bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel inhibitors and substrates of bilirubin: UDP‐glucuronosyltransferase Arylalkylcarboxylic acids
Authors: FOURNEL‐GIGLEUX Sylvie , SHEPHERD Steven R. P. , CARRE Marie‐Christiane , BURCHELL Brian , SIEST Gérard , CAUBERE Paul
Published: March 2005

The in vitro inhibitory potency of 20 structurally related alkanoic and arylalkanoic acids has been investigated on rat liver UDP‐glucuronosyltransferase. These compounds were tested on the microsomal and purified enzyme, and a cloned cDNA expressed in COS 7 cell cultures. Among all the acids tested, 7,7,7‐triphenylheptanoic acid was the most powerful inhibitor of bilirubin: UDP‐glucuronosyltransferase with a lower effect on 1‐naphtol, androsterone and testosterone glucuronidation. The inhibition was competitive towards the microsomal and purified bilirubin: UDP‐glucuronosyltransferases with Kiapp values of 12.0 μM and 1.6μM, respectively. Twenty analogues were examined, and the results showed that their inhibitory potency on bilirubin: UDP‐glucuronosyltransferase activity was a function of at least three structural features (a) the presence of a hydrophobic triphenyl moiety; (b) the length of the aliphatic chain and (c) the presence of a carboxylic group.

These inhibitors were also tested as possible substrates of UDP‐glucuronosyltransferases. The strongest inhibitors were poor substrates of rat liver microsomal UDP‐glucuronosyltransferases. However, 7,7,7‐triphenylheptanoic acid was actively glucuronidated by purified bilirubin: UDP‐glucuronosyltransferase, in contrast to its analogues with decreasing alkyl chain length. In addition, glucuronidation of this molecule was enhanced by clofibrate treatment but could not be detected in Gunn rats, which are deficient in bilirubin: UDP‐glucuronosyltransferase, further indicating that the glucuronidation of this compound was catalysed by bilirubin: UDP‐glucuronosyltransferase.

The results suggest that 7,7,7‐triphenylheptanoic acid may be a useful structural probe to investigate the molecular basis of glucuronidation of bilirubin and carboxylic acids.
Pairs of cyclic AMP analogs, that are specifically synergistic for type I and type II cAMP‐dependent protein kinases, mimic thyrotropin effects on the function, differentiation expression and mitogenesis of dog thyroid cells
Authors: VAN SANDE Jacqueline , LEFORT Anne , BEEBE Stephen , ROGER Pierre , PERRET Jason , CORBIN Jackie , DUMONT Jacques Emile
Published: March 2005

The role of the two different isozymes of the cAMP‐dependent protein kinase is still unclear. We have investigated the potential roles for each isozyme in dog thyroid cells, a model in which the function, expression of differentiation and proliferation are positively regulated by thyrotropin acting through cyclic AMP. The dog thyroid contains both type I and type II cAMP‐dependent protein kinases. These isozymes were selectively activated in vitro by type‐I‐directed and type‐II‐directed analog pairs. In thyroid slices, both type‐I directed and type II‐directed analog pairs synergistically activated thyroid hormone synthesis, as measured by incorporation of 131I into proteins and thyroid hormone secretion as determined by the release of butanol‐extractable 131I. In primary cultures of dog thyroid cells both isozyme‐directed analog pairs synergistically enhanced iodide trapping, a marker of differentiation, and DNA synthesis, as measured by the percentage of cells incorporating [3H]thymidine into their nuclei. However, DNA synthesis was more sensitive to type‐I‐directed pairs. The results demonstrate that both cAMP‐dependent protein kinase isozymes can mediate the action of cAMP on function, differentiation expression and cell proliferation in dog thyroid cells.
Intracellular Ca2+ potentiates Na+/H+ exchange and cell differentiation induced by phorbol ester in U937 cells
Authors: ALVAREZ Javier , GARCÍA‐SANCHO Javier , MOLLINEDO Faustino , SANCHEZ Ana
Published: March 2005

The human cell line U937 differentiates to monocyte macrophage‐like cells in response to tumour‐promoting phorbol esters. This effect is attributed to activation of protein kinase C. We show here that U937 cell differentiation induced by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) is associated with cytoplasmic alkalinization. Ethyl‐isopropyl‐amiloride (EIPA), a potent inhibitor of Na+/H+ exchange, blocked both cytoplasmic alkalinization and cell differentiation. Cell acidification by addition of 2–4 mM sodium propionate also blocked TPA‐induced U937 cell differentiation. These results suggest that a sustained cell alkalinization mediated by activation of Na+/H+ exchange is essential for TPA‐induced differentiation in U937 cells. The increase of cytoplasmic free calcium concentration ([Ca2+]i) by addition of the calcium ionophore ionomycin enhanced TPA‐induced alkalinization by increasing the apparent affinity of the Na+/H+ antiporter for intracellular H+. Treatment with ionomycin also potentiated differentiation of U937 cells induced by TPA. This synergism suggests that [Ca2+]i either potentiates the activation of protein kinase C or triggers additional transducing mechanisms. The key events of this interaction occur during the first 30 min of treatment, even though cell differentiation manifests much later.
Complete amino acid sequence of a new murine T‐cell growth factor P40
Authors: SIMPSON Richard J. , MORITZ Robert L. , RUBIRA Michael R. , GORMAN Jeffrey J. , VAN SNICK Jacques
Published: March 2005

A new murine T‐cell growth factor, designated P40, which supports growth of helper T‐cells in the absence of interleukin‐2, interleukin‐4 and antigen has been isolated from helper T‐cell lines in sufficient quantities (100 μg) to permit its complete amino acid sequence determination. This was achieved by a combination of sensitive peptide mapping using microbore reversed‐phase high performance liquid chromatography and automated microsequence analysis. Attempts to obtain N‐terminal sequence data on P40 were unsuccessful due to N‐terminal blockage of the native molecule. The nature of this N‐terminal blocking was established using a combination of amino acid analysis, fast‐atom‐bombardment mass spectrometry and peptide synthesis. The P40 molecule, a single polypeptide chain comprising 126 amino acid residues, is structurally distinct from other known T‐cell growth factors. No similarity was revealed when the amino acid sequence of P40 was compared with other proteins whose biochemical structure is known. The protein sequence data reported here predict four N‐linked glycosylation sites in the P40 molecule.
Thermodynamics and the structure of biological macromolecules
Authors: EISENBERG Henryk
Published: March 2005

In this review, I will discuss the role of thermodynamics in both the determination and evaluation of the structure of biological macromolecules. The presentation relates to the historical context, state‐of‐the‐art and projection into the future. Fundamental features relate to the effect of charge, exemplified in the study of synthetic and natural polyelectrolytes. Hydrogen bonding and water structure constitute basic aspects of the medium in which biological reactions occur. Viscosity is a classical tool to determine the shape and size of biological macromolecules. The thermodynamic analysis of multicomponent systems is essential fo the correct understanding of the behavior of biological macromolecules in solution and for the evaluation of results from powerful experimental techniques such as ultracentrifugation, light, X‐ray and neutron scattering. The hydration, shape and flexibility of DNA have been studied, as well as structural transitions in nucleosomes and chromatin. A particularly rewarding field of activity is the study of unusual structural features of enzymes isolated from the extreme halophilic bacteria of the Dead Sea, which have adapted to saturated concentrations of salt. Future studies in various laboratories will concentrate on nucleic‐acid–protein interactions and on the so‐called ‘crowding effect’, distinguishing the behavior in bacteria, or other cells, from simple test‐tube experiments.
Identification and characterization of the major chicken bone phosphoprotein
Authors: GOTOH Yozo , GERSTENFELD Louis C. , GLIMCHER Melvin J.
Published: March 2005

The major phosphoprotein synthesized by cultured chicken embryo osteoblasts had a molecular mass of approximately 66 kDa. The 32P label on the protein was cleaved by acid phosphatase treatment and O‐[32P]phosphoserine and O‐[32P]phosphothreonine could be identified after partial acid hydrolysis. The phosphoprotein contributed approximately 2.0% of the total protein synthesized by osteoblasts and was shown to be secreted, as shown by its presence in the culture media. Glycosylation was demonstrated by the fact that it could be labelled with [3H]galactosamine. The major ∼ 66‐kDa phosphoprotein was resolved by isoelectric focusing into three major variants with pI values ranging over 3.7–3.9; all three forms appear to be the result of variation in the extent of protein phosphorylation. An identical ∼ 66‐kDa phosphoprotein could be extracted from chicken bones which had both the same range of pI values and an identical elution position following DEAE‐Sephacel chromatography. Analysis of the protein isolated from bone demonstrated the presence of sialic acid and, while amino‐terminal sequence analysis and internal tryptic fragment sequence analysis of about 25% of the protein revealed little similarity to the rat phosphoprotein osteopontin, a conserved nine‐residue sequence spanning the Arg‐Gly‐Asp cell‐binding site of the rat protein osteopontin, was identified in the ∼ 66‐kDa chicken protein. Peptide mapping with Staphylococcus aureus V8 protease of the in vivo protein compared to the in vitro synthesized protein demonstrated identical peptide fingerprints. The two proteins also had comparable amino acid compositions.

Several smaller‐molecular‐mass phosphoproteins ranging in size over about 55–29 kDa were also observed in the HC1 extracts of bone. Peptide mapping of these species demonstrated that the ∼ 66‐kDa, ∼ 55‐kDa, and ∼ 45‐kDa species had a common core of peptide fragments. Pulse/chase experiments in culture revealed no evidence for a defined pathway of intracellular proteolysis associated with the ∼ 66‐kDa species since this phosphoprotein remained the prevalent species after a 24‐h chase. Because of the predominant association of all the smaller‐molecular‐mass forms with the cell layer and an absence of a quantitative conversion to any of the smaller forms over a 24‐h chase, these results suggested that the lower‐molecular‐mass species were not the result of proteolytic processing during synthesis or secretion, but rather represent proteolysis of the ∼ 66‐kDa component in the extracellular matrix. In summary, these results demonstrate that cultured osteoblasts synthesize a major ∼ 66‐kDa phosphoprotein that is identical to the major HCl‐extractable phosphoprotein present in postnatal chick bone.
Description of enzyme kinetics in reversed micelles
Authors: VERHAERT Raymond M. D. , HILHORST Riet , VERMUË Marian , SCHAAFSMA Tjeerd J. , VEEGER Cees
Published: March 2005

In the literature measurements of kinetic data of enzymes in reversed micelles have been interpreted in two ways. In the first, all enzyme parameters are expressed with respect to the total volume of the reversed micellar solution. In the second, the enzymatic conversion is related only to the fraction of the volume consisting of aqueous solution (pseudophase model). In this paper equations are derived describing the rate of an enzymatic reaction for three different kinds of enzymes: enzymes obeying Michaelis‐Menten kinetics, enzymes following a ping‐pong bi‐bi mechanism and enzymes which convert substrates according to an ordered mechanism. In deriving these equations, a distinction is made between intermicellar exchange reactions of substrate(s) and product(s) and the enzymatic reaction which takes place in the waterpool of a reversed micelle. In the description, all intrinsic rate constants of the enzyme are assumed to be independent of its environment. The rate equations show that the presence and efficiency of the intermicellar exchange reaction, which supplies the enzyme with substrate and removes product, can affect the rate of an enzymatic reaction under common experimental conditions.

Whereas kinetic parameters derived from double‐reciprocal plots often seem to be affected by enclosure in reversed micelles, these apparent deviations from kinetics in aqueous media can be explained by the model presented here as arising from exchange phenomena. Neither the experimentally determined maximum enzyme velocity, vmax, nor the Michaelis constants are affected by the incorporation of the enzyme in reversed micelles. The deviations of kinetic parameters from the aqueous values are shown to depend strongly on the concentration of reversed micelles, the intermicellar exchange rate and the volume fraction of water, a dependence in agreement with findings reported in the literature.
Ribofuranosyl‐benzimidazole derivatives as inhibitors of casein kinase‐2 and casein kinase‐1
Authors: MEGGIO Flavio , SHUGAR David , PINNA Lorenzo A.
Published: March 2005

5,6‐Dichloro‐1‐(β‐d‐ribofuranosyl)benzimidazole (DiCl‐RB) is a powerful inhibitor of casein kinase‐2 (CK‐2) [Zandomeni, R. et al. (1986) J. Biol. Chem. 261, 3414–3420]. Here a series of 17 analogues of DiCl‐RB has been employed for studying the specificity and the mode of action of this family of CK‐2 inhibitors. The two halogen substituents on the benzene ring are shown to play a prominent role in inhibition, the 5,6‐dibromo derivative (DiBr‐RB) being fivefold more effective than DiCl‐RB (Ki= 2 μ M cf. 10 μ M, with GTP as substrate), whereas the difluoro derivative (DiF‐RB) is nearly as ineffective as unsubstituted 1‐(β‐d‐ribofuranosyl)benzimidazole. On the other hand, although some modifications of the ribose group significantly decrease the inhibitory efficiency, the sugar moiety is not strictly required, since dichlorobenzimidazole itself (DiCl‐Bz) is an inhibitor almost as good as DiCl‐RB. Inhibition of CK‐2 by DiCl‐RB and by its analogues, DiCl‐Bz included, is of the competitive type with respect to the nucleotide substrate, the Ki values being lower with GTP than with ATP. The Ki values of the most potent inhibitor, DiBr‐RB, with ATP and GTP, are 6 μ M and 2 μ M, respectively, denoting an affinity for the enzyme higher than that of the physiological substrates, ATP and GTP.

DiBr‐RB has been assayed for its inhibitory capacity toward several protein kinase other than CK‐2. Protein kinase‐C, cAMP‐dependent protein kinase, the Ser/Thr protein kinase expressed by Pseudorabies virus, and four different tyrosine protein kinases from spleen, proved insensitive to DiBr‐RB concentrations capable of almost entirely suppressing the activity of rat liver and maize seedling CK‐2. Casein kinase‐1 however is nearly as sensitive as CK‐2 to DiBr‐RB. Inhibition of CK‐1 is also of the competitive type with respect to ATP (Ki= 14 μ M). Although the inhibitory spectrum of CK‐1 by the various analogues is reminiscent of that observed with CK‐2, a remarkable difference is revealed by 5′‐phosphorylation of ribose which increases the Ki with CK‐2 while decreasing that with CK‐1.
Interaction in situ of the cytoskeletal protein vinculin with bilayers studied by introducing a photoactivatable fatty acid into living chicken embryo fibroblasts
Authors: NIGGLI Verena , SOMMER Lukas , BRUNNER Josef , BURGER Max M.
Published: March 2005

The cytoskeletal protein vinculin, a putative actin–plasma‐membrane linker, has been shown by hydrophobic photo‐labeling to interact in vitro directly with bilayers of acidic phospholipids [Niggli et al. (1986) J. Biol. Chem. 261, 6912–6918]. In order to demonstrate that such an interaction occurs also in intact cells, chicken embryo fibroblasts were incubated for 2 h with a 3H‐labeled photoactivatable fatty acid, 11‐{4‐[3‐(trifluoromethyl)‐diazirinyl]phenyl}‐[2‐3H]undecanoic acid. This resulted in biosynthetic incorporation into cellular lipids of a fraction of the fatty acid added. Following photolysis, vinculin was immunoprecipitated from different subcellular fractions using a specific polyclonal anti‐vinculin antibody. The protein was recovered from both the cytosolic and the crude membrane fraction. Vinculin from both fractions incorporated label, but the membrane‐associated population was at least eight times more strongly photolabeled than the cytosolic protein. Moreover, photolysis increased only labeling of the membrane‐bound but not of the cytosolic protein. These results suggest that the direct interaction of vinculin with the hydrophobic core of the phospholipid layer observed in vitro may also be relevant in intact cells, and may be involved in its function as a linker protein.
Localization and characterization of three different β‐adrenergic receptors expressed in Escherichia coli
Authors: CHAPOT Marie‐Pierre , ESHDAT Yuval , MARULLO Stefano , GUILLET Jean‐Gérard , CHARBIT Alain , STROSBERG A. Donny , DELAVIER‐KLUTCHKO Colette
Published: March 2005

After fusion with the N‐proximal portion of the outer membrane protein LamB, three β‐adrenergic receptors, the human β1‐ and β2‐ and turkey β1‐adrenergic receptor, were expressed in Escherichia coli with retention of their own specific pharmacological properties. Molecular characterization and localization of the three receptors in bacteria and comparison of the behaviour of each hybrid protein are reported. The bacteria were lysed and fractionated on a sucrose gradient. Saturable [125I]iodocyanopindolol binding activity was found associated mainly with the inner membrane fraction, suggesting that the receptor is correctly folded in this membrane. Binding activity was also found in the outer membrane fraction but varied according to the receptor type. Photoaffinity labeling experiments revealed that the receptors exhibit binding activity only after proteolytic removal of the LamB moiety from the fusion protein. The three hybrid proteins, detected in immunoblots by anti‐peptide antibodies, were found mainly in the outer membrane fraction. Each of them exhibited different susceptibility to intrinsic bacterial proteolytic enzymes; sites of proteolytic cleavage were localized by the use of anti‐peptide antibodies.

The functional expression in E. coli of three β‐adrenergic receptors with similar structure but different amino acid sequences suggests that this expression system may be a general feature among similar receptors of the family of G‐protein‐coupled receptors. The level of expressed binding activity of a given receptor will be within the control of proteolytic degradation processes, depending on the primary sequence of the receptor. Constructions of new hybrid proteins, in combination with expression in protease mutants of E. coli, should help in controlling such processes.
Core histone‐DNA interactions in sea urchin sperm chromatin
Authors: HILL Caroline S. , THOMAS Jean O.
Published: March 2005

A three‐stage chemical modification procedure [Lambert, S. F. & Thomas, J. O. (1986) Eur. J. Biochem. 160, 191–201; Thomas, J. O. & Wilson, C. M. (1986) EMBO J. 5, 3531–3537] for selectively radiolabelling lysine residues that interact with DNA has been used to investigate core histone – DNA interactions in sea urchin sperm chromatin, in particular to determine the binding site of the long N‐terminal domain of sperm‐specific H2B. Comparison of the patterns of radiolabelling of core histones from extended chromatin and nucleosome core particles (which lack linker DNA) reveals the regions of the histones involved in interactions with the linker. The results show that the N‐terminal domain of H2B is bound to DNA outside the 146‐bp nucleosome core, presumably to the linker DNA. H2A and H4 make no substantial contacts with the linker in extended chromatin; the N‐terminal tail of H4 is bound within the core particle, but the N‐terminal tail of H2A is not bound in core particles or in extended chromatin, and may therefore have a role in higher‐order structure. H3, like H2B, makes contacts with DNA outside the 146‐bp nucleosome core in its N‐terminal region, as well as elsewhere, and probably interacts with the two 10‐bp extensions that complete the two turns of DNA in the nucleosome and/or with the linker.
The 2‐oxoglutarate dehydrogenase complex from Azotobacter vinelandii
Authors: SCHULZE Egbert , WESTPHAL Adrie H. , HANEMAAIJER Roeland , de KOK Arie
Published: March 2005

The nucleotide sequence of the gene encoding the 2‐oxoglutarate dehydrogenase component (E1o) of the 2‐oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined. The protein‐coding sequence consists of 2832 bp (944 codons, including the AUG start codon and the UAA stop codon). The predicted molecular mass (105687 Da) is in good agreement with that published for the isolated enzyme. The E1o gene is separated from the gene encoding the E2o component by a 42‐bp intergenic region. No Escherichia‐coli‐like promoter sequences are found in the sequenced 97 bp upstream from the E1o gene. A putative ribosome‐binding site is located 10–16 bp upstream from the start codon of the E1o gene. No terminator sequences could be detected downstream from the stop codon. Together with the identical situation for the E2o gene and the presence of terminating sequences downstream of the E3 gene, it can be assumed that all three genes of the 2‐oxoglutarate dehydrogenase multienzyme complex are transcribed as a single mRNA transcript under the control of a promoter, located more than 100 bp upstream of the E1o gene, analogous to the pyruvate dehydrogenase complex in E. coli. The similarity with the suc A gene of E. coli is high with 59% identity.
A structural basis of light energy and electron transfer in biology
Authors: HUBER Robert
Published: March 2005

Aspects of intramolecular light energy and electron transfer will be discussed for three protein cofactor complexes, whose three‐dimensional structures have been elucidated by X‐ray crystallography: components of light‐harvesting cyanobacterial phycobilisomes, the purple bacterial reaction centre and the blue multi‐copper oxidases. A wealth of functional data is available for these systems which allow specific correlations between structure and function, and general conclusions about light energy and electron transfer in biological materials to be made.
Thermostable β‐galactosidase from the archaebacteriumSulfolobus solfataricusPurification and properties
Authors: PISANI Francesca M. , RELLA Rocco , RAIA Carlo A. , ROZZO Carla , NUCCI Roberto , GAMBACORTA Agata , de ROSA Mario , ROSSI Mosè
Published: March 2005

A thermophilic and thermostable β‐galactosidase activity was purified to homogeneity from crude extracts of the archaebacteriumSulfolobus solfataricus, by a procedure including ion‐exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75°C witho‐nitrophenyl β‐galactopyranoside as substrate. Molecular mass studies demonstrated that theS. solfataricusβ‐galactosidase was a tetramer of 240 ± 8 kDa composed of similar or identical subunits. Comparison of the amino acid composition of β‐galactosidase fromS. solfataricuswith that fromEscherichia colirevealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not crossreact with β‐galactosidase fromE. coli.The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for β‐galactosidases from other mesophilic and thermophilic sources.
The blue oxidases, ascorbate oxidase, laccase and ceruloplasmin Modelling and structural relationships
Authors: MESSERSCHMIDT Albrecht , HUBER Robert
Published: March 2005

On the basis of the spatial structure of ascorbate oxidase [Messerschmidt, A., Rossi, A., Ladenstein, R., Huber, R., Bolognesi, M., Gatti, G., Marchesini, A., Petruzzelli, R. & Finazzi‐Agro, A. (1989) J. Mol. Biol. 206, 513–529], an alignment of the amino acid sequence of the related blue oxidases, laccase and ceruloplasmin is proposed. This strongly suggests a three‐domain structure for laccase closely related to ascorbate oxidase and a six‐domain structure of ceruloplasmin. These domains demonstrate homology with the small blue copper proteins. The relationships suggest that laccase, like ascorbate oxidase, has a mononuclear blue copper in domain 3 and a trinuclear copper between domain 1 and 3 and ceruloplasmin has mononuclear copper ions in domains 2, 4 and 6 and a trinuclear copper between domains 1 and 6.
Autoregulation of PurR repressor synthesis and involvement of purR in the regulation of pur B, purC, purL, purMN and guaBA expression in Escherichia coli
Authors: MENG Li Mei , KILSTRUP Mogens , NYGAARD Per
Published: March 2005

The pur R gene encodes a repressor (PurR) controlling the synthesis of the enzymes of purine biosynthesis. The subunit of PurR was identified as a 38‐kDa polypeptide by SDS/polyacrylamide gel electrophoresis. Analysis of a pur R–lacZ transcriptional fusion indicated that pur R expression is autoregulated. This was confirmed by gel retardation and DNasel footprinting experiments, where two PurR‐binding sites were identified in the transcribed part of pur R. Introduction of a purR mutation in wild‐type and pur – lac fusion strains was found to abolish purine repression of all genes of the purine biosynthetic pathway except for pur A.
Isolation and characterization of abaecin, a major antibacterial response peptide in the honeybee (Apis mellifera)
Authors: CASTEELS Peter , AMPE Christophe , RIVIERE Lise , van DAMME José , ELICONE Christopher , FLEMING Mark , JACOBS Frans , TEMPST Paul
Published: March 2005

Honeybees (Apis mellifera) are frequently exposed to and likely to be infected by plant‐associated bacteria. We mimicked this process by injecting bees with live bacteria and isolated five induced antibacterial substances by comparative liquid chromatographic mapping of the hemolymph. Three of these antibiotics belong to a unique family of small (18 amino acids) peptides: the apidaecins [Casteels et al. (1989) EMBO J. 8, 2387–2391].

We have now characterized a fourth bee immune response peptide. The complete sequence was established by Edman degradation of the peptide and fragments thereof. It is 34 amino acids long and contains 10 proline residues. The amino‐terminal half is related to the apidaecins; similar proline motifs are also present in the aminoterminal quarter of the much longer fly diptericins.

The newly identified peptide's broad spectrum, lower specific activities against Gram‐negative plant pathogens and its inability to inhibit bacterial growth at medium ionic strength are different from the apidaecins. Moreover, the highest observed specific activity was against an apidaecin‐resistant Xanthomonas strain. In contrast to the immediate action of apidaecins, bactericidal activity is delayed. We propose the name ‘abaecin’ for this new antibacterial response peptide.
Intraorganelle localization and substrate specificities of the mitochondrial acyl‐CoA:sn‐glycerol‐3‐phosphate O‐acyltransferase and acyl‐CoA:1‐acyl‐sn‐glycerol‐3‐phosphate O‐acyltransferase from potato tubers and pea leaves
Authors: FRENTZEN Margrit , NEUBURGER Michel , JOYARD Jacques , DOUCE Roland
Published: March 2005

The mitochondrial sn‐glycerol‐3‐phosphate and 1‐acyl‐sn‐glycerol‐3‐phosphate O‐acyltransferases from potato tubers and pea leaves were investigated with respect to their intraorganelle localization, their positional and substrate specificities, and their fatty acid selectivities. In mitochondria from potato tubers both enzymes were found to be located in the outer membrane. The 1‐acyl‐sn‐glycerol‐3‐phosphate O‐acyltransferase of pea mitochondria showed the same intraorganelle localization whereas the sn‐glycerol‐3‐phosphate O‐acyltransferase behaved like a soluble protein of the intermembrane space.

The sn‐glycerol‐3‐phosphate O‐acyltransferase of both potato and pea mitochondria used sn‐glycerol‐3‐phosphate but not dihydroxyaetone phosphate as acyl acceptor and exclusively catalyzed the formation of 1‐acyl‐sn‐glycerol‐3‐phosphate which subsequently served as substrate for the second acylation reaction at its C‐2 position. Both acyltransferases of potato as well as pea mitochondria showed higher activities with acyl‐CoA than with the corresponding acyl‐(acyl carrier protein) thioesters.

When different acyl‐CoA thioesters were offered separately, the sn‐glycerol‐3‐phosphate O‐acyltransferase of potato mitochondria displayed no fatty acid specificity whereas the enzyme of pea mitochondria revealed one for saturated acyl groups. On the other hand, the mitochondrial 1‐acyl‐sn‐glycerol‐3‐phosphate O‐acyltransferases from both potato tubers and pea leaves were more active on unsaturated than on saturated acyl‐CoA thioesters. Furthermore, these enzymes preferentially used oleoyl‐ and linoleoyl‐CoA when they were offered in a mixture with saturated ones, although the fatty acid selectivity of the pea enzyme was less pronounced than that of the potato enzyme. The sn‐glycerol‐3‐phosphate O‐acyltransferase of potato mitochondria displayed a slight preference for saturated acyl groups.
Effect of the N‐terminal hydrophobic sequence of hepatitis B virus surface antigen on the folding and assembly of hybrid β‐galactosidase in Escherichia coli
Authors: LEE Sang Chul , CHOI Young Chul , YU Myeong‐Hee
Published: March 2005

To investigate the mechanism of inclusion body formation and the effect of a hydrophobic sequence on the in vivo polypeptide folding, the aggregation caused by recombinant fusion β‐galactosidase in Escherichia coli was examined. Two plasmids were constructed: pTBG(H–) carried only the preS2 sequence of the hepatitis B virus surface antigen (HBsAg) in front of the β‐galactosidase gene (lacZ) while pTBG(H+) carried an additional sequence encoding the amino‐terminal hydrophobic sequence of the S region of HBsAg between preS2 and lacZ. Unlike cells expressing the fusion protein not containing the hydrophobic sequence, E. coli JM109/pTBG(H+) exhibited temperature‐sensitive production of β‐galactosidase. As the culture temperature increased the activity decreased dramatically. This decrease in activity was not due to a decrease in fusion polypeptide production, but rather the fusion polypeptides containing the hydrophobic sequence aggregated within the cells at high temperature. However once the fusion polypeptides folded into proper conformation at low temperature, they maintained the activity even at high temperature. The results indicate that aggregation is a consequence of incorrect folding and assembly of the polypeptides, and is not derived from the native structure. The aggregates of the pTBG(H+)‐encoded fusion polypeptides did not revert to active form when the culture temperature was lowered.
Lignin peroxidase from Phanerochaete‐chrysosporium
Authors: GLUMOFF Tuomo , HARVEY Patricia J. , MOLINARI Sergio , GOBLE Martin , FRANK Gerhard , PALMER John M. , SMIT Jan Derk G. , LEISOLA Matti S. A.
Published: March 2005

Five isozymes of lignin peroxidase from Phanerochaete chrysosporium were purified and their physical, molecular and kinetic properties determined. The isozymes differ from each other in terms of their isoelectric point, molecular mass, sugar content, spectral characteristics, substrate specificity and stability. The N‐terminal sequence of amino acids was different for each isozyme suggesting they are different gene products. The isozyme with the highest carbohydrate level was most sensitive to changes in environmental factors. The kinetic behaviour of the isozymes varied clearly when tert‐butyl hydroperoxide instead of hydrogen peroxide was used as the oxidant. Two out of five isozymes had very similar substrate specificity. The results are discussed in relation to the role which lignin peroxidase isozymes may play in lignin biodegradation.
A two‐dimensional 1H NMR study on Megasphaera elsdenii flavodoxin in the reduced state
Authors: van MIERLO Carlo P. M. , VERVOORT Jacques , MÜLLER Franz , BACHER Adelbert
Published: March 2005

Assignments for the 137 amino acid residues of Megasphaera elsdenii flavodoxin in the reduced state have been made using the sequential resonance assignment procedure. Several hydroxyl and sulfhydryl protons were observed at 41°C at pH 8.3. Spin systems were sequentially assigned using phase‐sensitive two‐dimensional‐correlated spectroscopy and phase‐sensitive nuclear Overhauser enhancement spectroscopy. Spectra of the protein in H2O and of protein preparations either completely or partly exchanged against 2H2O were obtained.

Use of the fast electron shuttle between the paramagnetic semiquinone and the diamagnetic hydroquinone state greatly simplified the NMR spectra, making it possible to assign easily the 1H resonances of amino acid residues located in the immediate neighbourhood of the isoalloxazine ring.

The majority of the nuclear Overhauser effect contacts between the flavin and the apoprotein correspond to the crystal structure of the flavin domain of Clostridium MP flavodoxin, but differences are also observed. The assignments provide the basis for the structure determination of M. elsdenii flavodoxin in the reduced state as well as for assigning the resonances of the oxidized flavodoxin.
Purification and characterization of Escherichia coli RNase I
Authors: MEADOR James , CANNON Barbara , CANNISTRARO Vincent J. , KENNELL David
Published: March 2005

The endoribonuclease, RNase I, was purified from the periplasm of Escherichia coli. Based on PAGE, it has molecular mass of approximately 27 kDa with a migration rate indistinguishable from that of the recently reported RNase M from E. coli. The amino acid sequence of the two enzymes must be very similar based on two‐dimensional mapping of their tryptic peptides and suggests either a post‐transcriptional modification to yield different proteins from the same gene or evolution of two genes by gene duplication. However, while RNase I could degrade each of the four ribonucleotide homopolymers, only poly(U) or poly(C) were good substrates for RNase M with possibly some hydrolysis of poly(A). The reaction rate for poly(C) hydrolysis with RNase M was about ten times faster than for poly(U), while for RNase I the rates were about equal.

Besides differences in specificity, RNase M was only located in the spheroplasts while RNase I found in the periplasm of growing cells. In terms of function, RNase I is known to cause degradation of rRNA during periods of stress or non‐growth, whereas it has been proposed that RNase M is the endonuclease for mRNA degradation in growing cells.
Induction of the 4‐aminobutyrate and urea‐catabolic pathways in Saccharomyces cerevisiae
Authors: VISSERS Stephan , ANDRE Bruno , MUYLDERMANS Francine , GRENSON Marcelle
Published: March 2005

In the yeast Saccharomyces cerevisiae, induction of the 4‐aminobutyrate‐catabolic pathway by 4‐aminobutyrate requires two positive regulatory factors, encoded by the UGA3 and the UGA35 genes respectively. In addition to this, expression of one gene of this pathway, namely the UGA4 gene encoding the 4‐aminobutyrate‐specific permease, is controlled negatively by the product of the UGA43 gene [Vissers, S., André, B., Muyldermans, F. & Grenson, M. (1989) Eur. J. Biochem. 181, 357–361].

We show here that the products of two of these regulatory genes, UGA35 and UGA43, also control the expression of the genes encoding the urea‐catabolic pathway, although the 4‐aminobutyrate and urea‐catabolic pathways are synthesised under specific conditions and do not share any enzymatic step or metabolite: the UGA35 gene is shown to be identical to the DURL gene which was previously identified as a positive regulatory factor of the urea‐catabolic pathway; the UGA43 gene product is shown to behave like a negative regulatory factor of this pathway.

In contrast to UGA35/DURL and UGA43, the positive regulatory factors encoded by the UGA3 gene and the previously identified DURM gene specifically control 4‐aminobutyrate and urea catabolisms respectively.

Northern hybridization experiments suggest that the UGA35/DURL and UGA43 common regulatory factors act at the transcriptional level.

Our results show that the expression of two biochemically distinct nitrogenous catabolisms, as triggered by their respective inducers, seems to involve multiple regulatory factors, some of which are common to the two catabolic pathways.
Development of the uncoupling protein in the rat brown‐adipose tissue during the perinatal period
Authors: PORRAS Almudena , PEÑAS Matilde , FERNÁNDEZ Margarita , BENITO Manuel
Published: March 2005

In this paper we report the development of the immunologically detected uncoupling protein (UCP) in brown adipose tissue during the perinatal period in the rat and its relationship with its functional activity expressed in terms of GDP‐binding capacity, GDP‐sensitive permeabilities and GDP‐sensitive respiration. Immunologically detected UCP increased during the last 2 days of foetal life (under euthermic conditions) and after birth (after postnatal hypothermia) during the early suckling period, reaching its maximum value on day 10 after birth. This increase in UCP is accompanied by parallel increases in the GDP‐binding capacity, GDP‐sensitive permeabilities to protons and chloride ions and GDP‐inhibitable respiration. During the suckling – weaning transition, there was a regression of the parameters related to the functional activity of the UCP (GDP‐binding capacity and nucleotide‐sensitive permeabilities and respiration) without changes in the immunologically detected UCP. These results suggest that the involution of this tissue in the rat commences in this period; the first parameters affected are those related to the functional activity of the UCP while the UCP is still present in its highest level. This seems to support the idea that, in this period of development of the rat, the UCP may exist in the brown fat mitochondria in a functional (unmasked) form and a non‐functional (masked) form.
Role of lysine‐67 in the active site of class C β‐lactamase from Citrobacter freundii GN346
Authors: TSUKAMOTO Kikuo , TACHIBANA Katsumi , YAMAZAKI Noriko , ISHII Yuko , UJHE Kumiko , NISHIDA Naoko , SAWAI Tetsuo
Published: March 2005

Citrobacter freundii GN346 produces a class C β‐lactamase exhibiting the substrate profile of a typical cephalosporinase. The structural and promoter regions of the cephalosporinase gene, comprising 1408 nucleotides, were completely sequenced. The amino acid sequence of the mature enzyme, comprising 361 amino acids, and its molecular mass, 39878 Da, were determined. The active site was confirmed to be Ser‐64. The amino acid sequence of the enzyme differs from that of the cephalosporinase of C. freundii OS60 by nine residues. The nucleotide sequence of the promoter region suggests a possible attenuator structure. Lys‐67, one of the most conserved residues found in class A and C β‐lactamases and penicillin‐binding proteins, was converted into arginine, threonine or glutamic acid through site‐directed mutagenesis. The Glu‐67 enzyme had lost the catalytic activity and the Thr‐67 enzyme only showed a trace of activity. The Arg‐67 enzyme, which retained a significant amount of the activity, was purified. The Km values of the Arg‐67 enzyme for cephalothin, cephaloridine and benzylpenicillin are 13–19 times those of the wild‐type enzyme; the kcat values for the three substrates are 37%, 3%, and 36% those of the wild‐type enzyme, respectively.
Heat capacity and entropy changes of the major isotype of the toad (Bufo) parvalbumin induced by calcium binding
Authors: TANOKURA Masaru
Published: March 2005

The possible structural changes in the major isotype of parvalbumin from the toad (Bufo bufo japonicus) skeletal muscle caused by Ca2+ and Mg2+ binding have been analyzed by microcalorimetric titrations. Parvalbumin was titrated with Ca2+ in both the absence and presence of Mg2+ and with Mg2+ in the absence of Ca2+, at pH 7.0, and at 5°, 15°, and 25° C. The two sites in a molecular were equivalent on Mg2+‐Ca2+ exchange, but distinguishable on Ca2+ and Mg2+ binding. The reactions of parvalbumin with Ca2+ are exothermic at every temperature in both the absence and presence of Mg2+, but those with Mg2+ are always endothermic except for the binding to site 1 at 25°C. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of parvalbumin of Ca2+ and Mg2+ binding and Mg2+ ‐Ca2+ exchange have been estimated by the empirical method of Strurtevant [Sturtevant, J. M. (1977) Proc. Natl Acad. Sci. USA 74, 2236–2240].

Although no major conformational changes were noted between Ca2+ ‐ and Mg2+‐ bound forms of toad pervalbumin, the conformational difference was larger in Ca2 (or Mg2+) binding to site 1 than site 2. This may indicate that the metal‐free form is much less stable than any with Ca2+ (or Mg2+) bound at one site at least. On Mg2+ exchange, the vibrational as well as hydrophobic entropy is only slightly increased in a parallel manner. In contrast, on Ca2+ (or Mg2+) binding, the hydrophobic entropy increases but the vibrational entropy decreases; the former inidcates the sequestering of nonpoloar groups from the surface to the interior of a molecule, and the latter suggests that the overall structures are tightened on Ca2+ (or Mg2+) binding but loosened on Mg2+ ‐Ca2+ exchange. Despite the clear distinctions in the thermodynamic features, the conformational changes of toad parvalbumin are essentially the same as those of the two isotypes of bullfrog parvalbumins on Ca2+ binding and Mg2+ ‐C2+ exchange.
Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding
Authors: SVENSSON Birte , CLARKE Anthony J. , SVENDESEN Ib , MØLLER Hanne
Published: March 2005

Functionally important carboxyl groups in glucoamylase G2 form Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose‐inhibited enzyme with 1‐ethyl‐3(4‐azonia‐4,4‐dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantions. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC‐labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme‐acarbose complex were also identified.

Asp176, Glu179 and Glu180 are invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and α‐amylase. The partially radiolabelled Asp112 corresponds in Taka‐amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed.
A gated pathway for electrophoretic Na+ fluxes in rat liver mitochondria
Authors: BERNARDI Paolo , ANGRILLI Alessandro , AZZONE Giovanni Felice
Published: March 2005

Addition of EDTA to mitochondria incubated aerobically in a phosphate‐supplemented medium containing Na+ ions results in activation of cation uptake which is accompanied by membrane depolarization and stimulation of respiration. The same results are obtained in media containing Li+ but not K+, indicating that this pathway for cation transport is selective. The activation of Na+ transport is not accompanied by changes of matrix Mg2+, indicating that cation transport is controlled by surface‐bound rather than intramitochondrial Mg2+, Na+ transport in respiring mitochondria is competitvely inhibited by Mg2+ with a Ki in the nanomolar range. A Na+ current can also be induced by a K+ diffusion potential in the absence of respiration. The K+‐diffusion‐driven Na+ current has the same magnitude in the absence or presence of inorganic phosphate, suggesting that Na+ transport is mediated by Na+ uniport rather than by electrogenic nNa+/H+ antiport with n > 1. Analysis of the flow/force relationship indicates that the putative Na+ uniporter has a conductance of about 0.2 nmol Na+× mg protein−1× min−1× mV−1, and that it is active only when the membrane potential exceeds about 150 mV.
The role of intracellular orthophosphate in triggering osmoregulation in the alga Dunaliella salina
Authors: BENTAL Michal , PICK Uri , AVRON Mordhy , DEGANI Hadassa
Published: March 2005

A new hypothesis is presented for the mechanism of metabolic response during osmoregulation in the alga Dunaliella salina. We propose that the osmotic response is initiated by differential volume change of the cytoplasm and the chloroplast (observed using the electron microscope) which alter the cytoplasmic orthophosphate concentration. This triggers a flow through the pi/triose‐phosphate shuttle, activating chloroplast enzymes in the direction of either starch or glycerol synthesis. The P1‐dependent response was investigated in vivo using NMR. The rates of glycerol synthesis or elimination following osmotic shocks were modulated by the intracellular Pi level as predicated by the hypothesis.
Nuclear magnetic relaxation studies of the role of the metal ion in Mn2+‐substituted aminoacylase I
Authors: HEESE Dirk , BERGER Stefan , RÖHM Klaus‐Heinrich
Published: March 2005

Substitution of the essential Zn2+ ions of porcine kidney aminoacylase I (EC 3.5.1.14) by Mn2+ did not markedly affect the kinetic properties of the enzyme. Using Mn2+as a paramagnetic probe, we were able to study the conformations of bound ligand by measuring the enhancement of ligand proton relaxation in 1H NMR. In addition, the effects of inhibitors on the paramagnetic enhancement of water proton relaxation rates were examined.

The results of both approaches, in agreement with kinetic evidence, suggest that the metal center of aminoacylase I is too distant from the ligand binding site to allow direct participation of the metal in substrate binding or catalysis. We, therefore, propose that the metal ion of aminoacylase I plays a purely structural role.
Lactate dehydrogenase from the extreme thermophile Thermotoga maritima
Authors: WRBA Alexander , JAENICKE Rainer , HUBER Robert , STETTER Karl Otto
Published: March 2005

Lactate dehydrogenase was isolated from the extreme thermophilic eubacterium Thermotoga maritima. The enzyme is stereospecific for L(+)‐lactate. It represents a homotetramer of 144 kDa molecular mass, with a sedimentation coefficient of s20,w∼ 7 S.

Under physiological temperature conditions, the enzyme shows high catalytic efficiency with a broad pH optimum at pH 7.0 ± 1.0, and long‐term stability up to 80°C. The coenzyme, NAD+, and the effector fructose 1,6‐bisphosphate [Fru(1,6]P2] increase the thermal stability: at 90°C (pH 6.0), the liganded enzyme exhibits a half‐life of thermal inactivation of 150 min. The enhanced rigidity of the enzyme at ambient temperature is reflected by an anomalously high stability toward guanidine denaturation: the midpoint of the equilibrium transition being 1.6 M guanidine hydrochloride. Under optimum conditions of the enzyme assay, the Michaelis constants (Km) for NADH, NAD+, pyruvate and L(+)‐lactate at 55°C, and in the absence of Fru (1,6)P2, are 0.03 mM, 0.09 mM and 410 mM, respectively; Fru(1,6)P2, as a positive shifts the Km values for pyruvate and L(+)‐lactate to 0.06 mM and 25 mM, respectively. The Km values for the coenzyme are not affected. Neither Mn2+ nor other divalent cations have any activating effect.

In contrast to lactate dehydrogenases from eukaryotes, the N‐terminus of the enzyme from Th. maritima is not acetylated. Comparison of the 30 N‐terminal amino acid residues with lactate dehydrogenase from Thermus aquaticus shows a high degree of similarity. This also holds if the two lactate dehydrogenases are compared with the glyceraldehyde‐3‐phosphate dehydrogenases from the same organisms.