be formed by tryptophanyl‐tRNA synthetase from E. coli [K. H. Muench (1969) Biochemistry 8, 4872–4879]. Its formation is accompanied by a fluorescence decrease which reaches a minimum in about 30 min. The nature of the fourth process depends on the reaction conditions employed. In sodium bicarbonate or potassium phosphate buffer, the fourth process corresponds to the non‐enzymatic hydrolysis of tryptophanyl‐ATP ester. This spontaneous hydrolysis competes with formation of the ester and limits its concentration. Eventually, the progressive exhaustion of ATP brings the fluorescence intensity of the reaction mixture back to its initial value. In contrast, in ammonium bicarbonate buffer the previous third process is no longer visible, as evidenced by the absence of a fluorescence decrease beyond the fast initial quenching linked to the formation of tryptophanyl‐adenylate. Instead, a fluorescence increase is observed. However, unlike the fourth process seen in sodium bicarbonate buffer, the fluorescence increase in ammonium bicarbonate is much larger than the initial fluorescence decrease linked to adenylate formation, the final fluorescence greatly surpassing the starting fluorescence signal. The reaction product of this process is tryptophanamide, as evidenced by high‐performance liquid chromatography. Tryptophanamide formation is faster than that of tryptophanyl‐ATP ester and is enzyme‐catalyzed with a Km of 1 mM for ammonia and a rate constant of 5.7 min−1 at pH 8.3, 25°C. The affinity of tryptophanamide for the protein is too weak to allow the formation of a significant concentration of enzyme‐product complex. Tryptophanamide is therefore released in the reaction medium and its concentration reaches that of the limiting substrate.
Ether glycerolipids: novel substrates for studying specificity of enzymes involved in glycerolipid biosynthesis in higher plants
Authors: WEBER Nikolaus , BENNING Hildegard
Published: March 2005

Ether glycerolipids, predominantly alkylacylglycerols and alkylacylglycerophosphocholines, are synthesized in photomixotrophic rape (Brassica napus) suspension cells from various exogenous monoalkylglycerols. The stereospecific distribution of acyl moieties was studied in these ether glycerolipids with regard to chain‐length and degree of unsaturation of alkyl moieties and compared with the distribution of acyl moieties in the corresponding endogenous acyl glycerolipids. The results show the following: (1) Alkylacylglycerophosphocholines replaced up to one‐half of the corresponding physiological membrane lipids, i.e. diacylglycerophosphocholines, without changing the total amount of cholineglycerophospholipids as compared to untreated cells. (2) The composition of acyl moieties in total lipids of rape cell was practically unaltered by fatty acids derived via oxidative cleavage from the various alkyl moieties of ether glycerolipids. (3) In 1‐O‐alkyl‐2‐acylglycerols derived from exogenous alkylglycerols and in endogenous 1,2‐diacylglycerols compositions of acyl moieties were found to be different indicating that different pathways were operative in the biosynthesis of these two neutral glycerolipids. (4) Enzymes involved in synthesizing molecular species of 1‐O‐alkyl‐2‐acylglycerophosphocholines or 2‐O‐alkyl‐1‐acylglycerophosphocholines as well as 1,2‐diacylglycerophosphocholines showed similar pecificities with regard to chain‐length and degree of unsaturation of both alkyl and corresponding acyl moieties. Thus, ether glycerolipids formed by plant cells from exogenous alkylglycerols are suitable metabolites for studying the specificity of enzymes involved in the biosynthesis of glyerolipids.
Biosynthesis and secretion of α1 acute‐phase globulin in primary cultures of rat hepatcytes
Authors: BAUER Joachim , KURDOWSKA Anna , TRAN‐THI Thuy‐Anh , BUDEK Werner , KOJ Aleksander , DECKER Karl , HEINRICH Peter C.
Published: March 2005

Experimental inflammation in rats led to a sevenfold increase in serum levels of α1 acute‐phase globulin. This increase is correlated with elevated levels of translatable mRNA for α1 acute‐phase globulin in the liver.

Biosynthesis and secretion of α1 acute‐phase globulin were studied in rat hepatocyte primary cultures. An intracellular form of α1 acute‐phase globulin with an apparent relative molecular mass of 63 500 and a secreted form of 68 000 were found. The intracellular form of α1 acute‐phase globulin could be deglycosylated by endoglucosaminidase H treatment indicating that its oligosaccharide chains were of the high‐mannose type. The secreted form of α1 acute‐phase globulin was not sensitive to endoglucosaminidase H, but was susceptible to the action of sialidase reflecting carbohydrate side‐chains of the complex type.

Pulse‐chase experiments revealed a precursor‐product relationship for the high‐mannose and the complex type α1 acute‐phase globulin. In the hepatocyte medium newly synthesized α1 acute‐phase globulin was detected 30 min after the pulse.

Unglycosylated α1 acute‐phase globulin was found in the cells as well as in the medium when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked delay in α1 acute‐phase globulin secretion.
The role of cholesteryl 14‐methylhexadecanoate in the function of eukaryotic peptide elongation factor 1
Authors: TUHÁČKOVÁ Zdena , HRADEC Jan
Published: March 2005

The binding of [3H]cholesteryl 14‐methylhexadecanoate by a highly purified peptide elongation factor 1 from rabbit reticulocytes is significantly enhanced by GTP and CTP, much less by guanosine 5′‐[β,γ‐methylene]‐triphosphate and not at all by ATP or UTP. Removal of endogenous cholesteryl 14‐methylhexadecanoate present in the molecule of the factor [Hradec, J. et al. (1971) Biochem. J. 123, 959–966] by digestion with immobilized cholesterol esterase resulted in an almost complete loss of GTPase activity and this could be restored to nearly normal values by the addition of the ester. The same holds true for the GTP‐dependent autophosphorylation of the protein‐synthesis factor. Cholesteryl 14‐methylhexadecanoate was bound only by the β subunit of the factor. Addition of the α subunit, which was inactive on its own, stimulated the binding of the ester to the β subunit in a sigmoid dependance. The binding of the ester was significantly stimulated by aminoacyl‐tRNA but this effect was fully abolished by sodium fluoride, indicating a relation of cholesteryl 14‐methylhexadecanoate to the dephosphorylation of the peptide elongation factor. Treatment of the factor with cholesterol esterase decreased its activity in the poly(U)‐dependent binding of phenylalanyl‐tRNA to ribosome and this activity was again restored by the addition of cholesteryl 14‐methylhexadecanoate. The ester thus interacts with the GTP‐dependent autophosphorylation of peptide elongation factor 1 and in this way modulates the activity of the factor. A putative scheme is presented explaining the mode of action of cholesteryl 14‐methylhexadecanoate.
Phosphoenolpyruvate‐dependent fructose phosphotransferase system in Rhodopseudomonas sphaeroides
Authors: LOLKEMA Juke S. , ROBILLARD George T.
Published: March 2005

The bacterial phosphotransferase systems are believed to catalyze the concomitant transport and phosphorylation of hexoses and hexitols. The transport is from the outside to the inside of the cell. An absolute coupling between transport and phosphorylation has however been questioned in the literature. We have tested the coupling by analysing the kinetics of fructose phosphorylation by inside‐out vesicles of Rhodopseudomonas sphaeroides. We conclude that fructose indeed has to enter the vesicle before it can be phosphorylated and therefore cannot be phosphorylated from the cytoplasmic side of the membrane. The Km of the phosphorylation reaction is 8 μM. The diffusion of fructose into the vesicle is a reaction that is also catalysed by the compoments of the phosphotransferase system. The undirectional flux from the cytoplasmic side of the membrane to the periplasmic side is a slow process with a Km of 4 mM and is rate‐limiting over a large external fructose concentration range. In summary there is no phosphorylation without transport, but there is transport without phosphorylation.
Characterization of transcriptionally active DNA‐protein complexes from chloroplasts and etioplasts of mustard (Sinapis alba L.)
Authors: REISS Thomas , LINK Gerhard
Published: March 2005

DNA‐protein complexes that are capable of RNA synthesis in vitro (transcriptionally active chromosomes) were isolated from both chloroplasts and etioplasts of mustard (Sinapis alba L.) seedlings. Analyses of the polypeptide pattern of these complexes indicate that they comprise a specific subset of plastid proteins, distinct from the overall pattern of either the soluble or membrane‐bound plastid proteins. DNA‐protein complexes from the two plastid types have polypeptides in common. However, at least several polypeptides are highly enriched in either the chloroplast or the etioplast DNA‐protein complex.

The EcoRI restriction endonuclease fragments of the DNA associated with the complexes from either plastid type are the same. They are identical with the fragments obtained from highly purified chloroplast DNA.

The transcriptional activity of the chloroplast complex is more than ten times higher than the activity of the etioplast complex. However, the complexes from either plastid type are capable of transcribing DNA regions containing genes for both the plastid rRNAs and for plastid proteins. In vitro transcripts were found to hybridize not only to DNA regions for mature in vivo RNA but also to adjacent regions, indicating synthesis of precursor RNA sequences by the transcriptionally active chromosomes.
Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrate‐treated mice
Authors: MEIJER Johan , DEPIERRE Joseph W.
Published: March 2005

We have characterized certain catalytic properties of cytosolic epoxide hydrolases purified from untreated and clofibrate‐treated mouse liver.

The enzyme activity was found to be sensitive to oxygen, but nitrogen‐saturated buffers containing dithiothreitol maintained high activity for at least 12 h at 0°C. Linearity of the hydration of trans‐stilbene oxide with time and protein was established, the pH optimum was broad (6.5 to 7.4) and the temperature optimum was close to 50°C for both forms. The activity was independent of ionic strength, with the exception of the control form in the absence of dithiothreitol, where a lower activity was observed at low ionic strength. The activity decreased when ethanol was replaced by acetone or acetonitrile as solvent for the substrate. Tetrahydrofuran was found to be highly inhibitory, while dimethylsulfoxide had less pronounced effects.

The apparent Km values were 4.9 μM, 73 μM and 1980 μM for the control form with trans‐stilbene oxide, cis‐stilbene oxide and styrene oxide as substrates, respectively. The Km values for the enzyme from clofibrate‐treated mice were in the same range, although the V values were higher for all three substrates with this form. The highest turnover was found for trans‐β‐propylstyrene oxide as substrate, followed by trans‐β‐ethylstyrene oxide. Little or no activity was observed with benzo[a]pyrene 4,5‐oxide or cholesterol 5,6α‐oxide.

The enzymes were found to be sensitive to 5,5′‐dithiobis(2‐nitrobenzoic acid) and a phenylmercuric salt. α‐Naphthoflavone, β‐naphthoflavone and chalcone derivatives also inhibited the activity, while none of the compounds known to activate microsomal epoxide hydrolase activated the cytosolic forms.
Isolation and biochemical characterization of maleic‐acid hydratase, an iron‐requiring hydro‐lyase
Authors: DREYER Jean‐Luc
Published: March 2005

A procedure for the isolation of maleic acid hydratase (D‐malete hydro‐lyase, EC 4.2.1.31) of about 95% purity from rabbit kidneys is described. The enzyme consists of a single polypeptide chain of 582 amino‐acid residues with an approximate molecular mass of 68 kDa. The enzyme is very unstable and has an absolute requirement for chloride ions. Addition of sodium sulphide during the purification process was essential to maintain the enzyme in an activatable state. The pure preparation has low activity but responds to activation with Fe2+ ions, Na2S and a thiol. The sequence of adding the activating reagents is critical to achieve optimal activity. Ni2+ and to a lesser extent Co2+ can replace iron in the activation process. The enzyme incorporates 4–5 mol iron/mol and 4.5–6 mol sulphide/mol during activation. In this process an [Fe‐S] cluster appears to be built up, as indicated by optical and electron paramagnetic resonance (EPR) spectroscopy. In activated samples exposed to air the [Fe‐S] cluster is EPR‐detectable through an axial signal with g⊥= 2.01 and g⊥= 2.029 whose temperature and power saturation characteristics were similar to those of other [3 Fe‐xS] clusters. The activated enzyme, however, is readily inactivated even upon minor manipulation with destruction of the iron‐sulfur core.
Cultural Safety and professional practice in occupational therapy: A New Zealand perspective
Authors: Gray Marion , McPherson Kathryn
Published: March 2005

This research examined occupational therapists’ attitudes to Cultural Safety for Maori clients. The main research tool was a semi‐structured qualitative research interview. A cross‐case analysis method was used to determine the major recurring themes. Thirteen participants with varied occupational therapy training backgrounds were interviewed. Current Cultural Safety education is designed to challenge health professionals’ attitudes towards those culturally different from themselves. Results suggest that maturity and personal experiences had significant bearing on these attitudes. Implications for occupational therapy include implementing a policy that stipulates compulsory Cultural Safety education as part of professional development. Findings may have relevance for other countries where significant ethnic differentials in health service success exist, such as Australia.
Resident and therapist views of animal‐assisted therapy: Implications for occupational therapy practice
Authors: Velde Beth P. , Cipriani Joseph , Fisher Grace
Published: March 2005

Animal‐assisted therapy is offered in a wide variety of settings. The literature contains few studies investigating animal‐assisted therapy from an occupational therapy perspective. More information is needed to describe the use of animals as a therapeutic modality in occupational therapy. Three qualitative case studies were analysed to describe the perceptions of clients and therapists regarding animal‐assisted therapy. This analysis was synthesised with an extensive literature review to produce a perspective of animal‐assisted therapy for occupational therapy. Animal‐assisted therapy could be a beneficial modality for occupational therapy. The Lifestyle Performance Model provides a useful framework for analysis and interpretation of the positive outcomes of animal‐assisted therapy in an occupational therapy context.
Older people: The enigma of satisfaction surveys
Authors: Atwal Anita , Caldwell Kay
Published: March 2005

Background and Aims:  Health and social care reforms have emphasised the need for health and social care professionals to monitor user satisfaction. Obtaining the opinions of clients is an important mechanism for improving quality in health and social care. This paper discusses methodological issues in measuring satisfaction among older service clients.

Methods and Results:  Examples will be used from the authors’ own experiences of conducting research with older people to ascertain opinions and levels of satisfaction. The strengths and weakness of methods used to collect the opinions of older clients using interview and non‐standardised questionnaires are critically considered. The authors suggest that occupational therapists need to understand the methodological issues that can impact upon older clients’ responses to satisfaction questions.

Conclusion:  If occupational therapists are committed to the ethos of client‐centred practice, then clients must not only be consulted and involved in service delivery, but also in the design and implementation of satisfaction surveys.
Developing referral and reassessment criteria for drivers with dementia
Authors: Lovell Robin K. , Russell Kay J.
Published: March 2005

Background and Aims:  Determining if a person with dementia should be referred for a driver assessment, at what stage of the illness and how often to repeat this are difficult decisions for health professionals. The aims of this study were to develop criteria for driver assessment referral and to explore the value of routine reassessment for drivers with dementia.

Methods and Results:  Twenty participants with a diagnosis of dementia were recruited from a memory clinic and underwent a standard occupational therapy driver assessment and reassessment 6 months later. Fifteen failed the initial assessment but 10 went on to pass local area assessments. Some participants in the early stages of the disease failed. Five of the nine participants suitable for reassessment at 6 months also failed.

Conclusion:  The results confirmed that routine referral following the diagnosis of dementia is appropriate. Change in performance was noted at reassessment, confirming that 6 months between assessments is an appropriate timeframe.
Regaining childhood: A case study
Authors: Scaletti Rowena
Published: March 2005

A case study is used to describe the use of play as a therapeutic tool to mediate the effects of domestic violence on the development of self. The value of a collaborative approach by family and therapist, including the collection of qualitative data, demonstrates how external systems such as supervised access and the legal system may be used to support the relationship between occupation, well‐being and health.