bluuebunny/crossref_metadata_2025 huggingface dataset:
Two‐dimensional 1H‐NMR studies of horseradish peroxidase C and its interaction with indole‐3‐propionic acid
Authors: VEITCH Nigel C. , WILLIAMS Robert J. P.
Published: March 2005

The binding of aromatic donor molecules to plant peroxidases has been investigated by examining the complex formed between horseradish peroxidase isoenzyme C and indole‐3‐propionic acid using two‐dimensional 1H‐NMR spectroscopy. Despite the relatively high molecular mass and paramagnetism of the protein, this technique can be successfully applied to provide new information on the structure of the complex. A number of relatively well‐resolved resonances in certain regions of the one‐dimensional spectrum are assigned to amino acid type on the basis of the two‐dimensional experiments. Two phenylalanine side chains are found to interact at positions close to the haem group as shown by nuclear Overhauser effect spectroscopy (NOESY). Furthermore, the NOESY spectrum of the complex reveals distinct interactions between these phenylalanine residues and the indole ring of the donor molecule. The binding site is found to comprise of these phenylalanine side chains and also the methyl group of a leucine or valine residue. On the basis of the model structure of horseradish peroxidase isoenzyme C proposed by Welinder and Nørskov‐Lauritsen and information from previous studies of the related turnip peroxidases, possible locations for this binding site are discussed. The NMR methods adopted here may be generally applicable to the study of peroxidase –aromatic‐donor interactions.
The iron‐sulfur centers of the soluble [NiFeSe] hydrogenase, from Desulfovibrio baculatus (DSM 1743)
Authors: TEIXEIRA Miguel , MOURA Isabel , FAUQUE Guy , DERVARTANIAN Daniel V. , LEGALL Jean , PECK H. D. , MOURA José J. G. , HUYNH Boi H.
Published: March 2005

The soluble (cytoplasmic plus periplasmic) Ni/Fe‐S/Se‐containing hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from cells grown in an 57Fe‐enriched medium, and its iron‐sulfur centers were extensively characterized by Mössbauer and EPR spectroscopies. The data analysis excludes the presence of a [3Fe‐4S] center, either in the native (as isolated) or in the hydrogen‐reduced states. In the native state, the non‐heme iron atoms are arranged as two diamagnetic [4Fe‐4S]2+ centers. Upon reduction, these two centers exhibit distinct and unusual Mössbauer spectroscopic parameters. The centers were found to have similar mid‐point potentials (∼– 315 mV) as determined by oxidation‐reduction titrations followed by EPR.
Dibucaine interaction with phospholipid vesicles
Authors: COUTINHO Ana , COSTA Júlia , FARIA Joaquim L. , BERBERAN‐SANTOS Mário N. , PRIETO Manuel J. E.
Published: March 2005

Resonance energy transfer between a local anaesthetic, dibucaine (donor) and a set of functionalized probes [n‐(9‐anthroloxy)stearic acids, n= 2, 3, 6, 7, 9 and 12 and 16‐(9‐anthroyloxy)palmitic acid] (acceptors) was found to be an efficient process with a critical radius of transfer Ro= 2.1 nm, this interaction being used to locate the drug in a model membrane system, small unilamellar vesicles of dipalmitoylglycerophosphocholine, both above and below the temperature of the gel‐to‐the‐liquid‐crystal transition of the phospholipid.

From the sequence of relative quenching efficiencies of dibucaine fluorescence upon incorporation of the probes, it was concluded that the drug intercalates in the membrane near the glycerol backbone of the lipid. In addition, it was found that dibucaine location is not significantly affected upon crossing the phase‐transition temperature of the phospholipid.

Dibucaine photophysics was also studied and the short lifetime of the neutral form of the anaesthetic with respect to that of the monoprtonated species was attributed to an intramolecular charge‐transfer interaction.

From the study of its partition coefficient between the membrane and the aqueous phase, it was concluded that the only significant species present in the membrane is the charged one.
De novo synthesis of uracil nucleotides in mouse liver and intestine studied using [15N]alanine
Authors: ZAHAREVITZ Daniel W. , ANDERSON Lawrence W. , STRONG John M. , HYMAN Roosevelt , CYSYK Richard L.
Published: March 2005

The amount of newly synthesized uracil nucleotides in mouse liver and intestine was determined by analysis of 15N incorporation into the uracil nucleotide pool of these tissues after intraperitoneal infusion of 15N‐labelled amino acids. The appearance of newly synthesized uracil nucleotides was linear with time, and essentially independent of the rate of infusion of l‐[15N]alanine. Varying the amino acid used in the infusion could affect the enrichment in the uracil ring nitrogens, but had no significant effect on the calculated amount of de novo synthesis. These results demonstrate the utility of this method in measuring de novo uracil nucleotide synthesis in mouse liver and intestine in vivo. The method should be a valuable tool in the effort to understand the regulation and pharmacological manipulation of de novo uracil nucleotide synthesis.
Role of the C‐terminal region in the allosteric properties of Escherichia coli phosphofructokinase‐1
Authors: SERRE Marie‐Claude , GAREL Jean‐Renaud
Published: March 2005

In order to investigate the role of the carboxy‐terminal segment in the catalytic, regulatory, and structural properties of the major allosteric phosphofructokinase (ATP: D‐fructose‐6‐phosphate‐1‐phosphotransferase: EC 2.7.1.11) from Escherichia coli, the corresponding gene has been modified at either of two sites using oligonucleotide‐directed mutagenesis: the codon at position 279 was changed from TAC (Tyr) into TAA (Ochre), and the codon at position 311 from TGG (Trp) into TAG (Amber). The gene mutated at position 279 is not expressed as an active enzyme, probably because a polypeptide chain lacking 41 C‐terminal residues cannot fold and/or assemble under the intracellular conditions. The gene mutated at position 311 is expressed as an active enzyme which has been purified to homogeneity. The fluorescence of this protein shows that it has no tryptophan, which confirms that the last nine residues at the carboxy terminal are missing. This derivative has almost the same specific activity and affinities for the two substrates (fructose 6‐phosphate and ATP) as intact phosphofructokinase; the saturation by fructose 6‐phosphate is also very cooperative. The last nine residues are thus not important for substrate binding, homotropic cooperativity, and catalytic efficiency. The activity of the mutant enzyme is still sensitive to activation by GDP or inhibition by phosphoenolpyruvate, but its affinity for the allosteric effectors is reduced. The carboxy‐terminal segment also appears to contribute to the stability of the interactions between subunits: the mutant protein is less stable than the wild type towards denaturation by heat or guanidinium hydrochloride.
Microscale sequencing of O‐linked oligosaccharides using mild periodate oxidation of alditols, coupling to phospholipid and TLC‐MS analysis of the resulting neoglycolipids
Authors: STOLL Mark S. , HOUNSELL Elizabeth F. , LAWSON Alexander M. , CHAI Wengang , FEIZI Ten
Published: March 2005

In the course of characterising neoglycolipid products derived from mucin oligosaccharide alditols after periodate oxidation and coupling by reductive amination to the aminolipid dipalmitoylglycerophospho‐ethanolamine, we have obtained evidence that oxidative cleavage occurs specifically at the C4–C5 bond of core N‐acetylgalactosaminitol. Two lipid‐linked fragments thus obtained from each oligosaccharide alditol are well resolved on thin‐layer chromatography and can be sensitively analysed by liquid‐secondary‐ion mass spectrometry to assign the sequence and branching patterns of oligosaccharides linked at C6 and C3 to the N‐acetylgalactosamitol. These conclusions have been reached from detailed studies of the neoglycolipid derivatives of several oligosaccharides (di‐ to hexasaccharides) which were isolated from human meconium and characterised previously by MS and NMR studies as the free alditols.
Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis
Authors: CHOMA Christin T. , SUREWICZ Witold K. , CAREY Paul R. , POZSGAY Marianne , RAYNOR Tina , KAPLAN Harvey
Published: March 2005

Trypsin is shown to generate an insecticidal toxin from the 130‐kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD‐73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C‐terminus of the protoxin and proceeding toward the N‐terminal region. At each step, C‐terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67‐kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5‐kDa N‐terminal fragment and a 32.3‐kDa C‐terminal fragment. Similar fragments could be generated by elastase and trypsin. The N‐terminal fragment corresponds to the conserved N‐terminal domain predicted from the gene‐deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C‐terminal fragment is the predicted hypervariable sequence domain. A double‐peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N‐ and C‐terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.
Identification of distinct NAD‐linked hydrogenase protein species in mutants and nickel‐deficient wild‐type cells of Alcaligenes eutrophus H16
Authors: HORNHARDT Sabine , SCHNEIDER Klaus , FRIEDRICH Bärbel , VOGT Beate , SCHLEGEL Hans G.
Published: March 2005

By crossed immunoelectrophoresis with antibodies against the NAD‐linked hydrogenase the presence of three hydrogenase protein species was demonstrated in crude extracts of Alcaligenes eutrophus H16. Protein 1 (antigen 1) exhibited NAD‐reducing activity and was shown to be identical with the native heterotetrameric enzyme. Protein 2 (antigen 2) was catalytically inactive in the antibody‐precipitated form and corresponded to the β subunit (56 kDa) of the holoenzyme. Protein 3 (antigen 3) was serologically distinct from antigen 2 and catalyzed NADH‐oxidizing (diaphorase) activity, suggesting that it either consists of the α peptide or of the α and γ subunits of the diaphorase dimer. Tandem immunoelectrophoresis revealed that antigen 2 was the predominant protein species in cells cultivated under nickel deficiency. Low concentrations of the diaphorase‐active antigen 3 were also detected under these conditions. Extracts from mutants defective in the catalytic activity of NAD‐reducing hydrogenase under these conditions. Extracts from mutants defective in the catalytic activity of NAD‐reducing hydrogenase still contained the four polypeptides. This was shown by immunodiffusion and immunoblotting with antibodies raised against the individual subunits. However, as observed with nickel‐deficient cells, no complete tetrameric protein could be identified, and the dominant subunit species (70–80%) was the β peptide.
Mechanism of stimulation of cyclic‐GMP level in a neuronal cell line mediated by serotonin (5‐HT3) receptors
Authors: REISER Georg
Published: March 2005

The mechanism by which serotonin (5‐HT3) receptors mediate a rise in cyclic‐GMP level was investigated in a neuronal cell line. Inhibitors of phospholipase A2 (mepacrine) and of lipoxygenase (eicosatetraynoic acid or nordihydroguaiaretic acid) suppressed the action of serotonin. On the other hand, inhibition by hemoglobin indicates a role for nitric oxide which could be in part responsible for the cyclic‐GMP effect as an intercellular stimulant. The suppression of the serotonin effect by the arginine analogues Nω‐methyl‐L‐arginine and canavanine is consistent with the notion that nitric oxide could be released from arginine. The serotonin‐induced rise of cyclic‐GMP level depends on the presence of extracellular Ca2+ with half‐maximal stimulation at 0.3 mM Ca2+. The serotonin‐stimulated rise of cyclic GMP was inhibited by (a) addition of inorganic blockers of Ca2+‐permeable channels (La3+, half‐maximal inhibitory concentration (IC50) 0.04 mM; Mn2+, IC50, 0.4 mM; Co2+, IC50, 0.9 mM; Ni2+, IC50, 1.2 mM) and (b) of organic blockers (diltiazem: IC50, 6 μM, methoxyverapamil: IC50, 3 μM and (c) intracellular application of the Ca2+ chelator bis‐(O‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (IC50, 2 μM). Thus, two pathways for the activation of soluble guanylate cyclase by serotonin are possible: (a) via lipoxygenase products of arachidonic acid and/or (b) via nitric oxide or a related nitroso compound. Serotonin mediates a rise of cytosolic Ca2+ activity due to entry of extracellular Ca2+. It still has to be investigated which step depends on a rise of cytosolic Ca2+ activity that appears to be a prerequisite for activation of guanylate cyclase.
Divalent cation biding to reduced and octanoyl acyl‐carrier protein
Authors: TENER David M. , MAYO Kevin H.
Published: March 2005

Mn(II) EPR binding studies with reduced acyl‐carrier protein (ACP‐SH) strongly suggest the presence of two relatively high‐affinity manganese‐binding sites (average Kd/site ∼ 80 μM) at physiological pH. Lowering the pH or titrating with sodium chloride reduces the average number of bound divalent cations and decreases the binding affinity. This is consistent with the idea that anionic ligand(s), e.g. the carboxylate of glutamic or aspartic acid, on the protein are involved in manganese ion coordination. At pH values above 8.0, binding affinity is also reduced, whereas the average number of bound metal ions increases to about five at pH 8.5. By interacting weakly with divalent cations (average Kd/site ∼ 1 mM), octanoyl acyl‐carrier protein (OcoACP) exhibits dramatically different metal‐ion‐binding properties compared to ACP‐SH. Calcium and magnesium can compete in either ACP species for manganese binding. Photochemically‐induced dynamic nuclear polarisation 1H‐NMR experments strongly suggest that ACP‐SH and OcoACP undergo at pH‐induced conformational change between pH 5.5 and pH 7.0, and that divalent cations stabilize the protein against such pH‐induced structural perturbations.
Reactivation of 3‐phosphoglycerate kinase from its unfolded proteolytic fragments
Authors: VAS Mária , SINEV Michael A. , KOTOVA Nina V. , SEMISOTNOV Gennady V.
Published: March 2005

Limited trypsinolysis of pig muscle 3‐phosphoglycerate kinase yielded a nicked enzyme without loss of catalytic activity [Jiang, S. X. & Vas, M. (1988) FEBS Lett. 231, 151–154]. The reactivation rate of the nicked enzyme after denaturation does not differ substantially from the reactivation rate of teh denatured intact enzyme; t1/2 varies between 70–110 s at 25°C, pH 7.0 in both cases. Thus, the absence of a covalent likage between the two proteolytic fragments of the enzyme molecule apparently does not affect the refolding.

The two proteolytic fragments can be separated by FPLC under denaturing conditions. Fluorescence spectra of the isolated fragments may indicate that the tryptic cleavage site is within the N‐terminal domain. Thus, the larger fragment (molecular mass about 30 kDa) probably contains the whole nucleotide‐binding C‐terminal domain plus a small part of the N‐terminal domain.

The inactive isolated fragments were used in renaturation experiments to study the reassembly of active 3‐phosphoglycerate kinase. Kinetic measurements revealed the presence of a bimolecular rate‐limiting step of reactivation. Separate preincubation of the fragments under renaturing conditions did not cause substantial acceleration of reactivation. This implies that assembly of the separate structural units (possibly domains) may limit the reactivation of the intact enzyme.
Structural and catalytic properties of oxidized and reduced chloroplast NADP–malate dehydrogenase upon denaturation and renaturation
Authors: SCHEIBE Renate , RUDOLPH Rainer , RENG Winfried , JAENICKE Rainer
Published: March 2005

Chloroplast NADP‐dependent malate dehydrogenase exists in two interconvertible forms: the inactive disulfide‐containing form and the active dithiol form. No major difference in secondary structure or conformation was found between the oxidized and the reduced enzyme as determined by circular dichroism and intrinsic protein fluorescence. The guanidine/HCl‐dependent unfolding of the enzyme is characterized by two transition midpoints: those of the reduced enzyme are lower by about 0.2 M guanidine/HCl compared to the oxidized enzyme. As shown by analytical ultracentrifugation, there was no effect of guanidine/HCl concentrations up to 0.25 M on the quaternary structure of the enzyme in its oxidized and reduced forms: both sedimentation coefficient (s20,w= 4.9 ± 0.1 S) and sedimentation equilibrium (75 ± 3 kDa) yield the dimer. In the oxidized state the enzyme undergoes guanidine‐dependent dissociation to the monomer with a midpoint of transition at 0.5 M. The kinetics of unfolding were found to be significantly faster for the reduced than for the oxidized enzyme. Renaturation and reactivation of reduced enzyme was more rapid and occurred with higher yields (100%) than for the oxidized enzyme (60–80% yield). Furthermore, the effect of denaturants on catalytic activity, and reductive activation of the oxidized form, were studied. Both increase in protein fluorescence and a stimulatory effect on the activities at low guanidine/HC1 concentrations were observed for the oxidized and the reduced form of the enzyme. Denaturants increase the rate of reductive activation of NADP–malate dehydrogenase.
The H+‐ATPase of the plasma membrane from yeast
Authors: WACH Achim , AHLERS Jan , GRÄBER Peter
Published: March 2005

The H+‐ATPase of the plasma membrane from Saccharmyces cerevisiae has been isolated, purified and reconstituted into asolectin liposomes. The kinetics of ATP hydrolysis have been compared for the H+‐ATPase in the plasma membrane, in a protein/lipid/detergent micelle (isolated enzyme) and in asolectin proteoliposomes (reconstituted enzyme). In all three cases the kinetics of ATP hydrolysis can be described by Michaelis‐Menten kinetics with Km= 0.2 mM MgATP (plasma membranes), Km= 2.4 mM MgATP (isolated enzyme) and Km= 0.2 mM MgATP (reconstituted enzyme). However, the maximal turnover decreases only by a factor of two during isolation of the enzyme and does not change during reconstition; the activation of the H+‐ATPase by free Mg2+ is also only slightly influenced by the detergent. The dissociation constant of the enzyme‐Mg2+ complex Ka, does not alter during isolation and the dissociation constant of the enzyme‐substrate complex, Ks, increases from Ks=30 μM (plasma membranes) to Ks= 90 μM (isolated enzyme). ATP binding to the H+‐ATPase (‘single turnover’ conditions) for the isolated and the reconstituted enzyme resulted in both cases in a second‐order rate constant k1= 2.6 · 104 M−1· s−1. From these observations it is concluded that the detergent used (Zwittergent TM 3‐14) interacts reversibly with the H+‐ATPase and that practically all H+‐ATPase molecules are reconstituted into the liposomes with the ATP‐binding site being directed to the outside of the vesicle.
Calcium‐dependent distance changes in binary and ternary complexes of troponin
Authors: SCHULZKI Horst‐Dieter , KRAMER Bernd , FLEISCHHAUER Jörg , MERCOLA Daniel A. , WOLLMER Axel
Published: March 2005

Calcium‐dependent distance changes have been determined by resonance energy transfer in binary and ternary troponin complexes in order to collect evidence for the structural rearrangements which are part of the hypothetical trigger mechanism of skeletal muscle contraction. Donor and acceptor fluorophores were either intrinisic tryptophans in subunits with a favourable sequence from different species, quasi‐intrinsic Tb3+ ions bound to troponin C or extrinsic labels attached to specific cysteine or methionine residues. All chemically modified subunits proved fully active in conferring calcium sensitivity onto myosin ATPase.

Nine distances were determined between five sites which allowed construction of a three‐dimensional lattice representing the spatial distribution of four sites in the ternary complex of troponin C, I and T. Distances in binary complexes were nearly unaltered upon addition of the third subunit. Regulatory calcium binding caused distance changes of the order of 0.7 – 1.1 nm. In view of the large displacements of the hypothetical mechanism, they turned out to be smaller than anticipated. The fluorophoric sites selected may be localized in a zone of the troponin complex which happens to be relatively little affected by the mechanism. Alternatively, amplification of the moderate changes seen here would require the complete set of thin filament proteins.
Human and murine urokinase cDNAs linked to the murine αA‐crystallin promoter exhibit lens and non‐lens expression in transgenic mice
Authors: MISKIN Ruth , AXELROD Jonathan H. , GRIEP Anne E. , LEE Eric , BELIN Dominique , VASSALLI Jean‐Dominique , WESTPHAL Heiner
Published: March 2005

cDNAs encoding either the human or the murine urokinase‐type plasminogen activator (uPA) were fused downstream from the promoter‐enhancer element of the murine gene encoding αA‐crystallin, a protein found exclusively in the ocular lens. The DNAs were microinjected into fertilized mouse eggs as linear fragments free of bacterial sequences, and for each construct one line of transgenic mice was generated. In both lines transgenic uPA activity was detected in the ocular lens, in agreement with previous results reported on transgenic mice bearing genes fused to the same regulatory region. Unexpectedly however relatively high levels of this activity were found also in the retina, and furthermore, human uPA activity was found also in different parts of the brain and in the bone marrow, and to a lesser extent in the spleen, thymus and optic nerve. Transgenic uPA transcript was found in the lens, retina, brain and thymus of mice carrying the murine cDNA. Such a pattern of expression was different from that exhibited by the endogenous murine uPA gene and, excluding the lens, it appeared to be conferred by the cDNAs. The putative regulation by uPA cDNAs is suggested to be mediated through an internal enhancer‐like element functioning in combination with the αA‐crystallin promoter in a fashion independent of the specific nature of the promoter.
Conformational studies on the N‐linked carbohydrate chain of bromelain
Authors: BOUWSTRA Jan B. , SPOELSTRA Ellen C. , de WAARD Pieter , LEEFLANG Bastiaan R. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G.
Published: March 2005

1H‐ and 13C‐NMR assignments for the carbohydrate part of the glycopeptide α‐d‐Man‐(1→6)‐[ß‐d‐Xyl‐(1→2)]‐ß‐d‐Man‐(1→4)‐ß‐d‐GlcNAc‐(1→4)‐[α‐l‐Fuc‐(1→3)]‐ß‐d‐ GlcNAc‐(1→N)‐Asn∼, derived from the proteolytic enzyme bromelain (EC 3.4.22.4), have been obtained using homo‐ and heteronuclear correlation spectroscopy, two‐dimensional homonuclear Hartmann‐Hahn and nuclear Overhauser enhancement experiments. A conformational model for the carbohydrate chain, deduced from the NMR data and consistent with hard‐sphere exo‐anomeric calculations shows that the rotamer population about the C‐5–C‐6 bond of ß‐Man is restricted to the Pω=180 rotamer, mainly.
Biosynthesis and processing of delta sleep‐inducing peptide‐like precursors in primary cultures of mouse anterior pituitary cells
Authors: BJARTELL Anders , EKMAN Rolf , LOH Y. Peng
Published: March 2005

The biosynthesis and processing of material resembling delta sleep‐inducing peptide (DSIP) have been studied in mouse anterior pituitary primary cell cultures. Cells were pulse/chase incubated with 3H‐labelled amino acids (Gly, Arg or Ala) and cell extracts were immunoprecipitated with DSIP antiserum. Labelled DSIP‐related proteins were resolved by SDS/PAGE. Multiple forms of DSIP‐immunoprecipitable material were observed, including three precursors of molecular mass 50–60 kDa which were processed to two major groups of intermediates of 35–45 kDa and 9–16.5 kDa. These intermediates appear to be processed to a DSIP‐related peptide (molecular mass < 3 kDa), which co‐ran on reversed‐phase HPLC with an endogenous form of DSIP in mouse anterior pituitary, but not with rabbit DSIP. This < 3‐kDa peptide incorporated [3H]Gly, but not [3H]Arg or [3H]Ala. In addition, it incorporated [3H]glucosamine, indicating that it was a glycopeptide. Secretion studies showed release of the < 3‐kDa DSIP‐like glycopeptide and the 9–16.5‐kDa group of intermediates into the medium. The present study demonstrates the biosynthesis of a small DSIP‐like glycopeptide in mouse anterior pituitary cells, which is not identical with, but has similarities to, rabbit DSIP.
Immunosuppressive activity of bovine seminal RNase on T‐cell proliferation
Authors: TAMBURRINI Maurizio , SCALA Giuseppe , VERDE Cinzia , RUOCCO Maria Rosaria , PARENTE Augusto , VENUTA Salvatore , D'ALESSIO Giuseppe
Published: March 2005

The interest in the immunosuppressive activity of mammalian seminal plasma depends largely on its putative role in the immunoregulation of both the male and female genital systems. We report here that the immunosuppressive action of bovine seminal plasma is based on the presence in this fluid of copious amounts of an immunosuppressive RNase, bovine seminal RNase. Studies of structure‐function relationships have revealed that the immunosuppressive activity of seminal RNase depends on the integrity of the dimeric structure of the enzyme, as well as on the integrity of its catalytic function. While bovine seminal RNase has no effect on the secretion of interleukin‐2 by T‐cell cultures, the enzyme has been found to decrease drastically the expression of the α‐chain of the interleukin‐2 receptor on the T‐cell membrane.
Differential biogenesis of photosystem‐II in mesophyll and bundle‐sheath cells of ‘malic’ enzyme NADP+‐type C4 plants
Authors: OSWALD Angela , STREUBEL Monika , LJUNGBERG Ulf , HERMANS Jürgen , ESKINS K. , WESTHOFF Peter
Published: March 2005

We have investigated the photosystem‐II organization in differentiating‐bundle‐sheath cells of the three malate dehydrogenase (oxaloacetate decarboxylating) (NADP+)‐type C4 species maize, Sorghum and Pennisetum. Using a set of nine different antisera raised against individual subunits of photosystem‐II, we demonstrate that photosystem‐II components constitute a substantial part of the thylakoid membranes of young bundle‐sheath chloroplasts. The abundance of subunits of the photosystem‐II core, i.e. the 47‐and 43‐kDa chlorophyll‐a‐binding proteins, polypeptides D1 and D2, cytochrome b559, and the 34‐kDa polypeptide, varies with the developmental state of the plant. However, the levels of the 23‐kDa, 16‐kDa and 10‐kDa extrinsic polypeptides of the water‐oxidation complex are drastically reduced in bundle‐sheath chloroplasts of all three species analyzed, regardless of their state of differentiation. The reduction in protein abundance is also reflected at the transcript level: only traces of the nuclear‐encoded mRNAs are found in differentiating bundle‐sheath cells of Sorghum, suggesting that the transcription of these genes has been switched off. Our data are compatible with the idea that the water‐oxidation complex is a prime site for initiating or maintaining the process leading to photosystem‐II depletion during differentiation of bundle‐sheath cells.
Form determination of the heads of bacteriophages
Authors: KELLENBERGER Edward
Published: March 2005

The shape of the DNA‐containing heads of many bacteriophages is not only determined by the properties of the protein subunits which build the shell (capsid) but also by the scaffolding core which is a transient structure of the prohead. The form‐determining properties of the scaffolding proteins have been characterized by genetic methods based on conditional mutants and site‐directed mutagenesis. The mechanism of form determination has been studied by in vitro assembly experiments. The theoretical background is discussed and different models for mechanisms of form determination are considered. Definitive decisions about the validity of a model is still limited by the difficulty of obtaining unambiguous answers on the stoichiometry and the fine structure of the scaffold because of their high instability.
Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method
Authors: HUMBERT Peter , NIROOMAND Feraydoon , FISCHER Gabriele , MAYER Bernd , KOESLING Doris , HINSCH Klaus‐Dieter , GAUSEPOHL Heinrich , FRANK Rainer , SCHULTZ Günter , BÖHME Eycke
Published: March 2005

Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C‐terminus of the 70‐kDa subunit of the enzyme. After anion‐exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparenet molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10–20 nmol · min−1· mg−1 and 80–100 nmol · min−1· mg−1, respectively. The enzyme exhibited an ultraviolet‐visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO‐containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2–2.4 μmol · min−1· mg−1 (half‐maximal effect of sodium nitroprusside at 1.3–1.9 μM) and 0.9–1.8 μmol · min−1· mg−1 (half‐maximal effect at 0.28–0.41 μM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large‐scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large‐scale purification of various proteins.
Renaturation of protein phosphatase 1 expressed at high levels in insect cells using a baculovirus vector
Authors: BERNDT Norbert , COHEN Patricia T. W.
Published: March 2005

The catalytic subunit of protein phosphatase 1 (PP1), a key enzyme in the regulation of many cellular functions, has been expressed in insect cells using a baculovirus vector containing PP1α cDNA. The expressed protein had the same apparent molecular mass as PP1 from rabbit skeletal muscle and comprised up to 25% of the total cellular protein. About 5% of expressed PP1α was present as a soluble active species, representing a 15‐fold increase over the endogenous activity. Insoluble protein, comprising about 95% of the expressed PP1 was dissolved in 6 M guanidinium chloride and could be fully reactivated by extensive and rapid dilution with buffers containing Mn2+. By a number of criteria (specific activity towards phosphorylase, interaction with inhibitor‐1, inhibitor‐2 and okadaic acid), this reactivated species was indistinguishable from authentic PP1, and could be concentrated and purified to homogeneity by a single chromatography on DEAE‐Sepharose. This procedure yielded about 10 mg active PP1/l culture, which will facilitate future structural analyses of native and mutant protein phosphatases.
Isolation and sequence of the canine pancreatic phospholipase A2 gene
Authors: KERFELEC Brigitte , LaFORGE K. Steven , VASILOUDES Panayiotis , PUIGSERVER Antoine , SCHEELE George A.
Published: March 2005

A genomic library has been constructed in EMBL3 λ phage using high molecular mass DNA isolated from canine spleen. A cDNA clone, shown to code for preprophospholipase A2 which is processed to the prosecretory form prior to release from secretory cells, was used to identify a λ clone which contains the complete phospholipase A2 gene. Restriction enzyme and DNA sequence analysis indicate that the primary transcriptional unit for the phospholipase gene, ∼ 9.0 kb, is organized into four exon sequences. Exon 1 encodes the 5′ nontranslated sequence, the ATG initiation codon, and the hydrophobic core of the signal peptide. Exons 2–4 encode regions of the peptide of residues −11 to 43, 43 to 86 and 86 to 124, respectively. The 5′ flanking region shows a TATA box at position −29 and multiple CAAT boxes at positions −279, −206, −183 and −159. Regions of the 5′ flanking sequence in the canine sequence, from nucleotides −47 to −74 and −91 to −129, show high similarity to similar regions in the human gene. However, an analysis of 400 nucleotides of the 5′ flanking sequence in transient expression studies was unable to identify tissue‐specific promoter or enhancer sequences. Within 5′ nontranslated regions the canine and human genes share a pyrimidine‐rich sequence which may be involved in differential regulation of mRNA translation.
Fine tuning of the catalytic properties of carbonic anhydrase
Authors: BEHRAVAN Gity , JONSSON Bengt‐Harald , LINDSKOG Sven
Published: March 2005

The active sites of carbonic anhydrases I contain a unique histidine residue at sequence position 200. To test the hypothesis that His200 is essential for the isoenzyme‐specific catalytic and inhibitor‐binding properties of carbonic anhydrases I, a variant of human carbonic anhydrase II, having His200 for Thr200, was prepared by oligonucleotide‐directed mutagenesis. The variant has a circular dichroic spectrum that is indistinguishable from that of the parent enzyme. The kinetics of CO2 hydration and HCO−3 dehydration has been investigated. The results show that the amino acid substitution has led to changes of catalytic parameters as well as Ki values for anion inhibition in the expected directions towards the values for isoenzyme I. However, the maximal 4‐nitrophenyl acetate hydrolase activity of the variant is higher than for any naturally occurring carbonic anhydrase studied so far.

A detailed analysis of the kinetic observations suggests that the modification has resulted in a change of the step that limits the maximal rate of CO2 hydration at saturating buffer concentrations. This rate‐limiting step is an intramolecular proton transfer in unmodified isoenzyme II and, presumably, HCO−3 dissociation in the variant and in human isoenzyme I. A free‐energy profile for the dominating pathway of CO2 hydration at high pH was constructed. The results suggest that the major effect of His200 is a stabilization of the enzyme–HCO−3 complex by about 7.5 kJ/mol (variant) and 6.1 kJ/mol (human isoenzyme I) relative to unmodified isoenzyme II, while proton transfer between the metal site and the reaction medium is only marginally affected by the amino acid replacement.
Architecture of bacterial lipid A in solution
Authors: LABISCHINSKI Harald , VORGEL Eberhard , UEBACH Willi , MAY Roland Peter , BRADACZEK Hans
Published: March 2005

The phase structure of isolated bacterial lipid A, the lipid anchor of the lipopolysaccharides of the outer membrane of Gram‐negative bacteria, has been investigated by neutron small‐angle scattering. The shape of the scattering curves obtained at different H2O/2H2O ratios revealed a lamellar organisation of the lipid A at neutral pH both above and below its main phase temperature (approximately 40–45°C). Analysis of the scattering curves and interpretation of the corresponding thickness distance distribution functions of the lamellar aggregates led to a model in which the lipid A molecules form a bilayer of about 5 nm in thickness. This value for the thickness of the bilayer, as well as the neutron‐scattering density profile across the bilayer, can be explained by a molecular model which shows interdigitation of the fatty acid chains of the lipid A.
pH‐Induced insertion of the amphiphilic α‐helical anchor of Escherichia coli penicillin‐binding protein 5
Authors: PHOENIX David A. , PRATT Julie M.
Published: March 2005

By treating vesicles prepared from Escherichia coli K12 with various reagents, we have investigated the mechanism by which penicillin‐binding protein 5 anchors to the inner membrane. The results indicate that there are two forms of anchoring; one which is inaccessible to urea and probably inserted into the bilayer and one which is accessible. Association of the accessible form with the membrane seems to involve significant hydrophobic interaction and this form is triggered to undergo reversible ‘insertion’ by a decrease in pH.
Structure of human myeloma IgG3 Kuc
Authors: RYAZANTSEV Sergey , TISHCHENKO Vladimir , VASILIEV Victor , ZAV'YALOV Vladimir , ABRAMOV Vyacheslav
Published: March 2005

An electron microscopy study of human myeloma IgG3 Kuc has shown that the hinge region in an intact molecule is in a compact state. The subunits are not fixed rigidly and are very mobile. These data are supported by results of ultracentrifugation and microcalorimetry.

Non‐extremal denaturating effects (pH 4.0, 20°C or pH 7.8, 65°C) lead to ‘unfolding’ of the hinge region which has a rod‐like shape in electron micrographs.
Effects of nutrients and hormones on transcriptional and post‐transcriptional regulation of fatty acid synthase in rat liver
Authors: KATSURADA Akihiko , IRITANI Nobuko , FUKUDA Hitomi , MATSUMURA Yohko , NISHIMOTO Naomi , NOGUCHI Tamio , TANAKA Takehiko
Published: March 2005

The effects of nutrients and hormones on transcriptional and post‐transcriptional regulation of fatty acid synthase in rat liver were investigated following cDNA cloning. When fasted rats were fed a carbohydrate/protein diet, the transcriptional rate was greatly increased even in 1 h. The transcriptional rate, mRNA concentration and enzyme induction reached maximum levels in 4 h, 8–16 h and 48 h, respectively. Although dietary carbohydrate increased each level more than protein did, both carbohydrate and protein were required to reach a high level. Corn oil feeding markedly decreased the transcriptional rate. In diabetic rats, the transcriptional rate, mRNA concentration and enzyme induction were very low in comparison with the normal. By treating the diabetic rats with insulin, however, the transcriptional rate was increased 5‐fold in 1 h and 15‐fold in 6 h, preceding a great increase in the mRNA and enzyme levels. On the other hand, fructose feeding or triiodothyronine treatment of diabetic rats abundantly increased the mRNA concentration and somewhat increased the transcriptional rate. Thus, it is suggested that insulin mainly stimulates the transcription of the fatty acid synthase gene, whereas triiodothyronine and fructose mainly increase the mRNA stability.
Potent inhibitory effects of the 5′‐triphosphates of (E)‐5‐(2‐bromovinyl)‐2′‐deoxyuridine and (E)‐5‐(2‐bromovinyl)‐1‐β‐D‐arabinofuranosyluracil on DNA polymerase γ
Authors: ONO Katsuhiko , NAKANE Hideo , De CLERCQ Erik
Published: March 2005

(E)‐5‐(2‐Bromovinyl)‐2′‐deoxyuridine 5′‐triphosphate (BrVdUTP) and (E)‐5‐(2‐bromovinyl)‐1‐β‐D‐arabinofuranosyluracil 5′‐triphosphate (BrVarafUTP), which are known as specific inhibitors of herpes simplex viral (type 1 and 2) DNA polymerase, were found to be strong inhibitors of DNA polymerase γ from human KB and murine myeloma cells. In fact BrVdUTP and BrVarafUTP were found to be stronger inhibitors of DNA polymerase γ than of other DNA polymerases having viral (herpes simplex virus or retrovirus) origin or cellular (eukaryotic α and β, or prokaryotic) origin. The mode of inhibition of DNA polymerase γ by BrVdUTP and BrVarafUTP was competitive with respect to dTTP, the normal substrate. Whereas BrVdUTP was an efficient substrate for DNA polymerase γ and other DNA polymerases that were examined, BrVarafUTP failed to serve as a substrate for DNA synthesis. Ki values for BrVdUTP (40 nM) and BrVarafUTP (7 nM) with DNA polymerase γ, as determined with (rA)n· (dT)12–18 as the template · primer, were much smaller than the Km values for dTTP (0.16 μM and 0.71 μM for murine and human DNA polymerase γ, respectively). Thus, the affinity of BrVdUTP or BrVarafUTP for DNA polymerase γ was much stronger than that of dTTP.
Differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular DNA and RNA polymerases
Authors: ONO Katsuhiko , NAKANE Hideo , FUKUSHIMA Masanori , CHERMANN Jean‐Claude , BARRÉ‐SINOUSSI Françoise
Published: March 2005

Four flavonoids, 5,6,7‐trihydroxyflavone (baicalein), 3,3′,4′,5,7‐pentahydroxyflavone (quercetin), 3,3′,4′,5,6,7‐hexahydroxyflavone (quercetagetin) and 3,3′,4′,5,5′,7‐hexahydroxyflavone (myricetin), were found to be potent inhibitors of reverse transcriptases from Rauscher murine leukemia virus (RLV) and human immunodeficiency virus (HIV). Under the reaction conditions employed, any one of these flavonoids almost completely inhibited the activity of RLV reverse transcriptase at a concentration of 1 μg/ml. HIV reverse transcriptase was inhibited by 100%, 100%, 90% and 70% in the presence of 2 μg/ml quercetin, myricetin, quercetagetin and baicalein, respectively. The mode of inhibition of these flavonoids was competitive (RLV reverse transcriptase) or partially competitive (HIV reverse transcriptase) with respect to the template primer complex, (rA)n· (dT)12–18, and noncompetitive with respect to the triphosphate substrate, dTTP. The Ki values for RLV reverse transcriptase were found to be 0.37 μM and 0.08 μM for baicalein and quercetin, respectively and those for HIV reverse transcriptase were 2.52 μM, 0.52 μM, 0.46 μM and 0.08 μM for baicalein, quercetin, quercetagetin and myricetin, respectively. Comparative studies with other flavonoids (hydroxyflavones, dihydroxyflavones and polyhydroxyflavones and flavanones) carried out to clarify the structure/activity relationships, revealed that the presence of both the unsaturated double bond between positions 2 and 3 of the flavonoid pyrone ring, and the three hydroxyl groups introduced on positions 5, 6 and 7, (i.e. baicalein) were a prerequisite for the inhibition of reverse transcriptase activity. Removal of the 6‐hydroxyl group of baicalein required the introduction of three additional hydroxyl groups at positions 3, 3′ and 4′ (quercetin), to afford a compound still capable of inhibiting the reverse transcriptase activity. Quercetagetin which contains the structures of both baicalein and quercetin, and myricetin which has the structure of quercetin with an additional hydroxyl group on the 5′ position also proved strong inhibitors of reverse transcriptase activity. The inhibition by baicalein of reverse transcriptase is highly specific, whereas quercetin and quercetagetin were also strong inhibitors of DNA polymerase β and DNA polymerase I, respectively. Myricetin was also a potent inhibitor of both DNA polymerase α and DNA polymerase I.
Characterization of the human cyclophilin gene and of related processed pseudogenes
Authors: HAENDLER Bernard , HOFER Erhard
Published: March 2005

The human cyclophilin gene was isolated from a genomic library derived from leucocyte DNA and sequenced. The gene contains five exons and four introns. The amino acid sequence deduced from the exons matches perfectly the one previously determined from the T‐cell cyclophilin cDNA. A TATA box is visible in the promoter region and putative Sp1 binding sites are also found there as well as in the first intron. Six members of the middle repetitive Alu gene family are present in one or other orientation in the non‐coding regions of the cyclophilin gene. Hybridisation of genomic DNA to probes derived from the promoter region or the first intron indicates that the cyclophilin gene is present as a single copy in the human haploid genome. Seven other cyclophilin‐related DNA clones isolated from the same library were also characterized. They show a high degree of similarity to the cyclophilin cDNA and are colinear to it. However, multiple genetic lesions, often including deletion and/or insertion events which modify the reading frame, are found in these clones which are therefore likely to represent processed pseudogenes.
Thermodynamic parameters for binding of fatty acids to human serum albumin
Authors: PEDERSEN Anders Overgaard , HONORÉ Bent , BRODERSEN Rolf
Published: March 2005

Binding of laurate and myristate anions to human serum albumin has been studied over a range of temperatures, 5–37°C, at pH 7.4. The binding curves indicate that the strength of binding of the first few molecules of fatty acid to albumin (r < 5) decreases with increasing temperature, whereas binding of the following molecules seems to proceed independently of temperature.

Binding data were analyzed according to the general binding equation yielding several sets of acceptable binding constants within a probability limit of 0.75. From the temperature dependence of the first step constant, it was possible to calculate values for the changes in enthalpy and entropy during the initial binding step. For the medium‐chain fatty acids, laurate and myristate, binding of the first molecule to albumin appeared to be enthalpic, with a tendency to an increasing contribution of entropy to binding energy with increasing chain length of the fatty acid.
Analysis of the regulatory region of the Escherichia coli nupG gene, encoding a nucleoside‐transport protein
Authors: MUNCH‐PETERSEN Agnete , JENSEN Nina
Published: March 2005

The S1‐mapping procedure, when applied to an 800‐bp fragment upstream of the nupG structural gene of Escherichia coli revealed one transcription‐initiation site. The corresponding promoter is negatively regulated by the cytR and the deoR repressors. The expression of the gene is activated by the complex between cAMP and its receptor protein. In strains lacking cAMP the promoter expression is reduced and it is no longer regulated by the cytR repressor, whereas the deoR regulation is retained.
Sites phosphorylated in bovine cardiac troponin T and I
Authors: SWIDEREK Kristine , JAQUET Kornelia , MEYER Helmut E. , SCHÄCHTELE Christoph , HOFMANN Franz , HEILMEYER Ludwig M. G.
Published: March 2005

Bovine cardiac troponin isolated in a highly phosphorylated form shows four 31P‐NMR signals [Beier, N., Jaquet, K., Schnackerz, K. & Heilmeyer, L. M. G. Jr (1988) Eur. J. Biochem. 176, 327–334]. Troponin I, which contains phosphate covalently linked to serine‐23 and/or‐24 [Swiderek, K., Jaquet, K., Meyer, H. E. & Heilmeyer, L. M. G. Jr (1988) Eur. J. Biochem. 176, 335–342], shows three resonances. Mg2+‐saturation of holotroponin shifts these troponin I resonances to higher fields. Direct binding of Mg2+ to the phosphate groups can be excluded. Both these serine residues of troponin I, 23 and 24, are substrates for cAMP‐ and cGMP‐dependent protein kinases as well as for protein kinase C.

Isolated bovine cardiac troponin T contains 1.5 mol phosphoserine/mol protein, indicating that minimally two serine residues are phosphorylated. One phosphoserine residue is located at the N‐terminus. An additional phosphoserine is located in the C‐terminal cyanogen bromide fragment, CN4, which contains covalently bound phosphate. Protein kinase C phosphorylates serine‐194, thus demonstrating exposure of this residue on the surface of holotroponin.