bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP‐citrate lyase
Authors: Kanao Tadayoshi , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki
Published: March 2003

The prokaryotic ATP‐citrate lyase is considered to be a key enzyme of the carbon dioxide‐fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP‐citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl‐ACL), an α4β4 heteromeric enzyme, revealed that the enzyme displayed typical Michaelis‐Menten kinetics toward ATP with an apparent Km value of 0.21 ± 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (nH) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 ± 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a Ki value of 0.037 ± 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl‐ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl‐ACL. His273 of the α subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl‐CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA.
A new conceptual framework for enzyme catalysis
Authors: Sutcliffe Michael J. , Scrutton Nigel S.
Published: March 2003

Recent years have witnessed high levels of activity in identifying enzyme systems that catalyse H‐transfer by quantum tunneling. Rather than being restricted to a small number of specific enzymes as perceived initially, it has now become an accepted mechanism for H‐transfer in a growing number of enzymes. Furthermore, H‐tunneling is driven by the thermally induced dynamics of the enzyme. In some of those enzymes that break stable C–H bonds the reaction proceeds purely by quantum tunneling, without the need to partially ascend the barrier. Enzymes studied that fall into this category include the flavoprotein and quinoprotein amine dehydrogenases, which have proved to be excellent model systems. These enzymes have enabled us to study the relationship between barrier shape and reaction kinetics. This has involved studies with ‘slow’ and ‘fast’ substrates and enzymes impaired by mutagenesis. A number of key questions now remain, including the nature of the coupling between protein dynamics and quantum tunneling. The wide‐ranging implications of quantum tunneling introduce a paradigm shift in the conceptual framework for enzyme catalysis, inhibition and design.
Differential regulation of telomerase activity by six telomerase subunits
Authors: Chang Joseph Tung‐Chieh , Chen Yin‐Ling , Yang Huei‐Ting , Chen Chi‐Yuan , Cheng Ann‐Joy
Published: March 2003

Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the ends of chromosomes. Six subunits composing the telomerase complex have been cloned: hTR (human telomerase RNA), TEP1 (telomerase‐associated protein 1), hTERT (human telomerase reverse transcriptase), hsp90 (heat shock protein 90), p23, and dyskerin. In this study, we investigated the role of each the telomerase subunit on the activity of telomerase. Through down‐ or upregulation of telomerase, we found that only hTERT expression changed proportionally with the level of telomerase activity. The other components, TEP1, hTR, hsp90, p23, and dyskerin remained at high and unchanged levels throughout modulation. In vivo and in vitro experiments with antisense oligonucleotides against each telomerase component were also performed. Telomerase activity was decreased or abolished by antisense treatment. To correlate clinical sample status, four pairs of normal and malignant tissues from patients with oral cancer were examined. Except for the hTERT subunit, which showed differential expression in normal and cancer tissues, all other components were expressed in both normal and malignant tissues. We conclude that hTERT is a regulatable subunit, whereas the other components are expressed more constantly in cells. Although hTERT has a rate‐limiting effect on enzyme activity, the other telomerase subunits (hTR, TEP1, hsp90, p23, dyskerin) participated in full enzyme activi`ty. We hypothesize that once hTERT is expressed, all other telomerase subunits can be assembled to form a highly active holoenzyme.
Molecular interaction of neutral trehalase with other enzymes of trehalose metabolism in the fission yeast Schizosaccharomyces pombe
Authors: Soto Teresa , Franco Alejandro , Padmanabhan S. , Vicente‐Soler Jero , Cansado Jose , Gacto Mariano
Published: March 2003

Trehalose metabolism is an essential component of the stress response in yeast cells. In this work we show that the products of the principal genes involved in trehalose metabolism in Schizosaccharomyces pombe, tps1+ (coding for trehalose‐6‐P synthase, Tps1p), ntp1+ (encoding neutral trehalase, Ntp1p) and tpp1+ (that codes for trehalose‐6‐P phosphatase, Tpp1p), interact in vitro with each other and with themselves to form protein complexes. Disruption of the gene tps1+ blocks the activation of the neutral trehalase induced by heat shock but not by osmotic stress. We propose that this association may reflect the Tps1p‐dependent requirement for thermal activation of trehalase. Data reported here indicate that following a heat shock the enzyme activity of trehalase is associated with Ntp1p dimers or trimers but not with either Ntp1p monomers or with complexes involving Tps1p. These results raise the possibility that heat shock and osmotic stress activate trehalase differentially by acting in the first case through an specific mechanism involving Tps1p–Ntp1p complexes. This study provides the first evidence for the participation of the catabolic enzyme trehalase in the structural framework of a regulatory macromolecular complex containing trehalose‐6‐P synthase in the fission yeast.
Metabolic control in integrated biochemical systems
Authors: de la Fuente Alberto , Snoep Jacky L. , Westerhoff Hans V. , Mendes Pedro
Published: March 2003

Traditional analyses of the control and regulation of steady‐state concentrations and fluxes assume the activities of the enzymes to be constant. In living cells, a hierarchical control structure connects metabolic pathways to signal‐transduction and gene‐expression. Consequently, enzyme activities are not generally constant. This would seem to compromise analyses of control and regulation at the metabolic level. Here, we investigate the concept of metabolic quasi‐steady state kinetics as a means ofapplying metabolic control analysis to hierarchical biochemical systems. We discuss four methods that enable the experimental determination of metabolic control coefficients, and demonstrate these by computer simulations. The best method requires extra measurement of enzyme activities, two others are simpler but are less accurate and one method is bound only to work under special conditions. Our results may assist in evaluating the relative importance of transcriptomics and metabolomics for functional genomics.
A structural basis for the pH‐dependence of cofilin
Authors: Blondin Laurence , Sapountzi Vasilia , Maciver Sutherland K. , Lagarrigue Emeline , Benyamin Yves , Roustan Claude
Published: March 2003

A marked pH‐dependent interaction with F‐actin is an important property of typical members of the actin depolymerizing factor (ADF)/cofilin family of abundant actin‐binding proteins. ADF/cofilins tend to bind to F‐actin with a ratio of 1 : 1 at pH values around 6.5, and to G‐actin at pH 8.0. We have investigated the mechanism for the pH‐sensitivity. We found no evidence for pH‐dependent changes in the structure of cofilin itself, nor for the interaction of cofilin with G‐actin. None of the actin‐derived, cofilin‐binding peptides that we had previously identified [Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C. (1999) J. Biol. Chem. 274, 28893–28899] bound cofilin in a pH‐sensitive manner. However, we have detected a conformational change in region 75–105 in the actin subdomain 1 by the use of a peptide‐directed antibody. A pH‐dependent conformational change has also been detected spectroscopically in a similar peptide (84–103) on binding to cofilin. These results are consistent with a model in which pH‐dependent motion of subdomain 1 relative to subdomain 2 (through region 75–105) of actin reveals a second cofilin binding site on actin (centered around region 112–125) that allows ADF/cofilin association with the actin filament. This motion requires salt in addition to low pH.
Localization of N‐linked carbohydrate chains in glycoprotein ZPA of the bovine egg zona pellucida
Authors: Ikeda Keiichi , Yonezawa Naoto , Naoi Keita , Katsumata Toshiyuki , Hamano Seizo , Nakano Minoru
Published: March 2003

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, consists of three glycoproteins, ZPA, ZPB and ZPC, and plays a role in sperm–egg interactions. In bovines, these glycoproteins cannot be separated unless the acidic N‐acetyllactosamine regions of the carbohydrate chains are removed by endo‐β‐Galactosidase digestion. Endo‐β‐Galactosidase‐digested ZPB retains stronger sperm‐binding activity than ZPC. It is still unclear whether ZPA possesses significant activity. Recently, we reported that bovine sperm binds to Man5GlcNAc2, the neutral N‐linked chain in the cow zona proteins. In this study, we investigated the localization of the sperm‐ligand active high‐mannose‐type chain and the acidic complex‐type chains in bovine ZPA. Three N‐glycopeptides of ZPA, containing an N‐glycosylation site at Asn83, Asn191 and Asn527, respectively, were obtained from endo‐β‐Galactosidase‐digested ZPA. Of these glycosylation sites, only Asn527 is present in the ZP domain common to all the zona proteins. The carbohydrate structures of the N‐linked chains obtained from each N‐glycopeptide were characterized by two‐dimensional sugar mapping analysis, while considering the structures of the N‐linked chains of the zona protein mixture reported previously. Acidic complex‐type chains were found at all three N‐glycosylation sites, while Man5GlcNAc2 was found at Asn83 and Asn191, but there was very little of this sperm‐ligand active chain at Asn527 in the ZP domain of ZPA.
Structure and function of N‐acetylglucosamine kinase
Authors: Berger Markus , Chen Hao , Reutter Werner , Hinderlich Stephan
Published: March 2003

N‐Acetylglucosamine is a major component of complex carbohydrates. The mammalian salvage pathway of N‐acetylglucosamine recruitment from glycoconjugate degradation or nutritional sources starts with phosphorylation by N‐acetylglucosamine kinase. In this study we describe the identification of two active site cysteines of the sugar kinase by site‐directed mutagenesis and computer‐based structure prediction. Murine N‐acetylglucosamine kinase contains six cysteine residues, all of which were mutated to serine residues. The strongest reduction of enzyme activity was found for the mutant C131S, followed by C143S. Determination of the kinetic properties of the cysteine mutants showed that the decreased enzyme activities were due to a strongly decreased affinity to either N‐acetylglucosamine for C131S, or ATP for C143S. A secondary structure prediction of N‐acetylglucosamine kinase showed a high homology to glucokinase. A model of the three‐dimensional structure ofN‐acetylglucosamine kinase based on the known structure of glucokinase was therefore generated. This model confirmed that both cysteines are located in the active site of N‐acetylglucosamine kinase with a potential role in the binding of the transfered γ‐phosphate group of ATP within the catalytic mechanism.
Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate
Authors: Sanghani Sonal P. , Davis Wilhelmina I. , Dumaual Natividad G. , Mahrenholz Alan , Bosron William F.
Published: March 2003

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis–Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 µm and specific activities between 3 and 8 nmol·min−1·mg−1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene‐specific oligonucleotide probing of multiple‐tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.
Altering the surface properties of baculovirus Autographa californica NPV by insertional mutagenesis of the envelope protein gp64
Authors: Spenger Alexandra , Grabherr Reingard , Töllner Lars , Katinger Hermann , Ernst Wolfgang
Published: March 2003

The envelope protein gp64 of the baculovirus Autographa californica nuclear polyhedrosis virus is essential for viral entry into insect cells, as the glycoprotein both mediates pH‐dependent membrane fusion and binds to host cell receptors. Surface modification of baculovirus particles by genetic engineering of gp64 has been demonstrated by various strategies and thus has become an important and powerful tool in molecular biology. To improve further the presentation of peptides on the surface of baculovirus particles, several insertion sites within the gp64 envelope protein were selected by their theoretical maximum surface probability and investigated for efficient peptide presentation. The ELDKWA peptide of the gp41 of HIV‐1, specific for the human mAb 2F5, was inserted into 17 different positions of the glycoprotein gp64. Propagation of viruses was successful in 13 cases, mutagenesis at four positions did not result in production of intact virus particles. Western blotting, FACS analysis and ELISA were used for characterization of the different binding properties of the mutants. Insertion of this peptide into the native envelope protein resulted in high avidity display on the surface of baculovirus particles. This approach offers the possibility of effective modification of surface properties in regard to host range specificity and antigen display.
Contribution of Lys276 to the conformational flexibility of the active site of glutamate decarboxylase from Escherichia coli
Authors: Tramonti Angela , John Robert A. , Bossa Francesco , De Biase Daniela
Published: March 2003

Glutamate decarboxylase is a pyridoxal 5′‐phosphate‐dependent enzyme responsible for the irreversible α‐decarboxylation of glutamate to yield 4‐aminobutyrate. In Escherichia coli, as well as in other pathogenic and nonpathogenic enteric bacteria, this enzyme is a structural component of the glutamate‐based acid resistance system responsible for cell survival in extremely acidic conditions (pH < 2.5). The contribution of the active‐site lysine residue (Lys276) to the catalytic mechanism of E. coli glutamate decarboxylase has been determined. Mutation of Lys276 into alanine or histidine causes alterations in the conformational properties of the protein, which becomes less flexible and more stable. The purified mutants contain very little (K276A) or no (K276H) cofactor at all. However, apoenzyme preparations can be reconstituted with a full complement of coenzyme, which binds tightly but slowly. The observed spectral changes suggest that the cofactor is present at the active site in its hydrated form. Binding of glutamate, as detected by external aldimine formation, occurs at a very slow rate, 400‐fold less than that of the reaction between glutamate and pyridoxal 5′‐phosphate in solution. Both Lys276 mutants are unable to decarboxylate the substrate, thus preventing detailed investigation of the role of this residue on the catalytic mechanism. Several lines of evidence show that mutation of Lys276 makes the protein less flexible and its active site less accessible to substrate and cofactor.
Electrochemical, FT‐IR and UV/VIS spectroscopic properties of the caa3 oxidase from T. thermophilus
Authors: Hellwig Petra , Soulimane Tewfik , Mäntele Werner
Published: March 2003

The caa3‐oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS and Fourier‐transform infrared (FT‐IR) spectroscopic approach. In this oxidase the electron donor, cytochrome c, is covalently bound to subunit II of the cytochrome c oxidase. Oxidative electrochemical redox titrations in the visible spectral range yielded a midpoint potential of −0.01 ± 0.01 V (vs. Ag/AgCl/3m KCl, 0.218 V vs. SHE′) for the heme c. This potential differs for about 50 mV from the midpoint potential of isolated cytochrome c, indicating the possible shifts of the cytochrome c potential when bound to cytochrome c oxidase. For the signals where the hemes a and a3 contribute, three potentials, = −0.075 V ± 0.01 V, Em2 = 0.04 V ± 0.01 V and Em3 = 0.17 V ± 0.02 V (0.133, 0.248 and 0.378 V vs. SHE′, respectively) could be obtained. Potential titrations after addition of the inhibitor cyanide yielded a midpoint potential of −0.22 V ± 0.01 V for heme a3‐CN– and of Em2 = 0.00 V ± 0.02 V and Em3 = 0.17 V ± 0.02 V for heme a (−0.012 V, 0.208 V and 0.378 V vs. SHE′, respectively). The three phases of the potential‐dependent development of the difference signals can be attributed to the cooperativity between the hemes a, a3 and the CuB center, showing typical behavior for cytochrome c oxidases. A stronger cooperativity of CuB is discussed to reflect the modulation of the enzyme to the different key residues involved in proton pumping. We thus studied the FT‐IR spectroscopic properties of this enzyme to identify alternative protonatable sites. The vibrational modes of a protonated aspartic or glutamic acid at 1714 cm−1 concomitant with the reduced form of the protein can be identified, a mode which is not present for other cytochrome c oxidases. Furthermore modes at positions characteristic for tyrosine vibrations have been identified. Electrochemically induced FT‐IR difference spectra after inhibition of the sample with cyanide allows assigning the formyl signals upon characteristic shifts of the ν(C=O) modes, which reflect the high degree of similarity of heme a3 to other typical heme copper oxidases. A comparison with previously studied cytochrome c oxidases is presented and on this basis the contributions of the reorganization of the polypeptide backbone, of individual amino acids and of the hemes c, a and a3 upon electron transfer to/from the redox active centers discussed.
Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N‐acyl‐d‐amino acid amidohydrolase responsible for d‐amino acid production
Authors: Lin Pei‐Hsun , Su Shiun‐Cheng , Tsai Ying‐Chieh , Lee Chia‐Yin
Published: March 2003

An N‐acyl‐d‐amino acid amidohydrolase (N‐D‐AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N‐acetyl‐d‐methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467‐bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN‐D‐AAase showed significant amino acid similarity to the N‐acyl‐d‐amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A‐6 (44–56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over‐expression of the N‐D‐AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel‐filtration chromatography. A homogenous protein with a specific activity of 516 U·mg−1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N‐D‐AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 °C, respectively. After 30 min heat treatment at 45 °C, between pH 6 and pH 8, 80% activity remained. The N‐D‐AAase had higher hydrolysing activity against N‐acetyl‐d‐amino acid derivates containing d‐methionine, d‐leucine and d‐alanine and against N‐chloroacetyl‐d‐phenylalanine. Importantly, the enzyme does not act on the N‐acetyl‐l‐amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N‐D‐AAase from V. paradoxus can be considered a chiral specific and metal‐dependent enzyme.
Identification of residues in the PXR ligand binding domain critical for species specific and constitutive activation
Authors: Östberg Tove , Bertilsson Göran , Jendeberg Lena , Berkenstam Anders , Uppenberg Jonas
Published: March 2003

The cytochrome P450 family of enzymes has long been known to metabolize a wide range of compounds, including many of today's most common drugs. A novel nuclear receptor called PXR has been established as an activator of several of the cytochrome P450 genes, including CYP3A4. This enzyme is believed to account for the metabolism of more than 50% of all prescription drugs. PXR is therefore used as a negative selector target and discriminatory filter in preclinical drug development.

In this paper we describe the design, construction and characterization by transient transfection of mutant receptors of the human and mouse PXR ligand binding domains. By modeling the human PXR ligand binding domain we have identified and mutated two polar residues in the putative ligand binding pocket which differ between the human and the mouse receptor. The first residue (Q285 in human/I282 in mouse) was mutated between the two species with the corresponding amino acids. These mutants showed that this residue is important for the species specific activation of PXR by the ligand pregnenolone‐16α‐carbonitrile (PCN), while having a less pronounced role in receptor activation by rifampicin. The second residue to be mutated (H407 in human/Q404 in mouse) unexpectedly proved to be important for the basal level of activation of PXR. The H407A mutant of the human receptor showed a high level of constitutive activity, while the Q404H mutant of the mouse receptor demonstrated a sharply decreased basal activity compared to wild‐type.
Mechanism of 1,4‐dehydrogenation catalyzed by a fatty acid (1,4)‐desaturase of Calendula officinalis
Authors: Reed Darwin W , Savile Christopher K. , Qiu Xiao , Buist Peter H. , Covello Patrick S.
Published: March 2003

The mechanism by which the fatty acid (1,4)‐desaturase of Calendula officinalis produces calendic acid from linoleic acid has been probed through the use of kinetic isotope effect (KIE) measurements. This was accomplished by incubating appropriate mixtures of linoleate and regiospecifically dideuterated isotopomers with a strain of Saccharomyces cerevisiae expressing a functional (1,4)‐desaturase. GC‐MS analysis of methyl calendate obtained in these experiments showed that the oxidation of linoleate occurs in two discrete steps since the cleavage of the C11‐H bond is very sensitive to isotopic substitution (kH/kD = 5.7 ± 1.0) while no isotope effect (kH/kD = 1.0 ± 0.1) was observed for the C8‐H bond breaking step. These data indicate that calendic acid is produced via initial H‐atom abstraction at C11 of a linoleoyl substrate and supports the hypothesis that this transformation represents a regiochemical variation of the more common C12‐initiated Δ12 desaturation process.
Ribosome‐associated factor Y adopts a fold resembling a double‐stranded RNA binding domain scaffold
Authors: Ye Keqiong , Serganov Alexander , Hu Weidong , Garber Maria , Patel Dinshaw J.
Published: March 2003

Escherichia coli protein Y (pY) binds to the small ribosomal subunit and stabilizes ribosomes against dissociation when bacteria experience environmental stress. pY inhibits translation in vitro, most probably by interfering with the binding of the aminoacyl‐tRNA to the ribosomal A site. Such a translational arrest may mediate overall adaptation of cells to environmental conditions. We have determined the 3D solution structure of a 112‐residue pY and have studied its backbone dynamic by NMR spectroscopy. The structure has a βαβββα topology and represents a compact two‐layered sandwich of two nearly parallel α helices packed against the same side of a four‐stranded β sheet. The 23 C‐terminal residues of the protein are disordered. Long‐range angular constraints provided by residual dipolar coupling data proved critical for precisely defining the position of helix 1. Our data establish that the C‐terminal region of helix 1 and the loop linking this helix with strand β2 show significant conformational exchange in the ms–µs time scale, which may have relevance to the interaction of pY with ribosomal subunits. Distribution of the conserved residues on the protein surface highlights a positively charged region towards the C‐terminal segments of both α helices, which most probably constitutes an RNA binding site. The observed βαβββα topology of pY resembles the αβββα topology of double‐stranded RNA‐binding domains, despite limited sequence similarity. It appears probable that functional properties of pY are not identical to those of dsRBDs, as the postulated RNA‐binding site in pY does not coincide with the RNA‐binding surface of the dsRBDs.
Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG‐1
Authors: Snellman Erick A. , Sullivan Elise R. , Colwell Rita R.
Published: March 2003

An extracellular lipase, LipA, extracted from Acinetobacter sp. RAG‐1 grown on hexadecane was purified and properties of the enzyme investigated. The enzyme is released into the growth medium during the transition to stationary phase. The lipase was harvested from cells grown to stationary phase, and purified with 22% yield and > 10‐fold purification. The protein demonstrates little affinity for anion exchange resins, with contaminating proteins removed by passing crude supernatants over a Mono Q column. The lipase was bound to a butyl Sepharose column and eluted in a Triton X‐100 gradient. The molecular mass (33 kDa) was determined employing SDS/PAGE. LipA was found to be stable at pH 5.8–9.0, with optimal activity at 9.0. The lipase remained active at temperatures up to 70 °C, with maximal activity observed at 55 °C. LipA is active against a wide range of fatty acid esters of p‐nitrophenyl, but preferentially attacks medium length acyl chains (C6, C8). The enzyme demonstrates hydrolytic activity in emulsions of both medium and long chain triglycerides, as demonstrated by zymogram analysis. RAG‐1 lipase is stabilized by Ca2+, with no loss in activity observed in preparations containing the cation, compared to a 70% loss over 30 h without Ca2+. The lipase is strongly inhibited by EDTA, Hg2+, and Cu2+, but shows no loss in activity after incubation with other metals or inhibitors examined in this study. The protein retains more than 75% of its initial activity after exposure to organic solvents, but is rapidly deactivated by pyridine. RAG‐1 lipase offers potential for use as a biocatalyst.
Oxidative deamination of lysine residue in plasma protein of diabetic rats
Authors: Akagawa Mitsugu , Sasaki Takeshi , Suyama Kyozo
Published: March 2003

The levels of α‐aminoadipic‐δ‐semialdehyde residue, the oxidative deamination product of lysine residue, in plasma protein from streptozotocin‐induced diabetic rats were evaluated. α‐Aminoadipic‐δ‐semialdehyde was converted to a bisphenol derivative by acid hydrolysis in the presence of phenol, and determined by high performance liquid chromatography. Analysis of plasma proteins revealed three times higher levels of α‐aminoadipic‐δ‐semialdehyde in diabetic subjects compared with normal controls. Furthermore, we explored the oxidative deamination via the Maillard reaction and demonstrated that the lysine residue of bovine serum albumin is oxidatively deaminated during the incubation with various carbohydrates in the presence of Cu2+ at a physiological pH and temperature. This experiment showed that 3‐deoxyglucosone and methylglyoxal are the most efficient oxidants of the lysine residue. When the reaction was initiated from glucose, a significant amount of α‐aminoadipic‐δ‐semialdehyde was also formed in the presence of Cu2+. The reaction was significantly inhibited by deoxygenation, catalase, and a hydroxyl radical scavenger. The mechanism we propose for the oxidative deamination is the Strecker‐type reaction and the reactive oxygen species‐mediated oxidation. Based on these findings, we propose a novel mechanism for the oxidative modification of proteins in diabetes, namely the oxidative deamination of the lysine residue via the Maillard reaction.
Anti‐ and pro‐oxidant effects of urate in copper‐induced low‐density lipoprotein oxidation
Authors: Filipe Paulo , Haigle Josiane , Freitas João , Fernandes Afonso , Mazière Jean‐Claude , Mazière Cécile , Santus René , Morlière Patrice
Published: March 2003

We reported earlier that urate may behave as a pro‐oxidant in Cu2+‐induced oxidation of diluted plasma. Thus, its effect on Cu2+‐induced oxidation of isolated low‐density lipoprotein (LDL) was investigated by monitoring the formation of malondialdehyde and conjugated dienes and the consumption of urate and carotenoids. We show that urate is antioxidant at high concentration but pro‐oxidant at low concentration. Depending on Cu2+ concentration, the switch between the pro‐ and antioxidant behavior of urate occurs at different urate concentrations. At high Cu2+ concentration, in the presence of urate, superoxide dismutase and ferricytochrome c protect LDL from oxidation but no protection is observed at low Cu2+ concentration. The use of Cu2+ or Cu+ chelators demonstrates that both copper redox states are required. We suggest that two mechanisms occur depending on the Cu2+ concentration. Urate may reduce Cu2+ to Cu+, which in turn contributes to  formation. The Cu2+ reduction is likely to produce the urate radical (UH·–). It is proposed that at high Cu2+ concentration, the reaction of UH·– radical with  generates products or intermediates, which trigger LDL oxidation. At low Cu2+ concentration, we suggest that the Cu+ ions formed reduce lipid hydroperoxides to alkoxyl radicals, thereby facilitating the peroxidizing chain reaction. It is anticipated that these two mechanisms are the consequence of complex LDL–urate–Cu2+ interactions. It is also shown that urate is pro‐oxidant towards slightly preoxidized LDL, whatever its concentration. We reiterate the conclusion that the use of antioxidants may be a two‐edged sword.
Characterization of an omega‐class glutathione S‐transferase from Schistosoma mansoni with glutaredoxin‐like dehydroascorbate reductase and thiol transferase activities
Authors: Girardini Javier , Amirante Alejandro , Zemzoumi Khalid , Serra Esteban
Published: March 2003

Glutathione S‐transferases (EC 2.5.1.18) (GSTs), are a family of multifunctional enzymes present in all living organisms whose main function is the detoxification of electrophilic compounds. GSTs are considered the most prominent detoxifying class II enzymes in helminths. We describe here the characterization of novel dehydroascorbate reductase and thiol transferase activities that reside in the human parasite Schistosoma mansoni GSTx. Protein sequence analysis of this parasite product showed lower identity to known GSTs. However, phylogenic analysis placed SmGSTx among the recently described omega class GSTs (GSTO1‐1). We report here that SmGSTO protein is a 28‐kDa polypeptide, detected in all life stages of the parasite, being highly expressed in adult worms. Like other omega class GSTs, SmGSTO showed very low activity toward classical GSTs substrates as 1‐chloro‐2,4‐dinitrobenzene, and no binding affinity to glutathione–agarose matrix but showed some biochemical characteristics related with thioredoxins/glutaredoxins. Interestingly, SmGSTO was able to bind S‐hexyl glutathione matrix and displayed significant glutathione‐dependent dehydroascorbate reductase and thiol transferase enzymatic activities.
Purification and catalytic properties of a CO‐oxidizing:H2‐evolving enzyme complex from Carboxydothermus hydrogenoformans
Authors: Soboh Basem , Linder Dietmar , Hedderich Reiner
Published: March 2003

From the membrane fraction of the Gram‐positive bacterium Carboxydothermus hydrogenoformans, an enzyme complex catalyzing the conversion of CO to CO2 and H2 was purified. The enzyme complex showed maximal CO‐oxidizing:H2‐evolving enzyme activity with 5% CO in the headspace (450 U per mg protein). Higher CO concentrations inhibited the hydrogenase present in the enzyme complex. For maximal activity, the enzyme complex had to be activated by either CO or strong reductants. The enzyme complex also catalyzed the CO‐ or H2‐dependent reduction of methylviologen at 5900 and 180 U per mg protein, respectively. The complex was found to be composed of six hydrophilic and two hydrophobic polypeptides. The amino‐terminal sequences of the six hydrophilic subunits were determined allowing the identification of the encoding genes in the preliminary genome sequence of C. hydrogenoformans. From the sequence analysis it was deduced that the enzyme complex is formed by a Ni‐containing carbon monoxide dehydrogenase (CooS), an electron transfer protein containing four [4Fe−4S] clusters (CooF) and a membrane bound [NiFe] hydrogenase composed of four hydrophilic subunits and two membrane integral subunits. The hydrogenase part of the complex shows high sequence similarity to members of a small group of [NiFe] hydrogenases with sequence similarity to energy conserving NADH:quinone oxidoreductases. The data support a model in which the enzyme complex is composed of two catalytic sites, a CO‐oxidizing site and a H2‐forming site, which are connected via a different iron–sulfur cluster containing electron transfer subunits. The exergonic redox reaction catalyzed by the enzyme complex in vivo has to be coupled to energy conservation, most likely via the generation of a proton motive force.
The group I‐like ribozyme DiGIR1 mediates alternative processing of pre‐rRNA transcripts in Didymium iridis
Authors: Vader Anna , Johansen Steinar , Nielsen Henrik
Published: March 2003

During starvation induced encystment, cells of the myxomycete Didymium iridis accumulate a 7.5‐kb RNA that is the result of alternative processing of pre‐rRNA. The 5′ end corresponds to an internal processing site cleaved by the group I‐like ribozyme DiGIR1, located within the twin‐ribozyme intron Dir.S956‐1. The RNA retains the majority of Dir.S956‐1 including the homing endonuclease gene and a small spliceosomal intron, the internal transcribed spacers ITS1 and ITS2, and the large subunit rRNA lacking its two group I introns. The formation of this RNA implies cleavage by DiGIR1 in a new RNA context, and presents a new example of the cost to the host of intron load. This is because the formation of the 7.5‐kb RNA is incompatible with the formation of functional ribosomal RNA from the same transcript. In the formation of the 7.5‐kb RNA, DiGIR1 catalysed cleavage takes place without prior splicing performed by DiGIR2. This contrasts with the processing order leading to mature rRNA and I‐DirI mRNA in growing cells, suggesting an interplay between the two ribozymes of a twin‐ribozyme intron.
Investigations into the mechanisms used by the C‐terminal anchors of Escherichia coli penicillin‐binding proteins 4, 5, 6 and 6b for membrane interaction
Authors: Harris Frederick , Brandenburg Klaus , Seydel Ulrich , Phoenix David
Published: March 2003

Escherichia coli low molecular mass penicillin‐binding proteins (PBPs) include PBP4, PBP5, PBP6 and PBP6b. Evidence suggests that these proteins interact with the inner membrane via C‐terminal amphiphilic α‐helices. Nonetheless, the membrane interactive mechanisms utilized by the C‐terminal anchors of PBP4 and PBP6b show differences to those utilized by PBP5 and PBP6. Here, hydrophobic moment‐based analyses have predicted that, in contrast to the PBP4 and PBP6b C‐termini, those of PBP5 and PBP6 are candidates to form oblique orientated α‐helices. Consistent with these predictions, Fourier transform infrared spectroscopy (FTIR) has shown that peptide homologs of the PBP4 and PBP5 C‐terminal regions, P4 and P5, respectively, both possessed the ability to adopt α‐helical structure in the presence of lipid. However, whereas P4 appeared to show a preference for interaction with the surface regions of dimyristoylglycerophosphoethanolamine and dimyristoylglycerophosphoglycerol membranes, P5 appeared to show deep penetration of both these latter membranes and dimyristoylglycerophosphocholine membranes. Based on these results, we have suggested that in contrast to the membrane anchoring of the PBP4 and PBP6b C‐terminal α‐helices, the PBP5 and PBP6 C‐terminal α‐helices may possess hydrophobicity gradients and penetrate membranes in an oblique orientation.
Presence of melanocortin (MC4) receptor in spiny dogfish suggests an ancient vertebrate origin of central melanocortin system
Authors: Ringholm Aneta , Klovins Janis , Fredriksson Robert , Poliakova Natalia , Larson Earl T. , Kukkonen Jyrki P. , Larhammar Dan , Schiöth Helgi B.
Published: March 2003

We report the cloning, expression, pharmacological characterization and tissue distribution of a melanocortin (MC) receptor gene in a shark, the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis showed that this receptor is an ortholog of the MC4 subtype, sharing 71% overall amino acid identity with the human (Hsa) MC4 receptor. When expressed and characterized by radioligand binding assay for the natural MSH (melanocyte‐stimulating hormone) peptides α‐, β‐, and γ‐MSH, the SacMC4 receptor showed pharmacological properties very similar to the HsaMC4 receptor. Stimulation of SacMC4 receptor transfected cells with α‐MSH caused a dose‐dependent increase in intracellular cAMP levels. The SacMC4 receptor has Ala in position 59 where all other cloned MC receptors have Glu. We confirmed that this was not due to individual polymorphism and subsequently mutated the residue ‘back’ to Glu but the mutation did not affect the pharmacological properties of the receptor. SacMC4 receptor mRNA was detected by RT‐PCR in the optic tectum, hypothalamus, brain stem, telencephalon and olfactory bulb but not in cerebellum or in peripheral tissues. This study describes the first characterization of an MC receptor in a cartilaginous fish, the most distant MC receptor gene cloned to date. Conservation of gene structure, pharmacological properties and tissue distribution suggests that this receptor may have similar roles in sharks as in mammals and that these were established more than 450 million years ago.
Cytotoxic activity of nucleoside diphosphate kinase secreted from Mycobacterium tuberculosis
Authors: Chopra Puneet , Singh Anubha , Koul Anil , Ramachandran S. , Drlica Karl , Tyagi Anil K. , Singh Yogendra
Published: March 2003

Pathogenicity of Mycobacterium tuberculosis is closely related to its ability to survive and replicate in the hostile environment of macrophages. For some pathogenic bacteria, secretion of ATP‐utilizing enzymes into the extracellular environment aids in pathogen survival via P2Z receptor‐mediated, ATP‐induced death of infected macrophages. A component of these enzymes is nucleoside diphosphate kinase (Ndk). The ndk gene was cloned from M. tuberculosis H37Rv and expressed in Escherichia coli. Ndk was secreted into the culture medium by M. tuberculosis, as determined by enzymatic activity and Western blotting. Purified Ndk enhanced ATP‐induced macrophage cell death, as assayed by the release of [14C]adenine. A catalytic mutant of Ndk failed to enhance ATP‐induced macrophage cell death, and periodate‐oxidized ATP (oATP), an irreversible inhibitor of P2Z receptor, blocked ATP/Ndk‐induced cell death. Purified Ndk was also found to be autophosphorylated with broad specificity for all nucleotides. Conversion of His117→Gln, which is part of the nucleotide‐binding site, abolished autophosphorylation. Purified Ndk also showed GTPase activity. Collectively, these results indicate that secreted Ndk of M. tuberculosis acts as a cytotoxic factor for macrophages, which may help in dissemination of the bacilli and evasion of the immune system.
The transthyretin‐related protein family
Authors: Eneqvist Therese , Lundberg Erik , Nilsson Lars , Abagyan Ruben , Sauer‐Eriksson A. Elisabeth
Published: March 2003

A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin‐related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three‐dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand‐binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double‐stranded RNA‐mediated interference techniques in order to determine the loss‐of‐function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.
Proteome analysis at the level of subcellular structures
Authors: Dreger Mathias
Published: March 2003

The targeting of proteins to particular subcellular sites is an important principle of the functional organization of cells at the molecular level. In turn, knowledge about the subcellular localization of a protein is a characteristic that may provide a hint as to the function of the protein. The combination of classic biochemical fractionation techniques for the enrichment of particular subcellular structures with the large‐scale identification of proteins by mass spectrometry and bioinformatics provides a powerful strategy that interfaces cell biology and proteomics, and thus is termed ‘subcellular proteomics’. In addition to its exceptional power for the identification of previously unknown gene products, the analysis of proteins at the subcellular level is the basis for monitoring important aspects of dynamic changes in the proteome such as protein transloction. This review summarizes data from recent subcellular proteomics studies with an emphasis on the type of data that can retrieved from such studies depending on the design of the analytical strategy.
Adaptive changes in the expression of nuclear and mitochondrial encoded subunits of cytochrome c oxidase and the catalytic activity during hypoxia
Authors: Vijayasarathy C , Damle Shirish , Prabu Subbuswamy K. , Otto Cynthia M. , Avadhani Narayan G.
Published: March 2003

The effects of physiologically relevant hypoxia on the catalytic activity of cytochrome c oxidase (CytOX), mitochondrial gene expression, and both nuclear and mitochondrial encoded CytOX mRNA levels were investigated in murine monocyte macrophages, mouse C2C12 skeletal myocytes and rat adrenal pheochromocytoma PC12 cells. Our results suggest a coordinated down regulation of mitochondrial genome‐coded CytOX I and II and nuclear genome‐coded CytOX IV and Vb mRNAs during hypoxia. Hypoxia also caused a severe decrease in mitochondrial transcription rates, and associated decrease in mitochondrial transcription factor A. The enzyme from hypoxia exposed cells exhibited altered subunit content as revealed by blue native gel electrophoresis. There was a generalized decline in mitochondrial function that led to a decrease in total cellular heme and ATP pools. We also observed a decrease in mitochondrial heme aa3 content and decreased levels of CytOX subunit I, IV and Vb, though the catalytic efficiency of the enzyme (TN for cytochrome c oxidase) remained nearly the same. Increased glycolytic flux and alterations in the kinetic characteristics of the CytOX might be the two mechanisms by which hypoxic cells maintain adequate ATP levels to sustain life processes. Reoxygenation nearly completely reversed hypoxia‐mediated changes in CytOX mRNA contents, rate of mitochondrial transcription, and the catalytic activity of CytOX enzyme. Our results show adaptive changes in CytOX structure and activity during physiological hypoxia.