bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin‐E2 9‐Reductase from Corpus Luteum of Pseudopregnant Rabbit is a Member of the Aldo‐Keto Reductase Superfamily Featuring 20α‐Hydroxysteroid Dehydrogenase Activity
Authors: Wintergalen Norbert , Thole Hubert Heinrich , Galla Hans‐Joachim , Schlegel Werner
Published: September 2004

The prostaglandin‐E2 9‐reductase (PGE2 9‐reductase) activity in the corpus luteum of rabbits corresponds to a cytosolic, NADPH‐dependent enzyme with a molecular mass of 36 kDa. This enzyme was purified from corpora lutea on day 12 of pseudopregnancy with a 266‐fold enrichment. The main purification step was affinity chromatography using Red Sepharose CL‐6B. The efficiency of this column was improved by elution with 1 mM NADH prior to elution of the active fractions with 1 mM NADPH. Amino acid sequence data demonstrate that the rabbit luteal PGE2 9‐reductase has to be classified as a member of the aldo‐keto reductase superfamily. The enzyme revealed a wide substrate specificity comprising the reduction of aldehydes, ketones, and quinones. Apparent kinetic constants were determined using methylglyoxal, DL‐glyceraldehyde, and 9,10‐phenanthrenquinone as substrates. The fully purified enzyme showed two catalytic activities of particular interest: PGE2 9‐reductase and 20α‐hydroxysteroid dehydrogenase (20α‐HSD) activities. The competitive inhibition of 20α‐HSD activity by PGE2 indicates that progesterone and PGE2 are substrates for the same enzyme. From these results, we conclude that prostaglandin and steroid metabolism are tightly linked to each other. For this reason the aldo‐keto reductase could be a key enzyme in the cascade of events leading to the regression of the corpus luteum in the rabbit.
Substrate Specificity of Flavin‐Dependent Vanillyl‐Alcohol Oxidase from Penicillium Simplicissimum
Authors: Fraaije Marco W. , Veeger Cees , Van Berkel Willem J. H.
Published: September 2004

The substrate specificity of the flavoprotein vanillyl‐alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl‐alcohol oxidase catalyzes besides the oxidation of 4‐hydroxybenzyl alcohols, the oxidative deamination of 4‐hydroxybenzylamines and the oxidative demethylation of 4‐(methoxy‐methyl)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed.

Vanillyl‐alcohol oxidase weakly interacts with 4‐hydroxyphenylglycols and a series of catechol‐amines. These compounds are converted to the corresponding ketones. Both enantiomers of (nor)epineph‐rine are substrates for vanillyl‐alcohol oxidase, but the R isomer is preferred.

Vanillyl‐alcohol oxidase is most active with chavicol and eugenol. These 4‐allylphenols are converted to coumaryl alcohol and coniferyl alcohol, respectively. Isotopic labeling experiments show that the oxygen atom inserted at the Cγ atom of the side chain is derived from water. The 4‐hydroxycinnamyl alcohol products and the substrate analog isoeugenol are competitive inhibitors of vanillyl alcohol oxidation.

The binding of isoeugenol to the oxidized enzyme perturbs the optical spectrum of protein‐bound FAD. pH‐dependent binding studies suggest that vanillyl‐alcohol oxidase preferentially binds the phenolate form of isoeugenol (pKa<6, 25°C). From this and the high pH optimum for turnover, a hydride transfer mechanism involving a P ‐quinone methide intermediate is proposed for the vanillyl‐alcohol‐oxidase‐catalyzed conversion of 4‐allylphenols.
Kinetic and Stereochemical Studies of Manno‐Oligosaccharide Hydrolysis Catalysed by β‐Mannanases from Trichoderma Reesei
Authors: HarjunpÄÄ Vesa , Teleman Anita , Siika‐Aho Matti , Drakenberg Törbjorn
Published: September 2004

The two β‐mannanases from Trichoderma reesei with pI of 4.6 and 5.4, respectively, have been characterised by NMR spectroscopy. Following the kinetics of manno‐oligosaccharide degradation with complete progress‐curve analysis the stereospecificity and degradation pattern have been delineated. It was found that degradation of mannotriose and mannopentaose proceeds with retention of the anomeric configuration. Mannotriose degradation proceeds by almost random release of mannose. For mannopentaose there is initially no mannose formed showing that only the two middle mannosidic linkages are attacked. Progress‐curve analysis shows that there is preference (70%) for cleavage of mannopentaose in such a way that mannobiose is released from the reducing end. The final product composition from the mannotriose degradation showed that transglycosylation has to be taken into account. Model calculation and progress‐curve analysis showed that the transglycosylation rate is the fastest of all the rates in this system, 15 s‐1 compared with mannohexaose and mannotetraose hydrolysis rates of 2 s‐1 and mannotriose hydrolysis rate of 0.03 s‐1 at 50°C.
Structurally and Functionally Distinct Ca2+ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa
Authors: Persson Egon , Petersen Lars C.
Published: September 2004

The structural and functional effects of Ca2+ binding to vitamin‐K‐dependent coagulation factor VIIa were investigated. Conformational changes with a midpoint around 0.7 mM Ca2+ quenched the intrinsic protein fluorescence of a fragment of factor VIIa comprising only the light chain and this coincided with an increase in factor Vila amidolytic activity in the absence of tissue factor. Ca2+ binding to sites in factor VIIa and in the fragment with an apparent dissociation constant of 1.3–1.4 mM induced binding to phospholipids. A similar Ca2+ dependency was not observed with factor Vila lacking the N‐terminal 38 or 44 residues of the light chain and the observed effects could thus be attributed to γ‐carboxyglutamic‐acid‐dependent Ca2+ binding. Mg2+ appeared to bind to the site(s) of relatively higher affinity since, although it was less efficient than Ca2+, it stimulated the amidolytic activity and induced quenching of the intrinsic fluorescence. In contrast, Mg2+ did not induce expression of the phospholipid‐interactive structure. The binding properties of two monoclonal antibodies that recognized epitopes in the γ‐carboxy‐glutamic‐acid‐rich domain of factor VIIa corroborated the occurrence of two Ca2+ ‐induced, sequential structural changes and only one of the antibodies recognized the Mg2+‐induced structure. Thus Ca2+ binding to the γ‐carboxyglutamic‐acid‐containing domain appeared to result in at least two distinct structural transitions with different functional consequences. The two (sets of) sites responsible for the observed effects could be distinguished based upon differences in Ca2+ affinity and metal ion selectivity. The interaction between factor VIIa and tissue factor was monitored by means of a direct binding assay and an amidolytic assay. In both systems, half‐maximal Ca2+ enhancement was observed at 0.25 mM. This coincided with a Ca2+‐induced conformational change in factor VIIa associated with fluorescence quenching. The same effect on amidolytic activity was observed with the two N‐terminally truncated forms of factor VIIa and it is presumably mediated by Ca2+ binding to a site located in the serine protease part.
Transcriptional Activation and Repression, Two Properties of the Lymphoid‐Specific Transcription Factor Oct‐2a
Authors: Friedl Erika M. , Matthias Patrick
Published: September 2004

The lymphoid‐specific transcription factor Oct‐2a contains two transcriptional activation domains which are located within the N‐terminal and C‐terminal regions. To study their differential activation properties, we linked the isolated effector domains to the GAL4 DNA‐binding domain. We have shown that both activating regions of Oct‐2a, isolated from their natural context, can activate transcription as promoter factors. In contrast to the C‐terminus, activation by the N‐terminal domain is dependent on a yet unidentified factor(s) binding to the simian virus 40 enhancer. The results obtained by duplication of activation domains or their mixed combination suggest that the domains are functionally independent. However, activation from a remote position could only be achieved with the C‐terminus of Oct‐2a in B cells. In lymphoid cells, higher activation levels were observed, suggesting that distinct B‐cell‐specific cofactors in concert with the effector domains of Oct‐2a might be involved in mediating transcription from proximal and remote positions. Furthermore, we identified a repression domain at the N‐terminus of Oct‐2a. When transferred to a potent activator, transcriptional stimulation was inhibited efficiently. These results underscore the modular structure of Oct‐2a with separable domains for activation and repression and suggest that Oct‐2a might have complex regulatory functions in vivo.
Different Susceptibility of Protein Kinases to Staurosporine Inhibition
Authors: Meggio Flavio , Deana Arianna Donella , Ruzzene Maria , Brunati Anna M. , Cesaro Luca , Guerra Barbara , Meyer Thomas , Mett Helmut , Fabbro Doriano , Furet Pascal , Dobrowolska Grazyna , Pinna Lorenzo A.
Published: September 2004

A systematic analysis reveals that out of 20 protein kinases examined, specific for either Ser/Thr or Tyr, the majority are extremely sensitive to staurosporine, with IC50 values in the low nanomolar range. A few of them however, notably protein kinases CK1 and CK2, mitogen‐activated protein (MAP) kinase and protein‐tyrosine kinase CSK, are relatively refractory to staurosporine inhibition, exhibiting IC50 values in the micromolar range. With all protein kinases tested, namely PKA, CK1, CK2, MAP kinase (ERK‐1), c‐Fgr, Lyn, CSK and TPK‐IIB/p38syk, staurosporine inhibition was competitive with respect to ATP, regardless of its inhibitory power. In contrast, either uncompetitive or noncompetitive kinetics of inhibition with respect to the phosphoacceptor substrate were exhibited by Ser/Thr and Tyr‐specific protein kinases, respectively, consistent with a different mechanism of catalysis by these two sub‐families of kinases. Computer modeling based on PKA crystal structure in conjunction with sequence analysis suggest that the low sensitivity to staurosporine of CK2 may be accounted for by the bulky nature of three residues, Val66, Phe113 and Ile174 which are homologous to PKA Ala70, Metl20 and Thr183, respectively. In contrast these PKA residues are either conserved or replaced by smaller ones in protein kinases highly sensitive to staurosporine inhibition. On the other hand, His160 which is homologous to PKA Glul70, appears to be responsible for the unique behaviour of CK2 with respect to a staurosporine derivative (CGP44171A) bearing a negatively charged benzoyl substituent: while CGP44171A is 10– 100‐fold less effective than staurosporine against PKA and most of the other protein kinases tested, it is actually more effective than staurosporine for CK2 inhibition, but it looses part of its efficacy if it is tested on a CK2 mutant (H160D) in which Hisl60 has been replaced by Asp.

It can be concluded from these data that the catalytic sites of protein kinases are divergent enough as to allow a competitive inhibitor like staurosporine to be fairly selective, a feature that can be enhanced by suitable modifications designed based on the structure of the catalytic site of the kinase.
Expression of a Recombinant Human Glycosyltransferase from a Synthetic Gene and its Utilization for Synthesis of the Human Blood Group B Trisaccharide
Authors: Seto Nina O. L. , Palcic Monica M. , Hindsgaul Ole , Bundle David R. , Narang Saran A.
Published: September 2004

A 1034‐bp synthetic gene encoding the human blood group B glycosyltransferase, which catalyzes the transfer of galactose from UDP‐Gal to Fucα(l‐2)Galβ‐OR to give the blood group B determinant Galα(l‐3)[Fucα(1‐2)]Gal β‐OR (where R is a glycoprotein or glycolipid), has been expressed in Escherichia coli by replacing its membrane‐anchoring domain with an omp A bacterial secretory signal. The active enzyme was purified from the periplasm using UDP‐hexanolamine affinity chromatography and used in the synthesis of preparative amounts of the human blood group B trisaccharide antigen. The substrate specificity and kinetics of the recombinant enzyme were comparable to the enzyme from human sera. Thus we have achieved the construction of a completely synthetic glycosyltransferase gene and its successful expression.
Molecular Cloning of the Human Glucose‐Regulated Protein ERp57/GRP58, a Thiol‐Dependent Reductase
Authors: Hirano Naoto , Shibasaki Futoshi , Sakai Ryuichi , Tanaka Tomoyuki , Nishida Junji , Yazaki Yoshio , Takenawa Tadaomi , Hirai Hisamaru
Published: September 2004

Recently it was shown that putative phospholipase C‐α cDNA does not code for an isotype of the phospholipase C superfamily but for one of the glucose‐regulated proteins (GRPs), ERp57/GRP58. We have isolated human ERp57/GRP58 cDNA from human placenta. Sequence analysis showed that ERp57/GRP58 has two Trp‐Cys‐Gly‐His‐Cys‐Lys motifs completely conserved among the mammals. Bacterially expressed recombinant ERp57/GRP58 protein contained a thiol‐dependent reductase activity which was completely abolished when Ser residues were substituted for Cys residues in both of the two motifs. Furthermore, we have identified a soluble form of ERp57/GRP58 by Western blotting and biosynthetic labeling. In v‐onc transformants of normal rat kidney cells, the expression level of ERp57/GRP58 was elevated at the protein level. In NIH3T3 cells transformed with v‐src, activated c‐src (Y527F) or c‐src, the expression level of ERp57/GRP58 was upregulated in proportion to their transforming abilities. These results indicate that a soluble form of ERp57/GRP58 exists and that this protein may control both extracellular and intracellular redox activities through its thiol‐dependent reductase activity. Moreover, it is likely that ERp57/GRP58 is involved in the oncogenic transformation.
Functional Properties of a Recombinant Chimeric Protein with Combined Thrombin Inhibitory and Plasminogen‐Activating Potential
Authors: Lijnen H. Roger , Wnendt Stephan , Schneider Johannes , Janocha Elke , Van Hoef Berthe , Collen Désiré , Steffens Gerd J.
Published: September 2004

A chimeric protein (rscu‐PA‐40‐kDa/Hir), consisting of the C‐terminal amino acids 53–65 of hirudin (Hir), fused via a 14‐amino‐acid linker sequence to the C‐terminal of a 40–kDa fragment (Ser47‐Leu411) of recombinant (r) single‐chain (sc) urokinase‐type plasminogen activator (rscu‐PA), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. The thrombin inhibitory potential of purified rscu‐PA‐40‐kDa/Hir was confirmed by complete inhibition of the coagulant activity of thrombin at 20–30‐fold molar excess of the chimera, and by the resistance of rscu‐PA‐40‐kDa/Hir to proteolytic cleavage by thrombin. rscu‐PA‐40‐kDa/Hir prolonged the thrombin time of normal human plasma in a dose‐dependent way (reduction of the apparent thrombin concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM hirudin), and inhibited thrombin‐mediated platelet aggregation (reduction of the apparent thrombin concentration to 50% with 40 nM chimeric protein).

The chimera had a specific activity on fibrin films of 57000 IU/mg as compared to 95000 IU/mg for rscu‐PA. The urokinase‐like amidolytic activity of the single‐chain protein was only 220 ILJ/mg but increased to 169000 ILJ/mg after treatment with plasmin, which resulted in quantitative conversion to a two‐chain (tc) derivative (rtcu‐PA‐40–kDa/Hir). Corresponding values for rscu‐PA were 270 and 226000 IU/mg. The catalytic efficiencies for plasmin‐mediated conversion to two‐chain molecules were comparable for rscu‐PA‐40–kDa/Hir and rscu‐PA (0.63 and 0.65 μM‐1 s‐1, respectively). The plasminogen‐activating potential of the single‐chain chimera was comparable to that of rscu‐PA; the catalytic efficiencies for plasminogen activation by their two‐chain counterparts were also similar (0.55 and 0.73 μM‐1 s‐1, respectively). In 2 h, 50% lysis of 125I‐fibrin‐labeled clots prepared from platelet‐poor human plasma and immersed in normal plasma was obtained with 1.3 μg/ml rscu‐PA‐40‐kDa/Hir and with 0.67 μg/ml rscu‐PA, with corresponding residual fibrinogen levels of 74% and 87%, respectively. In the absence of fibrin, 50% fibrinogenolysis in 2 h in normal human plasma required 2.1 μg/ml rscu‐PA, but 7.9 μg/ml rscu‐PA‐40‐kDa/Hir.

Thus, the chimera rscu‐PA‐40‐kDa/Hir has maintained the specific fibrinolytic and plasminogen activating activity of rscu‐PA as well as its fibrinolytic potency in plasma, whereas it displayed a similar or somewhat better fibrin specificity. In addition, the fibrinolytically active concentration in a plasma medium is severalfold lower than the concentration required for thrombin inhibition, which may limit systemic anticoagulant activity. Therefore, further evaluation of the thrombolytic and antithrombotic potential of such chimeric molecules seems to be warranted.
Phosphorylation of Myosin Regulatory Light Chains by the Molluscan Twitchin Kinase
Authors: Heierhorst Jörg , Probst William C. , Kohanski Ronald A. , Buku Angeliki , Weiss Klaudiusz R.
Published: September 2004

The unusually large (≈ 600 to > 3000 kDa) myosin‐associated proteins of the titin/twitchin superfamily are considered to be important cytoskeletal rulers for thick filament assembly in muscle. This function is maintained by approximately 60–240 modular fibronectin‐type‐III and immunoglobulin‐C2 repeats in these proteins which further contain a protein serine/threonine kinase domain of unknown function. In this study, the bacterially expressed kinase domain of Aplysia twitchin was used in order to identify a potential physiological substrate. Addition of the recombinant kinase to Aplysia actomyosin preparations resulted in the specific phosphorylation of the 19–kDa myosin regulatory light chains. The twitchin kinase phosphorylated purified light chains on Thr15 in a region which shared a high degree of similarity with the phosphorylation site for vertebrate smooth muscle myosin light chain kinase. Peptide analogs of the twitchin substrate sequence and the similar sequence in vertebrate smooth muscle myosin light chains were phosphorylated with good kinetic properties. These data reveal the first potential substrate for any of the giant protein kinases and support a dual role of twitchin in molluscan muscle as a cytoskeletal protein as well as a myosin light chain kinase.
High Efficiency of Glycerol 2‐Phosphate and sn ‐Glycerol 3‐Phosphate as Nucleotidyl Acceptors in Snake Venom Phosphodiesterase Esterifications
Authors: Vergeles José maría , García‐Díaz Miguel , Cameselle José Carlos
Published: September 2004

Snake venom phosphodiesterase (SVP) catalyzes the alcoholysis of ATP by primary R‐CH2OH alcohols with uncharged R residues, yielding AMP‐O‐CH2R esterification products. The alcohols compete with water for an SVP‐bound adenylyl intermediate. In this study, it has been shown that SVP also catalyzes the reactions of glycerol 2–phosphate and sn ‐glycerol 3–phosphate with ATP to yield AMP‐O‐glycerophosphoryl esters. The products were identified by HPLC, the dependency of the reactions on glycerol phosphates, ultraviolet spectroscopy, and conversion to AMP by phosphodiesterase, or to AMP‐O‐glyceryl esters by alkaline phosphatase. The results demonstrated that R‐CH2OH alcohols with negatively charged R residues, as well as secondary alcohols, act as adenylyl acceptors in SVP reactions, thus extending the usefulness of SVP as a tool to produce 5′‐nucleotide derivatives. The efficiencies (EA) of glycerol phosphates as adenylyl acceptors were very high at low, millimolar concentrations, but decreased abruptly when the acceptor concentration was increased and, for glycerol 2‐phosphate, when Pi or NaCl was present. In contrast, glycerol EA was independent of its own concentration, Pi, and NaCl. The responses of glycerol phosphates indicate that they act as adenylyl acceptors via a mechanism different from uncharged R‐CH2OH alcohols. The occurrence of an acceptor‐binding enzyme site, specific for negatively charged R residues, and its potential relevance to the in vivo role of 5′‐nucleotide phosphodiesterases as 5′‐nucleotidyl transferases are discussed.
Structural Elucidation of the O‐Antigenic Polysaccharide from Escherichia coli O44: H18
Authors: Staaf Mikael , Widmalm Göran , Weintraub Andrej , Nataro James P.
Published: September 2004

The O‐antigen polysaccharide of the lipopolysaccharide from the enteroaggregative Escherichia coli O44:H18 has been investigated. Sugar and methylation analysis, 1H‐ and 13C‐NMR spectroscopy revealed that the polysaccharide is composed of pentasaccharide repeating units. The sequence of sugar residues was determined by use of two‐dimensional nuclear Overhauser effect spectroscopy and heteronuclear multiple bond correlation experiments. The structure of the repeating unit of the O‐antigen from Escherichia coli O44:H18 is as follows.
image
Molecular Characterization of Api g 1, the Major Allergen of Celery (Apium graveolens), and Its Immumological and Structural Relationships to a Group of 17‐kDa Tree Pollen Allergens
Authors: Breiteneder Heimo , Hoffmann‐Sommergruber Karin , O'Riordain Gabriel , Susani Markus , Ahorn Horst , Ebner Christof , Kraft Dietrich , Scheiner Otto
Published: September 2004

Individuals suffering from immediate hypersensitivity (type‐I allergy) to a particular pollen frequently display intolerance to several foods of plant origin. In this respect, individuals sensitized to birch pollen and/or mugwort pollen frequently display type‐I allergic symptoms after ingestion of celery. In this study, we expressed the major allergenic protein of celery, Api g 1, which is responsible for the birch‐celery syndrome, in the form of a non‐fusion protein. The open reading frame of the cDNA of Api g 1 codes for a protein of 153 amino acids with a molecular mass of 16.2 kDa and 40% identity (60% similarity) to the major allergen of birch pollen, Bet v 1. Furthermore, Api g 1 exhibited similar characteristics to (a) two proteins in parsley induced by fungal infection, (b) the major tree pollen allergens and (c) pathogenesis‐related and stress‐induced proteins in other plant species. The reactivity of recombinant Api g 1 with IgE antibodies present in sera from celery intolerant patients was comparable to that of the natural celery allergen. Cross‐reactivity with Bet v 1 was proven by cross‐inhibition experiments, which provides further support for the existence of the birch‐celery syndrome and for the suggestion that allergies to some vegetable foods are epiphenomena to allergies caused by inhalation of tree pollen.
Purification and Characterization of Acid Cysteine Protease from Metacercariae of the Mammalian Trematode Parasite Paragonimus westermani
Authors: Yamakami Kazuo , Hamajima Fusanori , Akao Shinkichi , Tadakuma Takushi
Published: September 2004

Acid cysteine protease was purified from metacercariae of the mammalian trematode parasite Paragonimus westermani. The purified enzyme had a molecular mass of 27 kDa and was a monomeric polypeptide. The protease had an absolute requirement for a reducing agent for full activity towards fluorescein‐isothiocyanate‐labeled hemoglobin, and it was active in the acidic pH range, with an optimum pH of 4.0. While acidic proteolysis was insensitive to the aspartic protease inhibitor pepstatin A, activity was significantly inhibited by the cysteine protease inhibitors, leupeptin, chymostatin and L‐trans ‐epoxy‐succinyl‐L‐leucylamido(4‐guanidino)‐butane. The sensitivity of the enzyme to the inhibitors was similar to that of cathepsins B and L, but the specificity of the protease towards chromogenic substrates was slightly different from that of the cathepsins. The purified enzyme was highly specific for N‐substituted peptidyl substrates containing arginine in the P, position and phenylalanine in the P2 position, and the protease extensively degraded human native proteins, such as human serum albumin, immunoglobulins, complement components and also endogenous protease inhibitors. Since the protease hydrolyzes both soluble proteins and components of human defense systems, it may facilitate parasite nutrition and evasion of host defense mechanisms.
Bacterial Expression of the Catalytic Domain of 3–Hydroxy‐3–Methylglutaryl‐CoA Reductase (Isoform HMGR1) from Arabidopsis thaliana, and Its Inactivation by Phosphorylation at Ser577 by Brassica oleracea 3‐Hydroxy‐3‐Methylglutaryl‐CoA Reductase Kinase
Authors: Dale Susan , Arró Montserrat , Becerra Beatriz , Morrice Nick G. , Boronat Albert , Hardie D. Grahame , Ferrer Albert
Published: September 2004

The catalytic domain of 3–hydroxy‐3–methylglutaryl‐CoA reductase isoform 1 (HMGR1cd) from Arabidopsis thaliana has been expressed in Escherichia coli in a catalytically active form and purified. The high efficiency of the bacterial expression system together with the simplicity of the purification procedure used in this study resulted in the attainment of large quantities of pure enzyme (about 5 mg/l culture) with a final specific activity of up to 17 U/mg. This specific activity is higher than that reported to date for any 3–hydroxy‐3–methylglutaryl‐CoA reductase (HMGR) purified from a plant source. HMGR1cd activity was completely blocked by the HMGR inhibitor mevinolin (IC50= 12.5nM). No significant differences were observed between the Km values of HMGR1cd for NADPH (71 ± 7 μM) and (S)‐3–hydroxy‐3–methylglutaryl‐CoA (8.3 ± 1.5 μM) and those of pure HMGR preparations obtained from different plant sources. The purified HMGR1cd was reversibly inactivated by phosphorylation at a single site by Brassica oleracea HMGR kinase A, which is functionally related to the mammalian AMP‐activated protein kinase. The site of phosphorylation is Ser577 in the complete sequence of A. thaliana HMGR1. The results in this paper represent the first evidence that a higher plant HMGR is regulated by direct phosphorylation, at least in a cell‐free system. Our results also reinforce the view that the AMP‐activated protein kinase/SNF1 family is an ancient and highly conserved protein kinase system.
Urokinase‐Type Plasminogen Activator/Type‐2 Plasminogen‐Activator Inhibitor Complexes are not Internalized Upon Binding to the Urokinase‐Type‐Plasminogen‐Activator Receptor in THP‐1 Cells
Authors: Ragno Pia , Montuori Nunzia , Rossi Guido
Published: September 2004

The urokinase‐type plasminogen activator (uPA) and its inhibitor PAI‐2 form a covalent complex that, upon binding to the uPA receptor (uPA‐R), is cleaved into two fragments of molecular masses 70 kDa and 22 kDa. The 70‐kDa fragment results from the interaction of the B chain of uPA and PAI‐2 whereas the 22‐kDa fragment is the A chain of the enzyme [13].

We prove that, at 37°C, the 70–kDa fragment is released into the medium, whereas the 22–kDa fragment remains bound to the cell surface. uPA complexed with its other specific inhibitor, PAI‐1, is cleaved into fragments of identical sizes, but the 70–kDa component is internalized via the a2‐macroglobulin receptor.

At 4°C, both uPA/PAI‐2 complex degradation products remain bound to the uPA‐R. We propose that the 70–kDa molecule, which lacks the uPA binding region for uPA‐R, is bound to uPA‐R via a new binding site, unmasked only when uPA‐R is occupied by uPA/PAI‐2 complexes.
New Inhibitors of the Ubiquinol Oxidase of Higher Plant Mitochondria
Authors: Hoefnagel Marcel H. N. , Wiskich Joseph T. , Madgwick Sally A. , Patterson Zoe , Oettmeier Walter , Rich Peter R.
Published: September 2004

A screen has been performed of possible inhibitors of the ubiquinol oxidase of higher plant mitochondria by assaying their effects on cyanide‐insensitive NADH oxidase of mitochondria of Arum maculatum. A number of compounds which have powerful inhibitory effects have been identified.

Potent inhibition was found with compounds related to the previously described N‐propyl gallate, but with the N‐propyl sidechain replaced with alkyl chains of greater hydrophobicity. Titration of a range of partial reactions showed that the inhibitors act specifically on the ubiquinol oxidase. The concentrations of inhibitor required are dependent on the respiratory substrate and on the amount of mitochondria used in the assay. Octyl gallate also proved to be a potent inhibitor of the ubiquinol oxidase in tobacco cell suspensions.

A second class of compounds which strongly inhibit cyanide‐insensitive NADH oxidation is aurachin C and its analogues. Compounds related to aurachin D are much less effective. Titrations of a range of partial reactions indicate that inhibition is caused by a direct action on the ubiquinol oxidase. However, both types of aurachins also act strongly at the Qi site of the cytochrome bc1, complex, as already known to be the case in other systems, and so they are of more limited value for studies of the ubiquinol oxidase.

Titration of the oxidation of NADH via the ubiquinol oxidase in a purified mitochondrial fraction from the spadices of Arum maculatum with octyl gallate gave a half‐maximal effect at a concentration of around 6 nM when the protein concentration was 14 μg ml−1. A similar litre was obtained with a decyl derivative of aurachin C. This allowed us to estimate an upper limit for the concentration of ubiquinol oxidase in these mitochondria of 0.72 ± 0.15 nmol mg−1 protein, or a ratio of ubiquinol oxidase/cytochrome oxidase of about 15 ± 7:1. The measurements also provide a minimal turnover number for the ubiquinol oxidase of 186 ± 42 electrons s−1.

Titration of the ubiquinol oxidase in soybean cotyledon mitochondria with these compounds gave the concentration of inhibitor required to elicit 50% of the maximum observed effect (I50) values about one order of magnitude higher than those found with Arum mitochondria, and again the values depended on the respiratory substrate. An explanation for the variation in I50 values may be found in terms of differences in oxidase concentrations in the different mitochondrial membranes and in the differences in rate‐controlling steps with substrates of different activities.
Quantitative Analysis of the Pyrimidine Metabolism in Pheochromocytoma PC‐12 Cells
Authors: Slingerland Robbert J. , Van Gennip Albert H. , Bodlaender Jeroen M. , Voûte P. A. , Van Kuilenburg André B. P.
Published: September 2004

A detailed quantitative study of pyrimidine metabolism in exponentially growing rat pheochromocytoma PC‐12 cells has been performed. The sizes of ribonucleotide pools have been analysed and the pathways and the rates of metabolism of uridine, cytidine and aspartic acid have been determined, based on the incorporation of radioactive label. The fluxes of radioactive label through uridinecytidine kinase, cytidine deaminase, CTP synthetase, nucleoside monophosphate kinase and nucleoside diphosphate kinase were obtained, as well as the flux through the pyrimidine de novo pathway. Also, the fluxes of radioactive label towards UDP‐sugars, CDP‐compounds, DNA and RNA were quantified in situ under steady‐state conditions in intact PC‐12 cells. From these fluxes of radioactivity, distribution ratios at the branch points of the metabolism were obtained. The pyrimidines synthesised via the de novo pathway were preferentially used for the synthesis of UDP‐N‐acetylhexosamines and UDP‐hexoses, whereas the salvage of precursors from the medium contributed, to a large extent, to the synthesis of RNA. Therefore, we postulate that at least two different UTP pools exist in these cancer cells derived from the neural crest. Furthermore, after metabolism of radiolabeled cytidine and uridine into UTP, radiolabel was distributed in a similar manner from UTP towards UDP‐N‐acetylhexosamines, UDP‐hexoses and RNA‐UMP. Uridine, as well as cytidine, was channelled towards nucleic acids via small compartmented ribonucleotide pools.
Structural Characterization of an Intramolecular RNA Triple Helix by NMR Spectroscopy
Authors: Klinck Roscoe , Liquier Jean , Taillandier Eliane , Gouyette Catherine , Huynh‐Dinh Tarn , Guittet Eric
Published: September 2004

A chemically synthesized 29‐base RNA oligomer, designed to fold to form an intramolecular triple helix at acid pH, has been studied by NMR spectroscopy. The molecule consisted of seven U·A·U or C+·G·C base triples joined by two pyrimidine tetra‐loops. The fold was such that the third strand was Hoogsteen base‐paired in the major groove of a Watson‐Crick paired double helix. The nature and size of the molecule required the use of an assignment strategy using two‐ and three‐dimensional homonuclear methods, complemented by a natural abundance 13C correlation experiment. The assignment of the majority of the exchangeable and non‐exchangeable resonances is presented. The data suggest a C3′‐endo sugar puckering for all the nucleotides involved in base triples. A preliminary structural model consistent with the NMR data is presented.
Expression in Aspergillus niger of the Starch‐Binding Domain of Glucoamylase
Authors: Le Gal‐Coëffet Marie‐Françoise , Jacks Amanda J. , Sorimachi Kay , Williamson Michael P. , Williamson Gary , Archer David B.
Published: September 2004

Glucoamylase 1 from Aspergillus niger is an economically important enzyme in many industrial processes. It hydrolyses granular starch and comprises two distinct domains, a catalytic and a starch‐binding domain (SBD). We have transformed A. niger with an expression vector for the secretion of SBD for physicochemical studies. This was achieved by introducing into the glucoamylase gene a short sequence encoding an endoproteolytic cleavage recognition site such that free SBD was secreted at yields up to 200 mg/1. Free SBD was also obtained by proteolytic digestion of full‐length glucoamylase 1. Electrospray mass spectroscopy was used to determine the carbohydrate content of both SBDs. It revealed that the engineered one is more glycosylated: an average of three mannose residues compared to one for the proteolytically derived SBD. Sequencing results also suggest partial glycosylation for the three Thr residues involved (510, 511, 513). It is probable that the engineered SBD represents the true glycosylation level of the SBD in native glucoamylase. Binding of β‐cyclodextrin to the SBD was investigated. It was found that the stoichiometry and the spectral perturbation of Trp residues were identical for both SBDs, but the engineered SBD bound less strongly to the ligand. At high concentrations of β‐cyclodextrin relative to the estimated Kd values, the maximum absorbance changes were identical. The observed difference at low β‐cyclodextrin levels was probably due to the higher level of glycosylation of the expressed SBD. We conclude that the proteolytically derived and expressed starch binding domains both bind 2 mol β‐cyclodextrin/mol protein, but that the pattern of glycosylation and strength of binding are different.
1H and 15N Assignments and Secondary Structure of the Starch‐Binding Domain of Glucoamylase from Aspergillus niger
Authors: Jacks Amanda J. , Sorimachi Kay , Le Gal‐Coëffet Marie‐Francoise , Williamson Gary , Archer David B. , Williamson Michael P.
Published: September 2004

1H and 15N NMR resonance assignments of the granular starch‐binding domain (SBD) of glucoamylase from Aspergillus niger have been made by multi‐dimensional homonuclear and heteronuclear NMR techniques. Secondary structure analysis based on chemical shifts, 1H‐1H NOEs, coupling constants and backbone amide exchange data indicates the presence of a well‐defined β‐sheet structure. This consists of one parallel and five antiparallel pairs of β‐strands forming two β‐sheets. Cis‐trans isomerisation of proline residues and O‐glycosylation of threonine residues are observed and compared between the proteolytically derived SBD fragment and the recombinant protein. Structural features of the SBD in solution were compared to the X‐ray crystal structure of a homologous domain of cyclodextrin glyco‐syltransferase from Bacillus circulans. There are some differences in the locations of the start and end of β‐strands but overall the two structures are very similar. This study will form the basis for the structure determination of the granular SBD and of its complexes.
Evidence for Elongation of the Helical Pitch of the RecA Filament Upon ATP and ADP Binding Using Small‐Angle Neutron Scattering
Authors: Ellouze Christine , Takahashi Masayuki , Wittung Pernilla , Mortensen Kell , Schnarr Manfred , Nordén Bengt
Published: September 2004

Structural changes of the RecA filament upon binding of cofactors have been investigated by small‐angle neutron scattering. Both ATP and ADP increased the helical pitch of the RecA homopolymer, which is observed to be 7 nm in the absence of any cofactor. The binding of ATP altered the pitch to 9 nm, whereas the binding of ADP only produced a pitch of 8.2 nm. The pitch determined for the RecA complex with the ATP analog adenosine 5′‐[γ‐thio]triphosphate was similar to that found with ATP. Thus, at least three, somewhat different, RecA helical filamentous structures may form in solution. The binding of DNA to RecA did not alter the pitch significantly, indicating that the cofactor binding is the determining factor for the size of the helical pitch of the RecA filament. We also found that elongation of the helical pitch is a necessary, but not a sufficient condition, for the coprotease activity of RecA. The presence of acetate or glutamate ions is also required. The pitch of the ADP · RecA filament is in agreement with that found in the crystal structure. This correlation indicates that this structure corresponds to that of the ADP · RecA filament in solution, although this is not the species active in recombination.
Differential Functional Activities of Rainbow Trout and Human Estrogen Receptors Expressed in the Yeast Saccharomyces cerevisiae
Authors: Petit Fabrice , Valotaire Yves , Pakdel Farzad
Published: September 2004

The cDNA of rainbow trout estrogen receptor (rtER), highly and stably expressed in yeast, Saccharomyces cerevisirie, was used to analyse the biological activity of the receptor. The rtER mRNA encoded a 65‐kDa protein which was immunorevealed by a specific antibody and migrated with the authentic rtER major protein form detected in trout liver. Yeast rtER bound estradiol with high affinity and the dissociation constant (Kd= 1.35 nM) was very similar to the value measured from trout liver extracts but 3–5‐fold higher than the Kd found for human estrogen receptor (hER). This indicates therefore that the rtER has a lower estradiol affinity compared to the human receptor. While the hER Kd remained unchanged at both 4°C or 22°C, it was slightly modified at 30°C. The Kd measured for rtER at 22°C and 30°C were about 2‐fold, and 12‐fold higher, respectively, than the Kd obtained at 4°C suggesting an alteration of the rtER affinity for its ligand at elevated temperature. To examine the estrogen‐receptor‐mediated activation of transcription in yeast, reporter plasmids integrated or not in the yeast genome were used. The reporter genes consist of one, two, or three copies of estrogen‐responsive elements (ERE) upstream of the yeast proximal CYC1 or URA3 promoters fused to the lacZ gene of Escherichia coli coding for β‐galactosidase. The induction of β‐galactosidase activity for all reporter genes was strictly dependent on the presence of rtER and estrogens. The activation of transcription mediated by rtER responded in an estradiol‐dosedependent manner as in animal cells. However, compared to hER, the estradiol concentration necessary to achieve maximal activation was 10‐fold higher. This is probably a consequence of the lower estradiolaffinity for rtER compared to hER. The levels of induction of the reporter genes containing two or three ERE were strongly enhanced compared to the one ERE construct. This is in agreement with the synergistic effect previously described for multiple ERE. The magnitudes of transcriptional induction mediated by rtER and hER were similar when the reporter gene containing three ERE was used but changed when the one ERE construct was used. In this case transcriptional activation mediated by rtER was 10–20‐fold lower. This suggests that rtER requires protein/protein interaction for its stabilization on DNA. Antiestrogens were able to bind rtER and promote gene transcription. However, to produce effects comparable to those obtained with estrogens, much higher concentrations were required. This may imply nonetheless that antihormones were capable of provoking efficient interactions of rtER with the transcriptional machinery
The Covalent Linkage of a Viologen to a Flavoprotein Reductase Transforms it into an Oxidase
Authors: Bes M. Teresa , De Lacey Antonio L. , Peleato M. Luisa , Fernandez Victor M. , Gómez‐Moreno Carlos
Published: September 2004

Chemical cross‐linkage of the positively charged viologenN‐methyl‐N′‐(aminopropyl)‐4‐4′‐bipyridinium dibromide (APMV) to the enzyme ferredoxin‐NADP+reductase from the cyanobacteriumAnabaenaPCC 7119 has been performed using the carbodiimide 1‐ethyl[3‐(3‐dimethylaminopropyl)]carbodiimide. 0.5–1 mol, depending on the preparation, is introduced for each mol enzyme. The residue involved in the covalent linkage with the viologen, Glu139, has been identified using HPLC separation of the modified proteolytic peptides and subsequent sequencing. Modification of the enzyme changes its catalytic specificity since it is able to react directly with oxygen; this is observed by a high NADPH oxidase activity, which is completely absent in the native enzyme. More important, this new enzymic activity is indicative of the intramolecular electron transfer between the natural redox cofactor FAD and the artificially introduced viologen. Electrons can also flow in the reverse direction, from the viologen to the FAD group, then to NADP+, when the reaction is performed using glassy‐carbon electrodes to reduce the viologen. Cyclic voltammetry experiments have shown that there is a small catalytic current between the electrode and the enzyme which is not observed in the native enzyme.
Compressibility of the Heme Pocket of Substrate Analogue Complexes of CytochromeP‐450cam‐CO
Authors: Jung Christiane , Bon Hoa Gaston Hui , Davydov Dmitri , Gill Edward , Heremans Karel
Published: September 2004

The effect of hydrostatic pressure on the electronic absorption spectrum of the carbon monoxide complex of cytochrome P‐450cam (CYP101) in the presence of various substrates was studied.

With increasing pressure the wavenumber of the Soret band in the cytochromeP‐450–CO complex shifts linearily to lower values (red‐shift) and the half‐width increases (broadening). The microscopic theory of solvent–solute interaction discussed by Laird and Skinner is used to explain the observed pressure effects. According to this theory, the slope of the red‐shift of the Soret band is related to the compressibility of the chromophore environment, that is the heme moiety of the hemoproteins. It was found that the slope of the red‐shift and the slope of the broadening of the Soret band for the CO complex in the presence of various substrate analogues increase with the decrease of the initial high‐spin content at 0.1 MPa in the oxidized state. Variation of the high‐spin content reflects the changes in the number of water molecules and/or changes in the polarity of the heme environment. The higher compressibility of the cytochromeP‐450 complexes with the substrate analogues, which induce a lower degree of the high‐spin content in the oxidized protein, is explained by the ability of the water molecules in the heme moiety to transmit the pressure effect on the protein structure to the heme chromophore. Therefore, a larger pressure‐induced red‐shift of the Soret band in the CO complex of cytochromeP‐450cam might indicate a higher water content in the heme environment.
Man9‐Mannosidase from Human Kidney is Expressed in COS Cells as a Golgi‐Resident Type II Transmembrane N‐Glycoprotein
Authors: Bieberich Erhard , Bause Ernst
Published: September 2004

Man9‐mannosidase, an α1,2–specific exo‐enzyme involved in N‐linked oligosaccharide processing, has been cloned recently from a human kidney cDNA library [Bause, E., Bieberich, E., Rolfs, A., Volker, C. & Schmidt, B. (1993) Eur. J. Biochem. 217, 533–540], Transient expression in COS 1 cells of the enzyme resulted in a more than 20–fold increase of a catalytic activity cleaving specifically α1,2–mannosidic linkages in [14C]Man9‐GlcNAc2 or [14C]Man5‐GlcNAc2. Man9‐mannosidase is expressed as a N‐glycoprotein with a molecular mass of 73 kDa. Its enzymic activity is metal ion dependent and inhibited strongly by 1–deoxymannojirimycin (50% at 100 μM). Proteolytic studies with the membrane‐associated form of Man9‐mannosidase support the view that the enzyme is a type II transmembrane protein as predicted from its cDNA sequence. Several lines of evidence suggest that Man9‐mannosidase, as expressed, is N‐glycosylated at one of three potential Asn‐Xaa‐Thr/Ser/Cys acceptor sites. Approximately 50% of the N‐linked oligosaccharide chains are removed by endoglycosidase H treatment, whereas complete deglycosylation of the enzyme is observed, when transfected cells were cultured in the presence of the Golgi mannosidase II inhibitor swainsonine, indicating that the sugar moiety of Man9‐mannosidase is processed partially by Golgi‐resident enzymes. This observation is consistent with the results of indirect immunofluorescence studies, pointing to a localization of the Man9‐mannosidase predominantly in the juxtanuclear Golgi region. This localization clearly differs from that of pig liver Man9‐mannosidase which appears to be located in the endoplasmic reticulum and transient vesicles.
Solution Characterisation by NMR Spectroscopy of Two Horseradish Peroxidase Isoenzyme C Mutants with Alanine Replacing Either Phe142 or Phe143
Authors: Veitch Nigel C. , Williams Robert J. P. , Bone Neil M. , Burke Julian F. , Smith Andrew T.
Published: September 2004

Site‐directed mutagenesis of the horseradish peroxidase isoenzyme C (HRP C) gene has been undertaken in order to provide two recombinant enzymes where alanine replaces either Phe142 or Phe143 ([F142AJHRP C and [F143A]HRP C., respectively). These heme enzymes have been characterised in solution using proton NMR spectroscopy for both the high‐spin resting and low‐spin cyanide‐ligated states. Comparison of their NMR spectra with those recorded for wild‐type plant HRP C indicates that both the protein fold and the structure of the heme pocket are maintained. The structural integrity of the aromatic donor molecule binding site is altered as a result of the substitution of Phe142 by Ala, but not by the corresponding substitution at Phe143. This is evident from analysis of perturbations to the chemical shift and linewidth parameters of the proton resonances of two Phe side chains, Phe A and Phe B, that participate in this site. The resting and cyanide‐ligated states of [F142A]HRP C bind the aromatic donor molecule, benzhydroxamic acid, three to four times more weakly than the analogous states of wild‐type plant HRP C. A titration of cyanide‐ligated [F142A]HRP C with benzhydroxamic acid, monitored by NMR spectroscopy, further reveals that the dynamics of complex formation are considerably altered, in that only one of the two possible benzhydroxamic acid binding modes established for the cyanide‐ligated wild‐type enzyme is significantly populated. Although the assignment of Phe A and Phe B cannot be made to either Phe142 or Phe143, the results confirm that Phe142 is an important, although indirect, determinant of aromatic donor molecule binding and dynamics. The role of phenylalanine side chains in the binding of aromatic donor molecules by heme peroxidases is discussed in the light of these observations and a recent structural model for HRP C.