bluuebunny/crossref_metadata_2025 huggingface dataset:
Abalone (Haliotis tuberculata) hemocyanin type 1 (HtH1)
Authors: Keller Henning , Lieb Bernhard , Altenhein Benjamin , Gebauer Dagmar , Richter Stefanie , Stricker Sigmar , Markl Jürgen
Published: March 2003

We have identified two separate hemocyanin types (HtH1 and HtH2) in the European abaloneHaliotis tuberculata. HtH1/HtH2 hybrid molecules were not found. By selective dissociation of HtH2 we isolated HtH1 which, as revealed by electron microscopy and SDS/PAGE, is present as didecamers of a ≈ 400 kDa subunit. Immunologically, HtH1 and HtH2 correspond to keyhole limpet hemocyanin (KLH)1 and KLH2, respectively, the two well‐studied hemocyanin types of the closely related marine gastropodMegathura crenulata. On the basis of limited proteolytic cleavage, two‐dimensional immunoelectrophoresis, SDS/PAGE and N‐terminal sequencing, we identified eight different 40–60 kDa functional units in HtH1, termed HtH1‐ato HtH1‐h, and determined their linear arrangement within the elongated subunit. FromHaliotismantle tissue, rich in hemocyanin‐producing pore cells, we isolated mRNA and constructed a cDNA library. By expression screening with HtH‐specific rabbit antibodies, a cDNA clone was isolated and sequenced which codes for the three C‐terminal functional unitsf,gandhof HtH1. Their sequences were aligned to those available from other molluscs, notably to functional unit fand functional unit gfrom the cephalopodOctopus dofleini. HtH1‐f, which is the first sequenced functional unit of type ffrom a gastropod hemocyanin, corresponds to functional unit ffromOctopus. Also functional unitgfromHaliotisandOctopuscorrespond to each other. HtH1‐h is a gastropod hemocyanin functional unit type which is absent in cephalopods and has not been sequenced previously. It exhibits a unique tail extension of ≈ 95 amino acids, which is lacking in functional unitsatogand aligns with a published peptide sequence of 48 amino acids from functional unit hofHelix pomatiahemocyanin. The newHaliotissequences are discussed with respect to their counterparts inOctopus, the 15 Å three‐dimensional reconstruction of the KLH1 didecamer from electron micrographs, and the recent 2.3 Å X‐ray structure of functional unit gfromOctopushemocyanin.
Hydrolase and transferase activities of the β‐1,3‐exoglucanase of Candida albicans
Authors: Stubbs Hilary J. , Brasch Donald J. , Emerson George W. , Sullivan Patrick A.
Published: March 2003

The exo‐β‐1,3‐glucanase of Candida albicans (Exg) has a marked specificity for β‐1,3‐glucosidic linkages as judged by the kinetic constants for p‐nitophenyl β‐glucoside, β‐linked disaccharides of glucose (laminaribiose, gentiobiose, and cellobiose), oligosaccharides of the laminari series, laminarin and pustulan. The kcat/Km ratios for a series of laminari oligosaccharides from ‐biose to ‐heptaose showed that Exg has an extended substrate‐binding site which contains at least five binding sites for sugar residues. Binding at position +2 (the third sugar residue) increases the kcat twofold while positions +3 and +4 lower the Km value further and thereby increase the catalytic efficiency. Exg catalyses an efficient transglucosylation reaction with high concentrations of laminari‐oligosaccharides which specifically form β‐1,3 linkages and with yields up to 50%. The rate of the transglucosylation is concentration‐dependent and can be more than 10 times faster than the hydrolytic reaction with excess donor substrates such as laminaritriose and laminarihexaose. The kinetics of Exg and the predicted substrate‐binding site for up to five sugar residues are consistent with a recent structural analysis of the enzyme‐binding site.
New World, but not Old World, monkeys carry several genes encoding β‐microseminoprotein
Authors: Mäkinen Marjaana , Valtonen‐André Camilla , Lundwall Åke
Published: March 2003

It was shown by Southern hybridization that cotton‐top tamarin and common marmoset, New World monkeys, carry three or more genes encoding β‐microseminoprotein, also known as PSP94. In contrast, the genomes of Old World monkeys, as represented by rhesus macaque and sacred baboon, contain a single gene. Clones containing three different genes encoding β‐microseminoprotein were isolated from a cotton‐top tamarin genomic library. They carry two complete genes of four exons and a third gene lacking the first exon. The structure suggests that the three genes are functionally active and give rise to transcripts that are ≈ 86% similar in sequence. By sequencing one gene in full, it was shown that the introns carry an excess of interspersed repeats, on average 29% of the introns consist of Alu repeats. A phylogenetic analysis demonstrated that the genes probably arose in New World monkeys after the separation from Old World primates.
Electrostatics in the active site of an α‐amylase
Authors: Nielsen Jens E. , Beier Lars , Otzen Daniel , Borchert Torben V. , Frantzen Henrik B. , Andersen Kim V. , Svendsen Allan
Published: March 2003

The importance of electrostatics in catalysis has been emphasized in the literature for a large number of enzymes. We examined this hypothesis for theBacillus licheniformisα‐amylase by constructing site‐directed mutants that were predicted to change the pKavalues of the catalytic residues and thus change the pH–activity profile of the enzyme. To change the pKaof the catalytic residues in the active site, we constructed mutations that altered the hydrogen bonding network, mutations that changed the solvent accessibility, and mutations that altered the net charge of the molecule. The results show that changing the hydrogen bonding network near an active site residue or changing the solvent accessibility of an active site residue will very likely result in an enzyme with drastically reduced activity. The differences in the pH–activity profiles for these mutants were modest. pH–activity profiles of mutants which change the net charge on the molecule were significantly different from the wild‐type pH–activity profile. The differences were, however, difficult to correlate with the electrostatic field changes calculated. In several cases we observed that pH–activity profiles shifted in the opposite direction compared to the shift predicted from electrostatic calculations. This strongly suggests that electrostatic effects cannot be solely responsible for the pH–activity profile of theB. licheniformisα‐amylase.
Determinants in the N‐terminal domains of galectin‐3 for secretion by a novel pathway circumventing the endoplasmic reticulum–Golgi complex
Authors: Menon Rajesh P. , Hughes R. Colin
Published: March 2003

Galectin‐3 is a β‐galactoside‐binding protein that is secreted from many cells although the protein lacks a signal sequence for transfer into the endoplasmic reticulum and Golgi compartments and entry into classical secretory pathways. Previously it was shown that attachment of the first 120 amino acid residues of the N‐terminal sequence of hamster galectin‐3 to the cytoplasmic protein chloramphenicol acetyltransferase (CAT) supported the rapid secretion of the fusion protein from transiently transfected Cos cells under conditions in which CAT protein was not secreted. Here we report that progressive N‐terminal truncation gradually reduced secretion of the fusion proteins, eventually to very low levels compared with the starting product, but did not totally eliminate secretion until a significant majority of the sequence was removed. Mutant CAT fusion proteins containing internal deletions in residues 97–120 of the galectin‐3 N‐terminal sequence were also secreted to a similar extent to the starting product, but further deletion of residues 89–96 abolished detectable secretion. Proline to alanine mutagenesis of the sequence YP(90)SAP(93)GAY in two secretion‐competent CAT fusion proteins greatly reduced or abolished their secretion, whereas similar mutagenesis of proline pairings present elsewhere in the galectin‐3 N‐terminal segments of these proteins had no effect. The results indicate that this sequence is one essential determinant for secretion of galectin‐3–CAT fusion proteins and by inference galectin‐3, at least from transfected Cos cells. However, the short sequence of residues 89–96 by itself is insufficient to direct secretion of CAT fusion proteins and appears to be active only in the context of a larger portion of the galectin‐3 N‐terminal sequence.
Interaction of heme oxygenase‐2 with nitric oxide donors
Authors: Ding Yan , McCoubrey Jr William K. , Maines Mahin D.
Published: March 2003

Heme oxygenase‐2 (HO‐2) is the constitutive cognate of the heat‐shock protein‐32 family of proteins. These proteins catalyze oxidative cleavage of heme to CO and biliverdin, and release Fe. HO‐2 is a hemoprotein and binds heme at heme regulatory motifs (HRMs) with a conserved Cys‐Pro pair; two copies of HRM are present in HO‐2 (Cys264 and Cys281). The HO‐2 HRMs are not present in HO‐1 and are not involved in HO‐2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli‐expressed HO preparations, wild‐type HO‐2, Cys264/Cys281 → Ala/Ala HO‐2‐mutant (HO‐2‐mut) and HO‐1 preparations were used. A type II change (red shift) of the Soret band (405 nm → 413–419 nm) was observed when wild‐type HO‐2 was treated with sodium nitroprusside (SNP), S‐nitroglutathione (GSNO), S‐nitroso‐N‐acetylpenicillamine (SNAP) or 3‐morpholinosydnonimine (SIN‐1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN‐1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO‐2; superoxide dismutase (SOD) blocked the decrease. Binding of heme to HO‐2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO‐2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO‐2 inactivation. HO‐1 and HO‐2‐mut were not inactivated by NO donors. CD data suggest that the decrease in HO‐2 activity was not related to change by NO species of the secondary structure of HO‐2. Western blot analysis suggests that NO donors did not cause HO‐1 protein loss and Northern blot analysis of HeLa cells treated with SIN‐1 and SNP indicates that, unlike HO‐1 mRNA, which is remarkably responsive to the treatments, HO‐2 mRNA levels were modestly increased (≈ two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO‐2‐bound heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO‐2 and NO are trans‐inhibitors, whereby biological activity of NO is attenuated by interaction with HO‐2, serving as an intracellular ‘sink’ for the heme ligand, and NO inhibits HO‐2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.
Proteasome‐dependent cyst formation and stage‐specific ubiquitin mRNA accumulation in Entamoeba invadens
Authors: Gonzalez Jorge , Bai Guangxing , Frevert Ute , Corey E. J. , Eichinger Daniel
Published: March 2003

Proteases play an important role in the pathogenic mechanisms and differentiation events of protozoan parasites; the proteasome/ubiquitin system is essential for maintaining the differentiation state of many cell types. A single input of the specific inhibitor of proteasomes, lactacystin, prevented encystation of the protozoan parasite Entameoba invadens, whereas a cysteine protease inhibitor, E64, only delayed encystation. The ameba target of lactacystin was purified and it displayed the features typical of eukaryotic 20S proteasome complexes. In addition, transcripts encoding ubiquitin were detectable in trophozoites stage cells, disappeared immediately following transfer of amoebae to encystation induction medium, and reappeared at the same time during encystation as other encystation‐specific transcripts. These results demonstrate that proteasome function is required during the conversion of the disease‐causing trophozoite into the infectious cyst stage of Entamoeba parasites, and that ubiquitin transcript levels undergo an unusual decrease during the early stages of this differentiation process.
Structural roles of acetylcholinesterase variants in biology and pathology
Authors: Grisaru Dan , Sternfeld Meira , Eldor Amiram , Glick David , Soreq Hermona
Published: March 2003

Apart from its catalytic function in hydrolyzing acetylcholine, acetylcholinesterase (AChE) affects cell proliferation, differentiation and responses to various insults, including stress. These responses are at least in part specific to the three C‐terminal variants of AChE which are produced by alternative splicing of the single ACHE gene. ‘Synaptic’ AChE‐S constitutes the principal multimeric enzyme in brain and muscle; soluble, monomeric ‘readthrough’ AChE‐R appears in embryonic and tumor cells and is induced under psychological, chemical and physical stress; and glypiated dimers of erythrocytic AChE‐E associate with red blood cell membranes. We postulate that the homology of AChE to the cell adhesion proteins, gliotactin, glutactin and the neurexins, which have more established functions in nervous system development, is the basis of its morphogenic functions. Competition between AChE variants and their homologs on interactions with the corresponding protein partners would inevitably modify cellular signaling. This can explain why AChE‐S exerts process extension from cultured amphibian, avian and mammalian glia and neurons in a manner that is C‐terminus‐dependent, refractory to several active site inhibitors and, in certain cases, redundant to the function of AChE‐like proteins. Structural functions of AChE variants can explain their proliferative and developmental roles in blood, bone, retinal and neuronal cells. Moreover, the association of AChE excess with amyloid plaques in the degenerating human brain and with progressive cognitive and neuromotor deficiencies observed in AChE‐transgenic animal models most likely reflects the combined contributions of catalytic and structural roles.
Catalytic properties of an expressed and purified higher plant typeζ‐carotene desaturase fromCapsicum annuum
Authors: Breitenbach Jürgen , Kuntz Marcel , Takaichi Shinichi , Sandmann Gerhard
Published: March 2003

The ζ‐carotene desaturase fromCapsicum annuum(EC 1.14.99.‐) was expressed inEscherichia coli, purified and characterized biochemically. The enzyme acts as a monomer with lipophilic quinones as cofactors.Kmvalues for the substrate ζ‐carotene or the intermediate neurosporene in the two‐step desaturation reaction are almost identical. Product analysis showed that different lycopene isomers are formed, including substantial amounts of the all‐transform, together with 7,7′,9,9′‐tetracisprolycopene via the corresponding neurosporene isomers. The application of different geometric isomers as substrates revealed that the ζ‐carotene desaturase has no preference for certain isomers and that the nature of the isomers formed during catalysis depends strictly on the isomeric composition of the substrate.
Capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus
Authors: Gloaguen Vincent , Morvan Henri , Hoffmann Lucien , Plancke Yves , Wieruszeski Jean‐Michel , Lippens Guy , Strecker Gerard
Published: March 2003

The capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus has been subjected to a specific degradation with lithium in ethylenediamine. The released undecasaccharide attached to one unit of tetrahydroxycyclopentanecarboxylic acid has been characterized by a combination of 2D nuclear magnetic resonance spectroscopy, mass spectrometry, monosaccharidic composition and linkage analyses. From the overlap of the structure of this oligosaccharide with previously identified di‐, tri‐ and pentasaccharides released by mild acid hydrolysis, the capsular polysaccharide was deduced to have a pentadecasaccharide repeating unit with the following structure:
Characterization of a recombinant soluble form of human placental leucine aminopeptidase/oxytocinase expressed in Chinese hamster ovary cells
Authors: Matsumoto Hideko , Rogi Tomohiro , Yamashiro Kyoko , Kodama Shiho , Tsuruoka Nobuo , Hattori Akira , Takio Koji , Mizutani Shigehiko , Tsujimoto Masafumi
Published: March 2003

Serum levels of human placental leucine aminopeptidase/oxytocinase (P‐LAP) increase with gestation. cDNA cloning of P‐LAP revealed that the enzyme is a type II membrane‐bound protein containing the consensus HEXXH(X)18E motif found in the M1 family of zinc‐metallopeptidase proteins. In this study, a recombinant soluble form of P‐LAP found in maternal serum was expressed in Chinese hamster ovary cells, purified to homogeneity and then characterized. Although N‐terminal sequencing revealed a four‐amino‐acid deletion, the purified enzyme was active and was shown to be a zinc‐containing homodimeric protein with molecular mass of 280 kDa in solution. Using artificial substrates, it was shown that the enzyme has broad specificity and is inhibited by several compounds known as aminopeptidase inhibitors. Subsequently, sequential N‐terminal amino‐acid liberation of several peptide hormones by the enzyme was monitored and structures of the products were determined. Among the hormones having a cysteine residue at their N‐terminal end and intramolecular disulfide bonds, it was found that vasopressin and oxytocin, but not calcitonin and endothelins, were cleaved by the enzyme. Because the molecular properties of oxytocinase so far reported often conflict, our results provide an initial biochemical and enzymatic characterization of moleculary defined P‐LAP/oxytocinase.
Characterization of a sulfurtransferase from Arabidopsis thaliana
Authors: Papenbrock Jutta , Schmidt Ahlert
Published: March 2003

A database search for similarities between sequenced parts of the Arabidopsis thaliana genome with known sulfurtransferase sequences from Escherichia coli and mammals was undertaken to obtain information about plant sulfurtransferase‐like proteins. One gene and several homologous EST clones were identified. One of the EST clones was used for screening an Arabidopsis cDNA library. The isolated full‐length clone consists of 1134 bp and encodes a 42.6 kDa protein that includes a putative transit peptide sequence of about 7.1 kDa. Sequence comparisons with known sulfurtransferases from different organisms confirmed high homology between them and the existence of several highly conserved regions. Results of a Southern blot performed with genomic Arabidopsis DNA showed the occurrence of at least two sulfurtransferase‐like isozymes in Arabidopsis. Recombinant proteins with and without the putative transit peptide were expressed in E. coli with an N‐terminal His6‐tag, purified by affinity chromatography and tested for enzyme activity using different sulfur donors and acceptors. Both recombinant proteins catalyzed the formation of SCN− from thiosulfate and cyanide as a rhodanese per definition; however, both recombinant proteins preferred 3‐mercaptopyruvate to thiosulfate. A monospecific antibody produced by using the mature recombinant protein as an antigen recognized a single protein band in total extracts of Arabidopsis plants equating to the full‐length protein size. A single band equating to the size of the mature protein was detected from purified Arabidopsis mitochondria, but there was no antigenic reaction with any protein from chloroplasts. The function of the protein is still speculative. Now tools are available to elucidate the roles and substrates of this sulfurtransferase in higher plants.
A superoxide dismutase from the archaeon Sulfolobus solfataricus is an extracellular enzyme and prevents the deactivation by superoxide of cell‐bound proteins
Authors: Cannio Raffaele , D’Angelo Anna , Rossi Mosè , Bartolucci Simonetta
Published: March 2003

An oxygen‐induced iron superoxide dismutase was found in the culture fluid of the thermoacidophilic crenarchaeon Sulfolobus solfataricus during growth on glucose‐rich media.

This protein was also identified as being associated with the cell‐surface, with the amount of the released and cell‐bound protein fractions depending on the growth phase of the cells. The steady decrease in cell‐associated superoxide dismutase during continued growth correlated with the increase of free superoxide dismutase in the medium. Both enzyme fractions were purified to homogeneity and found to be active with different catalytic efficiency, with the released superoxide dismutase showing a fourfold lower specific activity.

Characterization in comparison with the cytosolic superoxide dismutase revealed identical N‐terminal sequences, electrophoretic mobility, isoelectric point, and molecular mass for all three differently located enzymes.

In order to clarify the physiological role of the cell‐associated superoxide dismutase, the prevention of cell‐bound protein deactivation by oxyradicals was also investigated. Glucose dehydrogenase, which was chosen as a model enzyme, was demonstrated to be located on the cell surface and to be inactivated by potassium superoxide by in vivo assays. The direct protective effect of superoxide dismutase on glucose dehydrogenase was demonstrated by in vitro assays on the free released enzyme.

Similarly, the prevention of deactivation by potassium superoxide was also demonstrated for the integral membrane protein succinate dehydrogenase by intact cell assay.

Superoxide dismutase added to cells was shown to moderately reduce the critical damaging peroxidation and hence play a major role in maintaining the integrity of the outer cell envelope components.
Midkine binds specifically to sulfatide
Authors: Kurosawa Nobuyuki , Kadomatsu Kenji , Ikematsu Shinya , Sakuma Sadatoshi , Kimura Terutoshi , Muramatsu Takashi
Published: March 2003

Midkine is a heparin‐binding polypeptide which is implicated in the control of development and repair of various tissues. Recognition of sulfate groups in glycosaminoglycans is important for its function. To elucidate further its mechanism of action, the interactions of midkine with sulfated glycolipids were studied. Of various glycolipids and lipids examined, midkine bound strongly to sulfatide and cholesterol‐3‐sulfate (CHO‐3‐SO4) in a dose‐dependent manner but failed to bind to other standard glycolipids and lipids. The properties of midkine binding to sulfatide and to CHO‐3‐SO4 differed in their sensitivity to inhibition by anionic polysaccharides, salt concentration and unlabeled midkine. Heparin inhibited midkine binding to sulfatide but weakly inhibited its binding to CHO‐3‐SO4. Liposomes bearing sulfatide carried out significant interactions with immobilized midkine, whereas those bearing CHO‐3‐SO4 did not. Incorporation of sulfatide into 32D cells and trypsinized COS cells enhanced 125I‐labelled midkine binding, whereas incorporation of ganglioside or galactosylceramide had no effect. Furthermore, sulfatide‐incorporated cells enhanced cell attachment to midkine‐coated coverslips. These results indicate that midkine binds to sulfatide under physiological conditions and the midkine–sulfatide interaction may be important in controlling cell attachment.
Plant fructans stabilize phosphatidylcholine liposomes during freeze‐drying
Authors: Hincha Dirk K. , Hellwege Elke M. , Heyer Arnd G. , Crowe John H.
Published: March 2003

Fructans have been implicated as protective agents in the drought and freezing tolerance of many plant species. A direct proof of their ability to stabilize biological structures under stress conditions, however, is still lacking. Here we show that inulins (linear fructose polymers) isolated from chicory roots and dahlia tubers stabilize egg phosphatidylcholine large unilamellar vesicles during freeze‐drying, while another polysaccharide, hydroxyethyl starch, was completely ineffective. Liposome stability was assessed after rehydration by measuring retention of the soluble fluorescent dye carboxyfluorescein and bilayer fusion. Inulin was an especially effective stabilizer in combination with glucose. Analysis by HPLC showed that the commercial inulin preparations used in our study contained no low molecular mass sugars that could be responsible for the observed stabilizing effect of the fructans. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid‐crystalline phase transition temperature of dry egg PtdCho by more than 20 °C in the presence of inulin. A direct interaction of inulin with the phospholipid in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum between samples with and without the polysaccharide.
Cytochrome cM from Synechocystis 6803
Authors: Cho Yoon Shin , Pakrasi Himadri B. , Whitmarsh John
Published: March 2003

Based on DNA sequence data a novel c‐type cytochrome, cytochrome cM, has been predicted to exist in the cyanobacterium Synechocystis 6803. The precursor protein consists of 105 amino acids with a characteristic heme‐binding motif and a hydrophobic domain located at the N‐terminal end that is proposed to act as either a signal peptide or a membrane anchor. For the first time we report the detection of cytochrome cM in Synechocystis 6803 using Western blot analysis. The soluble portion cytochrome cM has been overexpressed in Escherichia coli in two forms, one with a poly histidine tag to facilitate purification and one without such a tag. The overexpressed protein has been purified and shown to bind heme, exhibiting an absorption peak in the Soret band near 416 nm and a peak in the α band at 550 nm. The extinction coefficient of cytochrome cM is 23.2 ± 0.5 mm−1·cm−1 for the reduced minus oxidized α band peak (550–535 nm). The isoelectric point of cytochrome cM is 5.6 (without the histidine tag), which is significantly lower than the pI of 7.2 predicted from the amino acid sequence. The redox midpoint potential of cytochrome cM expressed in E. coli is 151 ± 5 mV (pH 7.1), which is quite low compared to other c‐type cytochromes in which a histidine and a methionine residue serve as the axial ligands to the heme. This work opens the way for determining the three‐dimensional structure of cytochrome cM and investigating its function in cyanobacteria.
Nucleotide and amino‐acid sequences of a new‐type pectate lyase from an alkaliphilic strain of Bacillus
Authors: Sawada Kazuhisa , Ogawa Akinori , Ozawa Tadahiro , Sumitomo Nobuyuki , Hatada Yuji , Kobayashi Tohru , Ito Susumu
Published: March 2003

A pectate lyase (pectate transeliminase; EC 4.2.2.2), designated Pel‐15E, was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. strain KSM‐P15. The purified enzyme had a molecular mass of ≈ 33 kDa, as determined by SDS/PAGE, and a pI of ≈ pH 9.2. Pel‐15E exhibited optimum activity at pH 10.5 and 50–55 °C in glycine/NaOH buffer. Pel‐15E had an absolute requirement for Ca2+ ions for manifestation of the enzymatic activity and trans‐eliminated poly(galacturonic) acid, most likely by endo‐type cleavage. A gene for the enzyme, which was cloned using the shotgun method and sequenced, contained a 960‐bp ORF encoding 320 amino acids. The mature enzyme (286 amino acids, 32 085 Da) from the deduced amino‐acid sequence showed quite low homology to known Pels from various microorganisms with 16.1–20.4% identity. Furthermore, we were not able to find any conserved regions in the sequence of Pel‐15E when aligned with the sequences of other enzymes from the established Pel superfamily. However, Pel‐15E had some regions that were homologous to PelA from Azospirillum irakense with 39.8% identity. Based on their amino‐acid sequence homology, Pel‐15E and PelA appear to belong to a new class of Pel family, although the enzymatic properties of both enzymes were quite different.
Roles of His205, His296, His303 and Asp259 in catalysis by NAD+‐specific D‐lactate dehydrogenase
Authors: Kochhar Sunil , Lamzin Victor S. , Razeto Adelia , Delley Michele , Hottinger Herbert , Germond Jacques‐Edouard
Published: March 2003

The role of three histidine residues (His205, His296 and His303) and Asp259, important for the catalysis of NAD+‐specific d‐lactate dehydrogenase, was investigated using site‐directed mutagenesis. None of these residues is presumed to be involved in coenzyme binding because Km for NADH remained essentially unchanged for all the mutant enzymes. Replacement of His205 with lysine resulted in a 125‐fold reduction in kcat and a slight lowering of the Km value for pyruvate. D259N mutant showed a 56‐fold reduction in kcat and a fivefold lowering of Km. The enzymatic activity profile shifted towards acidic pH by ≈ 2 units. The H303K mutation produced no significant change in kcat values, although Km for pyruvate increased fourfold. Substitution of His296 with lysine produced no significant change in kcat values or in Km for substrate. The results obtained suggest that His205 and Asp259 play an important role in catalysis, whereas His303 does not. This corroborates structural information available for some members of the d‐specific dehydrogenases family. The catalytic His296, proposed from structural studies to be the active site acid/base catalyst, is not invariant. Its function can be accomplished by lysine and this has significant implications for the enzymatic mechanism.
High expression of the human hepatocarcinoma‐intestine‐pancreas/pancreatic‐associated protein (HIPPAP) gene in the mammary gland of lactating transgenic mice
Authors: Christa Laurence , Pauloin Alain , Simon Marie‐Thérèse , Stinnakre Marie‐Georges , Fontaine Marie‐Louise , Delpal Serge , Ollivier‐Bousquet Michèle , Bréchot Christian , Devinoy Eve
Published: March 2003

The human hepatocarcinoma‐intestine‐pancreas/pancreatic‐associated protein (HIPPAP) gene was previously identified because of its increased expression in primary liver cancers and during the acute phase of pancreatitis. In normal tissues, HIP/PAP is expressed both in endocrine and exocrine cells of the intestine and pancreas. HIP/PAP is a lactose binding C‐type lectin which acts as an adhesion molecule for rat hepatocytes.

The aim of the work was to study the HIP/PAP secretory pathway and to produce high levels of HIP/PAP in the milk of lactating transgenic mice. In view of its lactose C‐type lectin properties, we have studied the consequences of the expression of HIP/PAP on mammary epithelial cells.

In homozygous mice, production reached 11.2 mg·mL−1 of milk. High levels of soluble and pure HIP/PAP (18.6 mg) were purified from 29 mL of milk. The purified protein was sequenced and the N‐terminal amino acid of the mature HIP/PAP was identified as Glu27, thus localizing the site of cleavage of the signal peptide. The HIP/PAP transgene was only expressed in the mammary gland of lactating transgenic mice. HIP/PAP was detected by immunofluorescence in the whole gland, but labelling was heterogeneous between alveolar clusters, with strongly positive sparse cells. Using immuno electron microscopy, HIP/PAP was observed in all the compartments of the secretory pathway within the mammary epithelial cells.

We provide evidence that HIP/PAP is secreted through the Golgi pathway. However, the number of distended Golgi saccules was increased when compared to that found in wild‐type mouse mammary cells. These modifications could be related to HIP/PAP C‐type lectin specific properties.
A hydrophobic sequence at position 313–316 (Leu‐Ala‐Phe‐Trp) in the fifth domain of apolipoprotein H (β2‐glycoprotein I) is crucial for cardiolipin binding
Authors: Mehdi Haider , Naqvi Asma , Kamboh M. Ilyas
Published: March 2003

Apolipoprotein H (apoH, protein; APOH, gene) binds to negatively charged phospholipids, which triggers the production of a subset of autoantibodies against phospholipid in patients with autoimmune diseases. We have demonstrated that two naturally occurring missense mutations in the fifth domain of apoH, Trp316Ser and Cys306Gly, disrupt the binding of native apoH to phosphatidylserine [Sanghera, D. K., Wagenknecht, D. R., McIntyre, J. A. & Kamboh, M. I. (1997) Hum. Mol. Genet.6, 311–316]. To confirm whether these are functional mutations, we mutagenized APOH cDNAs and transiently expressed them in COS‐1 cells. The cardiolipin ELISA of wild‐type and mutant recombinant apoH confirmed that the Gly306 and Ser316 mutations are responsible for abolishing the binding of recombinant apoH to cardiolipin. These mutations, however, had no effect on the levels of expression or secretion of recombinant apoH in transfected COS‐1 cells. While the Cys306Gly mutation disrupts a disulfide bond between Cys306 and Cys281, which appears to be critical for clustering positively charged amino acids, the Trp316Ser mutation affects the integrity of an evolutionarily conserved hydrophobic sequence at position 313–316 (Leu‐Ala‐Phe‐Trp), which is hypothesized to interact with anionic phospholipid. To test this hypothesis, we exchanged the remaining three hydrophobic amino acids with neutral amino acids by site‐directed mutagenesis (Leu313Gly, Ala314Ser and Phe315Ser). Binding of the Leu313Gly and Phe315Ser mutants to cardiolipin was significantly reduced to 25% and 13%, respectively, of that of the wild‐type. On the other hand, the Ala314Ser mutation showed normal cardiolipin binding. Taken together with our previous findings, these results strongly suggest that the configuration of the fifth domain of apoH, as well as the integrity of the highly conserved hydrophobic amino acids at positions 313–316, is essential for the binding of apoH to anionic phospholipid.
Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase
Authors: Rawls John , Knecht Wolfgang , Diekert Kerstin , Lill Roland , Löffler Monika
Published: March 2003

In animals, dihydroorotate dehydrogenase (DHODH) is a mitochondrial protein that carries out the fourth step in de novo pyrimidine biosynthesis. Because this is the only enzyme of this pathway that is localized to mitochondria and because the enzyme is cytosolic in some bacteria and fungi, we carried out studies to understand the mode of targeting of animal DHODH and its submitochondrial localization. Analysis of fractionated rat liver mitochondria revealed that DHODH is an integral membrane protein exposed to the intermembrane space. In vitro‐synthesized Drosophila, rat and human DHODH proteins were efficiently imported into the intermembrane space of isolated yeast mitochondria. Import did not alter the size of the in vitro synthesized protein, nor was there a detectable size difference when compared to the DHODH protein found in vivo. Thus, there is no apparent proteolytic processing of the protein during import either in vitro or in vivo. Import of rat DHODH into isolated yeast mitochondria required inner membrane potential and was at least partially dependent upon matrix ATP, indicating that its localization uses the well described import machinery of the mitochondrial inner membrane. The DHODH proteins of animals differ from the cytosolic proteins found in some bacteria and fungi by the presence of an N‐terminal segment that resembles mitochondrial‐targeting presequences. Deletion of the cationic portion of this N‐terminal sequence from the rat DHODH protein blocked its import into isolated yeast mitochondria, whereas deletion of the adjacent hydrophobic segment resulted in import of the protein into the matrix. Thus, the N‐terminus of the DHODH protein contains a bipartite signal that governs import and correct insertion into the mitochondrial inner membrane.
Structure of the human oxytocinase/insulin‐regulated aminopeptidase gene and localization to chromosome 5q21
Authors: Rasmussen Thomas E. , Pedraza‐Díaz Susana , Hardré Reneau , Laustsen Palle G. , Carríon Ana G. , Kristensen Torsten
Published: March 2003

The human oxytocinase/insulin‐regulated aminopeptidase (OTase/IRAP) is a 1024 amino acid type II integral membrane protein that is expressed mainly in fat, muscle and placenta tissues. It has been thought to be involved mainly in the control of onset of labour but recently rat OTase/IRAP was shown to participate in the regulation of glucose transporter isoform 4 vesicle trafficking in adipocytes as well. To approach an understanding of OTase/IRAP gene regulation the organization of the human gene was determined. Accordingly, three overlapping genomic clones were isolated and characterized. The human OTase/IRAP gene (OTASE) was found to span ≈ 75 kb containing 18 exons and 17 introns. The gluzincin aminopeptidase motif: GAMEN‐(31 amino acids)‐HELAH‐(18 amino acids)‐E associated with Zn2+‐binding, substrate binding and catalysis is encoded by exons 6 and 7. A major and a minor transcriptional initiation site in OTASE were identified by primer extension 514 bp and 551 bp, respectively, upstream of the translation start codon. Chloroamphenicol acetyltransferase‐reporter assays revealed a functional CpG‐rich promoter/enhancer region located between nucleotide −621 and the major transcriptional initiation site. Human OTASE was assigned to chromosome 5 by hybridization to genomic DNA from characterized somatic cell hybrids. Finally, the OTASE and the human aminopeptidase A gene were subchromosomally localized to 5q21 and 4q25, respectively, by in situ hybridization.
Glycerol kinase of Trypanosoma brucei
Authors: Králová Ivica , Rigden Daniel J. , Opperdoes Fred R. , Michels Paul A. M.
Published: March 2003

Trypanosoma brucei contains two tandemly arranged genes for glycerol kinase. The downstream gene was analysed in detail. It contains an ORF for a polypeptide of 512 amino acids. The polypeptide has a calculated molecular mass of 56 363 Da and a pI of 8.6. Comparison of the T. brucei glycerol kinase amino‐acid sequence with the glycerol kinase sequences available in databases revealed positional identities of 39.0–50.4%. The T. brucei glycerol kinase gene was overexpressed in Escherichia coli cells and the recombinant protein obtained was purified and characterized biochemically. Its kinetic properties with regard to both the forward and reverse reaction were measured. The values corresponded to those determined previously for the natural glycerol kinase purified from the parasite, and confirmed that the apparent Km values of the trypanosome enzyme for its substrates are relatively high compared with those of other glycerol kinases. Alignment of the amino‐acid sequences of T. brucei glycerol kinase and other eukaryotic and prokaryotic glycerol kinases, as well as inspection of the available three‐dimensional structure of E. coli glycerol kinase showed that most residues of the magnesium‐, glycerol‐ and ADP‐binding sites are well conserved in T. brucei glycerol kinase. However, a number of remarkable substitutions was identified, which could be responsible for the low affinity for the substrates. Most striking is amino‐acid Ala137 in T. brucei glycerol kinase; in all other organisms a serine is present at the corresponding position. We mutated Ala137 of T. brucei glycerol kinase into a serine and this mutant glycerol kinase was over‐expressed and purified. The affinity of the mutant enzyme for its substrates glycerol and glycerol 3‐phosphate appeared to be 3.1‐fold to 3.6‐fold higher than in the wild‐type enzyme. Part of the glycerol kinase gene comprising this residue 137 was amplified in eight different kinetoplastid species and sequenced. Interestingly, an alanine occurs not only in T. brucei, but also in other trypanosomatids which can convert glucose into equimolar amounts of glycerol and pyruvate: T. gambiense, T. equiperdum and T. evansi. In trypanosomatids with no or only a limited capacity to produce glycerol, a hydroxy group‐containing residue is found as in all other organisms: T. vivax and T. congolense possess a serine while Phytomonas sp., Leishmania brasiliensis and L. mexicana have a threonine.
Analysis of the molecular composition of Ro ribonucleoprotein complexes
Authors: Fabini Gustav , Rutjes Saskia A. , Zimmermann Christof , Pruijn Ger J. M. , Steiner Günter
Published: March 2003

Human Ro ribonucleoproteins (RNPs) are composed of one of the four small Y RNAs and at least two proteins, Ro60 and La; association of additional proteins including the Ro52 protein and calreticulin has been suggested, but clear‐cut evidence is still lacking. Partial purification of Ro RNPs from HeLa S100 extracts allowed characterization of several subpopulations of Ro RNPs with estimated molecular masses of between 150 and 550 kDa. The majority of these complexes contained Ro60 and La, whereas only a small proportion of Ro52 appeared to be associated with Ro RNPs. To identify novel Y RNA‐associated proteins in vitro, binding of cytoplasmic proteins to biotinylated Y RNAs was investigated. In these reconstitution experiments, several proteins with estimated molecular masses of 80, 68, 65, 62, 60 and 53 kDa, the latter two being immunologically distinct from Ro60 and Ro52, respectively, appeared to bind specifically to Y RNAs. Furthermore, autoantibodies to these proteins were found in sera from patients with systemic lupus erythematosus. The proteins bound preferentially to Y1 and Y3 RNA but, with the exception of the 53‐kDa protein, only weakly to Y4 RNA and not at all to Y5 RNA. Coprecipitation of the 80, 68, 65, and 53‐kDa proteins by antibodies to Ro60 and La was observed, suggesting that at least a proportion of the novel proteins may reside on the same particles as La and/or Ro60. Finally, the binding sites for these proteins on Y1 RNA were clearly distinct from the Ro60‐binding site involving a portion of the large central loop 2, which was found to be indispensable for binding of the 80, 68, 65 and 53‐kDa proteins, as well as the stem 3–loop 3 and stem 2–loop 1 regions. Interestingly, truncation of the La‐binding site resulted in decreased binding of the novel proteins (but not of Ro60), indicating La to be required for efficient association. Taken together, these results suggest the existence of further subpopulations of Ro RNPs or Y RNPs, consistent with the heterogeneous characteristics observed for these particles in the biochemical fractionation experiments.
Regulation of secretory protein gene expression in Paramecium
Authors: Galvani Angélique , Sperling Linda
Published: March 2003

In cells that possess a regulated secretory pathway, exocytosis can lead to transcriptional activation of genes encoding products stored in secretory granules as well as genes required for granule biogenesis. With the objective of understanding this response, we have examined the expression of Paramecium secretory protein genes in different physiological and genetic contexts. The genes belong to the trichocyst matrix protein (TMP) multigene family, encoding polypeptides that form the crystalline matrix of the secretory granules, known as trichocysts. Approximately 1000 trichocysts per cell are docked at pre‐formed cortical exocytotic sites. Their rapid and synchronous exocytosis can be triggered by vital secretagogues such as aminoethyldextran without harming the cells. Using this exocytotic trigger, we found that the transcription of TMP genes undergoes rapid, transient and co‐ordinate 10‐fold activation in response to massive exocytosis, leading to a 2.5‐fold increase in the pool of TMP mRNA. Experiments with exocytosis‐deficient mutants show that the secretagogue‐induced increase in intracellular free calcium implicated in stimulus/secretion coupling is not sufficient to activate TMP gene expression. We present evidence that the state of occupation of the cortical exocytotic sites can affect TMP gene expression and suggest that these sites play a role in gene activation in response to exocytosis.
Molecular characterization of a puromycin‐insensitive leucyl‐specific aminopeptidase, PILS‐AP
Authors: Schomburg Lutz , Kollmus Heike , Friedrichsen Sönke , Bauer Karl
Published: March 2003

The family M1 of Zn‐dependent aminopeptidases comprises members of closely related enzymes which are known to be involved in a variety of physiologically important processes. On the basis of two highly conserved peptide motifs, we have identified a new member of this family by PCR amplification and cDNA‐library screening. The longest ORF encodes a protein of 930 residues. It contains the HEXXH(X)18E Zn‐binding motif and displays high homology to the other M1 family members except for its N‐terminus for which a signal sequence of 20 residues can be predicted. This interpretation was supported by expressing fusion proteins formed with green fluorescent protein which localized to intracellular vesicles in COS‐7 and BHK cells. Northern‐blot analysis revealed ubiquitous expression of a major 3.1‐kb transcript. For enzymatic studies, the complete protein was expressed in Sf 9 insect cells. When aminoacyl β‐naphthylamides were used as substrates, efficient hydrolysis was only observed for Leu (and to a lesser extent Met). The activity was inhibited by chelators of bivalent cations and by other known aminopeptidase inhibitors, but surprisingly puromycin was without effect. This newly identified puromycin‐insensitive leucyl‐specific aminopeptidase is a signal‐sequence‐bearing member of family M1 and may be another example of the small subset of substrate‐specific peptidases.
Structure and chromosomal localization of human and mouse genes for hematopoietic prostaglandin D synthase
Authors: Kanaoka Yoshihide , Fujimori Ko , Kikuno Reiko , Sakaguchi Yuriko , Urade Yoshihiro , Hayaishi Osamu
Published: March 2003

Hematopoietic prostaglandin D synthase (H‐PGDS) is the key enzyme for the production of the D and J series of prostanoids, and the first recognized vertebrate homolog of sigma‐class glutathione S‐transferase (GST). We isolated the genes and cDNAs for human and mouse H‐PGDSs. The human and mouse cDNAs contained a coding region corresponding to 199 amino‐acid residues with calculated molecular masses of 23 343 and 23 226, respectively. Both H‐PGDS proteins recombinantly expressed in Escherichia coli showed bifunctional activities for PGDS and GST, and had almost the same catalytic properties as the rat enzyme. Northern analyses demonstrated that the H‐PGDS genes were expressed in a highly species‐specific manner. Whereas the human gene was widely distributed, in contrast, the mouse gene was detected only in samples from oviduct and skin. By fluorescence in situ hybridization, the chromosomal localization of the human and mouse H‐PGDS genes were mapped to 4q21–22 and 3D‐E, respectively. The human and mouse H‐PGDS genes spanned approximately 41 and 28 kb, respectively, and consisted of six exons divided by five introns. The exon/intron boundaries of both genes were completely identical to those of the sigma‐class GST subfamily, although the amino‐acid sequences of the latter were only 17.0–21.5% identical to those of either H‐PGDS. These findings suggest that the H‐PGDS genes evolved from the same ancestral gene as the members of the sigma‐class GST family.
Trypsin‐specific acyl‐4‐guanidinophenyl esters for α‐chymotrypsin‐catalysed reactions
Authors: Günther Robert , Thust Sven , Hofmann Hans‐Jörg , Bordusa Frank
Published: March 2003

The function of acyl‐4‐guanidinophenyl esters as substrate mimetics for the serine protease α‐chymotrypsin was investigated by protein‐ligand docking, hydrolysis, and acyl transfer experiments. On the basis of protein‐ligand docking studies, the binding and hydrolysis properties of these artificial substrates were estimated. The predictions of the rational approach were confirmed by steady‐state hydrolysis studies on 4‐guanidinophenyl esters derived from coded amino acids (which α‐chymotrypsin is not specific for), noncoded amino acids, and even simple carboxylic acid moieties. Enzymatic peptide syntheses qualify these esters as suitable acyl donors for the coupling of acyl components far from the natural enzyme specificity, thus considerably expanding the synthetic utility of α‐chymotrypsin.