bluuebunny/crossref_metadata_2025 huggingface dataset:
The NAD‐linked aromatic α‐hydroxy acid dehydrogenase from Trypanosoma cruzi
Authors: Cazzulo Franke María C , Vernal Javier , Cazzulo Juan J , Nowicki Cristina
Published: March 2003

Trypanosoma cruzi, the protozoan parasite causing Chagas disease, contains a novel aromatic α‐hydroxy acid dehydrogenase. This enzyme is responsible, together with tyrosine aminotransferase, for the catabolism of aromatic amino acids, which leads to the excretion of aromatic lactate derivatives into the culture medium. The gene encoding the aromatic α‐hydroxy acid dehydrogenase has been cloned through a combined approach using screening of an expression genomic library with antibodies, peptide sequencing and PCR amplification. Its sequence shows high similarity to the cytosolic malate dehydrogenases. However, the enzyme has no malate dehydrogenase activity. The gene seems to be present in a single copy per haploid genome and is differentially expressed throughout the parasite’s life cycle, the highest levels being found in the insect forms of T. cruzi. The purified recombinant enzyme, expressed in Escherichia coli, was unable to reduce oxaloacetate and had kinetic constants similar to those of the natural aromatic α‐hydroxy acid dehydrogenase. Sequence comparisons suggest that the aromatic α‐hydroxy acid dehydrogenase derives from a cytosolic malate dehydrogenase no longer present in the parasite, made redundant by the presence of a glycosomal malate dehydrogenase as a member of a shuttle device involving the mitochondrial isoenzyme.
The chimeric peptide [Lys(−2)‐Arg(−1)]‐sarafotoxin‐S6b, composed of the endothelin pro‐sequence and sarafotoxin, retains the salt‐bridge staple between Arg(−1) and Asp8 previously observed in [Lys(−2)‐Arg(−1)]‐endothelin
Authors: Aumelas André , Chiche Laurent , Kubo Shigeru , Chino Naoyoshi , Watanabe Takushi X. , Kobayashi Yuji
Published: March 2003

The chimeric peptide [Lys(−2)‐Arg(−1)]‐sarafotoxin‐S6b (KR‐SRTb) designed from the Lys–2‐Arg–1 dipeptide of the endothelin pro‐sequence and the sarafotoxin‐S6b sequence was synthesized. Its contractile activity was found to be decreased markedly when compared with that of the parent SRTb. In contrast, the extension by the Lys‐Arg dipeptide was found to increase the formation of the native disulfide isomer (82/18 versus 96/4) when the reaction was carried out in the presence of redox reagents. The solution structure of KR‐SRTb was determined by NMR as a function of pH. In the carboxylic acid state, the structure consists of the cystine‐stabilized α‐helical motif, with the α‐helical part spanning residues 9–15, and of an unstructured C‐terminal tail. In the carboxylate state, the structure is characterized by a salt‐bridge between Arg(−1) and Asp8, which we identified previously in the [Lys(−2)‐Arg(−1)]‐endothelin‐1 peptide (KR‐ET‐1). The fact that this salt‐bridge is commonly observed in KR‐SRTb and KR‐ET‐1, despite the 33% sequence difference between the corresponding parental peptides, highlights the remarkable adaptability of the Lys‐Arg extension for the formation of a special salt‐bridge. As a consequence, this salt‐bridge, which does not depend on either the 4–7 sequence of the loop or the C‐terminal sequence, appears to be particularly well suited to improve the stability of the cystine‐stabilized α‐helical motif. Therefore, because of its high yield in the native disulfide arrangement and its high permissiveness for sequence mutation in the 4–7 loop, such a stabilized cystine‐stabilized α‐helical motif could be a valuable scaffold for the presentation of a library of constrained short peptides.
Production and activation of recombinant hK2 with propeptide mutations resulting in high expression levels
Authors: Lövgren Janita , Tian Sha , Lundwall Åke , Karp Matti , Lilja Hans
Published: March 2003

Human glandular kallikrein 2 (hK2) is a serine protease expressed mainly by the prostate gland with 80% identity in primary structure to prostate specific antigen (PSA). hK2 has proven to be a useful marker of prostate cancer which can be used in combination with PSA to better discriminate between prostate cancer and benign prostate hyperplasia. The studies on hK2 have been hampered by its very low phyciological levels (6 µg·mL−1), its close similarity to PSA, and the low expression levels obtained using recombinant procedures to produce hK2 (0.7 mg·L−1). We have now generated propeptide mutations of hK2 which can be used to isolate stable, inactive prohK2 mutants. Compared with wild‐type hK2, expression of the propeptide hK2 mutants increases the expression levels up to 15–40‐fold giving 10–30 mg hK2·L−1. These results indicate that the low expression levels of wild‐type hK2 are related to the activation or autoactivation of the wild‐type enzyme and the instability of the active protease in cell culture and possibly also in tissue. The purified mutant hK2 may be activated by either enterokinase or factor Xa to generate an enzyme for use in functional studies with the characteristics of the original wild‐type protein. Further, the stable inactive mutant hK2 protein may be used for immunizations to generate novel monoclonal antibodies, used as standard material for clinical assays or in crystallization studies where large quantities of protein are required.
Molecular characterization of the Caenorhabditis elegans Rho GDP‐dissociation inhibitor
Authors: Yap Seow F. , Chen Weining , Lim Louis
Published: March 2003

GDP‐dissociation inhibitors (GDIs) form one of the classes of regulatory proteins that modulate the cycling of the Ras superfamily of GTPases between active GTP‐bound and inactive GDP‐bound states. We report here the characterization of the Caenorhabditis elegans RhoGDI (CeRhoGDI) as part of our investigations into Rho‐GTPase signalling pathways that are involved in nematode development. CeRhoGDI is a 23‐kDa protein that is localized predominantly in the cytosol. CeRhoGDI interacts only with the lipid‐modified forms of C. elegans Rho‐GTPases, CeRhoA, CeRac1 and Cdc42Ce, in vitro and is able to solubilize the membrane‐bound forms of these GTPases. CeRhoGDI recognizes the GTPases in both GTP‐ and GDP‐bound forms; hence it inhibits both the guanine‐nucleotide dissociation and GTP‐hydrolysis activities. The inhibitory activity towards the GTP‐bound GTPases is weak compared with that towards GDP‐bound GTPases. CeRhoGDI is expressed throughout development and is highly expressed in marginal and vulval epithelial cells, in sperm cells and spicules. Taken together, our results suggest that CeRhoGDI may be involved in specific morphogenetic events mediated by the C. elegans Rho‐GTPases.
Mutational analysis of Asp51 of Anabaena azollae glutamine synthetase.
Authors: Crespo José L. , Guerrero Miguel G. , Florencio Francisco J.
Published: March 2003

The role of Asp51 in the catalytic activity of glutamine synthetase from the cyanobacterium Anabaena azollae has been analyzed. Five mutant enzymes, D51S, D51A, D51E, D51N and D51R, were constructed by site‐directed mutagenesis and characterized. Asp51 appears to participate in the binding of ammonium ion, as affinity for this substrate was affected in all cases, although it varied according to the charge and/or size of the amino‐acid residue, decreasing in the order Glu > Asn > Ser > Ala. The replacement of Asp51 by Glu (D51E) conferred besides a high resistance to the herbicides l‐methionine‐dl‐sulfoximine and phosphinothricin, as a result of a decreased phosphorylation ability.
Growth factor‐induced promoter activation of murine phospholipase C δ4 gene
Authors: Fukami Kiyoko , Takenaka Kei , Nagano Kohji , Takenawa Tadaomi
Published: March 2003

Phospholipase C δ4 (PLCδ4) is one of the delta‐type PLC isozymes, the expression of which is induced in nuclei by treatment with serum and also in some cancer cells. We isolated and analyzed a promoter region of the murine PLCδ4 gene. DNA sequence analysis showed that this region is GC‐rich and has no TATA box, and the region from −143 to −127 was found, by luciferase activity and gel mobility‐shift assay, to be essential for transcription of PLCδ4. We also found that the promoter activity of PLCδ4 was stimulated by treatment with growth factors such as bradykinin, lysophosphatidic acid, and Ca2+ ionophore in addition to serum. In parallel, we detected PLCδ4 mRNA induction and an increase in complex formation of the promoter region and nuclear protein from HeLa cells on stimulation with these growth factors. Finally, we found that trapping the growth factor‐induced cytoplasmic Ca2+‐inhibited activation of the promoter activity and protein induction in nuclei. These results show that PLCδ4 may have an important role in nuclei in response to growth factors, and its expression may be partially regulated by an increase in cytoplasmic Ca2+.
The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms
Authors: van der Veen Bart A. , van Alebeek Gert‐Jan W. M. , Uitdehaag Joost C. M. , Dijkstra Bauke W. , Dijkhuizen Lubbert
Published: March 2003

Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme–intermediate complex (substituted‐enzyme intermediate). Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms. Disproportionation (cleavage of an α‐glycosidic bond of a linear malto‐oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping‐pong mechanism. Cyclization (cleavage of an α‐glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single‐substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow elucidation of the kinetic mechanism. Michaelis–Menten kinetics, however, have been observed using shorter amylose chains. Coupling (cleavage of an α‐glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto‐oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism.

In view of the different kinetic mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate. In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme. These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes.
Cloning of the guanylate kinase homologues AGK‐1 and AGK‐2 from Arabidopsis thaliana and characterization of AGK‐1
Authors: Kumar Vinod , Spangenberg Oliver , Konrad Manfred
Published: March 2003

Guanylate kinase is an essential enzyme for nucleotide metabolism, phosphorylating GMP to GDP or dGMP to dGDP. The low molecular mass cytosolic forms of guanylate kinase are implicated primarily in the regulation of the supply of guanine nucleotides to cell signalling pathways. The high molecular mass and membrane‐associated forms of guanylate kinase homologues, notably found in neuronal tissues, are assigned roles in cell junction organization and transmembrane regulation. Here, we describe the first plant guanylate kinase‐encoding genes, AGK1 and AGK2, from Arabidopsis thaliana. The nucleotide sequences of their genomic and cDNA clones predict proteins that carry N‐terminal and C‐terminal extensions of the guanylate kinase‐like domain. The amino acid sequences of this domain share 46–52% identity with guanylate kinases from yeast, Escherichia coli, human, mouse and Caenorhabditis elegans. Arabidopsis guanylate kinases (AGKs) exhibit a high degree of conservation of active site residues and sequence motifs in common with other nucleoside monophosphate kinases, which suggests overall structural similarity of the plant proteins. Although bacterially expressed AGK‐1 is enzymatically much less active than yeast guanylate kinase, its kinase domain is shown to complement yeast GUK1 recessive lethal mutations. AGKs are expressed ubiquitously in plant tissues with highest transcriptional activity detected in roots. The identification of AGKs provides new perspectives for understanding the role of guanylate kinases in plant cell signalling pathways.
Structure of the O‐specific polysaccharide of Proteus penneri 71 and classification of cross‐reactive P. penneri strains to a new proposed serogroup O64
Authors: Zych Krystyna , Kocharova Nina A. , Kowalczyk Marcin , Toukach Filip V. , Kaminska Dominika , Shashkov Alexander S. , Knirel Yuriy A. , Sidorczyk Zygmunt
Published: March 2003

A neutral O‐specific polysaccharide (O‐antigen) was isolated from the lipopolysaccharide (LPS) of the bacterium Proteus penneri 71. On the basis of sugar analysis and 1H‐ and 13C‐NMR spectroscopic studies, including two‐dimensional COSY, 13C,1H heteronuclear COSY and ROESY, the following structure of the trisaccharide repeating unit of the polysaccharide was established:

         →3)‐β‐d‐GlcpNAc‐(1→4)‐β‐d‐GlcpNAc‐(1→3)‐α‐d‐Galp‐(1→

The polysaccharide has the same carbohydrate backbone as the O‐specific polysaccharide of P. penneri 19 and both are similar to that of P. penneri 62 studied by us previously. A cross‐reactivity of anti‐P. penneri 71, 19 and 62 O‐antisera with 11 P. penneri strains was revealed and substantiated at the level of the O‐antigen structures. These strains could be divided into three subgroups within a new proposed Proteus O64 serogroup containing P. penneri strains only.
Structure of a 2‐aminoethyl phosphate‐containing O‐specificpolysaccharide of Proteus penneri 8 from a new serogroup O67
Authors: Knirel Yuriy A. , Zych Krystyna , Vinogradov Eugeny V. , Shashkov Alexander S. , Sidorczyk Zygmunt
Published: March 2003

An acidic O‐specific polysaccharide was obtained by mild acid degradation of the Proteus penneri 8 lipopolysaccharide and found to contain d‐glucose, d‐galacturonic acid, 2‐acetamido‐2‐deoxy‐d‐glucose, 2‐acetamido‐2‐deoxy‐d‐galactose, 2‐acetamido‐2,6‐dideoxy‐l‐galactose (l‐FucNAc) and 2‐aminoethyl phosphate (PEtn) in the ratios 2 : 1 : 1 : 1 : 1 : 1. 1H and 13C NMR spectroscopy was applied to the intact and dephosphorylated polysaccharides, and the following structure of the hexasaccharide repeating unit was established:

The O‐specific polysaccharide has a unique structure, and, accordingly, we propose for P. penneri 8 a new Proteus O67 serogroup, in which this strain is at present the single representative. The nature of epitopes on LPS of P. penneri 34, P. mirabilis O16, P. mirabilis O23 and P. vulgaris O22, which cross‐react with O‐antiserum against P. penneri 8, is discussed.
Identification of the site where the electron transfer chain of plant mitochondria is stimulated by electrostatic charge screening
Authors: Krab Klaas , Wagner Marijke J. , Wagner Anneke M. , Møller Ian M.
Published: March 2003

Modular kinetic analysis was used to determine the sites in plant mitochondria where charge‐screening stimulates the rate of electron transfer from external NAD(P)H to oxygen. In mitochondria isolated from potato (Solanum tuberosum L.) tuber callus, stimulation of the rate of oxygen uptake was accompanied by a decrease in the steady‐state reduction level of coenzyme Q, and by a small decrease in the steady‐state reduction level of cytochrome c. Modular kinetic analysis around coenzyme Q revealed that stimulation of the rate was due to stimulation of quinol oxidation via the cytochrome pathway (cytochrome bc1, cytochrome c and cytochrome c oxidase). It was not a consequence of any effect on quinone reduction (by external NADH or NADPH dehydrogenase). This explains the salt‐induced decrease in the steady‐state reduction level of coenzyme Q. Analysis around cytochrome c revealed that stimulation by salts was due to a dual effect on the respiratory chain. The kinetic curves for the oxidation and reduction pathways of cytochrome c revealed that they were both activated by salt, the simultaneity explaining the small variation observed in the steady‐state reduction level of cytochrome c. A simple kinetic core model is used to show that changes in the rate of dissociation of cytochrome c from the membrane can explain the observed kinetic changes in both cytochrome c reduction and cytochrome c oxidation. The stimulation is proposed to be the result of an increase in the rate constant of cytochrome c dissociation from the membrane induced by cation screening. We conclude that this type of modular kinetic analysis is a powerful tool to identify and quantitatively characterize multiple‐site effects on the mitochondrial respiratory chain.
High resolution X‐ray analysis of two mutants of a curaremimetic snake toxin
Authors: Gaucher Jean‐François , Ménez Renée , Arnoux Bernadette , Pusset Jacques , Ducruix Arnaud
Published: March 2003

A previous mutational analysis of erabutoxin a (Ea), a curaremimetic toxin from sea snake venom, showed that the substitutions S8G and S8T caused, respectively, 176‐fold and 780‐fold affinity decreases for the nicotinic acetylcholine receptor (AchR). In view of the fact that the side‐chain of Ser8 is buried in the wild‐type toxin, we wondered whether these affinity changes reflect a direct binding contribution of S8 to the receptor and/or conformational changes that could have occurred in Ea as a result of the introduced mutations. To approach this question, we solved X‐ray structures of the two mutants S8G and S8T at high resolution (0.18 nm and 0.17 nm, with R factors of 18.0% and 17.9%, respectively). The data show that none of the mutations significantly modified the toxin structure. Even within the site where the toxin binds to the receptor the backbone conformation remained unchanged. Therefore, the low affinities of the mutants S8T and S8G cannot be explained by a large conformational change of the toxin structure. Although we cannot exclude the possibility that undetectable structural changes have occurred in the toxin mutants, our data support the view that, although buried between loop I and II, S8 is part of the functional epitope of the toxin.
Expression and regulation of alkaline phosphatases in human breast cancer MCF‐7 cells
Authors: Tsai Lai‐Chen , Hung Mei‐Whey , Chen Yu‐Hou , Su Wen‐Cheng , Chang Gu‐Gang , Chang Tsu‐Chung
Published: March 2003

The effect of retinoic acid and dexamethasone on alkaline phosphatase (AP) expression was investigated in human breast cancer MCF‐7 cells. Cellular AP activity was induced significantly by retinoic acid or dexamethasone in a time‐dependent and dose‐dependent fashion. A marked synergistic induction of AP activity was observed when the cells were incubated with both agents simultaneously. Two AP isozymes, tissue‐nonspecific (TNAP) and intestinal (IAP), were shown to be expressed in MCF‐7 cells as confirmed by the differential rate of thermal inactivation of these isozymes and RT‐PCR. Based on the two‐isozyme thermal‐inactivation model, the specific activities for TNAP and IAP in each sample were analyzed. TNAP activity was induced only by retinoic acid and IAP activity was induced only by dexamethasone. Whereas dexamethasone conferred no significant effect on TNAP activity, retinoic acid was shown to inhibit IAP activity by ≈ 50%. Interestingly, TNAP was found to be the only isozyme activity superinduced when the cells were costimulated with retinoic acid and dexamethasone. Northern blot and RT‐PCR analysis were then used to demonstrate that the steady‐state TNAP mRNA level was also superinduced, which indicates that the superinduction is regulated at the transcriptional or post‐transcriptional levels. In the presence of the glucocorticoid receptor antagonist RU486, the dexamethasone‐mediated induction of IAP activity was blocked completely as expected. However, the ability of RU486 to antagonize the action of glucocorticoid was greatly compromised in dexamethasone‐mediated superinduction of TNAP activity. Furthermore, in the presence of retinoic acid, RU486 behaved as an agonist, and conferred superinduction of TNAP gene expression in the same way as dexamethasone. Taken together, these observations suggest that the induction of IAP activity by dexamethasone and the superinduction of TNAP by dexamethasone were mediated through distinct regulatory pathways. In addition, retinoic acid plays an essential role in the superinduction of TNAP gene expression by enabling dexamethasone to exert its agonist activity, which otherwise has no effect.
Characterization of the 5′‐flanking region of the human multidrug resistance protein 2 (MRP2) gene and its regulation in comparison withthe multidrug resistance protein 3 (MRP3) gene
Authors: Stöckel Birgit , König Jörg , Nies Anne T. , Cui Yunhai , Brom Manuela , Keppler Dietrich
Published: March 2003

The multidrug resistance proteins MRP2 (symbol ABCC2) and MRP3 (symbol ABCC3) are conjugate export pumps expressed in hepatocytes. MRP2 is localized exclusively to the apical membrane and MRP3 to the basolateral membrane. MRP2 mRNA is expressed at a high level under normal conditions, whereas MRP3 mRNA expression is low and increases only when secretion across the apical membrane by MRP2 is impaired. We studied some of the regulatory properties of the two human genes using transient transfection assays with promoter–luciferase constructs in HepG2 cells and cloned fragments of 1229 nucleotides and 1287 nucleotides of the MRP2 and MRP3 5′‐flanking regions, respectively. The sequence between nucleotides −517 and −197 was decisive for basal MRP2 expression. Basal promoter activity of MRP3 was only 4% of that measured for MRP2. At submicromolar concentrations, the histone deacetylase inhibitor trichostatin A reduced the MRP2 reporter gene activity and expression of the protein. Disruption of microtubules with nocodazole decreased gene and protein expression of MRP2 and increased MRP3 reporter gene activity. The genotoxic 2‐acetylaminofluorene decreased the activity of the human MRP2 reporter gene construct, but increased MRP3 gene activity and enhanced the amounts of mRNA and protein of MRP2 and MRP3. Thus, regulation of the expression of these ATP‐dependent conjugate export pumps is not co‐ordinate, but in part inverse. The inverse regulation of the two MRP isoforms is consistent with their distinct localization, their different mRNA expression under normal and pathophysiological conditions, and their different directions of substrate transport in polarized cells.
Reciprocal relationship between α1,2 mannosidase processing and reglucosylation in the rough endoplasmic reticulum of Man‐P‐Dol deficient cells
Authors: Duvet Sandrine , Chirat Frédéric , Mir Anne‐Marie , Verbert André , Dubuisson Jean , Cacan René
Published: March 2003

The study of the glycosylation pathway of a mannosylphosphoryldolichol‐deficient CHO mutant cell line (B3F7) reveals that truncated Glc(0–3)Man5GlcNAc2 oligosaccharides are transferred onto nascent proteins. Pulse‐chase experiments indicate that these newly synthesized glycoproteins are retained in intracellular compartments and converted to Man4GlcNAc2 species. In this paper, we demonstrate that the α1,2 mannosidase, which is involved in the processing of Man5GlcNAc2 into Man4GlcNAc2, is located in the rough endoplasmic reticulum. The enzyme was shown to be inhibited by kifunensine and deoxymannojirimycin, indicating that it is a class I mannosidase. In addition, Man4GlcNAc2 species were produced at the expense of Glc1Man5GlcNAc2 species. Thus, the trimming of Man5GlcNAc2 to Man4GlcNAc2, which is catalyzed by this mannosidase, could be involved in the control of the glucose‐dependent folding pathway.
Purification, cloning and sequence analyses for pro‐metalloprotease‐disintegrin variants from Deinagkistrodon acutus venom and subclassification of the small venom metalloproteases
Authors: Tsai Inn‐Ho , Wang Ying‐Ming , Chiang Tzen‐Yuh , Chen Yuh‐Ling , Huang Ruey‐Jen
Published: March 2003

Acidic and basic hemorrhagic metalloproteases were purified from the venom of Deinagkistrodon acutus (from Fujian Province, China) using gel filtration and anion exchange on FPLC and reversed‐phase HPLC. Their hemorrhagic activities and N‐terminal sequences were characterized. Extensive screening of the venom gland cDNA after PCR amplification resulted in the identification and sequencing of a total of seven cDNA clones encoding the multidomain precursors of six acidic and one alkaline low molecular mass metalloproteases. Two of the precursors contain a processable disintegrin domain. Disintegrins of 5 kDa were also purified from the venom. The partial amino‐acid sequences and molecular masses determined by electrospray ionization mass spectrometry of the purified proteins specifically match those deduced from two of the cDNA sequences. Moreover, phylogenetic analyses based on 30 complete sequences of low molecular mass venom metalloproteases revealed that they may be classified into three functional subtypes: acidic hemorrhagins, basic and moderate hemorrhagins, and nonhemorrhagic enzymes. Subtype‐specific amino‐acid substitutions in the C‐terminal regions of the enzymes were highlighted to explore the structure–activity relationships of the enzymes.
In vitro study of proteolytic degradation of rat histidine decarboxylase
Authors: Olmo María T. , Urdiales José L. , Pegg Anthony E. , Medina Miguel A. , Sánchez‐Jiménez Francisca
Published: March 2003

Mammalian ornithine decarboxylase (ODC) is a very unstable protein which is degraded in an ATP‐dependent manner by proteasome 26S, after making contact with the regulatory protein antizyme. PEST regions are sequences described as signals for protein degradation. The C‐terminal PEST region of mammalian ODC is essential for its degradation by proteasome 26S. Mammalian histidine decarboxylase (HDC) is also a short‐lived protein. The full primary sequence of mammalian HDC contains PEST‐regions at both the N‐ and C‐termini. Rat ODC and different truncated and full versions of rat HDC were expressed in vitro. In vitro degradation of rat ODC and rat 1–512 HDC were compared. Like ODC, rat 1–512 HDC is degraded mainly by an ATP‐dependent mechanism. However, antizyme has no effect on the degradation of 1–512 HDC. The use of the inhibitors MG‐132 and lactacystine significantly inhibited the degradation of 1–512 HDC, suggesting that a ubiquitin‐dependent, proteasome 26S proteolytic pathway is involved. Results obtained with the different modifications of rat HDC containing all three PEST regions (full version, 1–656 HDC), only the N‐terminal PEST region (1–512 HDC), or no PEST region (69–512 HDC), indicate that the N‐terminal (1–69) fragment, but not the C‐terminal fragment, determines that the HDC protein is a proteasome substrate in vitro.
New lessons in the regulation of glucose metabolism taught by the glucose 6‐phosphatase system
Authors: van de Werve Gérald , Lange Alex , Newgard Christopher , Méchin Marie‐Claire , Li Yazhou , Berteloot Alfred
Published: March 2003

The operation of glucose 6‐phosphatase (EC 3.1.3.9) (Glc6Pase) stems from the interaction of at least two highly hydrophobic proteins embedded in the ER membrane, a heavily glycosylated catalytic subunit of m 36 kDa (P36) and a 46‐kDa putative glucose 6‐phosphate (Glc6P) translocase (P46). Topology studies of P36 and P46 predict, respectively, nine and ten transmembrane domains with the N‐terminal end of P36 oriented towards the lumen of the ER and both termini of P46 oriented towards the cytoplasm. P36 gene expression is increased by glucose, fructose 2,6‐bisphosphate (Fru‐2,6‐P2) and free fatty acids, as well as by glucocorticoids and cyclic AMP; the latter are counteracted by insulin. P46 gene expression is affected by glucose, insulin and cyclic AMP in a manner similar to P36. Accordingly, several response elements for glucocorticoids, cyclic AMP and insulin regulated by hepatocyte nuclear factors were found in the Glc6Pase promoter. Mutations in P36 and P46 lead to glycogen storage disease (GSD) type‐1a and type‐1 non a (formerly 1b and 1c), respectively. Adenovirus‐mediated overexpression of P36 in hepatocytes and in vivo impairs glycogen metabolism and glycolysis and increases glucose production; P36 overexpression in INS‐1 cells results in decreased glycolysis and glucose‐induced insulin secretion. The nature of the interaction between P36 and P46 in controling Glc6Pase activity remains to be defined. The latter might also have functions other than Glc6P transport that are related to Glc6P metabolism.
Interaction and coordination geometries for Ag(I) in the two metal sites of hemocyanin
Authors: Holm Jens K. , Hemmingsen Lars , Bubacco Luigi , Salvato Benedetto , Bauer Rogert
Published: March 2003

111Ag(I) perturbed angular correlations of γ‐rays (PAC) has been used to investigate the binuclear metal site of 111Ag(I)‐substituted Carcinus aestuarii deoxyhemocyanin. The studies have shown that apo‐hemocyanin is able to bind 2 mol of Ag(I) per mol of protein and that the binding is specific for the metal ion sites. The PAC spectra show pronounced changes when the stoichiometry of Ag(I) to protein is increased from 0.1 to 2.0. These changes have been interpreted as evidence of interactions between the two sites in terms of a structural destabilization of the first occupied site caused by the occupation of the second site. The experimental data for the Ag(I)‐substituted metal sites do not agree well with the three‐coordinated structure found in the Cu(I) holo‐protein. However, if a water molecule is included as a coordinating ligand in the Ag(I) metal site a successful interpretation of the experimental data can be obtained.
Molecular cloning of the human and murine 2‐amino‐3‐ketobutyrate coenzyme A ligase cDNAs
Authors: Edgar Alasdair J. , Polak Julia M.
Published: March 2003

The conversion of l‐threonine to glycine in both prokaryotes and eukaryotes takes place through a two‐step biochemical pathway involving the enzymes l‐threonine dehydrogenase (EC 1.1.1103) and 2‐amino‐3‐ketobutyrate coenzyme A ligase (KBL; EC 2.3.1.29). The genes encoding these enzymes have been described in prokaryotes but not in eukaryotes. We report the cloning of transcripts for KBL, the second enzyme in the pathway, from human and murine lung and a partial transcript from bovine liver. Two peptide sequences from the purified bovine KBL protein, one from the N‐terminus and the other from the peptide containing the pyridoxal 5′‐phosphate‐binding lysine residue [Tong, H. & Davis, L. (1994) J. Biol. Chem.269, 4057–4064], are identical with regions of the conceptual translation of the transcript obtained from bovine liver. The partial transcript from bovine liver was very similar to the human transcript, being 91% and 92% identical at the nucleotide and amino‐acid levels, respectively. The human and murine KBL transcripts are 1.5 kb long, with ORFs encoding proteins of 419 and 416 residues, respectively. The mouse protein has 90% identity with the human protein. The human transcript is strongly expressed in heart, brain, liver and pancreas compared with the lung. The N‐termini of both human and mouse proteins have characteristics of mitochondrial import sequences. Both human and murine proteins have 54% identity with the well‐characterised prokaryote KLB protein from Escherichia coli. Database searches with the human cDNA sequence enabled us to identify the human KBL gene on chromosome 22q12‐13, consisting of nine exons over 9 kb, and a hypothetical Caenorhabditis elegans KLB gene on chromosome IV, consisting of five exons over 2 kb.
High‐molecular‐mass complexes formed in vivo contain smHSPs and HSP70 and display chaperone‐like activity
Authors: Smýkal Petr , Hrdý Ivan , Pechan Paul M
Published: March 2003

Stress can have profound effects on the cell. The elicitation of the stress response in the cell is often accompanied by the synthesis of high‐molecular‐mass complexes, sometimes termed heat shock granules (HSGs). The presence of the complexes has been shown to be important for the survival of cells subjected to stress. We purified these complexes from heat‐stressed BY‐2 tobacco cells. HSG complexes formed in vivo contain predominantly smHSPs, HSP40 and HSP70 and display chaperone‐like activity. Tubulins as well as other proteins may be part of the complex or its substrate. The proteins, except smHSPs and to some extent HSP70, were hypersensitive to proteolysis, suggesting that they were partially denatured and not an integral part of the HSG complexes. When citrate synthase was used as the substrate, in vivo generated HSG complexes exhibited strong nucleotide‐dependent in vitro chaperone activity. Measurable ATP‐mediated hydrolytic activity was detected. Isolated HSG complexes are stable until ATP is added, which leads to rapid dissociation of the complex into subunits. It is proposed that smHSPs form the core of the complex in association with ATP‐dependent HSP70 and HSP40 cochaperones. Implications of these findings are discussed.
The crystal structure of the Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor
Authors: Kontou Maria , Leonidas Demetres D , Vatzaki Efstratia H. , Tsantili Panayiota , Mamalaki Avgi , Oikonomakos Nikos G. , Acharya K. Ravi , Tzartos Socrates J.
Published: March 2003

The crystal structure of the Fab fragment of a rat monoclonal antibody, number 192, with a very high affinity (Kd = 0.05 nm) for the main immunogenic region of the human muscle acetylcholine receptor (AChR), has been determined and refined to 2.4 Å resolution by X‐ray crystallographic methods. The overall structure is similar to a Fab (NC6.8) from a murine antibody, used as a search model in molecular replacement. Structural comparisons with known antibody structures showed that the conformations of the hypervariable regions H1, H2, L1, L2, L3 of Fab192 adopt the canonical structures 1, 1, 2, 1, and 1, respectively. The surface of the antigen‐binding site is relatively planar, as expected for an antibody against a large protein antigen, with an accessible area of 2865 Å2. Analysis of the electrostatic surface potential of the antigen‐binding site shows that the bottom of the cleft formed in the center of the site appears to be negatively charged. The structure will be useful in the rational design of very high affinity humanized mutants of Fab192, appropriate for therapeutic approaches of the model autoimmune disease myasthenia gravis.
The ionization of a buried glutamic acid is thermodynamically linked to the stability of Leishmania mexicana triose phosphate isomerase
Authors: Lambeir Anne‐Marie , Backmann Jan , Ruiz‐Sanz Javier , Filimonov Vladimir , Nielsen Jens Erik , Kursula Inari , Norledge Brian V. , Wierenga Rik K.
Published: March 2003

The amino acid sequence of Leishmania mexicana triose phosphate isomerase is unique in having at position 65 a glutamic acid instead of a glutamine. The stability properties of LmTIM and the E65Q mutant were investigated by pH and guanidinium chloride‐induced unfolding. The crystal structure of E65Q was determined. Three important observations were made: (a) there are no structural rearrangements as the result of the substitution; (b) the mutant is more stable than the wild‐type; and (c) the stability of the wild‐type enzyme shows strong pH dependence, which can be attributed to the ionization of Glu65. Burying of the Glu65 side chain in the uncharged environment of the dimer interface results in a shift in pKa of more than 3 units. The pH‐dependent decrease in overall stability is due to weakening of the monomer–monomer interactions (in the dimer). The E65Q substitution causes an increase in stability as the result of the formation of an additional hydrogen bond in each subunit (ΔΔG ° of 2 kcal·mol−1 per monomer) and the elimination of a charged group in the dimer interface (ΔΔG ° of at least 9 kcal·mol−1 per dimer). The computated shift in pKa and the stability of the dimer calculated from the charge distribution in the protein structure agree closely with the experimental results.

The guanidinium chloride dependence of the unfolding constant was smaller than expected from studies involving monomeric model proteins. No intermediates could be identified in the unfolding equilibrium by combining fluorescence and CD measurements. Study of a stable monomeric triose phosphate isomerase variant confirmed that the phenomenon persists in the monomer.
M1 muscarinic acetylcholine receptors activate zif268 gene expression via small G‐protein Rho‐dependent and λ‐independent pathways in PC12D cells
Authors: Hirabayashi Toshiyuki , Saffen David
Published: March 2003

We have previously shown that stimulation of M1 muscarinic acetylcholine receptors (mAChRs) in neuronal PC12D cells rapidly induces the immediate‐early gene zif 268[Ebihara, T. & Saffen, D. (1997) J. Neurochem.68, 1001–1010]. Here we show that stimulation of M1 mAChRs in these cells activates four distal serum response elements (SREs) in the zif 268 promoter, and that this activation is strongly inhibited by Clostridium botulinum C3 exoenzyme (C3), which specifically inactivates the small G‐protein Rho. Even with high doses of C3, however, a portion of the activation remains intact, indicating that stimulation of M1 mAChRs activates zif 268 SREs via Rho‐dependent and Rho‐independent pathways. Moreover, the Rho‐independent activation of zif 268 SREs is inhibited by the dominant‐negative form of the small G‐protein Ras, suggesting that Rho‐independent activation of zif 268 SREs is mediated by Ras. To determine if muscarinic agonists activate RhoA, we also measured the translocation of RhoA from the cytosolic fraction to the particulate fraction. Translocation of RhoA to the particulate fraction was observed within 15 min following stimulation of M1 mAChRs, indicating that RhoA is activated with sufficient rapidity to participate in the induction of zif 268 mRNA. Together, these results suggest that RhoA is activated following stimulation of M1 mAChRs and functions in SRE‐dependent induction of the zif 268 gene within a Ras‐independent pathway.
Analysis of the thyrotropin‐releasing hormone‐degrading ectoenzyme by site‐directed mutagenesis of cysteine residues
Authors: Papadopoulos Theofilos , Heuer Heike , Bauer Karl
Published: March 2003

Thyrotropin‐releasing hormone‐degrading ectoenzyme is a member of the M1 family of Zn‐dependent aminopeptidases and catalyzes the degradation of thyrotropin‐releasing hormone (TRH; Glp‐His‐Pro‐NH2). Cloning of the cDNA of this enzyme and biochemical studies revealed that the large extracellular domain of the enzyme with the catalytically active site contains nine cysteine residues that are highly conserved among species. To investigate the functional role of these cysteines in TRH‐DE we used a site‐directed mutagenesis approach and replaced individually each cysteine by a serine residue. The results revealed that the proteolytically truncated and enzymatically fully active enzyme consists of two identical subunits that are associated noncovalently by protein–protein interactions but not via interchain S‐S bridges. The eight cysteines contained within this region are all important for the structure of the individual subunit and the enzymatic activity, which is dramatically reduced in all mutant enzymes. This is even true for the four cysteines that are clustered within the C‐terminal domain remote from the Zn‐binding consensus sequence HEICH. In contrast, Cys68, which resides within the stalk region seven residues from the end of the hydrophobic membrane‐spanning domain, can be replaced by serine without a significant change in the enzymatic activity. Interestingly, this residue is involved in the formation of an interchain disulfide bridge. Covalent dimerization of the subunits, however, does not seem to be essential for efficient biosynthesis, enzymatic activity and trafficking to the cell surface.
Characterization of three cloned and expressed 13‐hydroperoxide lyase isoenzymes from alfalfa with unusual N‐terminal sequences and different enzyme kinetics
Authors: Noordermeer Minke A. , van Dijken Anja J. H. , Smeekens Sjef C. M. , Veldink Gerrit A. , Vliegenthart Johannes F. G.
Published: March 2003

Three full‐length cDNAs from alfalfa seedlings coding for hydroperoxide lyases were cloned and expressed in Escherichia coli and characterized as cytochrome P450 enzymes. The isoenzymes were specific for 13‐hydroperoxy linoleic and linolenic acids and did not use the 9‐hydroperoxy isomers as substrates. Because alfalfa contains both specificities, this indicates the presence of two different types of hydroperoxide lyases, each specific for one kind of substrate. The enzymes contain 480 amino acids (54 kDa) and contain an unusual, nonplastidic N‐terminal sequence of 22 amino acids, which strongly reduces the enzyme activity. The only known presequence of a hydroperoxide lyase (from Arabidopsis thaliana) was considered to be a transit sequence. The reduced enzyme activity, however, indicates that the hydroperoxide lyases with N‐terminal extensions could be pro‐enzymes. This hypothesis is supported by the fast release of hydroperoxide lyase products by plants upon wounding. One of the isoenzymes showed a strongly decreased Vmax and Km compared to the other two. Because this is probably due to the substitution of Ser377 by Phe; the residue at position 377 seems to be important. This is the first time that sufficient quantities of hydroperoxide lyase have been obtained for characterization studies, by circumventing difficult purification procedures and degradation of the enzyme. The high expression level, easy purification, good stability and high specificity make these cloned hydroperoxide lyases excellent tools to study the reaction mechanism and structure. We postulate an integrated reaction mechanism, based on the known chemistry of cytochrome P450 enzymes. This is the first mechanism that unifies all observed features of hydroperoxide lyases.
Stability and folding of the protein complexes of barnase
Authors: Neira José L. , Vázquez Eugenio , Fersht Alan R.
Published: March 2003

Native‐like complexes of proteins, formed by the association of two complementary fragments comprising the entire sequence of the protein, can be used to gain insight into the stability and folding of the intact protein. We have studied the structural, thermodynamic and kinetic properties of four barnase complexes, with the cleavage site at different positions of the amino‐acid chain (CB36, at position 36; CB56, at position 56; CB68, at position 68; and CB79, at position 79). The four barnase complexes have native‐like structure as shown by fluorescence, far‐and near‐UV CD, size‐exclusion chromatography and NMR. The NMR characterization indicated that the structural changes were mainly located in regions close to the cleavage site. The main core of the protein was fully formed and the overall structure was similar to that of intact barnase. The thermal and chemical denaturation showed that all complexes were substantially destabilized. CB56 displayed two denaturation transitions, probably because of the presence of partially folded conformations around the cleavage site. The rate constant for the association/folding of fragments decreased with the decreasing length of the C‐terminal fragment. Thus, the larger the fragment (and, consequently, the larger the amount of residual native‐like structure), the faster the association. These findings are consistent with the proposed model of barnase folding.
Phosphorylation of phosphodiesterase‐5 by cyclic nucleotide‐dependent protein kinase alters its catalytic and allosteric cGMP‐binding activities
Authors: Corbin Jackie D. , Turko Illarion V. , Beasley Alfreda , Francis Sharron H.
Published: March 2003

In addition to its cGMP‐selective catalytic site, cGMP‐binding cGMP‐specific phosphodiesterase (PDE5) contains two allosteric cGMP‐binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem.265, 14971–14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP‐dependent protein kinase (PKG) or catalytic subunit of cAMP‐dependent protein kinase (PKA), MgATP, cGMP, 3‐isobutyl‐1‐methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50–70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP‐binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time‐dependent and was obtained with 0.2–0.5 μm PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the Km value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3‐isobutyl‐1‐methylxanthine. Phosphorylation reduced the cGMP concentration required for half‐maximum binding to the allosteric cGMP‐binding sites from 0.13 to 0.03 μm. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative‐feedback regulation of cGMP levels is discussed.
Characterization of the myo‐inositol transport system in Trypanosoma cruzi
Authors: Einicker‐Lamas Marcelo , Almeida Alessandra C. , Todorov Alex G. , de Castro Solange L. , Caruso‐Neves Celso , Oliveira Mecia M.
Published: March 2003

myo‐Inositol is a growth factor for mammalian cells as well as for the pathogenic protozoa Trypanosoma cruzi. Most of the cell surface molecules in this organism rely on myo‐inositol as the biosynthetic precursor for phosphoinositides and glycosylated phosphatidylinositols. The aim of this work was to investigate the process of myo‐inositol translocation across the parasite cell membrane. myo‐Inositol uptake was concentration‐dependent in the concentration range 0.1–10 µm with maximal transport obtained at 8 µm. Using sodium‐free buffers, where Na+ was replaced by choline or K+, myo‐inositol uptake was inhibited by 50%. Furosemide, an inhibitor of the ouabain‐insensitive Na+‐ATPase, inhibited the Na+‐dependent and Na+‐independent myo‐inositol uptake by 68 and 33%, respectively. In contrast, ouabain, an (Na++/K+) ATPase inhibitor, did not affect transport. Part of the myo‐inositol uptake is mediated by active transport as it was inhibited when energy metabolism inhibitors such as carbonyl cyanide p‐(trifluoromethoxy)‐phenylhydrazone (34%), 2,4‐dinitrophenol (50%), KCN (71%) and NaN3 (69%) were added to the medium, or the temperature of the medium was lowered to 4 °C. The addition of glucose (5–50 mm) or mannose (10 mm) did not change the myo‐inositol uptake, whereas the addition of 10 mm nonlabeled myo‐inositol totally inhibited this transport, indicating that the transporter is specific for myo‐inositol. Phloretin (0.3 mm) and phoridzin (5 mm), but not cytochalasin B, were efficient inhibitors of myo‐inositol uptake. A portion of the accumulated myo‐inositol is converted to inositol phosphates and phosphoinositides. These data show that myo‐inositol transport in T. cruzi epimastigotes is mediated by at least two specific transporters — one Na+‐dependent and the other Na+‐independent.