bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of a new acidic glutathione S‐transferase, GST‐Yn1 Yn1, with a high leukotriene‐C4 synthase activity from rat brain
Authors: TSUCHIDA Shigeki , IZUMI Takashi , SHIMIZU Takao , ISHIKAWA Takashi , HATAYAMA Ichiro , SATOH Kimihiko , SATO Kiyomi
Published: March 2005

A new acidic form of glutathione S‐transferase (GST, pI 6.2) was purified from rat brain by S‐hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20–25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (MT or 6‐6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the MT in isoelectric point, activity towards 1,2‐dichloro‐4‐nitrobenzene and immunological properties. On two‐dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn1) of two spots (Yn1 and Yn2) of the testis GST‐MT. Therefore, the brain acidic form is a homodimer, and named GST‐Yn1 Yn1. The activity was inhibited by sulfasalazine, an inhibitor of leukotriene‐C4 synthase. This form (GST‐Yn1Yn1) showed the highest leukotriene‐C4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The Km values for leukotriene A4 and glutathione were 26 μM and 3.5 mM respectively.

A major GST form of rat brain, occupying about 40% of the total activity, was identical with GST‐P (7–7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST‐P also showed the significant leukotriene‐C4 synthase activity, 67.2 nmol/mg protein in 5 min, but the Km for leukotriene A4 was 100 μM, fourfold higher than that of GST‐Yn1 Yn1. These results suggest that mainly GST‐Yn1 Yn1 may be involved in leukotriene‐C4 synthesis in rat brain.
A synthetic functional metabolic compartment
Authors: FOSSEL Eric T. , HOEFELER Herbert
Published: March 2005

A system was created to model the influence of microcompartments on linked enzymatic reactions. Creatine kinase and hexokinase were covalently attached to Sepharose beads. The gel could be perfused in a specially constructed chamber inside a 360‐MHz NMR spectrometer at different flow rates with solutions containing various concentrations of substrates. 31P NMR studies were carried out on the linked enzymatic reaction, creatine phosphate + glucose → creatine + glucose 6‐phosphate in two enzyme gels differing in only one aspect, the average distance between hexokinase and creatine kinase. At a distance on the order of 0.1 mm between the enzymes, the average bulk concentration of substrates and products in the perfusate determined the overall function of the linked system. At an average distance of the order of 10 nm, flux through the linked pair was much higher and much less dependent on the concentration of the intermediate substrate/product ADP/ATP. Even at adenine nucleotide concentrations far below the Km of hexokinase, substantial amounts of glucose 6‐phosphate were produced when the enzymes were near but not when they were distant. From saturation transfer measurements and turnover calculations, the lifetime of ATP in the system is estimated to be 0.14–0.5 s when the enzymes are near. This compares to 6 s for distant enzymes. From this it appears that the pair of linked enzymes comprise a functional compartment supported by propinquity in which hexokinase has preferential access to ATP produced by creatine kinase, and creatine kinase to ADP from the hexokinase reaction.
Microquantitative determination of intra‐acinar distribution profiles of low‐Km and high‐Km aldehyde dehydrogenase activity in rat liver
Authors: MALY Ireneusz Piotr , SASSE Dieter
Published: March 2005

Microquantitative measurements of total and of low‐Km aldehyde dehydrogenase (ALDH) activity with millimolar and micromolar concentrations of acetaldehyde and propionaldehyde were carried out on the livers of male and female rats. Lyophilized cryostat sections of liver parenchyma were microdissected along the entire sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ALDH activity was measured in a microbiochemical assay using the oil‐well technique with luminometric determination of NADH. On the basis of single measurements, mean values of total, low‐Km and high‐Km ALDH activity could be calculated and the specific distribution patterns graphically demonstrated.

The two substrates acetaldehyde and propionaldehyde yielded similar values of ALDH activity, the intra‐acinar distribution profiles of which showed characteristic sex differences. In the liver of the male rat high‐Km ALDH activity has two flat peaks in the periportal and the perivenous area, while low‐Km ALDH activity is almost evenly distributed throughout the acinus. In the livers of female rats, both high‐Km and low‐Km ALDH activity shows a continuous gradient which decreases from the periportal to the perivenous zone (pp/pv = 1.4:1). It was therefore possible to demonstrate that the maxima of alcohol dehydrogenase activity and of low‐Km ALDH activity are localized in opposite parts of the liver acinus of the female rat. This heterotopy should have consequences with respect to hepatotoxicity after alcohol ingestion.
A kinetic method for distinguishing whether an enzyme has one or two active sites for two different substrates
Authors: KELETI Tamás , LEONCINI Roberto , PAGANI Roberto , MARINELLO Enrico
Published: March 2005

We have elaborated a kinetic method which allows us to evaluate whether a Michaelis‐Menten‐type enzyme acting on two different substrates has one or two active sites. This method has been used with the rat liver l‐threonine dehydratase, which catalyzes the dehydrative deamination of both serine and threonine. The experimental data can be fitted to the theoretical plot obtained for the case of a single active site.
Influence of different 5′‐flanking sequences of tRNA genes on their in vivo transcription efficiencies in Saccharomyces cerevisiae
Authors: DINGERMANN Theodor , NERKE Käthe , MARSCHALEK Rolf
Published: March 2005

We have investigated the influence of 5′‐flanking sequences on the in vivo transcription activities in yeast. Since eukaryotic tRNA genes belong to multi‐copy gene families monitoring of the activity of a particular tRNA gene is not possible. We therefore used two different tRNA genes from the cellular slime mould Dictyostelium discoideum which are efficiently transcribed and processed in vivo in yeast. The original 5′‐flanking sequences of the two tRNA genes were replaced by random plasmid sequences. The modified tRNA genes were introduced into Saccharomyces cerevisiae and bulk tRNAs from the transformants were analyzed for the presence and the relative number of Dictyostelium tRNA gene transcripts. Substantial differences of steady‐state levels of RNA transcribed were detected dependent on the 5′‐flanking sequence of the tRNA gene. Minute structural changes, such as inserting two additional nucleotides in front of a tRNA gene, can lead to drastic activity changes. The efficiency of tRNA gene transcription can be conferred by sequences located more than 40 nucleotides upstream from the 5′ end of the mature tRNA coding region.
Prediction of ‘hot spots’ in SV40 enhancer and relation with experimental data
Authors: ĆOSIĆ Irena , NESIC Dobrila
Published: March 2005

A theoretical method for prediction of the points in the sequence which are relevant for its biological function, the so‐called ‘hot spots’, is presented. This method is based on significant correlation between the spectrum of numerical presentation of any genetic sequence and its biological function. One number corresponds to the particular nucleotide, thus forming a numerical sequence. The ‘hot spot’ prediction has been tested on the SV40 enhancer as a model system. The SV40 enhancer was chosen because of existing detailed data about activities of systematically obtained mutants. These results have been compared with results obtained theoretically.
Interactions of actin and tubulin with human deoxyhemoglobin
Authors: LEBBAR Ihsane , STETZKOWSKI‐MARDEN Françoise , MAUFFRET Olivier , CASSOLY Robert
Published: March 2005

Short actin filaments are an essential component of the red‐cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion‐binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high‐affinity complex with Hb. This is shown by measuring, by stopped‐flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an α,β‐tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3‐bisphosphoglycerate‐binding site of Hb and a negatively charged domain, most likely the highly acidic N and C‐terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.
1H‐NMR sequential assignments and cation‐binding studies of spinach plastocyanin
Authors: DRISCOLL Paul C. , HILL H. Allen O. , REDFIELD Christina
Published: March 2005

The essentially complete assignment of the 1H‐NMR spectrum of the Cu(I) form of spinach plastocyanin has been achieved using two‐dimensional NMR techniques and sequence‐specific resonance assignment procedures. A variety of pH and temperature conditions was utilised to overcome the problems of resonance overlap in the spectrum, degeneracy of CαH and solvent H2O chemical shifts, and cross‐saturation of labile NH resonances. A qualitative analysis of the long‐range nuclear Overhauser effects observed indicates that the backbone fold of spinach plastocyanin is very similar to that of poplar plastocyanin, whose structure has been solved by X‐ray crystallography and differs in 22 of its 99 amino acid residues. The assignments provide a basis for further investigations into the structural and ion‐ and protein‐binding properties of plastocyanin in solution.
Alkylamine derivatives of cytochrome c
Authors: WALLACE Carmichael J. A. , CORTHÉSY Blaise E.
Published: March 2005

For investigations of the functional roles of the lysine residues of cytochrome c, analogues in which these residues are modified without charge loss are highly desirable. The 19 lysine residues of the horse heart protein have been modified by reductive alkylation. Two analogues were prepared, using formaldehyde and acetone as the dialkylating and monoalkylating reagent respectively. Modification was shown to be clean and quantitative. Characterisation of the alkylamine derivatives by physicochemical measurements and biological activity determinations was carried out. The potential of these analogues in structure/function studies of cytochrome c is discussed. It is illustrated by their use, in conjunction with other lysine‐modified derivatives, to investigate the extent to which surface charge determines redox potential, and to study the physicochemical changes that accompany rising pH. In the latter case the observed phenomena are not as closely correlated as previously thought, suggesting that there is a complex set of rearrangements of structure underlying the functional changes.

The data confirm that modification of the lysine residues influences these changes. These residues participate in numerous surface intramolecular links, so the lack of correlation may be explained if each of the changing parameters were under the influence of a different set of residues. However, neither a lysine residue, nor a histidine residue directly displaces methionine from the sixth coordination position of the haem iron at alkaline pH.
Transamination catalysed by tyrosine phenol‐lyase from Citrobacter intermedius
Authors: DEMIDKINA Tatyana V. , MYAGKIKH Igor V. , AZHAYEV Alexei V.
Published: March 2005

The interactions of tyrosine phenol‐lyase with its substrates: l‐tyrosine and l‐serine, and the competitive inhibitors: l‐alanine, l‐phenylalanine, l‐m‐tyrosine, were studied. It was demonstrated that the enzyme catalyzed a half‐transamination reaction between substrates or inhibitors and the protein‐bound pyridoxal phosphate. The products of this side‐reaction, pyridoxamine phosphate and the respective keto acids, were identified. The kinetic parameters were determined for β‐elimination of l‐tyrosine and of l‐serine, and for the transamination of l‐serine and the inhibitors used. The transfer of the amino group to the coenzyme takes place in the direction from amino acid to pyridoxal phosphate, but not in the opposite direction, i.e. the transamination is irreversible.
Characterization of two d‐glyceraldehyde‐3‐phosphate dehydrogenases from the extremely thermophilic archaebacterium Thermoproteus tenax
Authors: HENSEL Reinhard , LAUMANN Silvia , LANG Jutta , HEUMANN Herrmann , LOTTSPEICH Friedrich
Published: March 2005

Thermoproteus tenax possesses two different glyceraldehyde‐3‐phosphate dehydrogenases, one specific for NADP+ and the other for NAD+. NADP(H) inhibits the NAD+‐specific enzyme competetively with respect to NAD+ whereas NAD(H) virtually does not interact with the NADP+‐specific enzyme. Both enzymes represent homomeric tetramers with subunit molecular masses of 39 kDa (NADP+‐specific enzyme) and 49 kDa (NAD+ ‐specific enzyme), respectively.

The NADP+‐specific enzyme shows significant homology to the known glyceraldehyde‐3‐phosphate dehydrogenases from eubacteria and eukaryotes as indicated by partial sequencing.

The enzymes are thermostable, the NADP+‐specific enzyme with a half‐life of 35 min at 100°C, the NAD+ ‐specific enzyme with a half‐line of ≥20min at 100°C, depending on the protein concentration. Both enzymes show conformational and functional changes at 60 – 70°C.
The role of the N‐terminus of the large subunit of ribulose‐bisphosphate carboxylase investigated by construction and expression of chimaeric genes
Authors: KETTLEBOROUGH Catherine A. , PARRY Martin A. J. , BURTON Steven , GUTTERIDGE Steven , KEYS Alfred J. , PHILLIPS Andrew L.
Published: March 2005

The genes for the large and small subunits of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Anacystis nidulans have been expressed in Escherichia coli under the control of the lac promoter to produce active enzyme. The enzyme can be purified from the cells to yield up to 200 mg Rubisco/l cultured bacteria, and is indistinguishable from the enzyme extracted from A. nidulans. In order to investigate the role of the N‐terminus of the large subunit in catalysis, chimaeric genes were constructed where the DNA coding for the 12 N‐terminal amino acids in A. nidulans was replaced by DNA encoding the equivalent, but poorly conserved, region of either the wheat or maize large subunit. These genes, in constructs also containing the gene for the A. nidulans small subunit, were expressed in E. coli and produced enzymes with similar catalytic properties to the wild‐type Rubisco of A. nidulans. In contrast, when the N‐terminal region of the large subunit was replaced by unrelated amino acids encoded by the pUC8 polylinker, enzyme activity of the expressed protein was reduced by 90% under standard assay conditions, due to an approximately tenfold rise in the Km for ribulose 1,5‐bisphosphate. This confirms that the N‐terminus of the large subunit has a function in catalysis, either directly in substrate binding or in maintaining the integrity of the active site.
Chemical modification of phenol hydroxylase by ethoxyformic anhydride
Authors: SEJLITZ Torsten , NEUJAHR Halina Y.
Published: March 2005

Phenol hydroxylase was inactivated by ethoxyformic anhydride. Part of the inactivation was related to modification of histidyl residues. The remaining part of the inactivation is proposed to be due to the modification of a lysyl residue which, we suggest, is identical with the one previously described, being essential for the binding of NADPH [Neujahr, H. Y. and Kjellén, K. G. (1980) Biochemistry 19, 4967–4972]. The overall inactivation reaction is biphasic and follows pseudo‐first‐order kinetics. Numerical analysis of kinetic data was applied to discriminate between simultaneous reactions at different sites. It is proposed that phenol hydroxylase contains two essential histidyl residues, located in or near the NADPH‐binding sites. Ethoxyformylation of the lysyl residue(s) caused tightening of the binding of phenol and perturbation of the FAD spectrum of phenol hydroxylase, similar to that caused by phenolic effectors.
Structure of the sidechain of lipopolysaccharide from Erwinia amylovora T
Authors: RAY Timothy C. , SMITH Anthony R. W. , WAIT Robin , HIGNETT Richard C.
Published: March 2005

The sidechain of lipopolysaccharide from Erwinia amylovora T was composed of d‐fucose, d‐galactose and d‐glucose in equimolar proportions. Using NMR spectroscopy, methylation analysis, mass spectrometry, Smith degradation and optical rotation data, the repeat unit was shown to have the following most probable structure:

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Isolation and characterization of 9‐hydroxy‐10‐trans,12‐cis‐octadecadienoic acid, a novel regulator of platelet adenylate cyclase from Glechoma hederacea L. Labiatae
Authors: HENRY Dominique Y. , GUERITTE‐VOEGELEIN Françoise , INSEL Paul A. , FERRY Nicolas , BOUGUET Jacqueline , POTIER Pierre , SEVENET Thierry , HANOUNE Jacques
Published: March 2005

We have identified and characterized a fatty acid, (9S,10E,12Z)‐9‐hydroxy‐10,12‐octadecadienoic acid (9‐HODE) as a regulator of adenylate cyclase activity of human platelet membranes. This fatty acid was isolated from a methanolic extract of the plant Glechoma hederacea L. Labiatae (commonly known as ‘lierre terrestre’, ‘ground ivy’ or ‘creeping Charlie’; it was identified by nuclear magnetic resonance and mass spectroscopy. This compound increased basal adenylate cyclase activity in platelet membranes about threefold and had an EC50 of 10–20μM. This increase in adenylate cyclase activity occurred without a temporal lag, was reversible, and represented an increase in Vmax without a substantial change in Km for ATP, Mg2+ or Mn2+. In addition, 9‐HODE additively or synergistically increased platelet adenylate cyclase activity in response to guanosine 5′‐[β,γ‐imido]triphosphate and forskolin, but the fatty acid failed to alter inhibition of adenylate cyclase activity mediated by epinephrine (α2‐adrenergic receptor). Studies of the interaction of 9‐HODE with activation of platelet adenylate cyclase activity mediated by prostaglandin El (PGE1) and prostaglandin D2 (PGD2) indicated that this fatty acid produced a parallel shift in the concentration/response curve of PGE1 and PGD2 without altering maximal response, which was substantially greater than that observed with 9‐HODE alone. From these results, we conclude that 9‐HODE appears to be a partial agonist at PGE1 and PGD2 receptors on human platelets. We believe that this is a novel example of a plant‐derived fatty acid which acts on cells to regulate adenylate cyclase via prostaglandin receptors.
Relations between plasma membrane and lysosomal membrane
Authors: DRAYE Jean‐Pierre , COURTOY Pierre J. , QUINTART Joël , BAUDHUIN Pierre
Published: March 2005

We have quantified, in cultured rat fibroblasts, the association to the lysosomal membrane of two classical plasma membrane markers, 5′‐nucleotidase and alkaline phosphodiesterase I. To isolate highly purified lysosomal preparations, lysosomes were loaded with horseradish peroxidase (2‐h cell uptake, 16‐h chase) and isolated by isopycnic centrifugation in linear Percoll gradients, followed by a 3,3′‐diaminobenzidine‐induced density shift in sucrose gradients. Purified lysosomal preparations contained up to 50% of N‐acetyl‐β‐glucosaminidase of the homogenate. This lysosomal enzyme was enriched 33‐fold in the most purified preparations. In the electron microscope, these preparations appeared to be highly purified and only contained organelles filled with diaminobenzidine reaction products. Analysis of purified preparations indicates that 0.5–0.8% of 5′‐nucleotidase, but as much as 10.9–14.3% of alkaline phosphodiesterase I activities of the homogenate, are associated with lysosomes. After freezing‐thawing, these activities remained essentially membrane‐associated. The larger value obtained for alkaline phosphodiesterase I could not be ascribed to other lysosomal enzymes, as no such activity was detected at acidic pH. These two plasma membrane markers are thus unevenly distributed in the lysosomal compartment.
Atrial peptide inactivation by rabbit‐kidney brush‐border membranes
Authors: OLINS Gillian M. , SPEAR Kerry L. , SIEGEL Ned R. , REINHARD Emily J. , ZURCHER‐NEELY Heidi A.
Published: March 2005

Atriopeptin (AP) 24, containing amino acids Ser103—Tyr126 of the carboxy‐terminal portion of the atrial natriuretic peptide prohormone, was degraded rapidly by rabbit kidney brush border membranes. The rate of degradation of AP24 measured by the loss of vasorelaxant activity followed a similar time course to the decrease in peptide peak area measured by high‐performance liquid chromatography. Inactivation of AP24 produced peptide fragments which were separated by HPLC. The major products were purified individually and their peptide sequences determined. Results indicate that AP24 was proteolytically cleaved at three peptide bonds: Ser103‐Ser104, Cys105‐Phe106 and Ser123‐Phe124. des‐Ser103‐AP24 had similar vasorelaxant activity to AP24, while AP24 cleaved at Cys105‐Phe106 was inactive. Regarding the proteolytic cleavage at Ser123‐Phe124, there was an accumulation of the C‐terminal tripeptide, Phe‐Arg‐Tyr, only at the later time points of the incubation. Degradation experiments were repeated with an amino‐ and carboxy‐terminal protected peptide, acetyl‐AP24‐amide. Peptide sequence analysis of the major degradation products of this peptide revealed that the critical peptide bond cleaved was Cys105‐Phe106. We conclude that the Cys‐Phe peptide bond renders atrial peptides highly susceptible to proteolysis by renal brush border membranes, resulting in inactivation.
Ester composition of carnitine in the perfusate of liver and in the plasma of donor rats
Authors: SANDOR Attila , KISPAL Gyula , MELEGH Bela , ALKONYI Istvan
Published: March 2005

When the carnitine pool of fed rats was labelled with tritium, in non‐recirculating perfusate of their liver 44% of acid‐soluble 3H activity was identified as free carnitine and 47% as short‐chain acylcarnitine. Of the latter component acetylcarnitine accounted for 30% and propionylcarnitine for 10% of total acid‐soluble.

In plasma the contribution of short‐chain acylcarnitines to total carnitine in fed, fasted and diabetic rats was 15.6%, 43.1% and 48.0%, respectively. Recirculating perfusion of livers from the same animals revealed that livers from fed rats released short‐chain acylcarnitines as much as 56.2% of total and this proportion did not increase further in the other two groups. At the same time, ketone bodies in the perfusate increased gradually in the fed, fasted and diabetic group, paralleling the plasma ketone levels. Although liver supplies the organism with carnitine the increment of plasma short‐chain acylcarnitines seen in ketosis is not a result of some extra output by the liver.
Biosynthesis of coenzyme F430 in methanogenic bacteria
Authors: PFALTZ Andreas , KOBELT André , HÜSTER Rudolf , THAUER Rudolf K.
Published: March 2005

Coenzyme F430 is a hydroporphinoid nickel complex present in all methanogenic bacteria. It is part of the enzyme system which catalyzes methane formation from methyl‐coenzyme M. We describe here that under certain conditions a second nickel porphinoid accumulates in methanogenic bacteria. The compound was identified at 15,173‐seco‐F430‐173‐acid. The structural assignment rests on 14C‐labelling experiments, fast‐atom‐bombardment mass spectra, 1H‐NMR spectra of the corresponding hexamethyl ester, and ultraviolet/visible spectral comparison with model compounds. In cell extracts and in intact cells of methanogenic bactera, 15,173‐seco‐F430‐173‐acid was converted to F430. These findings indicate that the new nickel‐containing porphinoid is an intermediate in the biosynthesis of coenzyme F430.
Cell‐wall lytic enzymes (autolysins) of Chlamydomonas reinhardtii are (hydroxy)proline‐specific proteases
Authors: JAENICKE Lothar , KUHNE Wolfgang , SPESSERT Rainer , WAHLE Ursula , WAFFENSCHMIDT Sabine
Published: March 2005

Two stage specific cell‐wall lytic enzymes (autolysins) from different strains of the unicellular, biflagellated green alga Chlamydomonas reinhardtii were isolated and purified to homogeneity. Quantitative and specific photometric assays for biological activity were worked out to follow fractionation and to establish lytic specificity and kinetics. The autolysins were studied for enzymatic properties and screened for biological activity towards several wall components obtained by salt extractions of sporangia and zoospores from C. reinhardtii. The autolysins are proteolytic enzymes, fragmenting proline‐ or hydroxyproline‐containing polypeptides in structures like connective tissue. They attack predominantly selected domains within the walls of zoosporangia or gametes. The sporangial autolysins are not only site‐ and strain‐specific but also stage‐specific, whereas the gamete autolysins lyse cell walls of gametes as well as those of sporangia and zoospores.
Developmental changes in rat hepatic casein kinases 1 and 2
Authors: PEREZ Mercè , GRANDE Jorge , ITARTE Emilio
Published: March 2005

Cytosolic histone kinase and casein kinase activities varied considerably in the late fetal and postnatal periods of liver development. Both activities showed a maximum at day 21 of gestation and decreased at birth to values close to those of adult rats. The changes in total casein kinase activity were due to variations of casein kinase 1 and casein kinase 2. Similarly the activities of both the cyclic‐AMP‐dependent protein (histone) kinase and the cyclic‐AMP‐independent histone kinase varied during development.

Besides the changes in total activity, the affinity of casein kinases 1 and 2 for casein also varied in fetal and postnatal development. The Km values of casein kinase 2 increased from day 18, reached a maximum at day 20 of gestation and then started to decrease until one day after birth. In contrast the Km values of casein kinase 1 decreased from day 18, reached its lowest value at day 21 of gestation and attained values similar to those in the adult at the day of birth. Changes in this parameter were also observed when insulin (3 IU/kg) was administered by intraperitoneal injection to one‐day‐old rats. The Km values of casein kinase 1 decreased while those of casein kinase 2 increased after administration of this hormone. On the other hand, the Km values for ATP of casein kinases 1 and 2 as well as their apparent molecular masses and sensitivity to heparin and GTP did not significantly change during ontogeny of rat liver.
Nucleotide sequence and linkage map position of the genes for ribosomal proteins L14 and S8 in the maize chloroplast genome
Authors: MARKMANN‐MULISCH Ulrich , SUBRAMANIAN Alap R.
Published: March 2005

The nucleotide sequence of a 1287‐base‐pair segment of the maize (Zea mays) chloroplast DNA, encoding chloroplast ribosomal proteins L14, S8 and the C‐terminal part of L16, has been determined using the dideoxy‐chain‐termination method. These data from a monocot plant are compared to the corresponding data from a dicot and a lower plant and from two bacteria. The deduced amino acid sequence of maize chloroplast L14 shows 80%, 81%, 51% and 52% and that of S8 shows 75%, 58%, 39% and 38% sequence identity, respectively, to the corresponding sequences of Nicotiana tabacum, Marchantia polymorpha, Bacillus stearothermophilus and Escherichia coli. The starting map coordinates of rpL14 and rpS8 in the physical map of the maize chloroplast DNA [Larrinua, I. M., Muskavitch, K. M. T., Gubbins, E. J. and Bogorad, L. (1983) Plant Mol. Biol. 2, 129–140] are 31.330 and 31.841. The gene order is rpL16‐spacer‐rpL14‐spacer‐rpS8. Shine‐Dalgarno sequences (GGA and AGGAGG) and computer‐derived stem‐loop structures of dyad symmetry are present in the spacers and the 3′ downstream region of rpS8, respectively, but a chloroplast promoter‐like sequence could not be detected suggesting that the latter might be located further upstream in this ribosomal protein gene cluster in maize chloroplast DNA.
Analysis of the aliphatic 1H‐NMR spectrum of plasminogen kringle 4
Authors: PETROS Andrew M. , GYENES Marianne , PATTHY László , LLINÁS Miguel
Published: March 2005

The aliphatic 1H‐NMR spectrum of the kringle 4 domain of human plasminogen has been studied via two‐dimensional chemical shift correlated (COSY) and nuclear Overhauser correlated (NOESY) experiments at 300 MHz and 620 MHz. A number of aliphatic proton spin systems have been identified and several definite assignments have been made. This was mainly achieved by comparison of the human kringle 4 spectrum with spectra of the porcine, bovine and chicken homologs and also with that of the kringle 1 from human plasminogen on which we have reported previously. The three valyl and two leucyl residues of human kringle 4 have been assigned. The eleven threonyl spin systems have been identified via a RELAYED‐COSY experiment and Thr17 has been assigned. The three alanyl spin systems have been identified and assigned. Six seryl spin systems have been identified and the signals from the seven glycyl residues of human kringle 4 have been located with Gly45 assigned. Furthermore, 24 AMX spin systems have been mapped in the COSY spectrum of human kringle 4 and Hα‐Hβ,β′ spin systems of Tyr2, Tyr41, Tyr50, Tyr74, Trp25 and Trp62 have been assigned. From the spectrum of a deglycosylated chicken homolog, the ɛ‐methyl singlets of Met28 and Met48 have been assigned. Finally, ligand effects on selected aliphatic resonances were observed which could be analyzed in terms of residues likely to neighbor the kringle lysine‐binding site.
ATP4, the structural gene for yeast F0F1 ATPase subunit 4
Authors: VELOURS Jean , DURRENS Pascal , AIGLE Michel , GUÉRIN Bernard
Published: March 2005

A plasmid containing the gene coding for the Saccharomyces cerevisiae F0Fl ATPase subunit 4 was isolated from a yeast genomic DNA library using the oligonucleotide probe procedure. The gene and the surrounding regions were cloned into M13 tg 130 and M13 tg 131 phage vectors. A 732‐base‐pair open reading frame encoding a 244‐amino‐acid polypeptide is described. The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein with a hydrophilic and basic 35‐amino‐acid leader sequence. Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 Da. This subunit presents amphiphilic behaviour with two distinct domains. A high α‐helix content of 77% was predicted from the sequence. Subunit 4 shows homology with the b subunit of Escherichia coli ATP synthase.
Purification in large scale and characterization of the human leukocyte adhesion glycoprotein GP90 (CD 18)
Authors: KANTOR Carmela , SUOMALAINEN‐NEVANLINNA Heli , PATARROYO Manuel , ÖSTERLUND Kaj , BERGMAN Tomas , JÖRNVALL Hans , SCHRÖDER Jim , GAHMBERG Carl G.
Published: March 2005

The leukocyte adhesion 90‐kDa glycoprotein GP90 (antigen CD18) is non‐covalently associated separately with cell‐surface glycoproteins GP160 (antigens CD11 a, TA‐1, LFA‐1), GP155 (antigens CD11 b, OKM1, M01) or GP130 (antigens CD 11c, Leu‐M5). Large amounts of these protein complexes were purified to homogeneity from blood mononuclear leukocytes by immunoaffinity chromatography using a monoclonal antibody. GP90 was further isolated by preparative gel electrophoresis in the presence of sodium dodecyl sulfate. Rabbit antiserum towards the complex inhibited phorbol‐ester‐induced adhesion of leukocytes. The antiserum towards purified GP90 reacted more strongly with the denatured GP90 protein, but showed reactivity also with GP160 protein, indicating structural homologies between GP90 and GP160. The amino acid composition of GP90 was determined. Its N‐terminus was found to be blocked. Treatment of GP90 with endo‐β‐N‐acetylglucosaminidase F, but not with endo‐β‐N‐acetylglucosaminidase H, reduced the apparent molecular mass to 75 kDa, indicating the presence of five or six N‐linked complex‐type oligosaccharides/molecule.
Further purification and characterization of the DNA 3′‐phosphatase from rat‐liver chromatin which is also a polynuclotide 5′‐hydroxyl kinase
Authors: HABRAKEN Yvette , VERLY Walter G.
Published: March 2005

The DNA 3′‐phosphatase activity of rat‐liver chromatin has been purified. A DNA 5′‐hydroxyl kinase activity comigrates at each step of purification. Both enzymes have the same molecular mass (79 kDa) and the same isoelectric point (8.6). It thus seems that the two activities are born by the same protein just as with the phage T4 enzyme which is, at the same time, a 5′‐hydroxyl kinase and a 3′‐phosphatase. An additional argument is that ATP, which does not influence the rate of the 3′‐phosphatase reaction but which is a cosubstrate of the 5′‐hydroxyl kinase, protects the 3′‐phosphatase activity against thermal denaturation and trypsin digestion.

The two active sites must, however, be largely independent within a common support: the thermal denaturation and trypsin inactivation rates are very different for the two activities; increasing the ionic strength activates the kinase and inhibits the phosphatase; polyvalent anions inhibit the phosphatase and have little effect on the kinase. The two active sites might belong to different domains of the protein; they could not however be separated by a partial trypsin digestion.

The rates of 3′‐dephosphorylation and 5′‐phosphorylation by the chromatin enzyme are the same in native and denatured DNA. The 3′‐phosphatase has no action on 3′‐monodeoxynucleotide, but it hydrolyzes the 3′‐phosphate in dinucleotides. The Km of the 3′‐phosphatase is 0.548 μM. The Km (5′‐OH) and Km (ATP) of the 5′‐hydroxyl kinase are about 3.9 μM and 0.69 μM respectively. The chromatin enzyme is unable to hydrolyze 3′‐phosphoglycolate ends in DNA.
Various kinds of lipoamino acids including a novel serine‐containing lipid in an opportunistic pathogen Flavobacterium
Authors: KAWAI Yohko , YANO Ikuya , KANEDA Kenji
Published: March 2005

Lipoamino acids were found to represent 60% of the total extractable cellular lipids in Flavobacterium meningosepticum and F. indologenes which are known to be opportunistic pathogens. The structures of the lipoamino acids were resolved by chemical and physicochemical methods. Three kinds of lipoamino acids were included in the lipid of Flavobacterium: 3‐hydroxyisoheptadecanoic acid amide‐linked to serine and esterified to isopentadecanoic acid, and 3‐hydroxyisoheptadecanoic acid amide‐linked to ornithine and esterified to isopentadecanoic acid or 2‐hydroxyisopentadecanoic acid. This type of serine‐containing lipid is a novel, rare substance and exhibited high hemagglutinating activity (minimum hemagglutinating concentration was 0.25–0.5 μg/ml) with very good, stable dispersion of its liposomes. We called the serine‐containing lipid ‘flavolipin’, based on the genus name of the bacteria. Of the two types of the ornithine‐containing lipids, the one that had a nonpolar terminal fatty acid showed higher hemagglutinating activity than the one that had a polar terminal fatty acid. The reconstituted liposomes whose lipid composition was similar to that of the original bacterial cell membrane exhibited definite hemagglutinating activity. As soon as a terminal fatty acid of the lipoamino acids was lost, their biological activity on erythrocytes changed from hemagglutinating to hemolytic, being accompanied by the disappearance of animal species specificity. The mechanism of both the hemagglutination and the hemolysis was discussed.
Cytochrome b6f complex is required for phosphorylation of light‐harvesting chlorophyll a/b complex II in chloroplast photosynthetic membranes
Authors: BENNETT John , SHAW Elizabeth K. , MICHEL Hanspeter
Published: March 2005

The light‐harvesting chlorophyll a/b complex (LHC II) and four photosystem II (PS II) core proteins (8.3, 32, 34 and 44 kDa) become phosphorylated in response to reduction of the intersystem electron transport chain of green plant chloroplasts. Previous studies indicated that reduction of the plastoquinone (PQ) pool is the key event in kinase activation. However, we show here that, unlike PS II proteins, LHC II is phosphorylated only when the cytochrome b6f complex is active. Two lines of evidence support this conclusion. (1) 2,5‐Dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone (DBMIB) and the 2,4‐dinitrophenyl ether of iodonitrothymol (DNP‐INT), which are known to block electron flow into the cytochrome complex, selectively inhibit LHC II phosphorylation in spinach thylakoids. (2) The hcf6 mutant of maize, which contains PQ but lacks the cytochrome b6f complex, phosphorylates the four PS II proteins but fails to phosphorylate LHC II in vivo or in vitro.
Purification and characterization of a casein‐kinase‐II‐type enzyme from Xenopus laevis ovary
Authors: MULNER‐LORILLON Odile , MAROT Jean , CAYLA Xavier , POUHLE Robert , BELLE Robert
Published: March 2005

A casein kinase of type II has been highly purified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four chromatographic steps: hydrophobic on tyrosine‐agarose, ion exchange on DEAE‐Sepharose, affinity on heparin‐Sepharose and fast protein liquid on Mono Q. The purification was > 20000, taking into account an inhibitor present in the starting material which masked the activity in the crude fraction. The overall yield was >20%. Full‐grown Xenopus oocytes contain 64 milliunits per oocyte corresponding to an intracellular concentration in the nanomolar range.

The enzyme shares the following features with the mammalian casein kinase II: (a) comparable subunit composition (42‐kDa doublet, 38 kDa and 26 kDa), (b) autophosphorylation of the 26‐kDa subunit, (c) ability to use GTP as well as ATP as phosphate donor, (d) inability to use Mn2+ instead of Mg2+ to support the activity, (e) phosphorylation of both threonine and serine residues of casein, (f) inhibition by low doses of heparin.

Biological effects of the highly purified enzyme have been investigated upon microinjection into Xenopus full‐grown oocytes. At nanomolar concentrations (∼ 3 nM) the enzyme inhibited progesterone induction of meiotic cell division whereas it facilitates meiotic maturation at the level of maturation‐promoting factor. These results suggest a role for the kinase in the phosphorylation cascade involved during the prophase/metaphase transition of meiotic cell division, both in the mechanism of the meiotic prophase arrest and in the activity of the cytoplasmic factor maturation‐promoting factor. When microinjected into oocytes above 45 nM, the kinase provoked complex changes in the profile of the in ovo32P‐labelled proteins indicating that its targets could be other kinase/phosphatase regulatory proteins.
Immunochemical properties of elongation factors 1 of plant origin
Authors: PULIKOWSKA Jadwiga , WOJTASZEK Przemyslaw , KORCZI Aleksandra , MICHALSKI Zbigniew , CANDRESSE Thierry , TWARDOWSKI Tomasz
Published: March 2005

Elongation factors 1 (EF‐1) have been isolated from different plants: wheat, yellow lupine, blue lupine, Chinese cabbage and Norway maple.

Antibodies for EF‐1 from yellow lupine have been obtained in rabbits; antibodies for wheat EF‐1 were elicited in mice. The immunological properties of EF‐1 were assayed by the following methods: western blotting, double immunodiffusion and rocket immunoelectrophoresis. Our results suggest that one antigenic site is similar for all plant elongation binding factors tested. This epitope probably overlaps the centre of biological activity of EF‐1, as was shown for wheat EF‐1.

The hypothesis concerning the potential presence of plant EF‐1 as a subunit of turnip yellow mosaic virus RNA replicase (similar to prokaryotic EF‐Tu in the Qβ RNA replicase system) has also been tested using immunotechniques as well as tests of biological activity, but has not been confirmed.
Conformational analysis of the tetranucleotides m62A‐m62A‐U‐m62A (m62A =N6‐dimethyladenosine) and U‐m62A‐U‐m62A and of the hybrid dA‐r(U‐A)
Authors: van den HOOGEN Yvonne Th. , TREURNIET Stevin J. , ROELEN Harlof C. P. F. , de VROOM Erik , van der MAREL Gijs A. , van BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

A 1H‐NMR investigation was carried out on the tetranucleotides U‐m62A‐U‐m62A and m62A‐m62A‐U‐m62A (m62=N6‐dimethyladenosine) as well as on the hybrid trinucleotide dA‐r(U‐A). An extensive comparison with m62A‐U‐m62A and other relevant compounds is made.

Previous proton NMR studies on trinucleotides have shown that purine‐pyrimidine‐purine sequences prefer to adopt a mixture of states which have as a common feature that the interior pyrimidine residue bulges out, whereas the flanking purine residues stack upon each other. A stacking interaction on the 3′ side of the bulge is known to have no measurable effect on the bulge population. Chemical‐shift data, ribose ring conformational analysis and information from NOE experiments now show unambiguously that the moderate U(1)‐m62A(2) stack in U‐m62A‐U‐m62A diminishes the population of bulged‐out structures in favour of a regular stack. This tendency towards conformational transmission in the downstream 5′→3′direction is fully confirmed by the fact that the strong m62A(1)‐m62A(2) stack in the tetranucleotide m62A‐m62A‐U‐m62A virtually precludes the formation of bulged‐out structures.

The conformational characteristics of dA‐r(U‐A) appear comparable with those of m62A‐U‐m62A, which indicates that the presence of a 2′‐hydroxyl group in the first purine residue is not a necessary prerequisite for the formation of a bulge.
Penicillin‐degrading activities of peptides from pneumococcal penicillin‐binding proteins
Authors: ELLERBROK Heinz , HAKENBECK Regine
Published: March 2005

Trypsin treatment of native penicillin‐binding proteins (PBPs) 1a, 2 b and 3 from Streptococcus pneumoniae resulted in the formation of stable peptides containing the β‐lactam‐binding site with molecular masses ranging from 26 kDa to 36 kDa.

Whereas the PBP 1a peptide (Ia) was enzymatically rather unstable, the PBP 2b peptide (IIb) and the PBP 3 peptide (III) were able to bind and release β‐lactams with similar rates compared to the intact PBP, the turnover rate of fragment IIb was even twice as fast as that observed with PBP 2b. Analysis of the turnover products by thin‐layer chromatography revealed that PBP 2b and 3 produced penicilloic acid as well as phenylacetylglycine. On the other hand, with the corresponding tryptic fragments only the hydrolysis product penicilloic acid was obtained.
Higher oxidation states of prostaglandin H synthase
Authors: KARTHEIN Rainer , DIETZ Reiner , NASTAINCZYK Wolfgang , RUF Hans Heinrich
Published: March 2005

Purified prostaglandin H synthase (EC 1.14.99.1), reconstituted with hemin, was reacted with substrates of the cyclooxygenase and peroxidase reaction. The resulting EPR spectra were measured below 90 K. Arachidonic acid, added under anaerobic conditions, did not change the EPR spectrum of the native enzyme due to high‐spin ferric heme. Arachidonic acid with O2, as well as prostaglandin G2 or H2O2, decreased the spectrum of the native enzyme and concomitantly a doublet signal at g= 2.005 was formed with maximal intensity of 0.35 spins/enzyme and a half‐life of less than 20 s at ‐12 °C. From the conditions for the formation and the effect of inhibitors, this doublet signal was assigned to an enzyme intermediate of the peroxidase reaction, namely a higher oxidation state.

The doublet signal with characteristic hyperfine structure was nearly identical to the signal of the tyrosyl radical in ribonucleotide reductase (EC 1.17.4.1). Hence the signal of prostaglandin H synthase was assigned to a tyrosyl radical. Electronic spectra as well as decreased power saturation of the tyrosyl radical signal indicated heme in its ferryl state which coupled to the tyrosyl radical weakly. [FeIVO(protoporphyrin IX)]…Tyr+· was suggested as the structure of this two‐electron oxidized state of the enzyme.

A hypothetical role for the tyrosyl radical could be the abstraction of a hydrogen at C‐13 of arachidonic acid which is assumed to be the initial step of the cyclooxygenase reaction.
Effective glucuronidation of 6α‐hydroxylated bile acids by human hepatic and renal microsomes
Authors: PARQUET Michel , PESSAH Marcia , SACQUET Edmond , SALVAT Colette , RAIZMAN Annie
Published: March 2005

The glucuronidation of bile acids is an established metabolic pathway in different human organs. The hepatic and renal UDP‐glucuronyltransferase activities vary according to the bile acids concerned. Thus, hyodeoxycholic acid is clearly differentiated from other bile acids by its high rate of glucuronidation and elevated urinary excretion in man. To determine whether such in vivo observations are related to variations in bile acid structure, human hepatic and renal microsomes were prepared and time courses of bile acid glucuronidation measured with the bile acids possessing hydroxyl groups in different positions. Eleven [24‐14C]bile acids were chosen or synthesized in respect of their specific combination of hydroxyl and oxo groups at the 3, 6, 7 and 12 positions and of their α or β hydroxyl configurations. The results clearly demonstrate that bile acids with an hydroxyl group in the 6a position underwent a high degree of glucuronidation. Apparent kinetic Km and Vmax values for UDP‐glucuronyltransferase activities ranged over 78–66 μM and 1.8–3.3 nmol · min−1· mg−1 protein in the liver and over 190–19 μM and 0.5–9.2 nmol · min−1· mg−1 protein in the kidney. All the other bile acids tested, each of which possessed a 3α‐hydroxyl group and whose second or third hydroxyl was bound at the 6β, 7 or 12 positions, were glucuronidated to a degree far below that of the 6α‐hydroxylated bile acids. We conclude that an active and highly specific UDP‐glucuronyltransferase activity for 6α‐hydroxylated bile acids exists in human liver and kidneys. Moreover, this activity results in the linkage of glucuronic acid to the 6α‐hydroxyl group and not to the usual 3α‐hydroxyl group of bile acids.
Engineered rat insulin I analogue having a B16 Tyr/Asp replacement exhibits unchanged susceptibility to cleavage by insulin proteinase
Authors: VARLEY Jennifer M. , DAVIES J. Gwynfor , SHIRE David , OFFORD Robin E. , TIMMIS Kenneth N.
Published: March 2005

An analogue of rat insulin I was produced by oligonucleotide‐directed mutagenesis of a cloned rat preproinsulin I cDNA, followed by expression of a resulting mutant gene in Escherichia coli K‐12 and proteolytic cleavage of mutant proinsulin isolated from this bacterium. The Tyr‐to‐Asp replacement at residue B16 in the insulin analogue had been expected to diminish the rate of cleavage of the molecule by the enzyme insulin proteinase, since the bond TyrB16‐LeuB17, invariant in all mammalian species, had been proposed by other authors as one of the early, major sites of proteolytic attack. In the event the substitution had no measurable effect on the rate of degradation by insulin proteinase. Thus we find no support in these experiments for the hypothesis that the site in question is of primary importance in the degradation of rat insulin I by the enzyme.