bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Form of Mouse Cytochrome P 450 3A (Cyp3a‐16)
Authors: Itoh Susumu , Satoh Mitsuharu , Abe Yoko , Hashimoto Hisashi , Yanagimoto Tom , Kamataki Tetsuya
Published: March 2005

A complementary DNA clone coding for a novel form of cytochrome P 450, Cyp3a‐16, in mouse fetal livers was isolated and completely sequenced. This clone encoded a polypeptide of 504 deduced amino acids and showed 87.3% and 66.6% amino acid identities with mouse Cyp3a‐11 and Cyp3a‐13, respectively. Cyp3a‐16 transcript was detectable before birth and remarkably diminished five weeks after birth in mice. We conclude that Cyp3a‐16 is a fetal‐ and puberty‐specific cytochrome P 450 in mice.
Reconstitution of adipokinetic hormone biosynthesis in vitro indicates steps in prohormone processing
Authors: RAYNE Richard C. , O'SHEA Michael
Published: March 2005

We have used a complete, synthetic precursor to adipokinetic hormone I (AKH I) and oligopeptides derived from this precursor as substrates for prohormone‐processing enzymes extracted from AKH‐synthesizing neurosecretory cells to reconstitute the post‐translational steps in AKH biosynthesis in vitro. The results demonstrate the existence of endoproteolytic activity which cleaves the precursor only at the appropriate processing site (at the C‐terminal side of Arg13). Further proteolytic processing of C‐terminally extended AKH I (AKH‐Gly‐Lys‐Arg) by a carboxypeptidase H‐like activity removes the basic residues producing AKH‐Gly‐Lys, followed by AKH‐Gly. Finally, a peptidylglycine‐α‐amidating‐monooxygenase activity produces the amidated bioactive product from the glycine‐extended peptide in a two‐step process, the first of which requires ascorbate and Cu2+. Our results show that all steps in AKH precursor processing can be reconstituted and studied in vitro, providing a system to characterize the processing enzymes and to investigate the development of enzyme inhibitors for use as potential insecticides.
Electron transfer reaction of stellacyanin at a bare glassy carbon electrode
Authors: IKEDA Osamu , SAKURAI Takeshi
Published: March 2005

Direct (unmediated) electrochemistry of Rhus vernicifera stellacyanin at a glassy carbon electrode has been briefly investigated in phosphate buffer. The voltammetry was practically independent of the buffer concentration, suggesting that the interaction between stellacyanin and the glassy carbon electrode is mainly realized through the hydrophobic interaction. The quasi‐reversible process was found to be diffusion controlled at a sweep rate < 80 mVs−1. From stellacyanin concentration dependence, a transformation from linear diffusion to radial diffusion was observed. Two‐step voltammetry was affected by translocations of the active site (rotation of the protein molecule on the electrode surface and diffusion). Activation energies for reduction and oxidation processes were determined to be 24 kJmol−1 and 54 kJmol−1, respectively, by the stationary method, and 15 kJmol−1 and 66 kJmol−1, respectively, by cyclic voltammetry. The considerable difference in the activation energies is supposedly due to the reduction and oxidation which are performed utilizing different electron‐transfer pathways or because only one electron‐transfer pathway (probably through His92) is significantly changed during the reorganization between the oxidized and reduced forms. The fact that the diffusion constant estimated from one‐step voltammetry (Dox= 4.2 × 10−9 cm2 s−1 for 484 μM stellacyanin) is much smaller than that determined from cyclic voltammetry (7.5 × 10−7 cm2 s−1) derives from the fact that motions of the stellacyanin molecule (rotation leading to translocation of the active site and diffusion) are not fast enough to allow data from potential step voltammetry to be treated as the reversible process, but are fast enough to allow data from cyclic voltammetry to be treated as a diffusion‐controlled process.
ATP‐dependent DNA renaturation and DNA‐dependent ATPase reactions catalyzed by the Ustilago maydis homologous pairing protein
Authors: KMIEC Eric B. , HOLLOMAN William K.
Published: March 2005

Purification of the ATP‐dependent homologous pairing activity from Ustilago maydis yields a protein preparation that is enriched for a 70‐kDa polypeptide as determined by SDS‐gel electrophoresis. The protein responsible for the ATP‐dependent pairing activity, using renaturation of complementary single strands of DNA as an assay, has a Stokes radius of 3.6 nm and a sedimentation coefficient of 4.3 S consistent with the interpretation that the activity arises from a monomeric globular protein of 70 kDa. Including heparin–agarose and FPLC gel filtration chromatography steps in the previously published protocol improves the purification of the protein. ATP and Mg2+ are necessary cofactors for optimal DNA renaturation activity. ADP inhibits the reaction. Analysis of the ATP‐dependent renaturation kinetics indicates the reaction proceeds through a first‐order mechanism. The protein has an associated DNA‐dependent ATPase as indicated by co‐chromatography with the purified ATP‐dependent renaturation activity through an FPLC gel‐filtration column. Single‐stranded DNA and Mg2+ are required for optimal ATP hydrolytic activity, although a number of other polynucleotides and divalent cations can substitute to varying degrees. Hydrolysis of ATP is activated in a sigmoidal manner with increasing amounts of the protein. At ATP concentrations below 0.1 mM the ATPase activity exhibits positive cooperativity as indicated from the Hill coefficient of 1.8 determined by steady‐state kinetic analysis of the reaction. ADP and adenosine 5′‐[β,γ‐imido]triphosphate are inhibitors of the ATPase activity although they appear to exert their inhibitory effects through different modes. These results are interpreted as evidence for protein–protein interactions.
Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain
Authors: RILEY Lisa G. , JUNIUS F. Keith , SWANTON Michael K. , VESPER Natalie A. , WILLIAMS Neal K. , KING Glenn F. , WEISS Anthony S.
Published: March 2005

Association of the human c‐Jun and c‐Fos proteins depends upon interactions involving their leucine zipper domains. We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs. With this goal in mind, we have developed a bacterial expression system for the efficient production of both unlabelled and isotopically labelled c‐Jun leucine zipper domain. A synthetic junLZ gene was created by annealing, ligation, and polymerase‐chain‐reaction amplification of overlapping synthetic oligonucleotides which comprised 132 bp of coding sequence encompassing residues Arg276–Asn314 of c‐Jun plus a total of five engineered non‐native residues at the N‐ and C‐termini. The junLZ gene was cloned into the pGEX2T vector from which recombinant c‐Jun leucine zipper domain (rJunLZ; 46 residues, 5.1 kDa) was overexpressed (∼15% total cell protein) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carboxy‐terminal portion of Schistosoma japonicum glutathione Stransferase. Two markedly different expression strategies have been devised which allow purification of rJunLZ from the soluble or inclusion‐body fraction of induced cells. We have used these strategies to produce unlabelled and uniformly 15N‐labelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled‐coil of parallel α‐helices. We also present 15N‐NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high‐resolution structure of the homodimeric Jun leucine zipper using heteronuclear three‐dimensional NMR spectroscopy.
Site‐specific mutagenesis of residues in the human C5a anaphylatoxin which are involved in possible interaction with the C5a receptor
Authors: BUBECK Peter , GRÖTZINGER Joachim , WINKLER Monica , KÖHL Jörg , WOLLMER Axel , KLOS Andreas , BAUTSCH Wilfried
Published: March 2005

To check and clarify existing data on receptor‐interacting residues in the human C5a anaphylatoxin, we tested mutant C5a proteins obtained by site‐directed mutagenesis of a recombinant human C5a (rhC5a) cDNA clone for structural and functional integrity. Amino acid positions in three different regions of the molecule were investigated: Arg74 at the C‐terminus, Arg40 and Pro45 located in the core region, and Lys14 and Lys19, Lys20 in the N‐terminus. Des‐Arg74‐rhC5a displayed only a residual 3–4% functional activity in the myeloperoxidase‐release assay from human granulocytes while retaining the three‐dimensional solution structure of wild‐type (wt)–rhC5a as shown by circular dichroism (CD) spectroscopy. Des‐Arg74‐rhC5a was able to activate the human C5a receptor transiently expressed in Xenopus oocytes, but was inactive in the heterologous guinea pig (gp) ileum‐contraction assay. These results reveal profound differences between the guinea pig and human C5a‐receptor ligand‐binding characteristics. Exchange of the core residue Arg40 by a glycine did not significantly affect functional C5a activity, in contrast to a previous observation [Mollison, K. W., Mandecki, W., Zuiderweg, E. P., Fayer, L., Fey, T. A., Krause, R. A., Conway, R. G., Miller, L., Edalji, R. P., Shallcross, M. A., Lane, B., Fox, J. L., Greer, J. & Carter, G. W. (1989) Identification of receptor‐interacting residues in the inflammatory complement protein C5a by site‐directed mutagenesis, Proc. Natl Acad. Sci. USA 86, 292–296], nor did exchange of the conserved Pro45 residue by the C3a analogue glutamic acid, a mutation expected to alter the whole geometry of the loop connecting helix III – helix IV (including Arg40) of the C5a molecule. Thus, participation of this loop in receptor interaction appears unlikely. While exchange of the N‐terminal Lys14 residue by alanine did not significantly affect functional activity, a double replacement of Lys19 and Lys20 by alanine residues reduced activity more than 30‐fold. These results confirm Lys19 and/or Lys20 as a putative receptor‐interacting site, although we could not obtain a CD spectrum of this important mutant due to poor expression.
Study of tissue‐type plasminogen activator binding sites on fibrin using distinct fragments of fibrinogen
Authors: GRAILHE Patrick , NIEUWENHUIZEN Willem , ANGLÉS‐CANO Eduardo
Published: March 2005

It is well established that tissue‐type plasminogen activator (t‐PA) binds to the D region of fibrin(ogen) and that two distinct CNBr fragments of fibrinogen (FCB), FCB‐2 and FCB‐5, comprising parts of this region, stimulate plasminogen activation by t‐PA. In the present work, ligand‐binding studies were performed to characterize the interactions between t‐PA and the corresponding fibrin regions using a well defined model of a fibrin surface and both FCB‐2 and FCB‐5 in liquid and solid phase. Binding isotherms showed a characteristic Langmuir adsorption saturation profile. The dissociation constants determined for the binding of t‐PA to immobilized FCB‐2 (Kd= 0.70±0.10 nM) and FCB‐5 (Kd= 0.47 ± 0.08 nM) were of the same order of magnitude as the Kd for fibrin binding (Kd= 1 ± 0.2 nM). The specificity of the binding was demonstrated by the ability of soluble FCB‐2 and FCB‐5 to inhibit t‐PA binding to solid‐phase fibrin (Ki= 3.3 μM and 6.4 μM, respectively). The binding of t‐PA to fibrin and to immobilized FCB‐2 was partially inhibited by the lysine analogue 6‐aminohexanoic acid (Ki= 123 ± 47 μM and 364 μM, respectively) but was not modified by carboxypeptidase B, thus indicating involvement of internal lysine residues. Removal of lysine residues by treatment with, successively, plasmin and carboxypeptidase B, produced only a partial inhibition of t‐PA binding, thus confirming the existence of both a lysine‐dependent and a lysine‐independent mechanism of binding of t‐PA to both fibrin and FCB‐2. In contrast, the binding of t‐PA to FCB‐5 was not significantly affected by 6‐aminohexanoic acid. Altogether, these data indicate that the mechanism of binding of t‐PA to fibrin involves mainly a lysine‐independent interaction with the D region which is contributed by sequences present in FCB‐5 and FCB‐2; contribution to binding by a lysine‐dependent interaction was detected only in FCB‐2 and is probably of minor relevance as suggested by the limited effect of 6‐aminohexanoic acid.
The catalytic properties of the reverse transcriptase of the lentivirus equine infectious anemia virus
Authors: RUBINEK Tami , LOYA Shoshana , SHAHARABANY Miriam , HUGHES Stephen H. , CLARK Patrick K. , HIZI Amnon
Published: March 2005

The reverse transcriptase (RT) of equine infectious anemia virus (EIAV) shares sequence similarity with the RTs of other lentiviruses, particularly with the RTs of human immunodeficiency viruses types 1 and 2 (HIV‐1 and HIV‐2, respectively), the causative agents of acquired immunodeficiency syndrome (AIDS). There is a 41–42% sequence identity between EIAV RT and both HIV RTs (which have 61% sequence identity to each other). We have compared the enzymic properties of EIAV RT with those of HIV‐1 RT. Several aspects of the activities of EIAV RT differ from the corresponding activities of HIV‐1 RT. There are significant differences in the inhibition of the DNA polymerase activities by the deoxynucleoside triphosphate analogs, 3′‐azido‐2,3′‐dideoxythymidine triphosphate, dideoxyTTP and dideoxyGTP and by the nonnucleoside inhibitor, tetrahydroimidazo[4,5,1‐jk‐1,4]benzodiazepin‐2‐(1H)‐one and thione; in the dependence of DNA polymerase and RNase H activities on pH; in the inhibition of the DNA polymerase activities by the thiol‐specific reagent N‐ethylmaleimide; in the specific DNA polymerase activity; in the inhibition of the ribonuclease H activity by the zinc chelator orthophenanthroline. However, there are several cases in which EIAV RT and HIV‐1 RT are more similar than was previously found for HIV‐1 RT and HIV‐2 RT. These include the Km values for the DNA polymerase activities, the heat stability of the DNA polymerase functions and the specific activity of the RNase H function.
Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea
Authors: VIDAL‐CROS Anne , VIVIANI Fabrice , LABESSE Gilles , BOCCARA Martine , GAUDRY Michel
Published: March 2005

During the biosynthesis of fungal melanin, tetrahydroxynaphthalene reductase catalyzes the NADPH‐dependent reduction of 1,3,6,8‐tetrahydroxynaphthalene (T4HN) into (+)‐scytalone and 1,3,8‐trihydroxynaphthalene into (–)‐vermelone. The enzyme from Magnaporthe grisea, the fungus responsible for rice blast disease, has been purified to homogeneity. It is a tetramer of four identical 30‐kDa subunits. A full‐length cDNA clone of about 1 kb encoding T4HN reductase has been isolated from a cDNA library constructed in the λZAP II vector and characterized. The clone contains a 846‐bp open reading frame. Translation of the DNA sequence gave a 282‐residue amino acid sequence with a calculated molecular mass of 29.9 kDa. Sequences corresponding to the amino‐terminal part and three internal proteolytic peptides were present in the translated sequence. T4HN reductase exhibits characteristics of the short‐chain alcohol dehydrogenase family. The reductase shares 56% identity with a putative ketoreductase involved in aflatoxin biosynthesis in Aspergillus parasiticus.
CMP‐N‐acetylneuraminic acid hydroxylase from mouse liver and pig submandibular glands
Authors: SHAW Lee , SCHNECKENBURGER Petra , SCHLENZKA Wiebke , CARLSEN Jens , CHRISTIANSEN Kirsten , JÜRGENSEN Dorthe , SCHAUER Roland
Published: March 2005

In this report, the nature of the protein components involved in the functioning of cytidine‐5′‐monophosphate‐N‐acetylneuraminic acid (CMP‐Neu5 Ac) hydroxylase in high‐speed supernatants of mouse liver has been investigated. Fractionation and reconstitution experiments showed that this enzyme system consists of NADH–cytochromeb5reductase, cytochromeb5and a 56‐kDa terminal electron acceptor having the CMP‐Neu5 Ac hydroxylase activity. This enzyme system is extracted in a soluble protein fraction; however, the amphipathic, usually membrane‐associated, forms of cytochromeb5and the reductase were found to predominate and are presumably the forms which support the turnover of the hydroxylasein vivo.Although the majority of cellular cytochromeb5and cytochromeb5reductase is membrane‐bound, the addition of intact microsomes elicited no significant increase in the hydroxylase activity of supernatants. Detergent‐solubilised microsomes, however, potently activated the hydroxylase, probably due to the greater accessibility of the cytochromeb5. Accordingly, in reconstitution experiments, pure hydrophilic cytochromeb5interacts more effectively with the hydroxylase than isolated amphipathic cytochromeb5.

Studies on the CMP‐Neu5Ac hydroxylase system in fractionated porcine submandibular glands and bovine liver suggest that the composition of this enzyme system is conserved in all mammals possessing sialoglycoconjugates containingN‐glycoloylneuraminic acid.
Inhibition of the synthesis of a cytochrome‐c‐oxidase subunit isoform by antisense RNA
Authors: SANDONÀ Dorianna , BISSON Roberto
Published: March 2005

To investigate the role of subunit VIIe, an oxygen‐regulated subunit isoform of Dictostelium discoideum cytochrome‐c oxidase, the full‐length cDNA was inserted into an expression vector under the control of an actin promoter in the sense and antisense orientation. The DNA constructs were used for stable transformation of the slime mold amoebae. In most of the 28 antisense clones tested, the concentration of cytochrome‐c oxidase was lowered compared to the wild type, while no significant changes were found in the sense mutants. Antisense RNA was abundantly expressed, leading to a drastic reduction of the steady‐state level of the endogenous subunit VIIe mRNA, which was decreased up to 20–30% the level observed in parent cells. In these transformants, the amount of the target polypeptide and cytochrome c oxidase was 40–50% and 60–70% of control, respectively. A similar decrease was found in the level of the remaining nuclear and mitochondrial subunits. Unexpectedly, these changes affected neither basal nor uncoupled cell respiration suggesting an increase of the enzyme specific activity. Hypoxia completely relieved the cytochrome‐c‐oxidase deficit. These results indicate that subunit VII is needed for an efficient assembly of the protein complex and provide evidence for its involvement in the modulation of the enzyme activity.
Rab4 is phosphorylated by the insulin‐activated extracellular‐signal‐regulated kinase ERK1
Authors: CORMONT Mireille , TANTI Jean‐François , ZAHRAOUI Ahmed , VAN OBBERGHEN Emmanuel , LE MARCHAND‐BRUSTEL Yannick
Published: March 2005

Rab4, a low‐molecular‐mass GTP‐binding protein, is associated with vesicles containing Glut 4 in adipocytes. Following insulin stimulation, the translocation of Glut 4 to the plasma membrane is associated with the movement of Rab4 to the cytosol. The same modifications are induced by the phosphatase inhibitor, okadaic acid. To establish a possible role for phosphorylation in Rab4 cycling, we searched for insulin‐stimulated cytosolic kinase(s) which could phosphorylate Rab4. In 3T3‐L1 adipocytes, insulin induced a rapid and transient activation of cytosolic kinase(s), which phosphorylated Rab4 in vitro. At least part of the Rab4 phosphorylation can be accounted for by ERK (extracellular‐signal‐regulated kinases) since immunopurified ERK1 from insulin‐stimulated cells phosphorylated Rab4 with a comparable time‐course. Both with cytosolic extracts and immunopurified ERK1, only serine residues were phosphorylated on Rab4. The phosphorylation site was localized in the C‐terminus of the molecule, and occurred very probably on Ser196. These results indicate that Rab4 is an in vitro substrate for ERK, and suggest that the insulin‐induced movement of Rab4 from the Glut‐4‐containing vesicles to the cytosol could result from phosphorylation of Rab4 by ERK.
Location of agonist‐dependent‐phosphorylation sites in the third intracellular loop of muscarinic acetylcholine receptors (m2 subtype)
Authors: NAKATA Hiroko , KAMEYAMA Kimihiko , HAGA Kazuko , HAGA Tatsuya
Published: March 2005

Muscarinic acetylcholine receptors (mAChR, human m2 subtype) expressed in Sf9 (Spodoptera frugiperda) cells using the baculovirus system were purified and subjected to phosphorylation by a mAChR kinase, which was partially purified from porcine cerebrum. Two bands with apparent molecular masses of 59 kDa and 39 kDa as determined by SDS/PAGE were found to be phosphorylated in an agonist‐dependent manner. Both bands were labeled by the irreversible muscarinic ligand [3H]propylbenzilylcholine mustard. Molecular masses of the [32P]phosphorylated or [3H]propylbenzilylcholine‐mustard‐labeled bands decreased following treatment with N‐glycanase. The 59‐kDa and 39‐kDa bands were converted to 52‐kDa bands 32‐kDa bands, respectively, indicating that both the 59‐kDa and 39‐kDa bands contain the amino‐terminal region where glycosylation sites are present. The ratio of incorporated [32P]phosphate and bound [3H]propylbenzilylcholine mustard was essentially the same for the 59‐kDa and 39‐kDa bands, indicating that all the phosphorylation sites reside in the sequence of 39 kDa from the amino‐terminal region. The amounts of incorporated [32P]phosphate were estimated to be 10–11/receptor, with 7–8 serine and 3–4 threonine, but no phosphorylated tyrosine residues. Further treatment of [32P]phosphorylated or [3H]propylbenzilycholine‐mustard‐labeled receptors with V8 protease indicated that the phosphorylation sites were not present in 30‐kDa amino‐terminal segment. These results indicate that the phosphorylation sites are localized in the range 30–39 kDa from the amino terminus, which consists of primarily the central part of the third intracellular loop. Consistent with this conclusion, a fusion protein containing glutathione S‐transferase linked to a peptide corresponding to residues 227–324 of the central part of the third intracellular loop was found to be phosphorylated by the mAChR kinase in a heparin‐sensitive manner.
Complex formation between protein C inhibitor and prostate‐specific antigen in vitro and in human semen
Authors: CHRISTENSSON Anders , LILJA Hans
Published: March 2005

Protein C inhibitor (PCI), a serine‐proteinase inhibitor first purified from human blood plasma, occurs at high concentrations (3–4 μM) in seminal fluid in both a high‐molecular‐mass and low‐molecular‐mass form. Immunochemical data have previously suggested that PCI in seminal plasma forms complexes with the most abundant serine proteinase in semen, prostate‐specific antigen (PSA). To provide a structural characterization of the PCI target, immunodetected as PSA, a procedure was developed to isolate low‐molecular‐mass and high‐molecular‐mass‐forms of PCI from seminal fluid. The high‐molecular‐mass form of PCI, recognized by monoclonal antibodies against PSA, was dissociated by alkaline treatment into the low‐molecular‐mass form of PCI and a 33‐kDa protein identified as PSA by 25 conclusive steps of N‐terminal sequence analysis. We developed a sensitive immunofluorometric assay (IFMA) to measure PCI‐PSA complexes in body fluids and investigated the rate at which purified PSA may form complexes with purified PCI. Formation of complexes detected by this IFMA and the appearance of SDS‐stable approximately 90‐kDa complexes paralleled loss of PSA activity recorded with chromogenic substrates. The rate of complex formation was slow compared to that reported for PCI and activated protein C, but was enhanced up to sixfold in the presence of heparin. Less than 10% of the initial PSA activity remained after 3 h incubation with a sevenfold molar excess of PCI and in the presence of heparin.

In freshly collected ejaculates, the rate of PCI‐PSA complex formation measured by IFMA was similar to that observed between the purified proteins, and paralleled the appearance of SDS‐stable complexes by immunoblotting. During gel dissolution in freshly collected ejaculates, approximately 40% of immunodetected PCI becomes complexed to PSA. Although PCI is a slow inhibitor of PSA, complexes between PCI and PSA are detected at levels that correspond to an inactivation of up to 5% of the PSA activity in the ejaculate.
Preparation of cyclic 2′,3′‐monophosphates of oligoadenylates (A2′p)nA > p and A3′p(A2′p)n‐1A>p
Authors: BUDOWSKY Edward I. , KAYUSHINA Elena N. , TARASOV Andrew K. , ORLENKO Svetlana A. , CHERKASOV Igor A. , GAVRILOV Alexander E. , STRELENKO Yuri A.
Published: March 2005

The action of the guanylyl‐preferring RNase from Bacillus intermedius (binase) on a mixture of oligoadenylates with randomly distributed 2′‐5′ and 3′‐5′ internucleotide bonds [(A2′/3′p)n] under conditions sufficient for complete hydrolysis of poly(A) results in a mixture containing a single circular oligoadenylate and two series of linear oligoadenylates ending in cyclic 2′,3′‐phosphate.

Individual compounds formed upon digestion of (A2′/3′p)n with binase have been isolated. Their structure was determined on the basis of their chemical and enzymatic conversions and confirmed by 1H‐, 13C‐ and 31P‐NMR spectra. According to these data, the circular triadenylate contains one 2′‐5′ and two 3′‐5′ internucleotide bonds, linear oligoadenylates of one series contain exclusively 2′‐5′ internucleotide bonds [(A2′p)nA>p], while each compound of the other series contains a single 3′‐5′ internucleotide bond connecting the 5′‐ultimate nucleotide residue with the penultimate one [A3′p(A2′p)n‐1A>p].

The incubation of compounds of the former series A3′p(A2′p)n>p at pH 1.0 and the subsequent action of phosphatase results in successive formation of compounds of two other new series: A3′p(A2′p)nA2′(3′)p and A3′p(A2′p)nA.
Induction by zinc of specific metallothionein isoforms in human monocytes
Authors: PAUWELS Martine , VAN WEYENBERGH John , SOUMILLION Ann , PROOST Paul , DE LEY Marc
Published: March 2005

A low‐molecular‐mass zinc‐containing protein was isolated by gel permeation and anion‐exchange chromatography of lysates of human monocytes induced with zinc acetate. Characterization by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and amino acid sequencing identified the two major charge‐separable fractions and an occasionally occurring third fraction as metallothionein‐1, metallothionein‐2 and metallothionein‐0, respectively. Metallothionein‐1 was shown to consist of a mixture of isoforms, confirmed as metallothionein‐1e, metallothionein‐1g and metallothionein‐11 by comparison with cDNA sequences obtained by screening a human monocyte cDNA library. We can find no previous observation in the literature of metallothionein‐1g at both the protein and RNA level in a non‐tumour cell, and of metallothionein‐0 in a non‐fetal cell or tissue. Since isoform‐specific polymerase‐chain‐reaction amplification showed the presence of metallothionein‐0 mRNA in zinc‐induced but not in untreated monocytes, these cells can be used as an in vitro system to investigate the expression of this previously considered fetal isoform.
Prothrombin activation on dioleoylphosphatidylcholine membranes
Authors: GOVERS‐RIEMSLAG José W. P. , JANSSEN Marie P. , ZWAAL Robert F. A. , ROSING Jan
Published: March 2005

Factor‐Xa‐catalyzed prothrombin activation is greatly accelerated by negatively charged phospholipids plus calcium ions. In 1990, we reported that neutral phosphatidylcholine membranes also stimulated prothrombin activation [Gerads, I., Govers‐Riemslag, J. W. P., Tans, G., Zwaal, R. F. A. & Rosing, J. (1990) Biochemistry 29, 7967–7974]. In the present study, we have performed a detailed analysis of the prothrombin‐converting activity of phosphatidylcholine membranes. Stimulation of prothrombin activation by phosphatidylcholine vesicles was particularly observed (a) with phosphatidylcholine molecules that contained unsaturated hydrocarbon side chains, (b) in the presence of factor Va, (c) at low ionic strength and (d) when Ca2+ were present in the reaction medium. It is unlikely that the prothrombinase activity of phosphatidylcholine preparations was due to contaminating aninic phospholipids. This is concluded from the fact that thin‐layer chromatographic analysis showed that dioleoylphosphatidylcholine [(Ole)2GroPCho] contained less than 0.1 mol/100 mol anionic phospholipid, and that incorporation of such amounts of anionic lipids in (Ole)2‐GroPCho membranes hardly increased their prothrombin‐converting activity. At low ionic strength and in the presence of factor Va and Ca2+ (Ole)2GroPCho membranes accelerated prothrombin activation about 100‐fold. At ionic strength (I) 0.06, prothrombin activation on 100 μM (Ole)2‐GroPCho was characterized by a Km for prothrombin of 2 μM, a Vmax of 3020 IIa min−1· Xa−1 and a Kd for factor Xa Va complex formation at the membrane surface of 7.5 nM. Prothrombin activation on (Ole)2GroPCho membranes was drastically reduced when the ionic strength was increased. The inhibition at high ionic strength could be explained by an effect on the Kd for Xa Va complex formation which increased from 7.5 nM at I = 0.06 to 100 nM at I = 0.22. Prothrombin activation on (Ole)2GroPCho required Ca2+ and was dependent on the presence of γ‐carboxyglutamic acid domains in prothrombin and factor Xa. This indicates that similar interactions may account for the assembly of prothrombinase complexes on phosphatidylcholine and on anionic lipid‐containing membranes.
Characterization of in‐vitro‐translated human regulatory and catalytic subunits of cAMP‐dependent protein kinases
Authors: FOSS Kari Bente , LANDMARK Brynjar , SKÅLHEGG Bjørn Steen , TASKÉN Kjetil , JELLUM Egil , HANSSON Vidar , JAHNSEN Tore
Published: March 2005

Full‐length human cDNAs for all the different regulatory (R) and catalytic (C) subunits of cAMP‐dependent protein kinases (PKA) were transcribed and translated in a cell‐free in vitro system. The resulting proteins were characterized with respect to molecular size, isoelectric focusing, immunoreactivity, cAMP binding, and to what extent the RII protein subunits revealed mobility shifts upon phosphorylation by catalytic subunit of PKA. We were able to express cDNAs for all the human R (RIα, RIβ, RIIα and RIIβ) and C (Cα, Cβ and Cγ) subunits in a wheat‐germ extract. [35S]Methionine‐labelled in‐vitro‐translated products were analyzed by SDS/PAGE and revealed distinct protein bands with apparent molecular masses of 49 (RIα), 54–55 (RIβ), 51 (RIIα) and 53 kDa (RIIβ) for the R subunits. In vitro transcription/translation of the cDNAs for the C subunits of PKA gave proteins with molecular masses of approximately 40 kDa for all the different C subunits. Phosphorylation of RIIα and RIIβ by the C subunit of PKA, revealed a distinct mobility shift of the RIIα subunit on one‐dimensional SDS/PAGE (51–54 kDa), but not of RIIβ (53 kDa). Further characterization of the R subunits by two‐dimensional SDS/PAGE revealed that RIα was more acidic than RIβ, with pIs of 6.1–6.0 and 6.4–6.2, respectively. Furthermore, the RIIα protein was more basic than RIIβ, with pIs of approximately 5.4–5.3 and 5.3–5.1, respectively. All the in‐vitro‐translated R subunits could be photoaffinity labelled by the cAMP‐analog 8‐azido‐[32P]cAMP and were also detected by immunoprecipitation with subunit‐specific antibodies.
Triose‐phosphate isomerase of Leishmania mexicana mexicana Cloning and characterization of the gene, overexpression in Escherichia coli and analysis of the protein
Authors: KOHL Linda , CALLENS Mia , WIERENGA Rik K. , OPPERDOES Fred R. , MICHELS Paul A. M.
Published: March 2005

The gene of triose‐phosphate isomerase in Leishmania mexicana has been cloned and characterized. The gene encodes a polypeptide of 251 amino acids, with a calculated molecular mass of 27561 Da and a net charge of +2. Only one gene could be detected, although the enzyme is present in two different compartments of the cell, in microbody‐like organelles called glycosomes and in the cytosol. The primary structure of the enzyme has many features in common with that of triosephosphate isomerase in the related organism Trypanosoma brucei. Their sequences are 68% identical. The residues consituting the subunit interface are highly conserved between the enzyme of L. mexicana and T. brucei, but are mostly different from those in the enzyme of other organisms. One major substitution was detected in the interface region of the L. mexicana protein: a glutamate was found at position 66, instead of glutamine in all other available 20 sequences. The glutamine is thought to be important for the stability of the dimeric enzyme.

L. mexicana triose‐phosphate isomerase has been overexpressed in Escherichia coli. Growth conditions were established to obtain high levels of soluble and active protein. The enzyme has been purified to near homogeneity. It appears a stable dimeric protein with a specific activity of 5500 units/mg protein, a subunit mass of 28 kDa and an isoelectric point of 9.0. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. Some kinetic properties of the recombinant protein have been compared with those of the promastigote enzyme and with the values previously reported for the T. brucei enzyme. The kinetics of the different enzyme preparations were very similar. For the recombinant enzyme the following values were measured: with glyceraldehyde 3‐phosphate as substrate Km= 0.30±0.05 mM and kcat= 2.5X105 min−1; with dihydroxyacetone phosphate as substrate Km= 1.3±0.3 mM and kcat= 2.8X104 min−1.
Endoglycosidic cleavage of branched polymers by poly(ADP‐ribose) glycohydrolase
Authors: BRAUN Stephan A. , PANZETER Phyllis L. , COLLINGE Margaret A. , ALTHAUS Felix R.
Published: March 2005

Post‐translational modification of nuclear proteins with poly(ADP‐ribose) modulates chromatin structure and may be required for DNA processing events such as replication, repair and transcription. The polymer‐catabolizing enzyme, poly(ADP‐ribose) glycohydrolase, is crucial for the regulation of polymer metabolism and the reversibility of the protein modification. Previous reports have shown that glycohydrolase digests poly(ADP‐ribose) via an exoglycosidic mechanism progressing from the protein‐distal end of the polymer. Using two independent approaches, we investigated the possibility that poly(ADP‐ribose) glycohydrolase also engages in endoglycosidic cleavage of polymers. First, partial glycohydrolase digestion of protein‐bound poly(ADP‐ribose) led to the production of protein‐free oligomers of ADP‐ribose. Second, partial glycohydrolase digestion of a fixed number of protein‐free poly(ADP‐ribose) polymers resulted in a transient increase in the absolute number of polymers while polymer size continuously decreased. Furthermore, endoglycosidic activity produced linear polymers from branched polymers although branch points themselves were not a preferential target of cleavage. From these data, we propose a mechanism whereby poly(ADP‐ribose) glycohydrolase degrades polymers in three distinct phases; (a) endoglycosidic cleavage, (b) endoglycosidic cleavage plus exoglycosidic, processive degradation, (c) exoglycosidic, distributive degradation.
Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C‐terminal processing at Arg537
Authors: ROSSMANN Reinhild , SAUTER Martin , LOTTSPEICH Friedrich , BÖCK August
Published: March 2005

Purification of the large subunit, HYCE, of Escherichia coli hydrogenase 3 revealed that it is a nickel‐containing polypeptide, which is subject to C‐terminal proteolytic processing. This processing reaction could be performed in vitro with partially purified components, yielding a low‐molecular mass C‐terminal peptide which was resolved in a Tricine/SDS/polyacrylamide gel. N‐terminal sequencing of this peptide revealed that proteolytic cleavage occurred at the C‐terminal side of the arginine residue at position 537, which corresponds to the histidine residue in the highly conserved motif, DPCXXCXXH, of other (NiFe) hydrogenases thought to be involved in active site nickel coordination. Nickel‐containing HYCE precursor for in vitro processing, was partially purified from strain HD708 (ΔhycH) in the presence of the reducing agent dithiothreitol. Using 2‐mercaptoethanol instead of dithiothreitol provided pure precursor, which was, however, no longer susceptible to in vitro processing; it proved to be devoid of nickel indicating that nickel incorporation into the HYCE precursor is a prerequisite for processing. This conclusion was supported by the finding that HYCE precursor from strain HD708 (ΔhycH) chromatographed with radioactivity from 63Ni incorporated in vivo and could be processed in vitro, whereas HYCE precursor from stain BEF314 (ΔhypB–E) lacking the nickel insertion system appeared to be devoid of nickel and was not sensitive to in vitro processing.
Spectrochemical evidence for the presence of a tyrosine residue in the allosteric site of glucosamine‐6‐phosphate deaminase from Escherichia coli
Authors: ALTAMIRANO Myriam M. , HERNANDEZ‐ARANA Andrés , TELLO‐SOLIS Salvador , CALCAGNO Mario L.
Published: March 2005

The interaction of the enzyme glucosamine 6‐phosphate deaminase from Escherichia coli with its allosteric activator, N‐acetyl‐d‐glucosamine 6‐phosphate, was studied by different spectrophotometric methods. Analysis of the circular‐dichroism differential spectra produced by the binding of the allosteric activator or the competitive inhibitor 2‐amino‐2‐deoxy‐d‐glucitol 6‐phosphate (a homotropic ligand displacing the allosteric equilibrium to the R conformer), strongly suggests the presence of tyrosine residues at or near the allosteric site, although a conformational effect cannot be ruled out. The involvement of a single tyrosine residue in the N‐acetyl‐d‐glucosamine‐6‐phosphate binding site of glucosamine‐6‐phosphate deaminase was supported by spectrophotometric pH titrations performed in the presence or absence of the homotropic and heterotropic ligand. In these experiments, a single titrated tyrosine residue is completely protected by saturation with the allosteric activator; this group is considerably acidic (pK 8.75). The analysis of the amino acid sequence of the deaminase using a set of indices for the prediction of surface accessibility of amino acid residues, suggests that the involved residue may be Tyr121 or Tyr254.
Resonance assignment, cysteine‐pairing elucidation and secondary‐structure determination of capsicein, an α‐elicitin, by three‐dimensional 1H NMR
Authors: BOUAZIZ Serge , VAN HEIJENOORT Carine , GUITTET Eric , HUET Jean‐Claude , PERNOLLET Jean‐Claude
Published: March 2005

Difficulties encountered in the interpretation of two‐dimensional NMR spectra of proteins exceeding roughly 100 amino acids, including resonance overlap and line broadening due to longer correlation times and/or aggregation phenomena, can be overcome by using three‐dimensional 1H‐NMR experiments. The improvement of spectral resolution using these experiments allows the size of molecules amenable to structure determination by NMR spectroscopy to be extended. A three‐dimensional non‐selective homonuclear Hartmann‐Hahn/nuclear Overhauser effect spectroscopy experiment was performed on capsicein, a 10161‐Da elicitin secreted by the Phytophthora capsici fungus. Sequential assignment and secondary structure determination is illustrated for β‐sheet, α‐helix and loop structures by analysis of planar cross sections perpendicular to the ω2 or ω3 axis at the amide proton resonance frequencies. Cysteine pairing was established in the course of the investigation. The secondary structure topology of the molecule is composed of five helices and an antiparallel β‐sheet. Four of the helices compose two pairs running antiparallel while the last one is parallel to the β‐sheet.
Tandem overproduction and characterisation of the nuclease domain of colicin E9 and its cognate inhibitor protein Im9
Authors: WALLIS Russell , REILLY Ann , BARNES Karen , ABELL Chris , CAMPBELL David G. , MOORE Geoffrey R. , JAMES Richard , KLEANTHOUS Colin
Published: March 2005

We report the overproduction of the non‐specific endonuclease domain of the bacterial toxin colicin E9 and its preliminary characterisation in vitro. The enzymatic colicins (61 kDa) are normally released from producing cells in a complex with their cognate inhibitors, known as the immunity proteins (9.5 kDa). Tryptic digestion of the purified ColE9 complex was found to generate two major components, a monomer derived from the N‐terminal and central regions of the toxin and a heterodimer comprising the catalytically active C‐terminal domain of the colicin bound to its intact immunity protein, Im9. N‐terminal amino acid sequencing, in conjunction with electrospray mass spectrometry, shows that preparations of the DNase domain isolated by this method are heterogeneous, thus making subsequent mechanistic and structural analysis difficult. This problem was circumvented by selectively overexpressing the C‐terminal 15‐kDa nuclease domain of colicin E9 in tandem with its cognate inhibitor in Escherichia coli. This tandem overexpression strategy allowed high‐level production of a 25‐kDa protein complex comprising the C‐terminal DNase domain of colicin E9 tightly bound to its specific inhibitor Im9, thus masking the anticipated toxicity of the nuclease. The DNase domain was then separated from Im9 under denaturing conditions, refolded by removal of the denaturant and the renatured protein shown to possess both endonuclease and Im9 binding activity. These results describe a novel method for the overproduction of a nuclease in bacteria by co‐expressing its specific inhibitor and lay the foundations for a full mechanistic, biophysical and structural characterization of the isolated DNase domain of the colicin E9 toxin.
Transport of citrate catalyzed by the sodium‐dependent citrate carrier of Klebsiella pneumoniae is obligatorily coupled to the transport of two sodium ions
Authors: LOLKEMA Juke S. , ENEQUIST Hans , VAN DER REST Michel E.
Published: March 2005

Aerobically grown Escherichia coli GM48 harboring plasmid pKScitS that codes for the sodium‐dependent citrate carrier from Klebsiella pneumoniae (CitS) allows initial‐rate measurements of citrate uptake in whole cells. The cation stoichiometry and selectivity of CitS was studied using this experimental system. The relationship between the initial rate of uptake of citrate and the Na+ concentration was sigmoidal at pH values between 5 and 7 suggesting a Na+ stoichiometry higher than 1. Rates of uptake increased quadratically in a range of non‐saturating Na+ concentrations showing that two Na+ are translocated/catalytic cycle. Symport of Na+ is absolutely required in the range pH 5–7 because no uptake could be detected in the absence of Na+. Protons cannot replace Na+ in the translocation step but the decrease in apparent affinity for Na+ towards lower pH suggests that protons can compete with Na+ for the cation‐binding sites.

Li+ can replace Na+ in the symport reaction but it takes about a 200‐fold higher concentration of Li+ over Na+ to achieve the same rate of uptake, showing that the affinity of CitS for Li+ is much lower than for Na+. Though high Li+ concentrations have an inhibitory effect on citrate uptake, the data suggest that the Li+ stoichiometry is also 2.
Characterization of aromatic aminotransferases from the hyperthermophilic archaeon Thermococcus litoralis
Authors: ANDREOTTI Giuseppina , CUBELLIS Maria Vittoria , NITTI Gianpaolo , SANNIA Giovanni , MAI Xuhong , MARINO Gennaro , ADAMS Michael W. W.
Published: March 2005

The hyperthermophilic archaeon (formerly archaebacterium) Thermococcus litoralis grows at temperatures up to 98°C using peptides and proteins as the sole sources of carbon and nitrogen. Cell‐free extracts of the organism contained two distinct types of aromatic aminotransferases (EC 2.6.1.57) which were separated and purified to electrophoretic homogeneity. Both enzymes are homodimers with subunit masses of approximately 47 kDa and 45 kDa. Using 2‐oxoglutarate as the amino acceptor, each catalyzed the pyridoxal‐5′‐phosphate‐dependent transamination of the three aromatic amino acids but showed virtually no activity towards aspartic acid, alanine, valine or isoleucine. From the determination of Km and kcat values using 2‐oxoglutarate, phenylalanine, tyrosine and tryptophan as substrates, both enzymes were shown to be highly efficient at transaminating phenylalanine (kcat/Km∼400 s−1 mM−1); the 47‐kDa enzyme showed more activity towards tyrosine and tryptophan compared to the 45‐kDa one. Kinetic analyses indicated a two‐step mechanism with a pyridoxamine intermediate. Both enzymes were virtually inactive at 30°C and exhibited maximal activity between 95–100°C. They showed no N‐terminal sequence similarity with each other (∼30 residues), nor with the complete amino acid sequences of aromatic aminotransferases from Escherichia coli and rat liver. The catalytic properties of the two enzymes are distinct from bacterial aminotransferases, which have broad substrate specificities, but are analogous to two aromatic aminotransferases which play a biosynthetic role in a methanogenic archaeon. In contrast, it is proposed that one or both play a catabolic role in proteolytic. T. litoralis in which they generate glutamate and an arylpyruvate. These serve as substrates for glutamate dehydrogenase and indolepyruvate ferredoxin oxidoreductase in a novel pathway for the utilization of aromatic amino acids.
The C‐terminus of the 14‐kDa subunit of ubiquinol‐cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisiae is involved in the assembly of a functional enzyme
Authors: HEMRIKA Wieger , DE JONG Marian , BERDEN Jan A. , GRIVELL Leslie A.
Published: March 2005

Disruption of QCR7, the gene encoding the 14‐kDa subunit of ubiquinol‐cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisie,, results in an inactive enzyme which lacks holo‐cytochrome b and has severely reduced levels of apo‐cytochrome b, the Rieske Fe‐S protein and the 11‐kDa subunit [Schoppink, P. J., Berden, J. A. & Grivell, L. A. (1989) Eur. J. Biochem. 181, 475–483].

An episomal system was developed to study the effect on complex III of transformation of in vitro mutagenised QCR7 genes to a QCR7° mutant. Transformation of a gene (TNT1) in which the 12 C‐terminal residues are replaced by 3 amino acids encoded by an oligonucleotide containing a stop codon in all three reading frames (STOP‐oligonucleotide), only leads to partial complementation of the respiratory capacity of the yeast strain. The amounts of apo‐cytochrome b, the Rieske Fe‐S protein and the 11‐kDa subunit are reduced and enzymic activity, together with the amount of holo‐cytochrome b, is lowered to about 40% of that of the wild type, indicating a normal turnover number of the mutant enzyme.

Transformation of the QCR7° mutant with another gene (TNT2) encoding the first 96 residues of the 14‐kDa subunit fused to 9 amino acids encoded by the STOP‐oligonucleotide, leads to a phenotype almost indistinguishable from that of the QCR7° mutant.

The role of the charged C‐terminus of the 14‐kDa (and the 11‐kDa) subunit in the assembly of a functional complex III is discussed.