bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Characterization and Physiological Regulation of A TATA‐less Gene Encoding Chicken Gastrin
Authors: Wu S. Vincent , Walsh John H. , Campbell Barry J. , Dimaline Rod
Published: March 2005

Avian gastrin is a gastric acid secretagogue, but is structurally more closely related to mammalian cholecystokinin, which is functionally distinct from gastrin. This apparently anomalous structure/activity relationship raises important issues for understanding the evolution of regulatory peptides and the mechanisms that control their expression. To clarify the possible mechanisms, we have determined the sequence of a 6.5‐kb Bam HI genomic DNA fragment that includes the entire avian gastrin transcriptional unit. The complete cDNA sequence, determined by anchored PCR, encoded a precursor of 105 amino acids. Conserved primary amino acid structures were limited to a decapeptide determining biological activity, and essential sites for post‐translational processing. Significantly, however, the genomic regulatory regions, and introns, were unlike those of any previously reported gastrin/cholecystokinin gene. The avian gastrin gene contained no recognizable TATA‐box motif, a feature unique to this gene family, but had a G+C‐rich region immediately upstream of the transcription initiation site, and a Z‐DNA purine‐pyrimidine repeat sequence. Moreover, physiological regulation of the avian gastrin gene differed markedly from that observed in mammals, in that the important local paracrine downregulation by antral somatostatin was absent; instead, evidence for a hormonal regulation was demonstrated. The data indicate that genomic regulatory elements within regulatory peptide families such as the gastrin/cholecystokinin family, and mechanisms of physiological control, are not conserved during evolution, even though biologically important amino acid sequence information is retained.
Distribution and Activity of Calcineurin in Rat Tissues
Authors: Su Qingxiang , Zhao Ming , Weber Elisabeth , Eugster Hans‐Pietro , Ryffel Bernhard
Published: March 2005

Calcineurin (CN), a Ca2+/calmodulin‐regulated phophatase 2B, plays an important role in many biological processes including T‐cell signal transduction. In the present study, the distribution and activity of CN were investigated in rat tissues. CN has a wide tissue distribution, as measured by enzyme‐linked immunosorbent assay. CN concentrations are 0.2–0.6 μg/mg protein in most tissues, while the brain contains 3–10‐fold higher concentrations. Immunohistochemical analyses using a monoclonal antibody to CN B subunit reveals that CN is not evenly distributed but concentrated in specific cells, especially in the brain, kidneys and testis. The specific enzymic activity of CN in tissues is around 10 pmol · min · mg protein−1, except in brain and liver (60 pmol · min−1· mg protein−1compared to 3.6 pmol · min−1· mg protein−1). The immunosuppressants cyclosporin A and tacrolimus, but not rapamycin, inhibit the phosphatase activity of CN derived from most tissues tested, while CN activity from liver was resistant to cyclosporin A. Furthermore, transcripts and protein of the common CN B subunit and of the testis‐specific form of CN B subunit were analyzed. The common CN B subunit transcripts and protein are detected in all tissues. Transcripts for the ‘testis‐specific’ CN B subunit are also found in brain, lung, thymus and heart, while the protein is only detected in testis. This indicates that the testis‐specific CN B subunit gene expression is regulated at both transcriptional and posttranscriptional levels. The findings demonstrate that CN is a widely distributed protein phosphatase and that its activity is regulated in a tissue‐specific manner.
Site‐directed Mutagenesis of Varicella‐zoster Virus Thymidylate Synthase
Authors: Harrison Patrick T. , Scott James E. , Hutchinson Michael J. , Thompson Russell
Published: March 2005

We have constructed a series of mutants to study the role of two structurally and functionally important regions of thymidylate synthase (TS) from varicella‐zoster virus (VZV). The first centres on a conserved glycine residue in the β‐kink of β‐strand i, a partially buried region of the protein that is important for dimer interactions and the formation of the active site. We show that the glycine residue located in β‐strand i is not essential for enzyme activity and that β‐strand i can readily accommodate several amino acid substitutions and also an insertion. A covariant residue that accommodates these changes was also identified. The second region of interest was the solvent‐exposed and highly mobile C‐terminal residue which is an essential component of the active site in TS from Lactobacillus casei and Escherichia coli. We demonstrate that removal of the C‐terminal residue from VZV TS does not completely inactivate the enzyme, implying that there are significant structural differences between the virus and bacterial enzymes. By combining site‐directed mutagenesis and molecular modelling we have identified these differences and propose a model that explains the contrasting activities.
cDNA Cloning, Overexpression in Escherichia coli, Purification and Characterization of Sheep Liver Cytosolic Serine Hydroxymethyltransferase
Authors: Jagath‐Reddy Junutula , Ganesan Kaliannan , Savithri Handanahal S. , Datta Asis , Rao Naropantul Appaji
Published: March 2005

A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransferase (SHMT) was isolated and its nucleotide sequence determined. The full‐length cDNA of SHMT was placed under the control of T7 promoter in pET‐3C plasmid and expressed in Escherichia coli. The overexpressed enzyme, present predominantly in the soluble fraction, was catalytically active. The recombinant SHMT was purified to homogeneity with a yield of 10 mg/1 bacterial culture. The recombinant enzyme was capable of carrying out tetrahydrofolate‐dependent and tetrahydrofolate‐independent reactions as effectively as the native enzyme. The Km values for serine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of the native enzyme. The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5′‐phosphate as an internal aldimine. The apoenzyme obtained upon removal of the cofactor was inactive and could be reconstituted by the addition of pyridoxal 5′‐phosphate demonstrating that the recombinant SHMT was functionally very similar to the native SHMT. This overexpression of eukaryotic tetrameric SHMT in E. coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme.
Distribution of Flux Control among the Enzymes of Mitochondrial Oxidative Phosphorylation in Calcium‐activated Saponin‐skinned Rat Musculus Soleus Fibers
Authors: Wisniewski Elke , Gellerich Frank N. , Kunz Wolfram S.
Published: March 2005

Metabolic control analysis was applied to describe the control of mitochondrial oxidative phosphorylation in calcium (approximately 2 μM free calcium) activated saponin‐skinned rat musculus soleus fibers oxidizing glutamate and malate. Under these circumstances approximately 80% of mitochondrial active‐state respiration was reached due to the activation of ATP turnover by actomyosin ATPase. The flux control coefficients of H+‐ATPase, adenine‐nucleotide translocase, phosphate transporter, NADH:ubiquinone oxidoreductase and cytochrome‐c oxidase were determined to be equal to 0.16 ± 0.08 (n= 6), 0.34 ± 0.12 (n= 5), 0.08 ± 0.03 (n= 5), 0.01 ± 0.006 (n± 4) and 0.09 ± 0.03 (n= 3) using inhibitor titrations with the specific inhibitors oligomycin, carboxyatractyloside, mersalyl, rotenone and cyanide, respectively, and applying non‐linear regression of the entire titration curve. The flux control coefficient of actomyosin ATPase was determined with vanadate to be equal to 0.50 ± 0.09 (n= 6), measuring independently the vanadate‐caused inhibition of fiber respiration and ATP‐splitting activity. In contrast to results with isolated rat skeletal muscle mitochondria reconstituted with soluble F1‐ATPase the decrease in phosphate concentration from 10 mM to 1 mM only slightly affected the distribution of flux control coefficients. This difference is caused by different kinetic properties of soluble F1‐ATPase and actomyosin ATPase. Therefore, phosphate seems to be in skeletal muscle in vivo only a modest modulator of control of oxidative phosphorylation.
Kinetic Analysis of Escherichia Coli Ribonuclease HI Using Oligomeric DNA/RNA Substrates Suggests an Alternative Mechanism for the Interaction between the Enzyme and the Substrate
Authors: Kanaya Eiko , Kanaya Shigenori
Published: March 2005

Escherichia coli ribonuclease HI mainly recognizes the DNA/RNA hybrid regions preceding the cleavage site. To understand the interaction between the enzyme and the substrate in more detail, the kinetic properties of the enzyme, as well as its variant with mutations in the basic protrusion, were studied using a series of oligomeric DNA/RNA hybrids as substrates. These substrates were prepared by hybridizing a 12‐b RNA (5′‐CGGAGAUGACGG‐3′) with DNA oligomers varying in size and sequence. The 12‐b RNA hybridized to the complementary 12‐b DNA was primarily cleaved at A9‐C10. Since an increase in the length of the RNA between the cleavage site and the 5′ end of the DNA/RNA hybrid, achieved using a longer DNA/RNA substrate, did not seriously affect the kinetic parameters of the enzyme, the 12‐bp DNA/RNA hybrid seems to be large enough to contact the entire substrate‐binding site of the enzyme. The kinetic data presented here suggest that the DNA residues complementary to the RNA residues located six or seven residues upstream from the cleavage site interact with the basic protrusion of the enzyme, regardless of whether or not it is hybridized to the RNA strand. Such an interaction is permitted only when the conformation of either the enzyme or the substrate, or both, is changed upon binding.
Prolonged Half‐Life in the Circulation of a Chemical Conjugate Between a Pro‐Urokinase Derivative and Human Serum Albumin
Authors: Breton Jérôme , Pezzi Nieves , Molinari Antonio , Bonomini Luisella , Lansen Jacqueline , De Buitrago Gonzalo Gonzalez , Prieto Ignacio
Published: March 2005

Pro‐urokinase is a natural plasminogen activator that displays a clot‐lysis activity through a fibrin‐dependent mechanism. It seems to be a promising agent for the treatment of coronary thrombosis. Like tissue‐type plasminogen activator and two‐chain urokinase‐type plasminogen activator, pro‐urokinase has a very short half‐life in circulation.

It has been described that conjugation of serum albumin with pro‐urokinase in plasma may occur that could protect this protein from degradation.

In this study we describe the insertion of an extra cysteine residue in the N‐terminal end of des‐(C11–K135)‐pro‐urokinase (Δ125‐proUK), a pro‐urokinase deletion mutant lacking amino acids 11–135. We have expressed and purified the new mutein [H5K, S9C, N10T]des‐(C11‐K135)‐pro‐urokinase (Cys‐Δ125‐pro‐urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys‐Δ125‐pro‐urokinase.

The purified conjugate obtained has a lower specific amidolytic activity (72000 U/mg) than unconjugated Cys‐Δ125‐pro‐urikinase (240000 U/mg) due to its higher molecular mass and has a similar fibrinolytic activity in a clot lysis test to that of Δ125‐pro‐urokinase.

We established an ELISA to measure the concentration of the conjugate in plasma and to follow the pharmacokinetics of the conjugate in monkeys after bolus injection.

The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half‐life in the circulation, with respect to pro‐urokinase and Δ125‐pro‐urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro‐urokinase.
Concerning the Measurement of Flux Control Coefficients by Enzyme Titration
Authors: Giersch Christoph
Published: March 2005

An established method to determine flux control coefficients is the enzyme titration method in which the change in pathway flux upon a change in the enzyme concentration is measured. In this study, the application of this method to a simple reconstituted pathway was investigated by simulated measurements. The pathway was assumed to be in the quasi‐steady‐state, which is the experimental realization of the mathematical construct ‘steady state’. It was shown that flux control coefficients, calculated in a way that mimics their experimental determination, were strongly time dependent. Initially, the calculated flux control coefficient was high for the enzyme adjacent to the reaction monitoring the flux, and the steady‐state value was overestimated. Likewise, flux control coefficients were underestimated for enzymes further away from the monitoring reaction. The observed time course of simulated flux control coefficients was shown to reflect the fact that experimental systems are not steady state but quasi‐steady‐state. For a pathway in the quasi‐steady‐state, some of the problems with enzyme titration experiments can be overcome by allowing the system to relax for a time interval that is large compared with the turnover time of the pooled pathway intermediates.
Epidermal Growth Factor (EGF) Induces Serine Phosphorylation‐Dependent Activation and Calcium‐Dependent Translocation of the Cytosolic Phospholipase A2
Authors: Schalkwijk Casper G. , Spaargaren Marcel , Defize Libert H. K. , Verkleij Arie J. , van den Bosch Henk , Boonstra Johannes
Published: March 2005

Phospholipase A2 (PLA2) is a key enzyme in the release of arachidonic acid and subsequent production of eicosanoids, which play an important role in a variety of biological processes, including mitogenic signalling by epidermal growth factor (EGF). In a previous study [Spaargaren, M. et al. (1992) Biochem J. 287, 37–43] we identified the EGF‐activated PLA2 as being similar to the recently cloned high‐molecular‐mass cytosolic phospholipase A2 (cPLA2), In the present study we demonstrate a rapid transient EGF‐induced activation of this cPLA2 and an EGF‐induced increase in phosphorylation of the cPLA2. The EGF‐induced activation of cPLA2 is reversed upon phosphatase treatment showing phosphorylation‐dependent activation of the cPLA2. No direct association of the cPLA2 to the EGF receptor was detected under conditions where such an association with phospholipase C‐γ was demonstrated. Phosphoamino acid analysis of this cPLA2 showed that EGF induced an increase in serine phosphorylation exclusively, no tyrosine phosphorylation being observed. EGF treatment of the cells resulted in a Ca2+‐dependent translocation of the cPLA2 from the cytosol to the membrane fraction. This is due to an EGF‐induced [Ca2+]irise which is dependent on the influx of extracellular Ca2+ via voltage‐independent Ca2+ channels. It is shown that the Ca2+‐dependent association of cPLA2 to membranes does not require accessory membrane molecules.
Structural Aspects of Bile Acids Involved in the Regulation of Cholesterol 7α‐Hydroxylase and Sterol 27‐Hydroxylase
Authors: Twisk Jaap , Hoekman Marco F. M. , Muller Linda M. , Iida Takashi , Tamaru Tamaaki , Ijzerman Ad , Mager Willem H. , Princen Hans M. G.
Published: March 2005

We have recently reported that coordinate down‐regulation of cholesterol 7α‐hydroxylase and sterol 27‐hydroxylase by bile acids results in suppression of bile acid synthesis in cultured rat hepatocytes [Twisk, J., De Wit, E. & Princen, H. M. G. (1995) Biochem. J. 305, 505–511]. In the current study, we have assessed the effects of a large group of different bile acids, both naturally occurring and synthetic, on these two key enzymes, to elucidate structural features which render bile acids potent as regulators of bile acid synthesis.

Addition of 50 μM deoxycholate or cholate, two relatively hydrophobic bile acids, to the culture medium of hepatocytes resulted in strong suppression of cholesterol 7α‐hydroxylase (suppression of 75% and 88%, respectively) and sterol 27‐hydroxylase activity (suppression of 76% and 72%, respectively). These effects were also reflected in the mRNA levels and the transcriptional activities of the two enzymes, showing a parallel suppression of both parameters in response to cholate (suppression of 78% and 43% for cholesterol 7α‐hydroxylase mRNA and transcription, respectively, and suppression of 76% and 42% for sterol 27‐hydroxylase mRNA and transcription, respectively). In contrast, no effects were observed with the two hydrophilic bile acids, β‐muricholate and ursocholate. Transient expression analysis in cultured rat hepatocytes, using a promoter‐reporter construct containing the proximal part of the cholesterol 7α‐hydroxylase promoter, demonstrated a reduction of transcriptional activity by cholate (reduction of 72%), but not by ursocholate.

Assessment of the effects of 27 different bile acids, varying in the number, position and orientation (α/β) of hydroxyl groups on the steroid nucleus of the molecule, on cholesterol 7α‐hydroxylase mRNA showed only a moderate correlation with the hydrophobicity index of the bile acid involved (r= 0.61; P < 0.0001). Analysis of the three‐dimensional structure of a number of these bile acids suggests that hydroxyl groups situated in close proximity to each other within the molecule, creating a hydrophilic environment, as in the case of cholate, may be a prerequisite for a strong inhibitory potency. Deviation from this situation leads to a markedly lesser effect on suppression of cholesterol 7α‐hydroxylase and sterol 27‐hydroxylase.
Actinomycin D Stimulates the Transcription of rRNA Minigenes Transfected into Mouse Cells
Authors: Hadjiolova Krassimira V. , Hadjiolov Asen A. , Bachellerie Jean‐Pierre
Published: March 2005

The in vivo hypersensitivity of eukaryotic rRNA gene transcription to actinomycin D has long beenknown, but this effect could not be reproduced in model systems and its molecular mechanisms remain uncertain. We studied the action of actinomycin D using mouse rRNA minigenes (with RNA polymerase I promoter and terminator signals), carrying truncated mouse or human rDNA inserts, which are faithfully transcribed upon transient transfection into mouse cells. Low concentrations (0.01–0.08 μg/ml) of actinomycin D caused within 1–2 h a 2–7‐fold stimulation of the transcription of rRNA minigenes which is inversely related to the size of the rDNA transcript. With transcripts longer than 3 kb the effect was reversed and at 4 kb a practically complete inhibition of the formation of full‐length transcripts was observed, accompanied, however, by an enhanced accumulation of unfinished rDNA transcripts. The dependence of actinomycin D action on transcript length was also observed with lacZ gene segments of different size inserted into the mouse rRNA minigenes. The transcription initiation of endogenous rRNA genes was also stimulated by the low doses of actinomycin D as indicated by the enhanced synthesis of unfinished rDNA transcripts (spanning mainly the 5′ external transcribed spacer), whereas the synthesis of full‐length transcripts was abolished.

Removal of actinomycin D from the medium caused within 8–24 h a dramatic increase of the transcription from all rRNA minigenes tested. This stimulation was also inversely related to the size of the transcripts and varied from twofold to fivefold for the 3–4‐kb transcripts to about 50–80‐fold for the basic minigene transcript (395 nucleotides). The amount of endogenous aborted rDNA transcripts was also markedly increased, but the synthesis of full‐length transcripts was not restored even 24 h after removal of the drug.

The present results reproduce in a model cellular system the in vivo hypersensitivity of rRNA gene transcription to actinomycin D and reveal that the major factor involved is the size of the rRNA gene transcript. This effect requires only the basic rRNA gene promoter and terminator signals and does not depend on the G+C content of the RNA polymerase I transcripts. We suggest that at low concentrations, the intercalation of actinomycin D changes the conformation of DNA in the promoter region in a manner that stimulates the transcription of both endogenous and transfected rRNA genes. However, transcriptiondriven positive supercoiling of rDNA is enhanced by actinomycin D and above a critical transcript length it causes a blockage of transcription elongation and the accumulation of aborted rDNA transcripts.
Overproduction in Escherichia Coli, Purification and Properties of Human Prothymosin α
Authors: Evstafieva Alexandra G. , Chichkova Nina V. , Makarova Tatyana N. , Vartapetian Andrey B. , Vasilenko Anna V. , Abramov Vjacheslav M. , Bogdanov Alexey A.
Published: March 2005

A bacterial strain overproducing human prothymosin α was constructed based on the efficient T7 RNA polymerase transcription of human prothymosin α cDNA. The highest yield of the human prothymosin α, up to 30% of the total bacterial protein, was achieved with constructions containing 6–10 nucleotides between the Shine‐Dalgarno sequence and initiation ATG codon. Unexpectedly, cells grown in the presence of inducer of T7 RNA polymerase synthesis produced substantially lower levels of prothymosin α than those grown in the absence of inducer. A simple procedure for prothymosin α isolation was elaborated, resulting in large amounts of electrophoretically pure and immunoactive protein.
The Molybdenum Nitrogenase from Wild‐type Xanthobacter autotrophicus Exhibits Properties Reminiscent of Alternative Nitrogenases
Authors: Schneider Klaus , Müller Achim , Krahn Eugen , Hagen Wilfred R. , Wassink Hans , Knüttel Karl‐Heinz
Published: March 2005

In the presence of molybdate (1 μM) 2–3.5% oxygen and with sucrose as carbon source, Xanthobacter autotrophicus GZ29, a microaerophilic nitrogen‐fixing hydrogen‐oxidizing bacterium, grew diazotrophically with a minimal doubling time of 2.5 h and a calculated absorbance of up to 52 (546 nm).

The maximal specific activity obtained was 145 nmol ethylene reduced ċ min−1· mg protein−1 (crude extract). The Mo nitrogenase was derepressed to a comparable level with methionine as nitrogen source. Vanadium compounds stimulated neither growth nor nitrogenase activity. Without added molybdate, diazotrophic growth and nitrogenase activity decreased to an extremely low level. The nitrogenase, responsible for the residual activity in molybdate‐starved cells, contained molybdate but no other heterometal atom. These results indicate that, in X. autotrophicus, a Mo‐independent nitrogenase does not exist. However, the molybdate‐containing nitrogenase exhibited some properties which are reminiscent of alternative nitrogenases.

The MoFe protein (component 1, Xa1) copurified with two molecules of a small, not previously detected polypeptide (molar mass 13.6 kDa) and was able to reduce acetylene not only to ethylene but also partly to ethane. Under certain conditions, i.e. in Tris/HCl buffer at alkaline pH values, with titanium (III) citrate as electron donor, at high component 1/component 2 ratios, and at low, non‐saturating acetylene concentrations, up to 5.5% ethane was measured. Parallel to the pH‐dependent increase of the relative yield of ethane, the total activity (both acetylene and nitrogen reduction rates) decreased and the S =3/2 FeMo cofactor ESR signal was split into three signals with different rhombicities [E/D values of 0.036 (signal I), 0.072 (signal II) and 0.11 (signal III)]. The intensities of the two new FeMo cofactor signals were more pronounced the more alkaline the pH. They could be further enhanced using titanium (III) citrate instead of Na2S2O4 as reductant.
14CO2 Exchange with Acetoacetate Catalyzed by Dialyzed Cell‐Free Extracts of the Bacterial Strain Bunn Grown with Acetone and Nitrate
Authors: Janssen Peter H. , Schink Bernhard
Published: March 2005

The nitrate‐reducing bacterial strain BunN is able to grow with acetone and nitrate under anoxic conditions. Dialyzed crude cell‐free extracts of acetone‐plus‐nitrate‐grown cells of strain BunN catalyzed the exchange of 14CO2 into acetoacetate in an ADP‐dependent reaction. The rates of exchange catalyzed by extracts of acetate‐grown or 3‐hydroxybutyrate‐grown cells were only 13% of that catalyzed by extracts of acetone‐grown cells. The activity was enzymic since it was destroyed by boiling and was proportional to the amount of added extract. The optimal acetoacetate concentration was 100 mM and the apparent Km was 11.1 mM. The pH optimum was 6.5, the exchange was not dependent on the addition of biotin, and the activity was not inhibited by avidin. The exchange activity was not stimulated (less than two fold) by a variety of metal ions or by a range of possible cofactors. Under optimal conditions (100 mM acetoacetate, 5 mM ADP, 10 mM NaHCO3, pH 6.5, under N2), the exchange activity was 2.7 nmol · min−1· mg protein−1; 2% of the in vivo carboxylation activity of acetone‐plus‐nitrate‐grown cultures. It is suggested that the exchange reaction is a partial reaction catalyzed by the enzyme (or enzyme complex) that carboxylates acetone, and that the methods developed in this study provide a means with which to investigate this reaction further.
Oligosaccharyl Transferase is a Constitutive Component of an Oligomeric Protein Complex from Pig Liver Endoplasmic Reticulum
Authors: Breuer Wilhelm , Bause Ernst
Published: March 2005

Oligosaccharyl transferase (OST), an intrinsic component of the endoplasmic reticulum membrane, catalyses the N‐glycosylation of specific asparagine residues in nascent polypeptide chains. We have purified the enzyme from crude pig liver microsomes by a procedure involving salt/detergent extraction, concanavalin‐A precipitation, S‐Sepharose, MonoP and concanavalin‐A–Sepharose chromatographies. A highly purified OST preparation exerting catalytic activity, contained two protein subunits of 48 kDa and 66 kDa, from which the 66‐kDa species was identified by immunoblotting as ribophorin I. The function of ribophorin I in this dimeric protein complex is unknown. The high degree of similarity between its transmembrane region and a putative dolichol‐recognition consensus sequence suggests that ribophorin I could be involved in glycolipid binding and delivery. Several lines of evidence indicate that the catalyti‐cally active 48‐kDa/66‐kDa polypeptides are associated in the endoplasmic reticulum membrane with other proteins, including ribophorin II and a 40‐kDa glycoprotein. The implication of ribophorins I and II in the translocation machinery and their apparent association with the OST activity point to a close relationship between polypeptide synthesis, translocation and N‐glycosylation, both spacially and temporally.

Kinetic studies with the MonoP‐purified Oligosaccharyl transferase showed that the enzyme transfers dolichyl‐diphosphate‐linked GlcNAc2 to synthetic tripeptides and hexapeptides, containing the Asn‐Xaa‐Thr motif, at a comparable rate. The glycosylation reaction was found to have a pH optimum close to 7 and to require divalent metal ions, with Mn2+ being most effective. Substitution of threonine in the N‐ glycosylation motif by serine impairs its function as an acceptor, measured by Vmax/Km, by approximately 17‐fold, consisting of a 7.3‐fold increase in Km and a 2.3‐fold decrease in Vmax. This indicates that the side chain structure of the hydroxyamino acid influences both binding and catalysis, consistent with previous studies highlighting its participation in the catalytic mechanism of transglycosylation. The Km values of peptide acceptors improved significantly when dolichyl‐phosphate‐bound oligosaccharides were used instead of lipid‐linked GlcNAc2 as the glycosyl donor. We conclude from this observation that the sugar residues on the outer branches of the glycolipid donor induce conformational changes in the active site of the Oligosaccharyl transferase, thus influencing the association constant of the peptide substrate.
Evidence for the Tissue Inhibitor of Metalloproteinases‐1 (TIMP‐1) in Human Polymorphonuclear Leukocytes
Authors: Triebel Susanne , Bläser Jörg , Gote Thomas , Pelz Günter , Schüren Elisabeth , Schmitt Manfred , Tschesche Harald
Published: March 2005

A tissue inhibitor of metalloproteinases (TIMP)‐1 was isolated from human polymorphonuclear leukocytes (PMNL) in a complex with latent 95‐kDa gelatinase (matrixmetalloproteinase, MMP‐9). It was separated from the enzyme by gel fitration in the presence of SDS. Using a competitive ELISA procedure, we determined that 10% of the isolated gelatinase was complexed with TIMP‐1. The presence of the inhibitor in isolated PMNL could also be demonstrated by indirect immunofluorescence using a specific antibody against TIMP‐1. Cellular mRNA was isolated from PMNL, which were highly purified by separation via formylMet‐Leu‐Pro‐stimulated chemotactic migration in a Boyden chamber. Using reverse‐transcription PCR and Nothern blotting, TIMP‐1 mRNA was shown to be present in PMNL, suggesting that these cells are also capable of synthesizing TIMP‐1.
The Gag Homologue of Retrotransposon Ty1 Assembles into Spherical Particles in Escherichia coli
Authors: Luschnig Christian , Hess Michael , Pusch Oliver , Brookman Jayne , Bachmair Andreas
Published: March 2005

Expression of TyA (reading frame A) of the yeast retrotransposon Ty1 in Escherichia coli is possible by using efficient transcriptional and translational initiation signals. When expressed in E. coli, the gag homologue of Ty1 assembles into spherical particles similar, but not identical to virus‐like particles in the natural host of Ty1, Saccharomyces cerevisiae. Deletion analysis reveals a domain in the C‐terminus of TyA that is essential for the assembly process. These findings indicate that an early step of the retroele‐ment life cycle, assembly of the gag homologue into spherical particles, does not depend on specific host factors. The experiments also demonstrate that Ty1 Gag fusion proteins, potential tools for immunization, can be produced in E. coli, an organism that lacks endogenous retrotransposons.
A Calmodulin‐binding Sequence in the C‐terminus of Human Cardiac Titin Kinase
Authors: Gautel Mathias , Morelli Maria A. Castiglione , Pfuhl Mark , Motta Andrea , Pastore Annalisa
Published: March 2005

The giant muscle proteins of the titin family, which are specific for the striated muscles of vertebrates and invertebrates, contain as a common feature a catalytic protein kinase domain of so far unclear function and regulation. In myosin light chain kinase, a family evolutionarily related to titin, kinase regulation is achieved by calmodulin binding to a region of the kinase C‐terminus which bears similarity to the substrate. A calmodulin‐binding sequence has also been identified in the C‐terminus of the Aplysia twitchin kinase. In analogy, we identified a putative calmodulin‐binding site in the titin kinase C‐terminal sequence. The expressed catalytic domain itself and a series of synthetic peptides from this region were tested for their ability to bind calmodulin. Biochemical data indicate that titin kinase as well as peptides from its C‐terminus bind to calmodulin in an equimolar complex in the presence of calcium. The interaction of truncated peptides with calmodulin is, however, weaker than that of myosin light chain kinase. Nuclear magnetic resonance studies showed that these peptides have a tendency to adopt α‐helical conformations in solution. Helicity increases upon binding of calmodulin in a calcium‐dependent fashion, as judged by circular dichroism spectra. We, therefore, propose that this calmodulin‐binding region of titin could play a regulatory role for the enzyme, the substrate of which still remains to be identified.
13C‐NMR Spectroscopy of Keratan Sulphates
Authors: Huckerby Thomas N. , Brown Gavin M. , Nieduszynski Ian A.
Published: March 2005

Skeletal keratan sulphate has been fragmented using the enzyme keratanase II, and the complete 13C chemical shift data are reported for the four tetrasaccharides which derived from the repeat region. They have the structures:

Galβ(1–4)GlcNAc(6S)β(1–3) Galβ(1–4)GlcNAc(6S)‐ol, Gal(6S)β(1–4)GlcNAc(6S)β(1–3)Gal(1–4)GlcNAc(6S)‐ol, Galβ(1–4)GlcNAc(6S)β(1–3)Gal(6S)β(1–4)GlcNAc(6S)‐ol, and Gal(6S)β(1–4)GlcNAc(6S)(1–3)Gal(6S)β(1–4)GlcNAc(6S)‐ol, where GlcNAc(6S)‐ol represents N ‐acetyl‐glucosaminitol‐6‐O ‐sulphate. The value of these data for microstructural analysis of undegraded keratan sulphate samples using 13C‐NMR spectroscopy is discussed with respect to galactose sulphation levels and the block sulphation structure.
Limited Proteolysis of Lysozyme in Trifluoroethanol
Authors: De Laureto Patrizia Polverino , De Filippis Vincenzo , Scaramella Elena , Zambonin Marcello , Fontana Angelo
Published: March 2005

Proteolysis of hen egg‐white lysozyme by thermolysin in 50% aqueous trifluoroethanol for 6–24 h at 40–52 °C produces a ‘nicked’ protein species which was purified to homogeneity by reverse‐phase HPLC and characterized. Protein chemistry analytical methods were used to establish that thermolysin cleaves the 129‐residue chain of lysozyme at peptide bond Lys97‐Ile98. Nicked lysozyme, which is therefore constituted by fragments 1–97 and 98–129 cross‐linked by disulfide bonds, was approximately 20% and 60% active towards Micrococcus luteus cells in respect to native intact lysozyme when assayed at 25°C or 5°C, respectively. Circular dichroic measurements provided evidence that nicked lysozyme in aqueous buffer at low temperature maintains the secondary structure content of native lysozyme, whereas the microenvironment of the aromatic chromophores, in particular of tryptophan residue(s), was somewhat perturbed. The stability to heat and urea denaturation of nicked lysozyme was dramatically reduced with respect to that of the intact protein. For example, the tm of the nicked species was 28°C in comparison with 73°C for the unmodified enzyme, both at pH 7.0. Inspection of the X‐ray structure of hen lysozyme reveals that thermolysin cleaves at the C‐terminus of α‐helix C (residues 88–98) located at the interface of the two structural domains of the protein, thus destabilizing the helix dipole and disrupting important tertiary interactions of the native enzyme. These results were interpreted considering that lysozyme in 50 % aqueous trifluoroethanol is an expanded and flexible protein species largely maintaining native‐like secondary structure, but lacking tertiary interactions [Buck, M., Radford, S. E. & Dobson, C. M. (1993) Biochemistry 32, 669–678]. Thus, whereas native lysozyme in its well‐packed and rigid structure is quite resistant to proteolysis and only upon thermal unfolding is degraded to many small peptides in an all‐or‐none process, lysozyme in the trifluoroethanol state is sufficiently flexible to act as a substrate for the protease, but maintains significant secondary structure (helix) precluding extensive proteolytic degradation.
Structures of the two Polymers Present in the Lipopolysaccharide of Burkholderia (Pseudomonas) Cepacia Serogroup O4
Authors: Cox Andrew D. , Taylor Catherine J. , Anderson Alistair J. , Perry Malcolm B. , Wilkinson Stephen G.
Published: March 2005

Like several other strains of Burkholderia (Pseudomonas) cepacia, the reference strain for serogroup 04 in the French typing scheme [Heidt, A., Monteil, H. & Richard, C. (1983) J. Clin. Microbiol. 18, 738–740] produces a lipopolysaccharide containing two distinct polymers. Attempts to separate the polymers chromatographically were unsuccessful, but the periodate‐resistant major polymer could be isolated by application of the Smith degradation technique to the mixture. By means of chemical and NMR spectroscopic analyses, the following structure could be assigned to the repeating unit of the major polymer:

→3)‐α‐d‐Galp ‐(1→3)‐β‐d‐Galp ‐(1→3)‐β‐d‐Galp NAc‐(1→.

The following structure of the repeating unit of the minor polymer was established from similar studies of its degradation product, resulting from the oxidation of L‐rhamnose (Rha), and of the original mixture:

→3)‐α‐d‐Galp NAc‐(1→3)‐β‐d‐Galp NAc‐(1→4)‐α‐L‐Rhap‐(1→.

Individually, the polymers have recently been found in related strains of B. cepacia. The minor polymer was identified as the O‐antigen in serotype A of a Canadian typing scheme [Beynon, L. M. & Perry, M. B. (1993) Biochem. Cell Biol. 71, 417–420], and the major polymer in serotype C of a Japanese typing scheme [Paramonov, N. A., Shashkov, A. S., Knirel, Y. A., Soldatkina, M. A. & Zakharova, I. Y. (1994) Bioorg. Khim. 20, 984–993]. In the case of the O4 strain studied here, both polymers were produced under a variety of growth conditions.
Peroxisome Proliferators Differentially Regulate Long‐chain Acyl‐CoA Thioesterases in Rat Liver
Authors: Svensson L. Thomas , Wilcke Mona , Alexson Stefan E. H.
Published: March 2005

We have investigated the effects of peroxisome proliferators on rat liver long‐chain acyl‐CoA thioesterase activities. Subcellular fractionations of liver homogenates from control, clofibrate‐ and di(2‐ethylhexyl)phthalate‐treated rats confirmed earlier studies which demonstrated that peroxisome‐proliferating drugs induce long‐chain acyl‐CoA thioesterase activity mainly in the mitochondrial and cytosolic fractions. The aim of the present study was to investigate whether the induced activities were due to increases in normally expressed enzymes, or due to induction of novel enzymes. To investigate whether structurally different peroxisome proliferators differentially induced thioesterase activities, we tested the effects of di(2‐ethylhexyl)phthalate (a plastisizer) and the hypolipidemic drug clofibrate. For this purpose, we established an analytical size exclusion chromatography method. Chromatography of solubilised mitochondrial matrix proteins showed that the activity in control mitochondria was mainly due to enzymes with molecular masses of about 50 kDa and 35 kDa. The activity in samples prepared from clofibrate‐ and di(2‐ethylhexyl)phthalate‐treated rats eluted as proteins of about 40 kDa and 110 kDa. Highly purified peroxisomes contained two peaks of activity, which were not induced, that corresponded to molecular masses of 40 kDa and 80 kDa. The 80‐kDa peak was shown to be due to dimerization by addition of glycerol. Chromatography of cytosolic fractions from control rat livers indicated the presence of long‐chain acyl‐CoA thioesterases with molecular masses of approximately 35 kDa and 125 kDa and a broad peak corresponding to a high‐molecular‐mass protein. The activity in cytosolic fractions from peroxisome‐proliferator‐treated rats eluted mainly as peaks corresponding to 40, 110 and 150 kDa. In addition, in the 110‐kDa peak, a different degree of induction and different chain‐length specificities were caused by clofibrate and di(2‐ethylhexyl)phthalate, suggesting that these peroxisome proliferators differentially regulate the cytosolic acyl‐CoA thioesterase activities. Western blot analysis showed that enzymes in the 40‐kDa peak of the peroxisomal and cytosolic fractions were structurally related, but not identical, to a 40‐kDa mitochondrial very‐long‐chain acyl‐CoA thioesterase.

Our data show that the increased acyl‐CoA thioesterase activities in mitochondria and cytosol were mainly due to induction of acyl‐CoA thioesterases which are not, or only weakly, expressed under normal conditions.
Purification and Molecular Cloning of a Major Antibacterial Protein of the Protozoan Parasite Entamoeba Histolytica with Lysozyme‐Like Properties
Authors: Jacobs Thomas , Leippe Matthias
Published: March 2005

A protein with potent antibacterial activity was purified to apparent homogeneity from pathogenic Entamoeba histolytica. It resembles lysozyme in that it is a basic protein which degrades cell walls of Micrococcus luteus, displays optimal activity at acidic pH, and shows a preference for Gram‐positive bacteria. The protein has a molecular mass of ≈23 kDa upon SDS/PAGE and is localized inside the cytoplasmic granules of the amoebae. The primary structure was elucidated by protein analysis and molecular cloning of the corresponding cDNA. It yielded a protein of 198 residues with structural similarity to the distinct class of lysozymes found in Streptomyces species and the fungus Chalaropsis.
Expression and Characterization of Geotrichum candidum Lipase I Gene
Authors: Bertolini Maria Celia , Schrag Joseph D. , Cygler Miroslaw , Ziomek Edmund , Thomas David Y. , Vernet Thierry
Published: March 2005

Despite tremendous progress in the elucidation of three‐dimensional structures of lipases, the molecular basis for their observed substrate preference is not well understood. In an effort to correlate the lipase structure with its substrate preference and to clarify the contradicting reports in the literature, we have compared the enzymic characteristics of two closely related recombinant lipases from the fungus Geotrichum candidum. These enzymes were expressed in the yeast Saccharomyces cerevisiae as fusions with an N‐terminal poly(His) tag and were purified in a single step by metal‐affinity chromatography. Their specific activities against a series of triacylglycerol substrates were compared using a titrimetric assay. The substrates varied in fatty acyl chain length, number of double bonds and their position along the chain. G. candidum lipases I and II (GCL I and GLC II) are markedly different with respect to their substrate preferences. For unsaturated substrates having long fatty acyl chains (C18:2cis ‐9, cis ‐12 and C18:3cis ‐9, cis ‐12, cis‐15), GCL I showed higher specific activity than GCL II, whereas GCL II showed higher specific activity against saturated substrates having short fatty acid chains (C8, C10, C12 and C14). We have constructed a hybrid molecule containing the N‐terminal portion of GCL I (including the flap covering the active site) linked to the C‐terminal portion of GCL II. The hybrid molecule showed a substrate preference pattern identical to that of GCL II. These results indicate that sequence variation within the N‐terminal 194 amino acids of G. candidum lipases do not contribute to the observed variation in efficiency by which the lipases hydrolyze their substrates. Moreover, it also shows that the flap region in GCL is not directly involved in substrate differentiation, even though this region is thought to be involved in recognition of the interface and in the activation of the enzyme.
Hypothyroidism Affects the Expression of the β‐F1‐ATPase Gene and Limits Mitochondrial Proliferation in Rat Liver at All Stages of Development
Authors: Izquierdo José M. , Jiménez Eugenio , Cuezva José M.
Published: March 2005

In order to analyze the role of thyroid hormones in mitochondrial biogenesis, we have studied the expression pattern of the β subunit of the mitochondrial ATP‐synthase complex in liver and in isolated mitochondria during postnatal development of hypothyroid rats. Chemically induced hypothyroidism promoted a significant reduction in body and liver masses at all stages of development. Furthermore, plasma 3,5,3′‐triiodo‐l‐thyronine (T3) and 3,5,3′,5′‐tetraiodo‐l‐thyronine (T4) concentrations were significantly reduced in hypothyroid animals when compared to euthyroid animals. Remarkably, steady‐state β‐F1‐ATPase mRNA levels in livers of hypothyroid animals showed an ≈ 50% reduction when compared to age‐matched euthyroid rats at all stages of development. The relative amounts of β‐F1‐ATPase protein determined in isolated mitochondria of 1‐day‐old and adult hypothyroid animals were similar to those determined in mitochondria of age‐matched euthyroids, indicating that hypothyroidism does not affect organelle differentiation in the liver of suckling and adult rats. In contrast, the relative amount of β‐F1‐ATPase protein in liver homogenates varied (0–30% reduction) due to the hypothyroid condition during development. These findings suggest the existence of compensatory mechanisms operating at the translational and/or post‐translational levels which promote proliferation of mitochondria in the hypothyroid liver. However, when the liver mass was considered, hypothyroidism significantly reduced overall mitochondrial proliferation in rat liver. Interestingly, the effects of thyroid hormones on the biogenesis of the ATP synthase complex at latter stages of development provide an example in which the hypothyroid condition limits the expression of the nuclear‐encoded gene with no apparent effect on the expression of the mitochondrial‐encoded genes (ATP synthase subunits 6–8).
The pH Dependence and Modification by Diethyl Pyrocarbonate of Isocitrate Lyase from Phycomyces blakesleeanus
Authors: Rúa Javier , Soler Joaquín , Busto Félix , De Arriaga Dolores
Published: March 2005

We determined the variation with pH of the kinetic parameters for the isocitrate cleavage reaction catalyzed by Phycomyces isocitrate lyase, with the aim of elucidating the role played by ionising amino acid residues in binding and catalysis. The log VmaXpH profile shows that the enzyme possesses two ionising groups with pK values of 6.1 and 8.3. The first group is also observed in the VmaxpH/KmpH and pKmpH profiles, so this group is involved in catalysis. The last two profiles exhibit a similar pK value of 16 on the basic side, which represents the sum of the pK values for two ionising groups with pK values that differ by less than two pH units.

Diethyl pyrocarbonate inactivated isocitrate lyase from Phycomyces with a second‐order rate constant of 18.58M−1 s−1 (at pH 6.0 and 20°C). The difference spectra of the modified enzyme revealed an absorption maximum at 242 nm, characteristic of N‐carbethoxyhistidine isocitrate lyase. No trough at around 280 nm due to O‐carbethoxytyrosine is observed. Quantification of the increase in absorbance to 242 nm due to N‐carbethoxyhistidine showed that ten histidine residues/active site were modified during total inactivation. However, only one of them was essential for catalysis. Treatment of the partially inactivated enzyme with hydroxylamine led to recovery of a substantial part of the original activity. The reactivity of isocitrate lyase towards diethyl pyrocarbonate declined with pH, following a titration curve for a group of pK 6.1. The presence of substrate decreased the rate of inactivation. Data‐protection analyses indicate that the reactive histidine residues are within the active site of the enzyme.
A New Approach to Isolate Genomic Control Regions
Authors: Joulin Virginie , Richard‐Foy Hélène
Published: March 2005

We have designed a new strategy to isolate unknown DNA regions interacting with one or several related regulatory proteins. It involves trapping such DNAs by their cognate binding proteins followed by PCR amplification, as described previously [Kinzler, K. & Vogelstein, B. (1989) Nucleic Acids Res. 17, 3645–3653]. To overcome the inability of such a procedure to discriminate between functional and non‐functional binding sites as well as to specifically trap short DNA motifs from the whole higher eukaryotic genome, we have used as starting material DNA isolated from transcriptionally competent chromatin fractions, instead of total genomic DNA. To test our strategy, we selected human DNA sequences that bind members of the GATA family, known to recognize similar WGATAR motifs. These proteins are expressed in different cell types in which they regulate the transcription of different sets of genes; thus, transcriptionally active chromatin containing GATA motifs should differ according to the cell type. We have trapped and analyzed DNA fragments isolated from an active chromatin fraction, from erythroid cells and lymphoid cells, using GATA‐1 and GATA‐3 proteins, respectively. We show that regulatory GATA sequences known to be in open chromatin in erythroid cells (typified by the HSIII fragment of the β‐globin locus control region) or in lymphoid cells (typified by a fragment of the CD2 locus control region) are dramatically enriched in a cell‐specific manner, demonstrating the potency of the method. The sequences of the erythroid or lymphoid DNA fragments isolated through the procedure described here were determined and display subset‐site preference for GATA‐1 and GATA‐3.