bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Human Immunodeficiency Virus Nef Protein with Actin is Myristoylation Dependent and Influences its Subcellular Localization
Authors: Fackler Oliver T. , Kienzle Norbert , Kremmer Elisabeth , Boese Annette , Schramm Birgit , Klimkait Thomas , Kücherer Claudia , Mueller‐Lantzsch Nikolaus
Published: July 2004

Human immunodeficiency virus (HIV) Nef functions are thought to be mediated via interactions with cellular proteins. Utilizing zone velocity sedimentation in glycerol gradients we found that recombinant HIV‐1 Nef non‐covalently associates with actin forming a high‐molecular‐mass complex of 150–300 kDa. This Nef/actin complex was present in human B and T lymphocytes but not in insect cells and was dependent on the N‐terminal myristoylation of Nef, whereas the SH3‐binding proline motif of Nef was not involved. Despite being myristoylated, HIV‐2 Nef did not associate with actin. This might reflect differences in the subcellular localization of Nef since cell‐fractionation experiments revealed that HIV‐ 1Nef was virtually exclusively localized in the cytoskeletal (detergent‐insoluble) fraction whereas HIV‐ 2 Nef had significantly reduced affinity for the cytoskeleton. Colocalization experiments in HIV‐1‐in‐fected CD4+ fibroblasts revealed that Nef/actin complexes may also exist in HIV‐infected cells. This novel interaction of HIV‐1 Nef with actin provides insight into the association of Nef with cellular structures and reveals general differences in the interactions of the Nef proteins from HIV‐1 and HIV‐2.
The 70‐kDa Heat‐Shock Protein/DnaK Chaperone System is Required for the Productive Folding of Ribulose‐Bisphosphate Carboxylase Subunits in Escherichia Coli
Authors: Checa Susana K. , Viale Alejandro M.
Published: July 2004

We have studied the in vivo requirements of the DnaK chaperone system for the folding of recombinant ribulose‐bisphosphate carboxylase/oxygenase in Escherichia coli. Expression of functional dimeric or hexadecameric ribulose‐bisphosphate carboxylase from different bacterial sources (including purple bacteria and cyanobacteria) was severely impaired in E. coli dnaK, dnaJ, or grpE mutants. These enzymes were synthesized mostly in soluble, fully enzymatically active forms in wild‐type E. coli cells cultured in the temperature range 20–42°C, but aggregated extensively in dnaK null mutants. Co‐expression of dnaK, but not groESL, markedly reduced the aggregation of ribulose‐bisphosphate carboxylase subunits in dnaK null mutants and restored the enzyme activity to levels found in isogenic wild‐type strains. Ribulose‐bisphosphate carboxylase expression in wild‐type E. coli cells growing at 30°C promoted an enhanced synthesis of stress proteins, apparently by sequestering DnaK from its negative regulatory role in this response. The overall results indicate that the DnaK chaperone system assists in vivo the folding pathway of ribulose‐bisphosphate carboxylase large subunits, most probably at its very early stages.
Diffusion‐Dependent Kinetic Properties of Glyoxalase I and Estimates of the Steady‐State Concentrations of Glyoxalase‐Pathway Intermediates in Glycolyzing Erythrocytes
Authors: Shih Mei J. , Edinger James W. , Creighton Donald J.
Published: July 2004

The diffusion‐dependent kinetic properties of the yeast glyoxalase I reaction have been measured by means of viscosometric methods. For the glyoxalase‐I‐catalyzed isomerization of glutathione (GSH)‐methylglyoxal thiohemiacetal to S‐d‐lactoylglutathione, the kcat/Km (3.5 × 106 M−1 S−1, pH7, 25°C) undergoes a progressive decrease in magnitude with increasing solution viscosity, using sucrose as a visco‐genic agent. The viscosity effect is unlikely to be due to a sucrose‐induced change in the intrinsic kinetic properties of the enzyme, as the magnitude of kcat/Km for the slow substrate GSH‐t‐butylglyoxal thiohemiacetal (3.5 × 103 M−1 s−1, pH 7, 25°C) is independent of solution viscosity. Quantitative treatment of the data by means of the Stokes‐Einstein diffusion law suggests that catalysis will be about 50% diffusion limited under conditions where [substrate] «Km; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate. In a related study, the steady‐state concentrations of glyoxalase‐pathway intermediates in glycolyzing human erythrocytes are estimated to be in the nanomolar concentration range, on the basis of published values for the activities of glyoxalase I and glyoxalase II in lysed erythrocytes and the steady‐state rate of formation of d‐lactate in intact erythrocytes. This is consistent with a model of the glyoxalase pathway in which the enzyme‐catalyzed steps are significantly diffusion limited under physiological conditions.
Characterization of Hexose Oxidase from the Red Seaweed Chondrus Crispus
Authors: Groen Barend W. , De Vries Simon , Duine Johannis A.
Published: July 2004

Hexose oxidase from the red seaweed, Chondrus crispus was purified to homogeneity. The enzyme appeared to be encapsulated in particles obtained after mechanical disintegration of the fronds. Liberation of the enzyme in soluble form required either waiting for the spontaneous development of a suitable microbial flora in the suspension, or treatment with a mixture of proteases (pronase). As deduced from (SDS/)PAGE, the enzyme has a molecular mass of 87 kDa and probably consists of subunits of 36 kDa and 25 kDa. The low isoelectric point of 2.8 and the presence of 25% (by mass) sugars indicate that the enzyme is a strongly acidic glycoprotein. The absorption spectrum of isolated enzyme minus that of the substrate‐reduced enzyme, and the EPR spectrum of the free radical observed in the reduced enzyme revealed the presence of a flavin. This cofactor is probably covalently bound since flavins were not released upon denaturation of the enzyme by heat or acid treatment. Taking free FAD as a reference compound, the enzyme contains 1 mol flavin/mol enzyme. EPR spectroscopy of the purified preparation showed the presence of Cu2+. However, since the amount was substoichiometric, substrate addition did not affect the signal, and the addition of chelator or Cu2+ did not affect the activity, the presence of this metal ion seems adventitious. It is concluded that the large discrepancies between the presently and the previously reported [Sullivan, J. D. & Ikawa, M. (1973) Biochim. Biophys. Acta 309, 11–22] characteristics of the enzyme probably originate from the characterization of a contaminating protein in the latter case.
Structures and Functions of Four Anabolic 2‐Oxoacid Oxidoreductases in Methanobacterium Thermoautotrophicum
Authors: Tersteegen Adrian , Linder Dietmar , Thauer Rudolf K. , Hedderich Reiner
Published: July 2004

Methanobacterium thermoautotrophicum (strain Marburg), which grows autotrophically on H2 and CO2, was found to contain 2‐oxoisovalerate oxidoreductase (Vor) and indolepyruvate oxidoreductase flor) besides pyruvate oxidoreductase (For) and 2‐oxoglutarate oxidoreductase (Kor). So far, Vor and lor have only been detected in peptide‐utilizing hyperthermophilic Archaea. The four 2‐oxoacid oxidoreductases were purified and characterized with respect to their subunit composition, N‐terminal amino acid sequences, and catalytic properties. For and Kor were composed of four different subunits, Vor was composed of three different subunits, and Ior of two different subunits. Comparisons of the N‐terminal amino acid sequences revealed that the four enzymes are structurally related to each other and to the respective enzymes from Pyrococcus and Thermococcus sp. Vor from M. thermoautotrophicum differed from Vor from Pyrococcus furiosus in being composed of only three instead of four different subunits. Evidence is presented that in the autotrophic methanogen the four 2‐oxoacid oxidoreductases have anabolic functions, Vor and Ior being involved in the biosynthesis of amino acids from fatty acids taken up from the growth medium, as shown by 14C‐labelling studies.
The Inactivation of Methionine Synthase in Isolated Rat Hepatocytes by Sodium Nitroprusside
Authors: Nicolaou Anna , Waterfield Catherine J. , Kenyon Susan H. , Gibbons William A.
Published: July 2004

Methionine synthase, the enzyme that catalyses the transfer of a methyl group from 5‐methyl tetrahydrofolate to homocysteine via the cofactor methylcobalamin, is one of the two established mammalian enzymes that utilise a biologically active vitamin B‐12 derivative. Through its substrates, products and downstream metabolites, methionine synthase is directly involved in the sulphur amino acid pathways, polyamine biosynthesis, biological methylations and one‐carbon‐unit transfers. Rat liver methionine synthase was shown to be inactivated by the nitric oxide donor sodium nitroprusside. The inactivation occurred during the treatment of isolated rat hepatocytes in a time‐dependent and dose‐dependent manner with an apparent IC50 value of 170 μM. Highly purified rat liver methionine synthase was inactivated in a partially irreversible manner with an apparent IC50 value of 10 μM. The inactivation has been attributed to nitric oxide released by sodium nitroprusside. Since biomolecules possessing transition state metals are targets for nitric oxide, the possibility of a nitric oxide‐cobalamin interaction could explain the observed inactivation. Nitric oxide is directly involved in different aspects of liver metabolic functions both under physiological and pathological conditions like sepsis and inflammation. The nitric‐oxide‐induced inactivation of methionine synthase could offer a rational explanation for the cellular and cyto‐toxic effects of this highly reactive molecule.
Evidence for a Cysteine‐Histidine Thioether Bridge in Functional Units of Molluscan Haemocyanins and Location of the Disulfide Bridges in Functional Units d and g of the βc‐Haemocyanin of Helix Pomatia
Authors: Gielens Constant , DE Geest Natalie , Xin Xue‐Qin , Devreese Bart , Van Beeumen Jozef , PrÉAux Gisèle
Published: July 2004

In functional units d and g from the βc‐haemocyanin of the gastropod Helix pomatia, amino acid analysis in the presence of dimethyl sulfoxide showed the occurrence of seven cysteine residues. Titration with 5,5′‐dithiobis(2‐nitrobenzoic acid), however, did not reveal any free thiol group. Pepsinolysis at pH 2.0 followed by amino acid analysis and partial sequencing of the cysteine‐containing peptides showed that six cysteine residues are involved in the formation of three disulfide bridges at corresponding positions. The results indicated that the remaining cysteine residue is linked by a thioether bridge to a histidine residue located two positions further in the sequence (H. pomatia d Cys60 His62; H. pomatia g Cys66 His68). This residue corresponds to one of the three histidine residues considered to be involved in the coordination of the copper A atom of the dinuclear copper group of the functional units.

The presence of the thioether bond was further evidenced by an absorption band at 255 nm and by molecular mass determinations with electrospray mass spectrometry on a peptic peptide from H. pomatia g and on peptides obtained by proteolysis of carboxymethylated H. pomatia d with trypsin and pronase. Upon sequence analysis the cysteine‐histidine thioether bridge was cleaved into didehydroalanine (polymers) and 2‐thiolhistidine.

A peptide with a cysteine‐histidine thioether bridge was isolated from pepsinolysates of functional units c, e, f, g and h of Sepia officinalis and unit g of Octopus vulgaris, obtained from haemocyanin of the cephalopods S. officinalis and O. vulgaris, A cysteine‐histidine thioether bridge, which was reported previously for tyrosinase of Neurospora crassa, thus seems to be a general feature for functional units of molluscan haemocyanins.
Generation of Inhibitory Mutants of Hepatocyte Nuclear Factor 4
Authors: Taraviras Stavros , SchüTz Günther , Kelsey Gavin
Published: July 2004

Hepatocyte nuclear factor 4 (HNF‐4) is a member of the nuclear‐receptor gene superfamily. HNF‐4 binds to response elements of several liver‐enriched genes and exhibits a restricted pattern of expression, suggesting an important role for HNF‐4 in tissue‐specific gene regulation. Here, we report the generation of three mutated forms of the HNF‐4 protein, their effects on the ability of the protein to transactivate through HNF‐4–response elements, and their ability to suppress transactivation by the wild‐type protein. Two mutated forms of the HNF‐4 protein, one in which the DNA‐binding domain has been deleted and another in which the HNF‐4 proximal box has been replaced by that of the glucocorticoid receptor, behaved as inhibitors of the wild‐type protein. The properties of a carboxy‐terminal‐deletion mutant allow us to propose a region of HNF‐4 involved in transactivation.
Studies on the ω Subunit of Escherichia Coli RNA Polymerase
Authors: Mukherjee Kakoli , Chatterji Dipankar
Published: July 2004

Highly purified Escherichia coli RNA polymerase contains a small subunit termed ω that has a molecular mass of 10105 Da and is comprised of 91 amino acids. To elucidate the function of ω, whose role is as yet undefined, the subunit was purified to over 95% purity from an overproducing strain [BL21 (pGP1‐2, pE3C‐2)]. Purified ω was then reconstituted with RNA polymerase isolated from an ω‐less mutant. Externally added ω inhibited promoter‐specific transcriptional activity at all promoters tested. Renaturation of fully denatured ω‐less RNA polymerase in the presence of excess ω yielded maximum recovery of activity suggesting a structural rather than functional role for ω.
Purification and Characterization of an Alcohol: N,N‐dimethyl‐4‐nitrosoaniline Oxidoreductase from the Methanogen Methanosarcina Barkeri DSM 804 Strain Fusaro
Authors: Daussmann Thomas , Aivasidis Alexander , Wandrey Christian
Published: July 2004

Cell‐free extracts of Methanosarcina barkeri DSM 804 showed alcohol dehydrogenase activity under aerobic conditions when N,N‐dimethyl‐4‐nitrosoaniline(NDMA) was used as an artificial electron acceptor. The NDMA‐dependent alcohol dehydrogenase (NDMA‐ADH) was purified to approximate homogeneity by column chromatography. It is most probably a homodimeric enzyme consisting of subunits of 45 kDa, the native molecular mass estimated by gel filtration being about 87 kDa. The purified protein had an isoelectric point of 4.3. It possesses a tightly but noncovalently bound NADP(H) cofactor. Each subunit contains 1 mol NADP(H)/mol, about 2 mol Zn2+/mol and significant amounts of magnesium.

The purified enzyme preferably oxidized primary alcohols (including benzyl alcohol). NDMA‐ADH from M. barkeri also catalyzed the stoichiometric dismutation of aldehydes, especially higher aliphatic aldehydes, to form equimolar amounts of the corresponding alcohol and acid without addition of an electron carrier. The enzyme did not catalyze the dehydrogenation of methanol or the disproportionation of formaldehyde and therefore is not directly involved in methanogenesis.

An alignment of the N‐terminal amino acid sequence of the enzyme with the sequences of other alcohol dehydrogenases from methanogenic and nonmethanogenic bacteria indicated no significant identity. Nevertheless there was a quite interesting sequence similarity in the first 30 N‐terminal amino acids to plant cinnamyl alcohol dehydrogenase. NDMA‐ADH from M. barkeri is a novel type of alcohol dehydrogenase in methanogenic bacteria.
Pro108 is Important for Folding and Stabilization of Adrenal Ferredoxin, but does not Influence the Functional Properties of the Protein
Authors: Uhlmann Heike , Iametti Stefania , Vecchio Giuseppe , Bonomi Francesco , Bernhardt Rita
Published: July 2004

The truncated mutant Met‐adrenodoxin‐(4–107)‐peptide of bovine adrenal ferredoxin was expressed as apoprotein in Escherichia coli BL21 and could be reconstituted to the holoform by chemical or enzymatic methods. The reconstituted protein had spectroscopic, functional and redox properties similar to the Met‐adrenodoxin‐(4–108)‐peptide of adrenal ferredoxin, into which the cluster was inserted upon expression in the same Escherichia coli strain. Rate of in vitro cluster insertion into the Met‐adrenodoxin‐(4–107) apoprotein was much lower than for the Met‐adrenodoxin(4–108) apoprotein under identical conditions. Comparative thermodynamic studies with the Met‐adrenodoxin‐(4–108)‐peptide indicated that removal of Pro108 resulted in an extensive decrease of the overall stability of the protein in either oxidation state. The Met‐adrenodoxin‐(4–107)‐peptide showed a higher sensitivity to urea denaturation and had a sensibly lower denaturation temperature, 44.8°C, compared with 51.7°C for mutant Met‐adrenodoxin‐(4–108). The stability of the reduced state of both mutants is slightly lower than that of the oxidized state indicating that this protein region does not undergo major structural changes upon reduction.
Identification and Initial Characterization of a N‐Benzyloxycarbonyl‐Prolyl‐Prolinal (Z‐Pro‐Prolinal)‐Insensitive 7‐(N‐Benzyloxycarbonyl‐Glycyl‐Prolyl‐Amido)‐4‐Methylcoumarin (Z‐Gly‐Pro‐NH‐Mec)‐Hydrolysing Peptidase in Bovine Serum
Authors: Cunningham Damian F. , O'connor Brendan
Published: July 2004

A group of enzymes exists that specifically recognises proline within proteins and peptides. Prolyl endopeptidase is one such enzyme, which cleaves on the carboxyl side of proline within peptide substrates. Its broad specificity towards bioactive peptides has led to its implication in various disease states including neurodegenerative and psychiatric disorders. This association has been based primarily on the abnormal levels of activity observed following the enzymes detection with the reportedly specific fluori‐metric substrate, 7‐(N‐benzyloxycarbonyl‐glycyl‐prolyl‐amido)‐4‐methylcoumarin (Z‐Gly‐Pro‐NH‐Mec). In this study, we report the discovery and preliminary characterisation of a Z‐Gly‐Pro‐NH‐Mec‐hydrolysing activity that is distinct from prolyl oligopeptidase (prolyl endopeptidase). Following the production of serum from bovine whole blood, Z‐Gly‐Pro‐NH‐Mec hydrolysis in serum was determined to be 7.2 U/mg protein. In the presence of 350 nM Z‐Pro‐prolinal, a specific inhibitor of prolyl endopeptidase, residual Z‐Gly‐Pro‐NH‐Mec hydrolysis of 2.6 U/mg protein was observed. This residual activity was resistant to inhibition by Z‐Pro‐prolinal at concentrations in excess of 200 times its reported Ki value for prolyl endopeptidase and could not be inhibited under conditions of prolonged incubation with the inhibitor. Following cation‐exchange chromatography, Z‐Gly‐Pro‐NH‐Mec‐hydrolysing activity was resolved into two distinct entities. The first of these activities was inhibited by Z‐Pro‐prolinal, demonstrated activity towards the thyroliberin analogue, 7‐(pyroglutamyl‐histidyl‐prolyl)‐4‐methylcoumarin (Glp‐His‐Pro‐NH‐Mec), and was catalytically enhanced under reduced assay conditions. This activity was subsequently designated prolyl endopeptidase. The second activity was totally resistant to Z‐Pro‐prolinal inhibition, demonstrated no activity towards Glp‐His‐Pro‐NH‐Mec, and was unaffected when assayed under reduced conditions. It was subsequently designated Z‐Pro‐prolinal‐insensitive Z‐Gly‐Pro‐NH‐Mec‐hydrolysing peptidase (ZIP).
The Kw Recombinase, an Integrase from Kluyveromyces Waltii
Authors: Ringrose Leonie , Angrand Pierre‐Olivier , Stewart A. Francis
Published: July 2004

Site‐specific recombinases of the integrase family share limited amino‐acid‐sequence similarity, but use a common reaction mechanism to recombine distinct DNA target sites. Here we report the characterisation of the Kw site‐specific recombinase, encoded on the 2μ‐like plasmid pKWS1 from the yeast Kluyveromyces waltii. Using in vitro‐translated Kw recombinase, we show that the protein is able to bind and to recombine its putative DNA target site. Recombination is conservative and the Kw target site has a spacer of seven base pairs. We show that Kw recombinase is able to mediate recombination in a mammalian cell line, thus, it has potential for use as a tool for genomic manipulation in heterologous systems.
Altered Methylation Substrate Kinetics and Calcium Binding of a Calmodulin with a Val136→Thr Substitution
Authors: Han Chang‐Hoon , Roberts Daniel M.
Published: July 2004

Calmodulin is trimethylated on Lys115 by a specific calmodulin methyltransferase. Previously, it was shown that the cam2 mutant (Ile136→Thr) of Paramecium has a decreased level of methylated Lys115 [Lukas, T. J., Friedman, M. W., Kung, C. & Watterson, D. M. (1989) Proc. Natl Acad. Sci. USA 86, 7331–7335]. To investigate how this substitution affects calmodulin structure, function and recognition by the calmodulin methyltransferase, a calmodulin with a Thr136 substitution ([Thr136]calmodulin) was expressed in Escherichia coli in an unmethylated form for in vitro enzyme activator, calcium binding and methylation kinetic analyses. [Thr136]calmodulin was indistinguishable from wild‐type calmodulin in saturating (1 mM) calcium in its ability to activate calmodulin‐dependent enzymes and in its steady‐state kinetic properties with isolated calmodulin methyltransferase. However, [Thr136]calmodulin did show two defects: a complete inability to be methylated in the absence of calcium; and defective calcium binding. As a result, an approximate 10‐fold shift in the K0.5 values for calcium dependence of enzyme activation (shifted from 1.1 μM to 9.1 μM of Ca2+ for NAD kinase) and methylation (from 0.71 μM to 7.2 μM of Ca2+ in 0.15 M K+, 2 mM Mg2+) were observed. Non‐denaturing electrophoresis and Tyr138 spectroscopic measurements suggest a difference in the conformation of the calcium‐depleted structures of normal calmodulin and [Thr136]calmodulin. Overall, the results suggest that the mutation in this conserved position in the COOH‐terminal hydrophobic core lowers calcium‐binding affinity and alters the calcium‐depleted structure leading to decreased methylation at physiological Ca2+ concentrations.
Efficient Photoaffinity Labeling of the Rat V1a, Vasopressin Receptor using a Linear Azidopeptidic Antagonist
Authors: Carnazzi Eric , Aumelas André , Phalipou Sylvie , Mouillac Bernard , Guillon Gilles , Barberis Claude , Seyer René
Published: July 2004

We have synthesized and fully characterized by fast‐atom‐bombardment‐mass, NMR and ultraviolet spectroscopies the vasopressin antagonist 3‐azidophenylpropionyl‐D‐Tyr(Me)‐Phe‐Gln‐Asn‐Arg‐Pro‐Arg‐Tyr(3I)‐NH2. Easily radioiodinatable just before use, it has a high affinity for the natural rat liver V1a receptor [dissociation constant (Kd) = 54+/‐220 pM; Carnazzi, E., Aumelas, A., Barberis, C., Guillon, G. & Seyer, R. (1994) J. Med. Chem. 37, 1841–1849] and for both the rat vasopressin V1a receptor expressed in Spodoptera frugiperdu 9 cells (Sf9 cells, Kd= 688+/−35 pM) and in COS‐7 cells (Kd= 320+/−220 pM). This probe labels specifically the V1a receptors in an ultraviolet‐dependent manner, and binds covalently to about 12% of the receptors with high stability over several days, even in dissociation or solubilization conditions. SDS/PAGE studies and autoradiographic analyses of the photolabeled receptors reveal a single band (49.5 kDa) and two bands (63 kDa and 93.6 kDa) for receptor‐probe associations obtained in Sf9 and COS‐7 cells respectively. These molecular masses are consistent with non‐glycosylated and highly glycosylated forms of the receptor, according to each expression system. In rat liver membranes, we have identified apparent molecular masses of about 32, 45 and more than 67 kDa. We finally demonstrated a proteolysis of the receptor that appeared to be Zn2+ and leupeptin sensitive. The high potency of this ligand is promising for the monitoring of the purification of the V1a receptor and for mapping its antagonist‐binding site.
Fourier Transform Infrared Spectroscopic Studies of Proton Transfer Processes and the Dissociation of Zn2+‐Bound Water in Alcohol Dehydrogenases
Authors: Nadolny Carsten , Zundel Georg
Published: July 2004

The following complexes were investigated by Fourier transform difference spectroscopy: binary complexes of alcohol dehydrogenases from yeast (YADH) and horse liver (LADH) with nicotinamide adenine dinucleotide (NAD+) and adenosine (5′)‐diphospho(5)‐β‐D‐ribose (ADP‐Rib); the binary complex of Zn2+)free YADH with NAD+, the ternary complex of LADH with NAD′ and 2,2,2‐trifluoroethanol. After addition of NAD+ to YADH and LADH, protonation of the N1 atom of the adenine ring of NAD+ is observed. It is shown that this proton arises from the dissociation of the Zn2+‐bound water. The interaction of the Zn2+ ion with water is very strong, since this interaction is not just an electrostatic interaction. If the Zn2+ ions are in a tetrahedral environment, a large covalent contribution also occurs. If ADP‐Rib is present instead of NAD+, no protonation of the N1 atom of the adenine ring of ADP‐Rib is found, which demonstrates that the positively charged nicotinamide ring favors the conduction of the positive charge. All these results confirm the mechanism of Brändén et al. (1975): the Zn2+‐bound water is split and the arising (OH)− deprotonates the alcohol. In the case of the ternary complex of LADH with NAD+ and 2,2,2‐trifluoroethanol, we demonstrate that the alcohol is deprotonated and the alcoholate ion is bound directly to the Zn2+ion. The conduction of the proton from the active site to the N1 atom of adenine occurs via a hydrogen‐bonded chain with large proton polarizability due to collective proton motion. The nature and mechanism of this pathway are discussed on the basis of data from previous studies.
Polarised Expression of Human Intestinal N‐benzoyl‐l‐tyroxyl‐p‐aminobenzoic Acid Hydrolase (Human Meprin) α and β Subunits in Madin‐Darby Canine Kidney Cells
Authors: Eldering Joyce A. , Grünberg Jürgen , Hahn Dagmar , Croes Huib J. E. , Fransen Jack A. M. , Sterchi Erwin E.
Published: July 2004

N‐Benzoyl‐l‐tyrosyl‐p‐aminobenzoic acid hydrolase (PPH, human meprin), is a peptidase found in the microvillus membrane of human small intestinal epithelial cells. PPH belongs to the astacin family of zinc‐metalloendopeptaidases and is a protein complex composed of two glycosylated subunits, α and β. The present report describes the cloning of the complete β subunit and the remaining N2‐terminal end of the a subunit for analysis of their primary structures in addition to the examination of their biogenesis in transfected cell cultures. The complete open reading frame of the PPHβ cDNA translates into 700 amino acid residues compared with 746 residues for the PPHα cDNA. The primary structure of β and α subunits are 44% identical and 61% similar. As predicted from their primary structure, the two subunits of PPH have identical modular structures; starting at the N2‐terminus both contain a signal peptide, a propeptide, a protease domain containing the astacin signature, a meprin A5 protein tyrosine phospatase μ (MAM) and a meprin and TRAF homology domain (MATH) domain, an epidermal growth factor(EGF)‐like domain, a putative transmembrane anchor domain and a short cytosolic tail. Pulsekhase labelling and immuno‐Gold lectronmicroscopy of recombinant PPH β and α subunits expressed in transfected Madin‐Darby canine kidney (MDCK) cells show that post‐translational processing and transport of the two subunits are very different. When expressed alone, the β subunit acquired complex glycan residues, readily formed homodimers and was transported to the plasma membrane. Small amounts of PPHP were found in the culture medium. In contrast, the cell‐bound a subunit, when expressed alone, remained primarily in the high‐mannose form, was aggregated and not expressed at the cell surface. However, the bulk of mostly endo‐β‐N‐acetylglucosaminidase H‐resistant α subunit was found in the filtered culture medium. The proteolytic event that leads to the formation of this soluble transport‐competent form occurs in the endoplasmic reticulum (ER). Coexpression of the α subunit with the β subunit allowed the localisation of the a subunit to the plasma membrane. These studies indicate that assembly of the two subunits of PPH is required for the localisation of the α subunit to the plasma membrane. In contrast to rodent meprin, both PPH subunits are apically secreted from MDCK cells.
Immunoreactive α‐Melanotropin as an Autocrine Effector in Human Melanoma Cells
Authors: Loir Béatrice , Bouchard Brigitte , Morandini Renato , Marmol Véronique DEL , Deraemaecker Rika , Garcia‐Borron José Carlos , Ghanem Ghanem
Published: July 2004

Melanotropin is a peptide having several functions, including the stimulation of melanogenesis and the modulation of proliferation of melanocytes and melanoma cells. It acts through binding to high‐affinity receptors of the melanocortin‐1 subtype, exclusively expressed in cells of the melanocytic lineage.

Elevated levels of immunoreactive α‐melanotropin were previously reported in melanoma cell lines, tumours and plasma from patients with melanoma. Here, we show that this high ectopic production of melanotropin is restricted to melanoma and non‐pituitary tumours with the same neuroectodermic origin.

The occurrence of a melanotropin‐specific autocrine loop was further investigated in human melanoma cells. Immunoreactive α‐melanotropin was spontaneously released from a melanoma cell line (HBL) expressing melanotropin receptors on the cell surface. This release was significantly increased in the presence of melanotropin‐related peptides such as corticotropin‐(4–10) peptide and β‐melanotropin, competing for binding to the melanotropin receptor and was directly correlated to the displacement potential of these peptides. Both spontaneous and induced releases of immunoreactive α‐melanotropin could be blocked at low temperatures, suggesting the involvement of intracellular protein movement in the release mechanism.

The release of immunoreactive α‐melanotropin was not significant in melanoma cells expressing very low levels of melanotropin receptors (IGR3) or in non‐melanoma cells (SCC1). However, upon expression of the melanocortin‐1 receptor cDNA into IGR3 cells, spontaneous and competition‐induced releases of immunoreactive α‐melanotropin were both increased and also blocked at low temperatures. This observation further underlines a role for the melanotropin receptor in the release of immunoreactive α‐melanotropin. These experiments indicate that an autocrine loop between the melanocortin‐1 receptor and immunoreactive α‐melanotropin may be functional in human melanoma cells.
Inactivation Kinetics of the Reduced Spinach Chloroplast Fructose‐1,6‐bisphosphatase by Subtilisin
Authors: Chen Ye , Wu Jia‐Wei , Xu Gen‐Jun , Tsou Chen‐Lu , Wang Zhi‐Xin
Published: July 2004

The course of inactivation of the reduced spinach chloroplast fructose‐1,6‐bisphosphatase by digestion with subtilisin has been followed by the progress curve method [Tsou, C. L. (1988) Adv. Enzymol. 61, 381–4361 and found to follow first‐order kinetics. On the basis of the hydrolysis of the substrate, fructose 1,6‐bisphosphate, at different concentrations during proteolysis by subtilisin, the first‐order inactivation rate constants for the free enzyme and the enzyme‐substrate complex can both be determined. The ratio between the inactivation rate constants for the free enzyme and the enzyme‐substrate complex indicates strong protection against subtilisin proteolysis by the substrate. It is proposed that the above ratio can be used as a quantitative measure of substrate protection for enzyme inactivation generally. As it has been found that the site of proteolysis is located in a loop region near the N‐terminus and well away from the active site, the substrate protection indicates a conformation change of the enzyme away from the substrate binding site.
Study of the Substrate‐Binding Properties of Bovine Liver Adenosine Kinase and Inhibition by Fluorescent Nucleoside Analogues
Authors: Pelicano Helene , Maury Georges , Elalaoui Abdelaziz , Shafiee Manijeh , Imbach Jean‐Louis , Goody Roger S. , Divita Gilles
Published: July 2004

Adenosine kinase (AK) catalyzes the phosphorylation of adenosine to AMP with ATP as phosphate donor. Intrinsic fluorescence of bovine liver AK was shown previously to be a sensitive probe to quantify the binding of substrates to the enzyme [Elaloui, A., Divita, G., Maury, G., Imbach, J.‐L. & Goody, R. S. (1994) Eur. J Biochem. 221, 839–8461] AK contains two catalytic, sites: a high‐affinity site, which binds adenosine and AMP selectively; and a site for ATP and ADP. In the present work, these two sites were characterized by combining the quenching of protein fluorescence induced by the binding of the ligands and the fluorescence enhancement observed upon binding of the N‐methylanthraniloyl‐derivated nucleotides or adenosine. A new fluorescent analog of adenosine, 5′‐N‐methylanthraniloyl‐adenosine, was synthesized and shown to bind selectively to the high‐affinity adenosine‐binding site with an affinity similar to that of adenosine (Kd 1 μM). In contrast, 2′(3′)‐N‐methylanthraniloyl derivatives of ATP, adenosine (5′)tetraphospho(5′)adenosine (Ap4A), and adenosine (5′)pentaphospho(5′)adenosine (Ap5A), bind to the enzyme at the ATP site. Methylantraniloyl derivatives of ATP and adenosine were used as tools for selective characterization of a series of adenosine analogues. The bisubstrate inhibitors Ap4A and Ap5A bind to the ATP site with high affinity and apparently not to the adenosine site, thus acting more as ATP analogues than true bisubstrate ligands. The binding properties of a series of adenosine analogues were strongly dependent on the structural modifications on adenosine. The analogues modified at positions 2′ or 3′ show similar affinities for AK as that of adenosine, whereas adenosine analogues modified at the base present a relatively low affinity for the enzyme.
Purification of a Head‐Activator Receptor from Hydra
Authors: Franke Inga , Buck Fritz , Hampe Wolfgang
Published: July 2004

Head activator (HA) is a neuropeptide conserved from hydra to humans. It acts in the development of neuronal cells and is, in hydra, an important factor in head regeneration. Here we report the solubilization and purification of one head activator receptor (Kd≈ 1 nM) from a multiheaded mutant of Chlorohydra viridissima using HA affinity chromatography. Functional solubilization of the HA receptor from hydra membranes was best performed with Triton X‐100 or Chaps. The addition of salt or urea and the protein concentration were important parameters in determining the yield of solubilized receptor. For affinity chromatography HA was coupled to Sepharose. The length of the spacer was optimized with respect to binding of the solubilized HA receptor. After rigorous washing a 200‐kDa protein was eluted from HA Sepharose but not from control Sepharoses coupled to bradykinin or without peptide. Ligand binding was preserved in the eluate from the HA Sepharose, and a 200‐kDa protein could be photoaffinity labeled. The 200‐kDa protein was shown to be glycosylated mainly of the N‐linked type. By Edman degradation of the purified protein sequence information was obtained for the N‐terminus and after protease digestion for several internal peptides.
Chloroplast Import of Four Carotenoid Biosynthetic Enzymes In Vitro Reveals Differential Fates Prior to Membrane Binding and Oligomeric Assembly
Authors: Bonk Michael , Hoffmann Benedikt , Von Lintig Johannes , Schledz Michael , Al‐Babili Salim , Hobeika Elias , Kleinig Hans , Beyer Peter
Published: July 2004

The precursor proteins of the carotenogenic enzymes geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase and lycopene cyclase were imported into isolated pea chloroplasts. Geranylgeranyl diphosphate synthase remained soluble in the stroma in a free form and phytoene synthase associated to thylakoid membranes upon import, both as expected. Surprisingly, phytoene desaturase and lycopene cyclase, which strongly depend on membrane association for enzymatic activity, also remained soluble in the chloroplast stroma. The soluble forms of these enzymes were, however, still competent for membrane‐association, e.g. with protein‐free liposomal membranes. Indeed the soluble forms of phytoene synthase, phytoene desaturase and lycopene cyclase occurred as ATP‐ and cold‐sensitive high‐molecular‐mass complexes. Gel‐filtration experiments and blue native‐PAGE plus autoradiography and western blot analysis indicated a participation of the chloroplast 60‐kDa chaperonin (Cpn60) in the soluble high‐molecular‐mass complexes of imported carotenogenic enzymes. Finally, it was inferred that a membrane‐bound regulatory factor plays a decisive role in membrane‐binding.
Enzymatic Properties of Phage‐Displayed Fragments of Human Plasminogen
Authors: Lasters Ignace , Van Herzeele Nathalie , Lijnen H. Roger , Collen Désiré , Jespers Laurent
Published: July 2004

Two low‐molecular‐mass forms of human plasminogen, plasminogen‐(543–791)‐peptide (micro‐plasminogen), comprising the serine protease domain, and plasminogen‐(444–791)‐peptide (mini‐plasminogen), which in addition contains kringle 5, were displayed on filamentous phage by fusion to the N‐terminus of the minor coat protein pill, to levels of 0.5 molecules micro‐plasminogen–pIII/phage particle and 0.1 molecules mini‐plasminogen–pIII/phage particle. The proenzymes, quantitatively activated by urokinase, showed catalytic efficiencies that were virtually identical to their soluble counterparts, and activity remained associated with the phage as demonstrated by phage ELISA and biopanning with human α2‐antiplasmin or the inhibitor Phe‐Pro‐Arg‐CH2Cl. Micro‐plasminogen–pIII was activated by streptokinase and staphylokinase, two non‐enzymatic plasminogen activators, to the same extent as by urokinase. Activated forms of mini‐plasminogen–pIII, micro‐plasminogen–pIII and mini‐plasminogen dissolved 125I‐labelled fibrin films in a dose‐dependent time‐dependent manner, with 50% lysis in 20 h requiring 0.52, 3.2 and 0.46 nM active plasmin, respectively. Thus, proenzyme moieties derived from plasminogen can be successfully displayed on phage with maintenance of their enzymatic properties. The micro‐plasminogen and mini‐plasminogen phage‐display systems may be useful to study mechanisms of plasminogen activation.
Regulation of the Wound‐Induced Myrosinase‐Associated Protein Transcript in Brassica Napus Plants
Authors: Taipalensuu Jan , Andreasson Erik , Eriksson Susanna , Rask Lars
Published: July 2004

Two slightly differing cDNA clones corresponding to the wound‐inducible form of a previously characterized seed myrosinase‐associated protein (MyAP) have been isolated from Brassica nupus L. The transcripts corresponding to the induced MyAP (iMyAP) were found to be developmentally regulated in various vegetative organs. Both young and old leaves exhibited wound‐inducible iMyAP expression. Furthermore, in the young plant, wounding resulted in a systemic increase in leaves located both acropetally and basipetally to the wounded leaf. Also, the iMyAP transcripts were induced by methyl jasmonate, jasmonic acid and abscisic acid. The different inductions could be antagonized by salicylic acid. A general responsiveness in methyl‐jasmonate‐treated leaves was demonstrated by in situ hybridization. No effect on the amount of iMyAP transcript was detected after feeding the plants with the ethylene precursor 1‐aminocyclopropane‐1‐carboxylic acid. The similarity between MyAP and a lipase from Arubidopsis thuli‐ana indicated a possible function in liberating acylated glucosinolates from their acyl group, thereby making them available for hydrolysis by the myrosinase enzymes. We also report on a reduction in the amount of myrosinase transcripts derived from the vegetatively expressed MB‐gene family after treatment with exogenously applied salicylic acid or abscisic acid.
Cytosolic Aspartate Aminotransferase Encoded by the AAT2 Gene is Targeted to the Peroxisomes in Oleate‐Grown Saccharomyces Cerevisiae
Authors: Verleur Nicolette , Elgersma Ype , Van Roermund Carlo W. T. , Tabak Henk F. , Wanders Ronald J. A.
Published: July 2004

Fatty acid β‐oxidation in peroxisomes requires the continued uptake of fatty acids or their derivatives into peroxisomes and export of β‐oxidation products plus oxidation of NADH to NAD. In an earlier study we provided evidence for the existence of an NAD(H) redox shuttle in which peroxisomal malate dehydrogenase plays a pivotal role. In analogy to the NAD(H)‐redox‐shuttle systems in mitochondria we have investigated whether a malate/aspartate shuttle is operative in peroxisomes. The results described in this paper show that peroxisomes of oleate‐grown Saccharomyces cerevisiae contain aspartate aminotransferase (AAT) activity. Whereas virtually all cellular AAT activity was peroxisomal in oleate‐grown cells, we found that in glucose‐grown cells most of the AAT activity resided in the cytosol. We demonstrate that the gene AAT2 codes for the cytosolic and peroxisomal AAT activities. Disruption of the AAT2 gene did not affect growth on oleate. Furthermore β‐oxidation of palmitate was normal. These results indicate that AAT2 is not essential for the peroxisomal NAD(H) redox shuttle.
Two Ribosomal DNA‐Binding Factors Interact with a Cluster of Motifs on the 5′ External Transcribed Spacer, Upstream from the Primary Pre‐rRNA Processing Site in a Higher Plant
Authors: Caparros‐Ruiz David , Lahmy Sylvie , Piersanti Sarah , Echeverría Manuel
Published: July 2004

In radish the primary processing site in pre‐rRNA has been mapped to a TTTTCGCGC sequence (motif P) in the 5′ external transcribed spacer (5′ ETS) of the ribosomal DNA (rDNA) [Delcasso‐Tremousaygue, D., Grellet, R, Panabiéres, R, Ananiev, E. & Delseny, M. (1988) Eur. J. Biochem. 172, 767–776]. The processing site is just downstream of four similar motifs named A1, A2, A3 and B. The five motifs constitute cluster A123BP. We have described previously that in radish extracts a nuclear protein, nuclear factor B (NF B) specifically binds to motif B [Echeverría, M., Penon, P. & Delseny, M. (1994) Mol. Gen. Genet. 243, 442–452], Here, by means of electrophoretic‐mobility‐shift assays, we describe an rDNA‐binding activity, nuclear factor D (NF D), that interacts with the A123BP cluster. Using various rDNA probes and competitors we show that NF D binds specifically to the A123 clustered motifs but not to similar B or P motifs. We used sequence‐specific DNA‐affinity chromatography to separate NF D from NF B. DNase I footprinting was used to map the binding site of NF D on the A123BP cluster and we compared it with that of NF B on the same probe. The footprint of NF D extends from the A1 motif to the 5′ end of the NF B‐binding site and includes motifs A2 and A3 on each strand. The footprinting of NF B is restricted to motif B and adjacent nucleotides. Thus the NF D‐binding and NF B‐binding sites are distinct but overlap. These two factors bind with a high specificity to the A123BP cluster in the radish 5′ ETS. The possibility that these factors regulate rDNA transcription elongation at the level of the primary pre‐rRNA processing site in crucifers is discussed.
Guanosine 3′,5′‐bis(diphosphate) (ppGpp)‐Dependent Inhibition of Transcription from Stringently Controlled Escherichia Coli Promoters can be Explained by an Altered Initiation Pathway that Traps RNA Polymerase
Authors: Heinemann Marianne , Wagner Rolf
Published: July 2004

An in vitro analysis was performed to investigate the inhibitory mechanism of the global regulatory substances guanosine 3′,5′‐bis(diphosphate) (ppGpp) and guanosine 3′‐diphosphate 5′‐triphosphate (pppGpp) during initiation of transcription. Three promoters with well known differential ppGpp sensitivities in vivo were studied: the Escherichia coli rrnB P2 promoter that is only weakly ppGpp dependent; a P2 base change variant (P2F) that confers both stringent and growth rate regulation; and the completely unregulated PtacI promoter. The in vivo ppGpp dependency for all three promoters was verified in vitro in multiple round transcription reactions, reflecting a combination of the effects at initiation, promoter clearance, and elongation. In the main part of our study, we concentrated on the contribution of initiation complex formation to the overall inhibition of transcription. Kinetic measurements of complex association and dissociation revealed that at sensitive promoters (p)ppGpp triggered an alternative initiation pathway by RNA polymerase. This involved the stabilization of the initial closed complexes, and impeded open complex formation. Subsequently formed ternary complexes were structurally altered. Based on the above findings, we propose a model which suggests that ppGpp‐altered RNA polymerases are preferentially bound and enter the alternative pathway. Thus, discrimination is obtained at early steps of initiation, which causes efficient inhibition at later steps of the transcription cycle probably involving promoter clearance and elongation.
Expression, Purification, Mass Spectrometry, Crystallization and Multiwavelength Anomalous Diffraction of Selenomethionyl PvuII DNA Methyltransferase (cytosine‐N4‐specific)
Authors: O'Gara Margaret , Adams Gail M. , Gong Weimin , Kobayashi Ryuji , Blumenthal Robert M. , Cheng Xiaodong
Published: July 2004

The type II DNA‐methyltransferase (cytosine N4‐specific) M.PvuII was overexpressed in Escherichia coli, starting from the internal translation initiator at Met14. Selenomethionine was efficiently incorporated into this short form of M.PvuII by a strain prototrophic for methionine. Both native and selenomethionyl M.PvuII were purified to apparent homogeneity by a two‐column chromatography procedure. The yield of purified protein was approximately 1.8 mg/g bacterial paste. Mass spectrometry analysis of selenomethionyl M.PvuII revealed three major forms that probably differ in the degree of selenomethionine incorporation and the extent of selenomethionine oxidation. Amino acid sequencing and mass spectrometry analysis of selenomethionine‐containing peptides suggests that Met30, Met51, and Met261 were only partially replaced by selenomethionine. Furthermore, amino acid 261 may be preferentially oxidized in both native and selenomethionyl form. Selenomethionyl and native M.PvuII were crystallized separately as binary complexes of the methyl donor S‐adenosyl‐L‐methionine in the monoclinic space group P21. Two complexes were present per asymmetric unit. Six out of nine selenium positions (per molecule), including the three that were found to be partially substituted, were identified crystallographically.
cDNA Cloning, Recombinant Expression, and Site‐Directed Mutagenesis of Bovine Liver Carnitine Octanoyltransferase
Authors: Cronin Ciarán N.
Published: July 2004

The cDNA for bovine liver carnitine octanoyltransferase (COT) has been cloned by a combination of γgtl 1 library screening and 3′ rapid amplification of cDNA ends (3′‐RACE). The cDNA comprises 338 bases of 5′ non‐coding sequence, a reading frame of 1839 bases including the stop codon, and 820 bases of 3′ non‐coding DNA. The deduced amino acid sequence of 612 residues predicts a protein with a calculated mass of 70263 Da and pI 6.28. The enzyme was expressed in recombinant soluble form in Escherichia coli and was purified by a two‐step procedure to near‐homogeneity with a yield of purified protein of 2–3 mg/l culture. Recombinant COT had similar kinetic properties to those of the enzyme isolated directly from beef liver. Arg505 in COT, conserved in all reported camitine acyltransferase sequences but replaced by asparagine or isoleucine in the choline acetyltransferases, was converted to asparagine by site‐directed mutagenesis. This single mutation resulted in a greater than 1650‐fold increase in the Km, value for COT towards carnitine, but had little effect on the value of kcat or the Km value for the acyl‐CoA substrate. In addition, although choline was an extremely poor substrate for COT, the kcat/Km, ratio towards this substrate was increased fourfold as a result of the mutation. These data support the notion that Arg505 in COT, and other carnitine acyltransferases, contributes to substrate binding by forming a salt bridge with the carboxylate moiety of carnitine.