bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymatic reduction of benzoyl‐CoA to alicyclic compounds, a key reaction in anaerobic aromatic metabolism
Authors: KOCH Jürgen , FUCHS Georg
Published: March 2005

Different anaerobic bacteria can oxidize a variety of aromatic compounds completely to CO2 via one common aromatic intermediate, benzoyl‐CoA. It has been postulated that anaerobically the aromatic nucleus of benzoyl‐CoA becomes reduced. An oxygen‐sensitive enzyme system is described catalyzing the reduction of benzoyl‐CoA to trans‐2‐hydroxycyclohexanecarboxyl‐CoA in a denitrifying Pseudomonas species grown anaerobically on benzoate plus nitrate. The assay mixture consists of cell extract, [U‐14C]benzoyl‐CoA, a [U‐14C]benzoyl‐CoA‐generating system (consisting of [U‐14C]benzoate, purified benzoate‐CoA ligase, Mg2+‐ATP, coenzyme A), an ATP‐regenerating system (consisting of phosphoenolpyruvate, pyruvate kinase, myokinase), and a low‐potential reductant [titanium(III) citrate]. The optimal pH is about 7, the specific activity 10 nmol benzoyl‐CoA reduced min−1× mg−1 protein. The apparent Km for benzoyl‐CoA is below 50 μM. Five major products were found. One product is cyclohex‐1‐enecarboxyl‐CoA which must have been formed by a benzoyl‐CoA reductase. The other product is probably trans‐2‐hydroxycyclohexanecarboxyl‐CoA rather than the cis‐stereoisomer; this product must have been formed by a cyclohex‐1‐enecarboxyl‐CoA hydratase. Two other products are likely to be intermediates of benzoyl‐CoA reduction to cyclohex‐1‐enecarboxyl‐CoA, suggesting that the reduction reaction is more complex. An early formed fifth product is more polar than cyclohexanecarboxyl‐ or cyclohex‐1‐enecarboxyl‐CoA.

The enzyme system is under oxygen control since it was not found in cells grown aerobically on benzoate. It is induced by aromatic compounds since its activity is low in cells grown anaerobically on acetate. The actual inducer is probably benzoyl‐CoA rather than benzoate. This conclusion is drawn from the fact that the system is also present in cells grown anaerobically on phenol, phenylacetate, 4‐hydroxybenzoate, or 2‐aminobenzoate; the anaerobic metabolism of these compounds has been shown in this organism to proceed directly via benzoyl‐CoA rather than via free benzoate.
Molecular characterization of a Leishmania donovani infantum antigen identified as histone H2A
Authors: SOTO Manuel , REQUENA Jose María , GOMEZ Luis Carlos , NAVARRETE Ignacio , ALONSO Carlos
Published: March 2005

A Leishmania donovani infantum promastigote cDNA expression library was screened with a serum obtained from a dog naturally infected with this parasite. One of the positive clones obtained revealed nucleotide sequence similarities with the histone H2A genes from various organisms. Northern blot analyses and sequence data of three independently isolated cDNA clones indicated that the Leishmania H2A mRNAs are polyadenylated, as are the basal histone mRNAs of higher eukaryotes and the histone mRNAs of yeast. The analysis of the genomic distribution of the DNA coding for histone H2A suggested that, in L. d. infantum, there are at least four genes coding for the H2A protein. It is likely that there is a simultaneous expression of at least two of the H2A genes since differences in nucleotide sequence between two of the sequenced cDNAs were observed. Affinity‐purified antibodies against the β‐galactosidase‐fused H2A protein recognize specifically a Leishmania protein band with a molecular mass of 14 kDa.
High‐affinity and low‐affinity calcium binding and stability of the multidomain extracellular 40‐kDa basement membrane glycoprotein (BM‐40/SPARC/osteonectin)
Authors: MAURER Patrik , MAYER Ulrike , BRUCH Marcel , JENÖ Paul , MANN Karlheinz , LANDWEHR Ruth , ENGEL Jürgen , TIMPL Rupert
Published: March 2005

Reversible binding of calcium ions to a single high‐affinity binding site in the 40‐kDa basement membrane protein (BM‐40) caused a 33% increase of α‐helicity, an about 60% change in intrinsic fluorescence and a dramatic increase of the rate of cleavage by α‐chymotrypsin. All these effects exhibited identical dependencies on calcium concentration from which a dissociation constant Kd= 0.6 μM was determined. Calcium release was accompanied by an increase of the frictional ratio in solution but not by denaturation which occurred at about equal guanidine · HCl concentration for both calcium‐saturated and calcium‐depleted protein (midpoint 1.5 M). The cleavage sites for α‐chymotrypsin are located in or near to the EF‐hand domain IV of calcium‐depleted BM‐40 (also known as SPARC, i.e. secreted protein acidic and rich in cysteine, and osteonectin). These and other data indicate that binding occurs in the EF‐hand domain from which a large conformational change is transmitted. Low‐affinity calcium‐binding sites in the N‐terminal glutamic‐acid‐rich domain I of BM‐40 were identified by human leukocyte elastase which was found to cleave very specifically in the middle of this domain. From the increase of cleavage rate with increasing calcium concentration a Kd≥ 10 mM was estimated. It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM‐40 such as collagen binding and that high‐affinity binding is important for stabilization, folding and secretion during biosynthesis.
Structure of the N‐linked oligosaccharides of the human transferrin receptor
Authors: ORBERGER Georg , GEYER Rudolf , STIRM Stephan , TAUBER Rudolf
Published: March 2005

Human transferrin receptor was isolated from placenta and from the hepatocarcinoma cell line Hep G2. Asparagine‐linked oligosaccharides were released by treatment of tryptic glycopeptides with endo‐β‐N‐acetylglucosaminidase H or peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F. Oligosaccharide alditols were fractionated by anion‐exchange high‐performance liquid chromatography and by high‐pH anion‐exchange chromatography. Glycans from placental transferrin receptor were further characterized, after desialylation, by methylation analysis and, in part, by liquid secondary‐ion mass spectrometry. Sialylation of placental transferrin receptor was examined by lectin affinity blotting with Sambucus nigra agglutinin and Maackia amurensis agglutinin. In order to trace possible inter‐individual differences in N‐glycosylation of the receptor, two preparations of placental transferrin receptor purified from two donors were compared.

The results demonstrate that human transferrin receptor from placenta predominantly carries diantennary and triantennary N‐acetyllactosaminic glycans as well as hybrid‐type species, the galactose residues of which being almost completely substituted with (α2–3)‐linked sialic acid residues. Distinct differences were noted in the glycosylation pattern of the receptor from different individuals. Transferrin receptor from donor A carried predominantly diantennary and triantennary complex‐type glycans, in part fucosylated at the innermost N‐acetylglucosamine residue, in addition to small amounts of bisected and of incomplete diantennary species. Placental transferrin receptor from donor B predominantly carried triantennary N‐acetyllactosaminic glycans without fucose and hybrid‐type oligosaccharides with four or five mannose residues. Distinct from placental transferrin receptor, the receptor from Hep G2 cells contained larger amounts of oligomannosidic glycans with six to nine mannose residues and tetrasialylated complex‐type oligosaccharides apart from mono‐, di‐ and trisialylated species.
Characterization of a cis‐acting regulatory element involved in human‐aromatase P‐450 gene expression
Authors: TODA Katsumi , MIYAHARA Kaoru , KAWAMOTO Takeshi , IKEDA Hisao , SAGARA Yusuke , SHIZUTA Yutaka
Published: March 2005

The characteristics of a cis‐acting regulatory region involved in the human‐aromatase P‐450 gene have been examined by transient expression analysis. The region spans from −242 –−166 relative to the cap site of the gene. A fragment containing the region excised from the gene enhances heterologous promoter activity as well as its own promoter activity in a position‐independent and orientation‐independent manner. The fragment exerts its enhancer activity in human BeWo choriocarcinoma cells in which the aromatase P‐450 gene is expressed, but not in other cell lines tested. Deletion of 38 bp from the 3′ end of the fragment results in a complete loss of enhancer activity. A gel‐retardation assay with nuclear extracts from BeWo cells suggests the existence of a nuclear factor(s) which interacts with the fragment. These results suggest that the regulatory element in the fragment is involved in efficient transcription of the human‐aromatase P‐450 gene.
Microdiversity of human‐plasma‐membrane calcium‐pump isoform 2 generated by alternative RNA splicing in the N‐terminal coding region
Authors: HEIM Roger , HUG Martin , IWATA Tomoko , STREHLER Emanuel E. , CARAFOLI Ernesto
Published: March 2005

cDNA species covering the entire coding sequence of the human homologue of the rat plasma membrane Ca2+‐ATPase (PMCA) isoform 2 have been isolated and characterized. The deduced amino acid sequence shows 99% identity with that of the rat protein and can be aligned with the latter without gaps except for one 14‐amino‐acid‐residue insert in the region immediately preceding the putative phospholipid‐sensitive domain in the human pump. cDNA clones isolated by anchored polymerase‐chain reaction revealed additional microheterogeneity in the same N‐terminal PMCA2‐coding region. Alternative RNA splicing involving a region of 135 nucleotides generates three types of cDNA. One does not contain any of the 135 bp, and the other two contain 42 bp or the entire 135 bp of the optional sequence. Analysis of genomic DNA indicates that this sequence is encoded by three separate exons of 33, 60 and 42 bp. Although each of these exons could be inserted into the mRNA without changing the reading frame, polymerase‐chain amplifications using cDNA libraries from several human tissues show that the 33‐bp and the 60‐bp exons are never independently used during splicing. The unequal distribution of the splice variants suggests tissue‐specific regulation of the alternative‐splicing pathways and indicates a functional specialization of the encoded isoform subtypes.
Incorporation and degradation by kidney lysosomes of cystatin α injected intravenously
Authors: OHSHITA Takeyuki , IKE Yoshimasa , KATUNUMA Nobuhiko
Published: March 2005

Cystatin α was purified from Escherichia coli transfected with a recombinant cystatin α gene and injected into the tail vein of rats. Its fate was then followed using a sensitive enzyme immunoassay for cystatin α. Results showed that it was rapidly removed from the blood and taken up by the kidney. Percoll density‐gradient analysis showed that it was rapidly incorporated into lysosomes in the kidney. Its level in the kidney was maximal 30 min after its injection then rapidly decreased. The activity of cathepsin H in the kidney lysosomal fraction was markedly decreased 30 min after injection of cystatin α but recovered rapidly. Anti‐(cystatin α) antibody precipitated cathepsin H and anti‐(cathepsin H) antibody precipitated cystatin α in an extract of the lysosome‐rich fraction of the kidney 30 min after injection of cystatin α. These results indicate that cystatin α was taken up by kidney lysosomes, formed a complex with cathepsin H and initially decreased the activity of cathepsin H, but that later the level of cystatin α in the kidney rapidly decreased.
Subcellular distribution in rat liver of a novel broad‐specificity (α1 → 2, α1 → 3 and α1 → 6) mannosidase active on oligomannose glycans
Authors: BONAY Pedro , ROTH Jürgen , HUGHES R. Colin
Published: March 2005

Recently, the purification to homogeneity was reported of a novel broad‐specificity α‐mannosidase from rat liver microsomal membranes [P. Bonay and R. C. Hughes (1991) Eur. J. Biochem. 197, 229–238]. The enzyme catalyzed the ordered removal of α1 → 2‐, α1 → 3‐ and α1 → 6‐linked mannose residues from MannGlcNAc oligosaccharide substrates where n= 4–9. We now show by subcellular fractionation and immunocytochemistry that the novel mannosidase is present in the endoplasmic reticulum, Golgi apparatus and endosomes. Enzyme activity is enriched in a heavy Golgi membrane fraction and to lesser extent in an intermediate density Golgi membrane fraction containing GlcNAc transferase I activity and in a ‘late’ endosomal fraction. Low levels of enzyme activity were detectable in endoplasmic reticulum membranes and in ‘early’ endosomes but not in receptor‐enriched and ligand‐free endosomes. Assays of enzymic activity using Golgi membrane fractions in the absence and presence of Triton X‐100 showed that the active site of the enzyme faces the lumen of the membrane vesicles. Antibodies directed against the purified mannosidase showed no immunological cross‐reaction to known endoplasmic reticulum and Golgi mannosidases. Conversely, the purified mannosidase was not recognized by antibodies directed against endoplasmic reticulum mannosidase nor Golgi mannosidase IA.
Leucine aminopeptidase from Arabidopsis thaliana
Authors: BARTLING Dieter , WEILER Elmar W.
Published: March 2005

Leucine aminopeptidases are exopeptidases which are presumably involved in the processing and regular turnover of intracellular proteins; however, their precise function in cellular metabolism remains to be established. Towards this goal, a full‐length complementary DNA encoding a plant leucine aminopeptidase was isolated from a cDNA library of Arabidopsis thaliana and sequenced. The nucleotide sequence showed 49.5% identity to the Escherichia coli xerB‐encoded leucine aminopeptidase. Sequence analysis revealed that the cDNA encodes a polypeptide of 520 amino acids with a calculated molecular mass of 54506 Da. The C‐terminal part (amino acids 200–520) of the deduced amino acid sequence showed 43.8% sequence identity to the xerB‐encoded leucine aminopeptidase and 42.6% sequence identity to the amino acid sequence of bovine lens leucine aminopeptidase (EC 3.4.11.1). No sequence similarity (not even over short sequence elements) was observed with any other known peptidase or proteinase sequence. The cDNA was expressed as a fusion protein from the lacZ promoter in E. coli. Enzymatic analysis proved that the cloned cDNA encoded an active leucine aminopeptidase. The properties of this enzyme, including metal requirements, inhibitor sensitivity, pH optimum and the remarkable temperature stability, are very similar to those reported for leucine aminopeptidases from other tissues. Amino acids involved in metal and substrate binding in bovine lens aminopeptidase are completely conserved in the plant enzyme as well as in the XerB protein. Our results show that leucine aminopeptidases form a superfamily of highly conserved enzymes, spanning the evolutionary period from the bacteria to animals and higher plants. This is the first aminopeptidase cloned from a plant.
M‐phase‐specific histone H1 kinase in fish oocytes
Authors: YAMASHITA Masakane , FUKADA Sachiko , YOSHIKUNI Michiyasu , BULET Philippe , HIRAI Toshiaki , YAMAGUCHI Akihiko , YASUDA Hideyo , OHBA Yoshiki , NAGAHAMA Yoshitaka
Published: March 2005

We demonstrate, for the first time in fish, that a Ca2+‐independent and cyclic‐nucleotide‐independent histone H1 kinase activity oscillates according to the cell cycle of the oocyte, peaking at the first and the second meiotic metaphase with a transient drop between them. The kinase, M‐phase‐specific histone H1 kinase (M‐H1K), was purified from mature carp oocytes by using two exogenous substrates for assaying its activity: histone H1 and a synthetic peptide (SP peptide, KKAAKSPKKAKK) containing the sequence KSPKK, which includes the consensus sequence of the site phosphorylated by a serine/threonine‐specific protein kinase encoded by the fission yeast cdc2+ gene (cdc2 kinase). The M‐H1K and maturation‐promoting factor (MPF) activities coincided closely throughout four steps of purification, strongly suggesting the identity of M‐H1K and MPF. The final preparation was purified 5000‐fold with a recovery of 4%, when histone H1 was used for the kinase assay, and 10000‐fold with a recovery of 7% when SP peptide was used. The purified molecular mass of the kinase was estimated to be 100 kDa by gel filtration and contained four proteins of 33,34,46 and 48 kDa. Anti‐PSTAIR antibody recognizing cdc2 kinase cross‐reacted with the 33‐kDa and 34‐kDa proteins, while the 46‐kDa and 48‐kDa bands cross‐reacted with monoclonal antibodies raised against cyclin B. The 33‐kDa protein was also recognized by an antibody against a goldfish cdk2 (Eg1) kinase, a cdc2‐related kinase which has the PSTAIR sequence and binds to p13suc1 but does not form a complex with cyclin B. M‐H1K activity corresponded well to the 34‐kDa, 46‐kDa and 48‐kDa proteins but not to the 33‐kDa protein. These results strongly suggest that M‐H1K consists of cdc2 kinase forming a complex with cyclin B, and that cdk2 kinase is not a component of M‐H1K, although it is found in the highly purified M‐H1K. The purified M‐H1K utilized Mg2+, Mn2+, ATP and GTP, and had a wide pH optimum ranging over 8.0–10.5. The kinase was thermolabile and sensitive to freezing/thawing.
Structural comparison of the chicken genes for α1(VI) and α2(VI) collagen
Authors: WÄLCHLI Chantal , KOLLER Erich , TRUEB Judith , TRUEB Beat
Published: March 2005

The chicken α(VI) polypeptide is encoded by a single gene spanning 21 kbp of genomic DNA. This gene is composed of 34 exons and 33 introns. Its structure is closely related to that of the α2(VI) collagen gene, suggesting that the two genes evolved by gene duplication. Both genes contain 19 exons coding for the triple‐helical domain. These exons are multiples of 9 bp (27, 36, 45, 54, 63 and 90 bp) and encode an integral number of collagenous Gly‐Xaa‐Yaa triplets. Since there is no convincing correlation to a building block of 54 bp, it is unlikely that type VI collagen has evolved from a primordial 54‐bp module as suggested for all fibrillar collagens.
Models of the actin monomer and filament from fluorescence resonance‐energy transfer
Authors: O'DONOGHUE Séan I. , HAMBLY Brett D. , dos REMEDIOS Cristobal G.
Published: March 2005

We have developed algorithms for combining fluorescence resonance‐energy transfer (FRET) efficiency measurements into structural models which predict the relative positions of the chemical groups used in FRET. We used these algorithms to construct models of the actin monomer and filament derived solely from FRET measurements based on seven distinct loci. We found a mirrorimage pair of monomer models which best fit the FRET data. One of these models agrees well with the atomic‐resolution crystal structure recently published by Kabsch et al. in Heidelberg [Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F. & Holmes, K. C. (1990) Nature 347, 37–44]. The root‐mean‐square deviation between this FRET model and the crystal structure was about 0.9 nm. Other macromolecular models assembled from FRET measurements are likely to have a similar resolution. The largest discrepancy was for the Cys10 locus which deviated 1.44 nm from the crystal position. We discuss the limitations of the FRET method that may have contributed to this discrepancy, and conclude that the Cys10 FRET data have probably located Cys10 incorrectly in the FRET monomer model. Using the FRET monomer models, we found three orientations in the filament which best fit the intermonomer FRET data. These orientations differ substantially from the atomic‐resolution filament model proposed by the Heidelberg group [Holmes, K., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature 347, 44–49], largely because of the discrepancies in the Cys10 data. These data should probably be excluded from the analysis; however, this would leave too few measurements to assemble a filament model. In the near future, we hope to obtain additional FRET measurements to other actin loci so that the filament modelling can be done without the Cys10 data.
Proton NMR studies of bovine serum albumin
Authors: SADLER Peter J. , TUCKER Alan
Published: March 2005

A variety of one‐ and two‐dimensional 1H‐NMR methods have been applied to the study of defatted 66.5‐kDa bovine serum albumin in solution.

The majority of the protons gave rise to broad unresolved resonances and spectral enhancement methods for one‐dimensional spectra were investigated in detail. A combination of exponential and sine‐bell functions was particularly effective.

The presence of contaminating glycoproteins in some commercial samples of bovine serum albumin was readily detectable from their N‐acetyl resonances at about 2.1 ppm.

The release of bound Cys (from mixed disulphide at Cys34) was observed after addition of dithiothreitol.

Through the use of two‐dimensional shift‐correlated spectroscopy, assignments of some 80 spin systems to amino acid type were made.

The pKa of the N‐terminal Asp was measured as 7.8 (0.1 M phosphate buffer. 310 K).

1H NMR spectra of bovine, human, porcine and rat serum albumins have been compared. Using sequence comparisons, specific assignments have been made for the N‐terminal residues of bovine (Asp‐Thr‐His), human (Asp‐Ala‐His), porcine (Asp‐Thr‐Tyr) and rat (Glu‐Ala‐His) albumins, and for Thr189, Tyr155 and His59/377 of bovine albumin.

These NMR data suggest that certain local regions of bovine serum albumin are highly mobile yet structured in solution, and demonstrate that the application of both one‐ and and two‐dimensional NMR methods will allow more detailed investigations of structural transitions in serum albumins induced by, for example, pH, drug and metal binding.
Amino acid sequence of calmodulin from Euglena gracilis
Authors: TODA Hiroko , YAZAWA Michio , YAGI Koichi
Published: March 2005

The complete amino acid sequence of calmodulin from Euglena gracilis was determined by isolation and sequence analyses of peptides derived from calmodulin by digestion with trypsin and Staphylococcus aureus V8 protease. Euglena calmodulin consists of 148 amino acid residues; it lacks tryptophan and cysteine and contains one tyrosine, three histidine and two Nε‐trimethyllysine residues molecule of the protein. Its N‐terminus was blocked with an acetyl group and C‐terminal lysine was trimethylated. Euglena calmodulin is the first calmodulin so far examined in which the C‐terminal lysine is trimethylated. The comparison of amino acid sequences between Euglena and human brain calmodulins indicated 17 amino acid substitutions in Euglena calmodulin.
Sequential assignment of the proton NMR spectrum of isolated α(CO) chains from human adult hemoglobin
Authors: MARTINEAU Laure , CRAESCU Constantin T.
Published: March 2005

In this paper we report proton two‐dimensional NMR experiments on isolated α chains from human hemoglobin A (HbA) in the monocarboxylated state. Several J‐correlated and NOE spectra in water or deuterium water and phosphate buffer (100 mM) at 310 K and pH 5.6 were acquired and analysed for the sequential assignment of the proton resonances. In addition, we used the topological data obtained from the crystal structure of α subunits in the monocarboxylated HbA tetramer. The assigned resonances correspond to 70% of the amino acid residues. The present results provide information on the tertiary structure of isolated α chains in solution, particularly in the heme region. This structure may be compared with that of the a subunits in the tetrameric HbA(CO) in crystal by comparison of observed chemical shifts and those calculated from the X‐ray atomic coordinates. Overall, the global folding of the two forms are highly similar. However, this analysis points out several local conformational differences in the heme pocket and the neighboring of the unique Trp residue. Possible explanations of these differences are discussed.
Evidence for controlled autoproteolysis of alkaline protease
Authors: PHADATARE Sangita U. , SRINIVASAN Mandyam C. , DESHPANDE Vasanti V.
Published: March 2005

The alkaline serine protease of Conidiobolus coronatus was shown to be involved in its conidial discharge [Phadatare, S., Srinivasan, M. C., Deshpande, M. (1989) Arch. Microbiol. 153, 47–49]. To understand the regulation of conidial discharge, the mechanism of control of protease activity was investigated, which revealed the presence of two electrophoretically separable intracellular proteases (protease I and protease II). The formation of smaller and less‐active protease II coincided with the decrease in conidial discharge. In order to trace the origin of protease II, the corresponding purified extracellular enzymes were compared with respect to their biochemical, physicochemical and immunological properties. The biochemical properties, such as optimum pH and temperature, stability, sensitivity to metal ions and substrate specificity were closely similar for both proteases. Amino acid analysis revealed that protease II is completely similar to protease I, though protease I contains an additional portion which is not contained in protease II. Western‐blot ELISA, immunotitration and determination of antigenic valencies also revealed the structural similarity between the two proteases. Purified protease I showed partial degradation to protease II in vitro, the process being sensitive to phenylmethylsulfonyl fluoride, indicating its proteolytic nature. These results suggest that the formation of a less‐active protease by autoproteolysis represents a novel means of physiological regulation of protease activity, which in turn regulates the conidial discharge in C. coronatus.
Biochemical properties of recombinant single‐chain urokinase‐type plasminogen activator mutants with deletion of Asn2 through Phe157 and/or substitution of Cys279 with Ala
Authors: LIJNEN Henri R. , LI Xian‐Kui , NELLES Luc , HU Mei‐Hao , COLLEN Désiré
Published: March 2005

The contribution of the NH2‐terminal polypeptide chain and of the Cys148–Cys279 interchain disulphide bond to the enzyme activity of urokinase‐type plasminogen activator (u‐PA) was studied using site‐specific mutagenesis. Recombinant single‐chain u‐PA (rscu‐PA) variants were produced by transfecting Chinese hamster ovary cells with cDNA encoding des(Asn2–Phe157)rscu‐PA (rscu‐PA with deletion of Asn2–Phe157), [Ala279]rscu‐PA (rscu‐PA with Cys279→Ala mutation) or des(Asn2–Phe157)[Ala279]rscu‐PA [des(Asn2–Phe157)rscu‐PA with Cys279→Ala mutation].

Des(Asn2–Phe157)rscu‐PA, [Ala279]rscu‐PA and des(Asn2–Phe157)[Ala279]rscu‐PA, purified from conditioned cell culture medium, were obtained as nearly homogeneous single‐chain molecules with Mr approximately 30000, 54000 and 30000, and specific fibrinolytic activities on fibrin plates of (mean ± SD; n= 3) 860 → 150 IU/mg, 43.0 → 2.5 IU/μg and 240 → 20 IU/mg, respectively, compared to 69.0 → 4.3 IU/μg for wild‐type rscu‐PA obtained in the same expression system. The plasminogen activating potential in a buffer milieu of [Ala279]rscu‐PA was somewhat lower than that of rscu‐PA, but that of both deletion mutants was virtually abolished. In a human plasma milieu in vitro, consisting of a radiolabelled human plasma clot submerged in plasma, 50% clot lysis in 2 h required 6.5 μg/ml [Ala279]rscu‐PA or 3.4 μg/ml rscu‐PA, whereas with both deletion mutants no significant clot lysis was observed with up to 16 μg/ml. Treatment of [Ala279]rscu‐PA or rscu‐PA with plasmin resulted in quantitative conversion to two‐chain molecules and was associated with an increase in specific amidolytic activity from about 600 IU/mg to 62.5 IU/μg for [Ala279]rscu‐PA as compared to an increase from about 0.3 IU/μg to 75.0 IU/μg for rscu‐PA. In contrast, no significant amidolytic activity could be generated by treatment of des(Asn2–Phe157)rscu‐PA or des(Asn2–Phe157)[Ala279]rscu‐PA with plasmin.

The u‐PA B‐chain, isolated from plasmin‐treated [Ala279]rscu‐PA, had enzymic properties which were comparable to those of rtcu‐PA, with respect to specific fibrinolytic activity, amidolytic activity, kinetics of plasminogen activation and clot‐lysis activity in a human plasma milieu in vitro. Following bolus injection into hamsters, the plasma clearances were comparable (0.7–1.1 ml/min) for wild‐type rscu‐PA and for the three truncated rscu‐PA mutants.

These results indicate that (a) deletion of residues Asn2–Phe157 results in abolition of the enzyme activity of rscu‐PA, (b) the interchain disulphide bond in u‐PA is not required for the enzymic activity of scu‐PA, (c) all the determinants required for the enzymic activity of two‐chain u‐PA are contained within the B‐chain, and (d) the region comprising residues Asn2–Phe157 of u‐PA is not required for the rapid in vivo clearance.
Purification and characterization of membrane‐bound endoglycoceramidase from Corynebacterium sp.
Authors: ASHIDA Hisashi , YAMAMOTO Kenji , KUMAGAI Hidehiko , TOCHIKURA Tatsurokuro
Published: March 2005

Endoglycoceramidase catalyzes the hydrolysis of the linkage between oligosaccharides and ceramides of various glycosphingolipids. We found that a bacterial strain Corynebacterium sp., isolated from soil, produced endoglycoceramidase both intracellularly and extracellularly. The intracellular enzyme bound to the cell membrane was solubilized with 1% Triton X‐100 and purified to homogeneity about 170‐fold with 60% recovery. The molecular mass of the enzyme was approximately 65 kDa. The enzyme is most active at pH 5.5–6.5 and stable at pH 3.5–8.0. Various neutral and acidic glycosphingolipids were hydrolyzed by the enzyme in the presence of 0.1% Triton X‐100. Ganglio‐ and lacto‐type glycosphingolipids were readily hydrolyzed, but globo‐type glycosphingolipids were hydrolyzed slowly.
Human hemi‐myeloperoxidase
Authors: ZUURBIER Karin W. M. , van den BERG Joop D. , Van GELDER Bob F. , MUIJSERS Anton O.
Published: March 2005

Human neutrophilic myeloperoxidase (MPO) is involved in the defence mechanism of the body against micro‐organisms. The enzyme catalyses the generation of the strong oxidant hypochlorous acid (HOCI) from hydrogen peroxide and chloride ions. In normal neutrophils MPO is present in the dimeric form (140 kDa). The disulphide‐linked protomers each consist of a heavy subunit and a light one. Reductive alkylation converts the dimeric enzyme into two protomers, ‘hemi‐myeloperoxidase’.

We studied the initial activities of human dimeric MPO and hemi‐MPO at the physiological pH of 7.2 and found no significant differences in chlorinating activity. These results indicate that, at least at neutral pH, the protomers of MPO function independently. The absorption spectra of MPO compounds II and III, both inactive forms concerning HOCI generation, and the rate constants of their formation were the same for dimeric MPO and hemi‐MPO, but hemi‐MPO required a slightly larger excess of H2O2 for complete conversion. Hemi‐MPO was less stable at a high temperature (80°C) as compared to the dimeric enzyme. Furthermore, the resistance of the chlorinating activity of hemi‐MPO against its oxidative product hypochlorous acid was somewhat lower (IC50= 32 μM HOCI) compared to dimeric MPO (IC50= 50 μM HOCI). The higher stability of dimeric MPO in the presence of its oxidative product compared to that of monomeric MPO might be the reason for the occurrence of MPO as a dimer.
Characterization of the chylomicron‐remnant‐recognition sites on parenchymal and Kupffer cells of rat liver Selective inhibition of parenchymal cell recognition by lactoferrin
Authors: van DIJK Marc C. M. , ZIERE Gijsbertus J. , van BERKEL Theo J. C.
Published: March 2005

Upon injection of chylomicrons into rats, chylomicron remnants are predominantly taken up by parchenymal cells, with a limited contribution (8.6% of the injected dose) by Kupffer cells. In vitro storage of partially processed chylomicron remnants for only 24 h leads, after in vivo injection, to an avid recognition by Kupffer cells (uptake up to 80% of the total liver‐associated radioactivity). Lactoferrin greatly reduces the liver uptake of chylomicron remnants, which appears to be the consequence of a specific inhibition of the uptake by parenchymal cells. Kupffer‐cell uptake is not influenced by lactoferrin. In vitro studies with isolated parenchymal and Kupffer cells show that both contain a specific recognition site for chylomicron remnants. The Kupffer‐cell recognition site differs in several ways from the recognition site on parenchymal cells as follows. (a) The maximum level of binding is 3.7‐fold higher/mg cell protein than with parenchymal cells. (b) Binding of chylomicron remnants is partially dependent on the presence of calcium, while binding to parenchymal cells is not. (c) β‐Migrating very‐low‐density lipoprotein is a less effective competitor for chylomicron‐remnant binding to Kupffer cells compared to parenchymal cells. (d) Lactoferrin leaves Kupffer‐cell binding uninfluenced, while it greatly reduces binding of chylomicron remnants to parenchymal cells. The properties of chylomicron‐remnant recognition by parenchymal cells are consistent with apolipoprotein E being the determinant for recognition.

It can be concluded that the chylomicron‐remnant recognition site on Kupffer cells possesses properties which are distinct from the recognition site on parenchymal cells. It might be suggested that partially processed chylomicron remnants are specifically sensitive to a modification, which induces an avid interaction with the Kupffer cells. The recognition site for (modified) chylomicron remnants on Kupffer cells might function as a protection system against the occurrence of these potential atherogenic chylomicron‐remnant particles in the blood.
The carbohydrate chains of the β subunit of human chorionic gonadotropin produced by the choriocarcinoma cell line BeWo
Authors: HÄRRD Karl , DAMM Jan B. L. , SPRUIJT Mark P. N. , BERGWERFF Aldert A. , KAMERLING Johannis P. , VAN DEDEM Gijs W. K. , VLIEGENTHART Johannes F. G.
Published: March 2005

The N‐linked carbohydrate chains of the β subunit of human chorionic gonadotropin (hCG‐β) isolated from the culture fluid of the choriocarcinoma cell line BeWo were released enzymatically by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F. Subsequently, the O‐linked oligosaccharides were split off from the N‐deglycosylated protein by mild alkaline borohydride treatment. The carbohydrate chains were purified in their intact sialylated forms by FPLC anion‐exchange chromatography on Mono Q, HPLC on Lichrosorb‐NH2, and high‐pH anion‐exchange chromatography on CarboPac PA1.

1H‐NMR spectroscopic analysis of the major fractions demonstrates the occurrence of the following sialylated diantennary and triantennary N‐linked oligosaccharides. Residues not written in bold letters are variably present.

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The incidence of triantennary carbohydrate chains is much higher than in normal urinary hCG‐β (26% vs 2%). The same holds for the α1–6‐fucosylation of the asparagine‐bound GlcNAc (95% vs 42%). The presence of a bisecting GlcNAc and the occurrence of α2–6‐linked Neu5Ac in the most abundant N‐glycans, are new features for hCG‐β.

The major O‐linked carbohydrate chains identified are the tetrasaccharide Neu5Acα2–3Galβ1‐3(Neu5Acα2–6)GalNAc‐ol and the hexasaccharide Neu5Acα2–3Galβ1–4GlcNAcβ1–6(Neu5‐Acα2–3Galβ1–3)GalNAc‐ol, both also found in normal urinary hCG. In addition, two novel O‐glycans were characterized:

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Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens
Authors: GERWIG Gerrit J. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. , MORAG Ely , LAMED Raphael , BAYER Edward A.
Published: March 2005

The multiple cellulase‐containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O‐linked mainly to a 230‐kDa subunit. The oligosaccharide chains were liberated by alkaline‐borohydride treatment and fractionated as oligosaccharide alditols via gel‐permeation chromatography and HPLC. The fractions were investigated by one‐ and two‐dimensional (correlation, homonuclear Hartmann‐Hahn, rotating‐frame nuclear Overhauser enhancement) 500‐MHz 1H‐NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast‐atom‐bombardment mass spectrometry. The following carbohydrate structures could be established:

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and

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The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B. cellulosolvens and of Clostridium thermocellum [Gerwig, G. J., Kamerling, J. P., Vliegenthart, J. F. G., Morag (Morgenstern), E., Lamed, R. & Bayer, E. A. (1991) Eur. J. Biochem. 196, 115–122], having 3, 5 and 6 as common elements.

The furanose form of a terminal α‐d‐galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome‐like entity of B. cellulosolvens.
13C‐NMR study of autotrophic CO2 fixation pathways in the sulfur‐reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus
Authors: STRAUSS Gerhard , EISENREICH Wolfgang , BACHER Adelbert , FUCHS Georg
Published: March 2005

The unresolved autotrophic CO2 fixation pathways in the sulfur‐reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus have been investigated. Autotrophically growing cultures were labelled with [1,4‐13C1]succinate, and the 13C pattern in cell constituents was determined by 1H‐ and 13C‐NMR spectroscopy of purified amino acids and other cell constituents.

In both organisms succinate contributed to <10% of cell carbon, the major part of carbon originated from CO2. All cell constituents became 13C‐labelled, but different patterns were observed in the two organisms. This proves that two different cyclic CO2 fixation pathways are operating in autotrophic carbon assimilation in both of which succinate is an intermediate.

The 13C‐labelling pattern in T. neutrophilus is consistent with the operation of a reductive citric acid cycle and rules out any other known autotrophic CO2 fixation pathway. Surprisingly, the proffered [1,4‐13C1]succinate was partially converted to double‐labelled [3,4‐13C2]glutamate, but not to double‐labelled aspartate. These findings suggest that the conversion of citrate to 2‐oxoglutarate is readily reversible under the growth conditions used, and a reversible citrate cleavage reaction is proposed.

The 13C‐labelling pattern in C. aurantiacus disagrees with any of the established CO2 fixation pathways; it therefore demands a novel autotrophic CO2 fixation cycle in which 3‐hydroxypropionate and succinate are likely intermediates. The bacterium excreted substantial amounts of 3‐hydroxypropionate (5 mM) and succinate (0.5 mM) at the end of autotrophic growth. Autotrophically grown Chloroflexus cells contained acetyl‐CoA carboxylase and propionyl‐CoA carboxylase activity. These enzymes are proposed to be the main CO2‐fixing enzymes resulting in malonyl‐CoA and methylmalonyl‐CoA formation; from these carboxylation products 3‐hydroxypropionate and succinate, respectively, can be formed.
Molecular cloning and nucleotide sequence of the gene encoding a H202‐forming NADH oxidase from the extreme thermophilic Thermus thermophilus HB8 and its expression in Escherichia coli
Authors: PARK Ho‐Jin , KREUTZER Roland , REISER Christian O. A. , SPRINZL Mathias
Published: March 2005

The sequence of the 32 N‐terminal amino acids of the NADH oxidase from the extreme thermophile, Thermus thermophilus HB8, was used to synthesize oligonucleotides to probe for the respective gene in a genomic library of T. thermophilus HB8. The gene encoding the NADH oxidase, designated nox, was cloned, its nucleotide sequence was determined and found to be colinear with the N‐terminal sequence of the enzyme. The molecular mass of 26835 Da, as deduced from the nox gene, agrees with that of the purified NADH oxidase from T. thermophilus HB8 (25000 Da), as estimated by polyacrylamide gel electrophoresis under denaturing conditions. The nox gene was overexpressed in Escherichia coli and a protocol for the rapid purification of the enzyme was developed. The E. coli‐borne T. thermophilus HB8 NADH oxidase has properties identical to those of the authentic T. thermophilus HB8 enzyme and possesses a high thermal stability.
The primary structure of the antenna polypeptides of Ectothiorhodospira halochloris and Ectothiorhodospira halophila
Authors: WAGNER‐HUBER Regula , BRUNISHOLZ René A. , BISSIG Iwan , FRANK Gerhard , SUTER Franz , ZUBER Herbert
Published: March 2005

Antenna polypeptides from two species of the family Ectothiorhodospiraceae have been investigated. By means of gel filtration and subsequent high‐performance liquid chromatography, at least five polypeptides were isolated from each of Ectothiorhodospira halochloris and Ectothiorhodospira halophila. The majority of their primary structures was identified by Edman degradation. Comparison of these polypeptide sequences with the known primary structures of antenna polypeptides from various purple non‐sulfur bacteria revealed interesting new aspects with regard to the structure of the core‐peripheral antenna system. E. halochloris and E. halophila contain two pairs of α‐ and β‐polypeptides each with typical primary structure elements of core complexes, indicating a modified antenna complex organization.
Structure of the genes encoding the rod‐core linker polypeptides of Mastigocladus laminusus phycobilisomes and functional aspects of the phycobiliprotein/linker‐polypeptide interactions
Authors: GLAUSER Manuel , STIREWALT Veronica L. , BRYANT Donald A. , SIDLER Walter , ZUBER Herbert
Published: March 2005

The 3′ portion of the cpc operon in Mastigocladus laminom encloses the genes 5′‐cpcF‐cpcGI‐ cpcG2‐cpcG3 3′. The three cpcG genes encode different phycocyanin‐associated rod‐core linker poly‐ peptides of the phycobilisomes with predicted 279, 247 and 254 amino acids in length. The gene products CpcG show a high similarity at their N‐terminal domains (190 amino acids) and an overall identity of 47–53% to one another. Each of the three CpcG polypeptides is highly related to one of the four CpcG gene products of Anabaena sp. PCC 7120 (66–81% identity). It is suggested that these pairs of rod‐core linker polypeptides mediate the same specific type of phycocyanin → allophycocyanin interaction in the similar phycobilisomes of M. laminosus and Anabaena sp. PCC 7120. The similarity of the CpcG1, CpcG2 and CpcG3 polypeptides to the single CpcG rod‐core linker polypeptide of Synechococcus sp. PCC 7002 (36–41% identity) is lower. The rod‐core linker polypeptides are more distantly related to the rod linker polypeptides associated with phycocyanin or phycoerythrin. However, six conserved domains were identified within the N‐terminal 190 amino acids of these linker proteins, which bear similar amino acid sequences, including highly conserved basic amino acids. A similar amino acid sequence but with conserved acidic amino acids can be found in the β subunits of phycocyanin, phycoerythrin and phycoerythracyanin, which is protruding into the central cavity of the phycobiliprotein hexamers. It is suggested that these domains are sites of phycobiliprotein‐hexamer/rod and rodcore linker interactions.
Uroporphyrinogen decarboxylase in Saccharomyces cerevisiae
Authors: GAREY James R. , LABBE‐BOIS Rosine , CHELSTOWSKA Anna , RYTKA Joanna , HARRISON Lyle , KUSHNER James , LABBE Pierre
Published: March 2005

The HEM12 gene from Saccharomyces cerevisiae encodes uroporphyrinogen decarboxylase which catalyzes the sequential decarboxylation of the four acetyl side chains of uroporphyrinogen to yield coproporphyrinogen, an intermediate in protoheme biosynthesis. The gene was isolated by functional complementation of a hem12 mutant. Sequencing revealed that the HEM12 gene encodes a protein of 362 amino acids with a calculated molecular mass of 41348 Da. The amino acid sequence shares 50% identity with human and rat uroporphyrinogen decarboxylase and shows 40% identity with the N‐terminus of an open reading frame described in Synechococcus sp. We determined the sequence of two hem12 mutations which lead to a totally inactive enzyme. They correspond to the amino acid changes Gly33 → Asp and Gly300 → Asp, located in two evolutionarily conserved regions. Each of these substitutions impairs binding of substrates without affecting the overall conformation of the protein. These results argue that a single active center exists in uroporphyrinogen decarboxylase.
Structure of the differentially expressed mouse U3A gene
Authors: MAZAN Sylvie , GULLI Marie‐Pierre , JOSEPH Nicole , BACHELLERIE Jean‐Pierre
Published: March 2005

Two markedly different forms of U3 RNA are present in mouse, the relative abundance of which largely depends upon the tissues. In all cases studied so far, the more abundant form is U3B, encoded by four previously characterized genes. We report here the isolation and analysis of the unique gene encoding the U3A variant, which completes the characterization of the mouse U3 multigene family. Comparisons with rat U3 genes indicate that the diversification of the A and B forms has predated the mouse/rat separation. The two forms of U3 RNA are submitted to similar, but not identical, primary and secondary structure constraints. As for the sequences flanking the RNA coding region, similar observations emerge for both types of genes: for each type, the 5′ flanks are strongly conserved between mouse and rat, over at least the proximal 500 bp, whereas only about 30 bp of proximal 3′ flanks are preserved, which include a signal for the formation of vertebrate U small nRNA 3′ end. By constrast the 5′ flanks of the two types of genes diverge extensively from each other, either in mouse or in rat, and could be involved in the differential expression of the two forms. Even over the few conserved motifs thought to be involved in the basic transcriptional control of vertebrate U small nRNA genes, the A and B forms of U3 genes exhibit specific differences maintained in the two rodent species.
An investigation of large inhibitors binding to phosphoglycerate kinase and their effect on anion activation
Authors: JOAO Heidi C. , WILLIAMS Robert J. P. , LITTLECHILD Jennifer A. , NAGASUMA R. , WATSON Herman C.
Published: March 2005

This study extends, to a series of larger anions, our earlier investigation of the interaction of the trypanocidal drug suramin and other small negatively charged molecules with yeast phosphoglycerate kinase. 1H‐NMR structural studies of phosphoglycerate kinase in the presence of varying concentrations of these large molecules (designed to mimic, at one end, the anionic charge distribution in the substrate 3‐phosphoglycerate, while possibly being able to interact across the cleft of the enzyme) including inositol 1,4,5‐triphosphate, 4‐amino‐6‐trichloroethenyl‐l,3‐benzenedisulphonamide, gallic acid and sulphasalazine are described. The anion activation and/or inhibition of the enzyme by these molecules are also reported.

Evidence that binding to the general anion site in the ‘basic patch’ region of the protein may be responsible for either the activating or inhibiting effects, while binding at the hydrophobic (catalytic) site leads to inhibition only is presented. A reaction scheme which explains these observations is given.