bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification, Characterization and Biological Evaluation of Recombinant Leech‐Derived Tryptase Inhibitor (rLDTI) Expressed at High Level in the Yeast Saccharomyces Cerevisiae
Authors: Pohlig Gabriele , Fendrich Gabriele , Knecht Rene , Eder Beate , Piechottka Gerd , Sommerhoff Christian P. , Heim Jutta
Published: July 2004

An efficient expressiordpurification procedure has been developed which allows the production of pure, biologically active recombinant leech‐derived tryptase inhibitor (rLDTI), originally found in the leech Hirudo medicinalis. The gene for LDTI was generated synthetically from three overlapping oligonu‐cleotides by PCR synthesis. LDTI was expressed in the yeast Saccharomyces cerevisiae under the control of the copper‐inducible CUPI promoter and fused to the invertase signal sequence (SUCZ). The entire expression cassette was inserted into the yeast high‐copy vector pDP34. Appropriate host strains transformed with the expression plasmid secreted rLDTI into the medium upon copper addition. Proteinchemi‐cal analysis of the secreted rLDTI revealed exclusively inhibitor with the correct N‐terminal sequence. Up to 60% of the rLDTI, however, appeared to be modified by glycosylation and the unglycosylated material showed heterogeneity at the C‐terminus. Besides full‐length rLDTI, truncated rLDTI species lacking either the terminal Asn46 or in addition the penultimate Leu45 were isolated. The C‐terminally truncated variants were eliminated using a S. cerevisiae host strain disrupted in the structural genes of carboxypeptidases yscY and yscα, thus identifying these proteases as being responsible for the degradation of rLDTI.

Mature rLDTI was purified in high yields from the culture supernatant of the carboxypeptidase‐deficient yeast strain by cation‐exchange chromatography and reverse‐phase HPLC. The recombinant protein is at least 98% pure, based on HPLC and capillary electrophoresis, and is fully biologically active. Structural identity with the authentic leech protein was confirmed by sequence analysis and molecular‐mass determination. The purified protein was tested for its ability to inhibit tryptase and trypsin in vitro and to interfere with the tryptase‐induced proliferation of human fibroblasts and keratinocytes. Recombinant LDTI appears to be as potent as the authentic leech protein, exhibiting Ki‐values of ≈1.5 nM and ≈1.6 nM against human tryptase and bovine trypsin, respectively. The tryptase‐induced proliferation of human fibroblasts and keratinocytes was inhibited with half‐maximum values of ≈0.1 nM and ≈1 nM, respectively. The availability of the recombinant material will allow evaluation of the concept of tryptase inhibition in various disease models and to test the therapeutic potential of LDTI in mast‐cell‐related disorders.
Differential Requirement of the Yeast Sugar Kinases for Sugar Sensing in Establishing the Catabolite‐Repressed State
Authors: De Winde Johannes H. , Crauwels Marion , Hohmann Stefan , Thevelein Johan M. , Winderickx Joris
Published: July 2004

Addition of rapidly fermentable sugars to cells of the yeast Saccharomyces cerevisiae grown on non‐fermentable carbon sources causes a variety of shortterm and longterm regulatory effects, leading to an adaptation to fermentative metabolism. One important feature of this metabolic switch is the occurrence of extensive transcriptional repression of a large group of genes. We have investigated transcriptional regulation of the SUC2 gene encoding repressible invertase, and of HXKI, HXK2 and GLKI encoding the three known yeast hexose kinases during transition from derepressed to repressed growth conditions. Comparing yeast strains that express various combinations of the hexose kinase genes, we have determined the importance of each of these kinases for establishing the catabolite‐repressed state. We show that catabolite repression involves two distinct mechanisms. An initial rapid response is mediated through any kinase, including Glk1, which is able to phosphorylate the available sugar. In contrast, long‐term repression specifically requires Hxk2 on glucose and either Hxkl or Hxk2 on fructose. Both HXKl and GLKI are repressed upon addition of glucose or fructose. However, fructose repression of HXKl is only transient, which is in line with its preference for fructose as substrate and its requirement for longterm fructose repression. In addition, expression of HXKI and GLKI is regulated through CAMP‐dependent protein kinase. These results indicate that sugar sensing and establishment of catabolite repression are controlled by an interregulatory network, involving all three yeast sugar kinases and the Ras‐CAMP pathway.
Identification of the Multidrug‐Resistance Protein (MRP) as the Glutathione‐S‐Conjugate Export Pump of Erythrocytes
Authors: Pulaski Lukasz , Jedlitschky Gabriele , Leier Inka , Buchholz Ulrike , Keppler Dietrich
Published: July 2004

The identification of the multidrug resistance protein (MRP) as a conjugate export pump in several cell types suggested its involvement in the long‐known glutathione‐S‐conjugate transport across erythrocyte membranes. We investigated the ATP‐dependent transport of glutathione S‐conjugates in human erythrocyte and erythroleukemia cell membrane vesicles using the endogenous conjugate leukotriene C4 (LTC4), known to be a high‐affinity substrate for MRP, in addition to S‐(2,4‐dinitrophenyl)glutathione. The kinetic parameters, including the Km, value for LTC4 of 118 ± 5 nM and the inhibition constants for transport of both substrates for the quinoline‐based inhibitor MK 571, were similar to those obtained for transport mediated by recombinant MRP. Direct photoaffinity labeling of human erythrocyte membranes with [3H]LTC4 revealed a major binding protein of about 190 kDa which was immunoprecipitated by an anti‐MRP serum. The radiolabeling of this protein was specifically suppressed by the transport inhibitor MK 571. Several additional anti‐MRP sera detected the protein of about 190 kDa in human erythrocyte and erythroleukemia cell membranes. These data identify for the first time the glutathione‐S‐conjugate transporting protein in erythrocyte membranes.
Expression of the Human Cytochrome c1 Gene is Controlled through Multiple Sp1‐Binding Sites and an Initiator Region
Authors: Li Ronggui , Luciakova Katarina , Nelson B. Dean
Published: July 2004

It is widely accepted that nuclear genes that encode proteins of the oxidative‐phosphorylation system are regulated by nuclear factors believed to be specific for such genes. In the present study we show that the promoter for the human cytochrome c1 gene is an exception, in that it involves only conserved Spl core elements and an initiator region. Maximal promoter activity within a 1.4‐kb 5′ flanking region of the cytochrome c1 gene is contained in a fragment (‐72 to +18) that lacks TATA and CCA AT elements. The transcriptional start site was mapped to an initiator region by RNase protection of mRNA from human HepG2 cells, and by primer extension of in vitro‐generated transcripts, to a sequence that is highly similar to the dihydrofolate reductase family of initiators. Deletion of this region (+1 to +18) severely impairs transcription initiation. Sp1 core elements centered at nucleotides‐21 and‐39 define the activation domain of the proximal promoter. Only the‐39 element is protected from DNase I in the presence of crude nuclear extracts. However, transfection, gel‐mobility‐shift, supershift and in vitro‐transcription experiments show that the‐21 element binds Sp1 protein and contributes to transcription activation. No other functional oxidative‐phosphorylation‐specific response elements have been identified. These data implicate Sp1 as a single activating factor for an oxidative‐phosphorylation gene.
Overproduction of a Foreign Membrane Protein in Escherichia Coli Stimulates and Depends on Phospholipid Synthesis
Authors: Nieboer Maarten , Vis Albert‐Jon , Witholt Bernard
Published: July 2004

When the Pseudomonas oleovorans alk system, consisting of the alkBFGHJKL and alkST genes, is expressed in Escherichia coli W3110, significant changes in phospholipid metabolism of the host are observed. A major role seems to be played by the cytoplasmic membrane protein alkane hydroxylase (AlkB), which is synthesized as up to 10–15% of the total protein in this strain [Nieboer, M., Kingma, J. & Witholt, B. (1993) The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110[pGEc47] affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subfraction, Mol. Microbiol. 8, 1039–1051]. In the present paper, we have studied the link between synthesis of the membrane protein and the synthesis of phospholipids and fatty acids by examining the kinetics of these processes.

Using [35S]methionine labeling, it was shown that induction of AlkB was maximal within 30–60 min after addition of inducer, when up to 15% of all newly synthesized protein is AlkB. Phospholipid synthesis was followed by measuring the incorporation of 14C‐labeled acetate and 33P‐labeled phosphoric acid into phospholipids. Despite a negative effect of the inducer on the growth rate of W3110[pGEc47], net phospholipid synthesis was significantly enhanced as a result of the expression of alkB. Synthesis of all three major phospholipids were stimulated to comparable extents by the induction of alkB. Induction did not increase 33P incorporation into lipids in the control recombinant alk+ strain which lacked alkB.

Simultaneous with AlkB synthesis, the conversion of unsaturated 9‐hexadecenoic acid (C,6:1) into 9,10‐methylene hexadecanoic acid (C17:ocyc) was reduced in the alk+ recombinant. Overall, these data show that the production of a foreign membrane protein in E. coli can engender a response of the phospholipid‐synthesizing system of the host. In the absence of such a response, induction of the alk system would be much more toxic to the cells. Apparently, the increased phospholipid synthesis plays an important role in enabling the AlkB overproducing strain to grow.
Cloning of the Gene Encoding the Antibacterial Peptide Drosocin Involved in Drosophila Immunity
Authors: Charlet Maurice , Lagueux Marie , Reichhart Jean‐Marc , Hoffmann Danièle , Braun Anne , Meister Marie
Published: July 2004

A potent inducible antibacterial peptide carrying an O‐glycosylated substitution has recently been isolated from Drosophila[Bulet, P., Dimarcq, J. L., Hetru, C., Lagueux, M., Charlet, M., Hegy, G., Van Dorsselaer, A. and Hoffmann, J. A. (1993)J. Biol. Chem. 268, 14893–14897]. Here we report cloning studies that show that Drosophila contains a single, intronless gene, located at position 51C1–6, which encodes the precursor protein from which drosocin is processed. The upstream and the downstream sequences of the drosocin gene contain putative cis‐regulatory elements similar to mammalian regulatory motifs, namely three κB‐related decameric sequences. The drosocin gene is silent in naive animals, and is strongly induced with acute phase kinetics after immune challenge in larvae and in adults. We have established several transgenic fly lines in which reporter genes were placed under the control of various drosocin promoter sequences. Our results indicate that 2.5 kb of upstream sequences confer inducibility and tissue specificity to the transgene, but that the level of its expression in the fat body after immune challenge is low. Addition of genomic regions downstream of the drosocin transcribed sequences results in increased transcription levels, which are similar for the fusion and the resident drosocin genes upon infection. Analysis of transgenic fly lines showed that the drosocin reporter gene is constitutively expressed in the oviducts of egg‐laying females.
A Nuclear Protein factor Binds Specifically to the 3′‐Regulatory Module of the Long‐Interspersed‐Nuclear‐Element‐Like Dictyostelium Repetitive Element
Authors: Geier Anja , Horn Jens , Dingermann Theodor , Winckler Thomas
Published: July 2004

The Dictyostelium repetitive element DRE integrates in a position‐specific manner upstream of tRNA genes in the Dictyostelium discoideum genome. DRE has structural similarities to the group of long interspersed nuclear elements, whose replication mechanism is poorly understood. The C‐module at the 3′ end of DRE encodes a regulatory cis ‐acting sequence that contains an RNA polymerase II promoter. This promoter directs the synthesis of RNAs that are thought to play a critical role in DRE transposition. In this study, we describe the identification of a nuclear protein factor that binds to the C‐module in a sequence‐specific manner. The C‐module‐binding factor (CMBF) recognizes three DNA sequence motifs that contain homopolymeric (dT) stretches of variable lengths, but does not bind to a standard RNA polymerase II promoter from D. discoideum. Analysis of highly CMBF‐enriched fractions and glycerol gradient sedimentation of CMBF suggest that the factor exists as a monomeric 115‐kDa protein. Possible roles of CMBF in DRE transposition are discussed.
NMR Studies of Cooperativity in the Tetrahaem Cytochrome c3 from Desulfovibrio vulgaris
Authors: Turner David L. , Salgueiro Carlos A. , Catarino Teresa , Legall Jean , Xavier António V.
Published: July 2004

The thermodynamic properties of the Desulfovibrio vulgaris (Hildenborough) tetrahaem cytochrome c3 (Dvc3) are rationalised by a model which involves both homotropic (e−/e−) and heterotropic (e−/H+) cooperativity. The paramagnetic shifts of a methyl group from each haem of the DVc3 have been determined in each stage of oxidation at several pH values by means of two‐dimensional exchange NMR. The thermodynamic parameters are obtained by fitting the model to the NMR data and to redox titrations followed by visible spectroscopy. They show significant positive cooperativity between two of the haems whereas the remaining interactions appear to be largely electrostatic in origin. These parameters imply that the protein undergoes a proton‐assisted two‐electron transfer which can be used for energy transduction. Comparison with the crystal structure together with measurement of the kinetics of proton exchange suggest that the pH dependence is mediated by a charged residue(s) readily acessible to the solvent and close to haem I.
The A‐Domain of Integrin α2 Binds Specifically to a Range of Collagens but is not a General Receptor for the Collagenous Motif
Authors: Tuckwell Danny S. , Reid Kenneth B. M. , Barnes Michael J. , Humphries Martin J.
Published: July 2004

Integrin α2β1 is a major cellular receptor for collagens, but the molecular basis of its function is unknown. The α2 subunit contains a von Willebrand factor A‐domain (I‐domain) in its N‐terminal region, and it has been demonstrated recently that this domain binds specifically to collagen I. This interaction requires divalent cations (e.g., Mg2+) and native collagen conformation, as does binding of the parent integrin to collagen. The α2 A‐domain therefore has a number of functional similarities to the parent integrin, α2β1. However, while sequence specificity has been demonstrated for the parent integrin, no such observations have been made for the A‐domain. In particular, it is not known whether the A‐domain is responsible for sequence‐specific recognition of collagens or whether it binds to the generic collagenous motif. To investigate the ligand specificity of the α2 A‐domain, its binding to a range of collagenous ligands has been studied, with cation dependence, collagen triple‐helicity, and inhibition by function‐blocking antibodies as criteria for specificity. Binding of the parent integrin was examined for comparison. The α2 A‐domain was found to bind specifically to collagens I, II, IV and XI. The complement component C1q has a collagenous domain but this was unable to support specific binding of α2 A‐domain or α2β1. Furthermore, synthetic triple‐helical collagenous peptides failed to act as specific ligands. In conclusion, the α2 A‐domain binds specifically to a range of extracellular matrix collagens, but it is not a receptor for all collagenous triple helices. By inference, these findings indicate the existence of an integrin‐specific sequence motif within collagenous ligands recognised by the α2 A‐domain.
An Immune Response to Ice Crystals in North Atlantic Fishes
Authors: Verdier Jean‐Michel , Ewart K. Vanya , Griffith Marilyn , Hew Choy L.
Published: July 2004

In mammals, the presence of crystals composed of small organic molecules, including urate and related compounds, has been shown to trigger an inflammatory response and the subsequent production of specific immunoglobulins (Ig's). Many fishes that are exposed to ice crystals in cold temperate and polar oceans may harbour ice crystals internally. Here, we report evidence for a specific immune response to ice crystals in cold‐ocean marine fishes. Using ice nucleation activity as an assay, anti‐ice Ig's were detected in the sera of the cold‐ocean marine fish species, ocean pout (Macrozoarces americanus) and Atlantic herring (Clupea harengus harengus), but not in the sera of species that are not exposed to ice. Purified Ig's isolated from ocean pout serum using two different protocols showed ice nucleation activity, thus demonstrating the presence of ice binding specificity among these Ig's.
Disulphide Bonding in a Stylar Self‐Incompatibility Ribonuclease of Nicotiana Alata
Authors: Oxley David , Bacic Antony
Published: July 2004

Many flowering plants have developed a self‐incompatibility mechanism, which is controlled by a single polyallelic locus (the S‐locus), to prevent inbreeding. The products of the S‐locus in the styles of solanaceous plants are an allelic series of glycoproteins with RNase activity [McClure, B. A., Haring, V., Ebert, P. R., Anderson, M. A., Simpson, R. J., Sakiyama, F. & Clarke, A. E. (1989) Nature 342, 955–957]. These S‐RNases show some amino‐acid‐sequence similarity with two fungal RNases (T2 and Rh), including the presence of two active‐site His residues, which suggests a common three‐dimensional structure. Disulphide bonding is important in the maintenance of the three‐dimensional structure of the fungal RNases [Kurihara, H., Mitsui, Y., Ohgi, K., Irie, M., Mizuno, H. & Nakamura, T. (1992) FEBS Lett. 306, 189–192] and the S‐RNases [Tsai, D. S., Lee, H.‐S., Post, L. C., Kreiling, K. M. & Kao, T.‐H. (1992) Sex. Plant Reprod. 5, 256–263]. We have used the S2‐allele RNase of Nicotiana alata, which has nine Cys residues, to establish the pattern of disulphide bonding. The disulphide bonds Cys16–Cys21, Cys45–Cys94, Cys153–Cys182 and Cys165–Cys176 are consistent with the S2‐RNase having a similar three‐dimensional structure to RNase Rh. A free Cys residue (Cys95) adjacent to Cys45‐Cys94 promotes a rapid specific disulphide migration when the protein is exposed to denaturing conditions.
Computational Method to Predict Mitochondrially Imported Proteins and their Targeting Sequences
Authors: Claros Manuel G. , Vincens Pierre
Published: July 2004

Most of the proteins that are used in mitochondria are imported through the double membrane of the organelle. The information that guides the protein to mitochondria is contained in its sequence and structure, although no direct evidence can be obtained. In this article, discriminant analysis has been performed with 47 parameters and a large set of mitochondrial proteins extracted from the SwissProt database. A computational method that facilitates the analysis and objective prediction of mitochondrially imported proteins has been developed. If only the amino acid sequence is considered, 75–97% of the mitochondrial proteins studied have been predicted to be imported into mitochondria. Moreover, the existence of mitochondrial‐targeting sequences is predicted in 76–94% of the analyzed mitochondrial precursor proteins. As a practical application, the number of unknown yeast open reading frames that might be mitochondrial proteins has been predicted, which revealed that many of them are clustered.
The Mechanism of Porcine Pancreatic α‐Amylase
Authors: Alkazaz Modhafar , Desseaux Véronique , Marchis‐Mouren Guy , Payan Françoise , Forest Eric , Santimone Marius
Published: July 2004

Kinetics of inhibition of the two porcine pancreatic α‐amylase components (PPA I and PPA II) by acarbose were performed using reduced DP18‐maltodextrin and amylose as substrates. Similar Line‐weaver‐Burk primary plots were obtained. Two mixed non‐competitive models are proposed. X‐ray crystallographic data [Qian, M., Buisson, G., Duée, E., Haser, R. & Payan, F. (1994)Biochemistry 33, 6284–6294] are in support of the mixed non‐competitive inhibition model which involves abortive complexes. Secondary plots are different; inhibition of reduced DP18‐maltodextrin hydrolysis gives straight‐lines plots while amylose gives parabolic curves. These results, confirmed by Dixon‐plot analyses, allow us to postulate that, in inhibition of reduced DP18‐maltodextrin hydrolysis, one molecule of acarbose is bound amylase molecule. In contrast, using amylose as a substrate, two molecules of acarbose are bound. These kinetically determined binding sites might correspond to surface sites found by X‐ray crystallography [Qian, M., Haser, R. & Payan, F. (1995)Protein Sci. 4, 747–7551; the glucose site close to the active site and the maltose site, 2 nm away.

In conclusion, no significant difference between PPA I and PPA II has been observed, either from molecular mass or from kinetic behaviours; this suggests multiple forms of the enzyme. A general mechanism of PPA action is proposed; in addition to the active site, long‐chain substrate hydrolysis requires the glucose‐binding site and the maltose‐binding site, while only one site is necessary for the hydrolysis of short chain substrate.
Oxidation of Tienilic Acid by Human Yeast‐Expressed Cytochromes P‐450 2C8, 2C9, 2C18 and 2C19
Authors: Jean Pascale , Lopez‐Garcia Pilar , Dansette Patrick , Mansuy Daniel , Goldstein Joyce L.
Published: July 2004

Oxidation of tienilic acid by human cytochromes P‐450 (CYP) 2C9, 2C18, 2C8 and 2C19 was studied using recombinant enzymes expressed in yeast. CYP 2C9 was the best catalyst for 5‐hydroxylation of tienilic acid (Km= 5 ±1 μM, kcat= 1.7 ± 0.2 min−1), 30‐fold more potent in terms of kcat/Km than CYP 2C18 (Km= 150 ± 15 μM, kcat= 1.8 ± 0.2 min−1 and 300‐fold more potent than CYP 2C8 (Km= 145 ± 15 μM, kcat= 0.2 ± 0.1 min−1). CYP 2C19 was unable to catalyze this hydroxylation under our experimental conditions. During this study, a marked effect of the ionic strength on the activities (hydroxylations of tienilic acid and tolbutamide) of these cytochromes P‐450 expressed in the yeast strain 334 was observed. The effect was particularly great in the case of CYP 2C18, with a tenfold decrease of activity upon increasing ionic strength from 0.02 to 0.1. Specific‐covalent binding of tienilic acid metabolites to cytochrome P‐450 (incubations in the presence of 5 mM glutathione) was markedly higher upon tienilic acid oxidation by CYP 2C9 than by CYP 2C18 and CYP 2C8. Mechanism‐based inactivation of cytochrome P‐450 during tienilic acid oxidation was observed in the case of CYP 2C9 but was not detectable with CYP 2C18 and CYP 2C8. Tienilic acid thus appears to be a mechanism‐based inhibitor specific for CYP 2C9 in human liver. Experiments performed with human liver microsomes confirmed that tienilic acid 5‐hydroxylase underwent a time‐dependent inactivation (apparent t½= 10 ± 5 min) during 5‐hydroxylation of tienilic acid.
Redox Control of RNA Synthesis in Potato Mitochondria
Authors: Wilson S. Brian , Davidson Gary S. , Thomson Laura M. , Pearson Colin K.
Published: July 2004

This study shows that the incorporation of radiolabelled UTP into RNA in Percoll‐gadient‐purified potato mitochondria is regulated by the redox state of the mitochondrial electron transport chain. An early indication that there might be a redox effect on RNA synthesis was a decrease in UTP incorporation in incubates containing an oxidisable substrate, such as succinate or malate. Subsequent use of a variety of electron transport inhibitors acting at different points in the electron transport chain established that the redox state of the Rieske iron‐sulphur protein was the major determinant of UTP incorporation. Inhibitors acting on the substrate side of the Rieske iron‐sulphur protein, and causing oxidation of components on the oxygen side of their site of action, increased UTP incorporation into RNA. These included antimycin A, myxothiazole, and undecylhydroxydioxobenzothiazole at 500 nM. Inhibitors acting on the oxygen side of the Rieske iron‐sulphur protein, and causing a reduction of components on the substrate side of the block, decreased UTP incorporation. These inhibitors were undecylhydroxydioxobenzothiazole at 25 nM and KCN. When phenazine methosulphate was present as an auto‐oxidisable electron sink the effect of KCN was diminished.

The conclusion from the inhibitor experiments that the redox state of the Rieske iron‐suphur protein was important was supported when RNA synthesis was measured at a range of redox potentials. This gave a measured redox potential for the control of UTP incorporation into RNA of +270 mV and the slope of the curve indicated an n= 1 carrier. This value is close to the reported value of the Rieske iron‐suphur protein.

UTP incorporation was decreased by some 50% in the presence of low concentrations of okadaic acid (5 nM), an inhibitor of protein phosphatases PP1 and PP2A, and α‐naphthyl acid phosphate, a broad‐spectrum phosphatase inhibitor, indicating that the redox effect on RNA synthesis may be mediated via protein phosphorylation. We did not, however, detect an expected increase in RNA synthesis when protein kinase inhibitors were used, so the involvement of protein phosphorylation in the redox regulation of RNA synthesis is as yet uncertain.
Expression and Characterization of Human Perlecan Domains I and II Synthesized by Baculovirus‐Infected Insect Cells
Authors: Groffen Alexander J. A. , Buskens Carin A. F. , Tryggvason Karl , Veerkamp Jacques H. , Monnens Leo A. H. , Van Den Heuvel Lambert P. W. J.
Published: July 2004

We present the in vitro expression and purification of N‐terminal fragments of human perlecan in insect cells. Three tailored fragments of human perlecan cDNA were introduced into the polyhedrin locus of baculovirus expression vectors (BEVs) encoding amino acids 1–196 (domain I), 1–404 (domain I + IIa) and 1–506 (domain I+Iab). The integrity of the BEVs was checked by DNA sequencing, polymerase chain reaction, restriction enzyme analysis and Southern blotting. Northern hybridization and metabolic labeling with [35S]methionine showed that expression of the perlecan‐(l–404)‐ and the ‐(1–506)‐peptide was successful, but in the case of the perlecan‐(1–196)‐peptide no recombinant protein was produced. Immunoblotting showed that both the (1–404)‐peptide and (1–506)‐peptide are recognized by 95J10, a monoclonal antibody that was previously raised against perlecan‐(24–404)‐peptide expressed in Escherichia coli. Gel permeation and anion‐exchange chromatography were applied to purify the recombinant proteins. Glycosaminoglycans were demonstrated to be present. Deglycosylation with chondroitinase ABC showed that the perlecan‐(1–404)‐peptide was glycosylated with chondroitin sulfate residues. Consistent with these results, glycosaminoglycans isolated from the perlecan‐(1–404)‐peptide were identified as chondroitin sulfate by agarose gel electrophoresis. Furthermore the perlecan‐(1–404)‐peptide showed affinity to immobilized basic fibroblast growth factor. The availability of baculovirus‐derived recombinant perlecan fragments will facilitate domain‐specific investigation of the structural and functional properties of perlecan in the future.
Separation and Characterization of the Metal‐Thiolate‐Cluster Domains of Recombinant Sea Urchin Metallothionein
Authors: Wang Yunjuan , Hess Daniel , Hunziker Peter E. , Kägi Jeremias H. R.
Published: July 2004

Partial metal depletion and 113Cd‐NMR studies have suggested that the recombinant Cd‐containing metallothionein of the sea urchin Strongylocentrutus purpurutus (Cd7‐MTA) binds its metal ions in a four‐metal (Cd4Cys11) and a three‐metal (Cd3Cys9) cluster associated with the N‐terminal and C‐terminal halves of the protein, respectively [Wang, Y., Mackay, E. A., Zerbe, O., Hess, D., Hunziker, P. E., Vasak, M. & Kägi, J. H. R. (1995)Biochemistry 34, 7460–7467]. This partitioning has now been confirmed by bisecting native Cd7‐MTA with subtilisin into products bearing only a single metalthiolate cluster. Their separation by reverse‐phase HPLC and on‐line electrospray mass spectrometry in combination with sequence analysis revealed selective cleavage of the protein into a set of N‐terminal polypeptides containing 37–39 residues with four Cd ions and a set of C‐terminal polypeptides containing 24 and 25 residues with three Cd ions. Thus, sea urchin MTA like its mammalian counterparts is made up of two separate cluster‐harboring domains. The fragmentation pattern indicated that the sites of cleavage are located in the peptide loop interspaced between the first two metal‐bound cysteine residues of the C‐terminal domain. Accordingly, with cleavage, one of the putative nine thiolate ligands of the three‐metal cluster was lost to the N‐terminal fragment. The coordinational consequences of this repartition were reflected in massive chiroptical changes accompanying the cleavage process. While the liberated N‐terminal domain retained the CD profile of the four‐metal cluster in the parent protein and thereby indicated preservation of its structure, the CD features attributable to the intact three‐metal cluster were largely lost on cleavage. The vanished features bear strong resemblance to the large biphasic ellipticity signal at 250 nm which dominates the CD spectrum of native Cd7‐MTA, and allow us thus to attribute this signal to excitonic coupling interactions of Cd‐thiolate chromophores in the three‐metal cluster.
Characterization of the murine plasminogen/urokinase‐type plasminogen‐activator system
Authors: Lijnen H. Roger , Van Hoef Berthe , Collen Désiré
Published: July 2004

The murine plasminogen/urokinase‐type plasminogen‐activator (u‐PA) system was studied using purified proteins, plasma and endothelioma cells. Recombinant murine u‐PA was obtained as a single‐chain molecule of 45 kDa which was converted to two‐chain u‐PA with plasmin by cleavage of the Lys159‐Ile1 60 peptide bond. Murine plasminogen, purified from plasma as a single‐chain protein of 95 kDa, was resistant to quantitative activation with murine recombinant two‐chain u‐PA: only 15% activation within 1 h at 37°C was obtained in mixtures of 1 μM plasminogen and 5 nM recombinant two‐chain u‐PA, whereas quantitative activation was observed in the autologous human system. Addition of 6‐aminohexanoic acid to native murine plasminogen resulted in quantitative activation within 1 h. In murine plasma in vitro, plasminogen was also resistant to quantitative activation with u‐PA (50% activation within 1 h at 37°C with 50 nM recombinant two‐chain u‐PA, whereas in the human system nearly quantitative activation was obtained). Murine plasma clots submerged in murine plasma were resistant to lysis with u‐PA; ≤2% clot lysis in 2 h was obtained with 80nM recombinant two‐chain u‐PA in the autologous murine system whereas 50% clot lysis in 2 h required only 15 nM recombinant two‐chain u‐PA in the autologous human system.

Saturable binding of murine recombinant two‐chain u‐PA was observed to murine endothelioma cells that are genetically deficient in u‐PA (u‐PA−/− End cells). Binding was characterized by a Kd of 5.5 nM and 800000 binding sites/cell. However, u‐PA−/− End cells did not significantly stimulate the activation rate of murine plasminogen by murine recombinant two‐chain u‐PA and did not enhance the plasmin‐mediated conversion rate of murine recombinant single‐chain u‐PA to its two‐chain derivative. Murine recombinant two‐chain u‐PA bound to murine endothelioma cells was quantitatively inhibited by murine plasminogen‐activator inhibitor‐1 (PAI‐1).

Thus, the interactions between murine plasminogen, u‐PA and PAI‐1 are qualitatively similar to those between their human counterparts. However, quantitative differences were observed both in the presence of cells and in plasma which may contribute to a reduced u‐PA‐mediated fibrinolytic activity in the murine systems.
Location of the Active Site and Proposed Catalytic Mechanism of Pterin‐4A‐Carbinolamine Dehydratase
Authors: Köster Sandra , Stier Gunter , Ficner Ralf , Hölzer Manuela , Curtius Hans‐Christoph , Suck Dietrich , Ghisla Sandro
Published: July 2004

Based on the recently solved three‐dimensional structure of pterin‐4a‐carbinolamine dehydratase from rat/human liver the involvement of the proposed active‐site residues Glu57, Asp60, His61, His62, Tyr69, His79, Arg87 and Asp88 was examined by site‐directed mutagenesis. Most of the mutants showed reduced activity, and only the Glu57→Ala mutant and the His61→Ala, His62→Ala double mutant were fully devoid of activity. The dissociation constants of quinonoid 6,6‐dimethyl‐7,8‐dihydropterin were significantly increased for binding to the Glu57→Ala, His61→Ala, His62→Ala single mutants and the His61→Ala, His62→Ala double mutant, confirming that His61 and His62 are essential for substrate binding and catalysis. The mechanism of dehydration is proposed to involve base catalysis at the N(5)‐H group of the substrate by His61.
Expression of the ADP/ATP Carrier and Expansion of the Mitochondrial (ATP + ADP) Pool Contribute to Postnatal Maturation of the Rat Heart
Authors: Schönfeld Peter , Schild Lorenz , Bohnensack Ralf
Published: July 2004

The role of the ADP/ATP carrier (AAC), a key protein of the mitochondrial ATP‐generating system, is not clear during postnatal rat heart development. To elucidate this role, the phosphorylating respiration (state 3), the activity and the content of AAC, the size of the exchangeable mitochondrial (ATP + ADP) pool and the control of AAC over respiration at state 3 were measured in mitochondria isolated from rat hearts at various postnatal ages.

There was a 5‐fold increase in the AAC activity from newborn to aged rat hearts, which was paralleled by a 1.5‐fold increase in state 3 respiration. At birth, the AAC and the F0F1‐ATP synthase exerted about 80% of the control over phosphorylating respiration (state 3: flux control coefficients 0.39 ± 0.04 and 0.38 ± 0.08). The strong increase in the AAC activity was partly caused by the doubling of the protein content. In addition, the turnover number of AAC increased by a factor of 2.5 due to the expansion of the (ATP + ADP) pool from 3.4 ± 0.9 to 10.6 ± 1.5 nmol · mg protein−1. The data strongly indicate that the increase in the AAC activity is an essential step in the postnatal maturation of rat heart mitochondria.
In vivo Phosphorylation of Actin in Physarum Polycephalum
Authors: De Corte Veerle , Gettemans Jan , Waelkens Etienne , Vandekerckhove Joël
Published: July 2004

Actin‐fragmin is a heterodimeric protein complex from Physarum polycephalum microplasmodia that is phosphorylated in vitro at residues Thr203 and Thr202 of the actin subunit by the endogenous actin‐fragmin kinase. Following phosphorylation, the F‐actin capping activity of the complex becomes Ca2+‐dependent, suggesting a fundamental regulatory role in controlling F‐actin growth [Gettemans, J., De Ville, Y., Waelkens E. and Vandekerckhove, J. (1995)J. Biol. Chem. 270, 2644–26511.

In this study we analysed actin phosphorylation in vivo. We demonstrate that the actin‐fragmin complex constitutes the only substrate of the actin‐fragmin kinase in plasmodia. Monomeric actin is not phosphorylated. Immunoprecipitation of actin‐fragmin reveals that approximately 405% of the actin sub‐unit of the complex is phosphorylated in vivo. However, using purified substrate and kinase, the complex can be quantitatively phosphorylated as judged by two‐dimensional gel electrophoresis. Through comparative phosphopeptide fingerprinting, we show that the phosphorylation sites in vivo are identical to those identified in vitro.

We additionally characterized a complex of actin and the NH2‐terminal half of fragmin (residues 1–168) that is also phosphorylated by the same kinase. In contrast to actin‐fragmin, phosphorylation of the complex between actin and residues 1–168 of fragmin is independent of Ca2+ because the second Ca2+‐dependent regulatory actin‐binding domain is missing.

By artificially varying the actin‐fragmin concentration or the actin‐fragmin kinase activity present in microplasmodia cytosolic extracts, we attempted to detect alternative protein substrates for the actin‐fragmin kinase. The fact that none could be identified suggests that the control and properties of actin‐fragmin phosphorylation observed in vitro may stand as a model for F‐actin growth control in Physarum cells.
Vanadate Triggers the Transition from Chromosome Condensation to Decondensation in a Mitotic Mutant (tsTM13)
Authors: Ajiro Kozo , Yasuda Hideyo , Tsuji Hideo
Published: July 2004

At the nonpermissive temperature (39°C), chromosomes remain condensed in a temperature‐sensitive cell mutant (tsTM13) arrested in the late stage of mitosis. Highly increased activity of histone H1 kinase, hyperphosphorylation of histone H1, and mitosis‐specific histone H3 phosphorylation are maintained, even in telophase. In the present study, the defect of chromosome decondensation in tsTM13 cells was found to be partially normalized by a tyrosine phosphatase inhibitor, vanadate, with induction of chromosome decondensation and the formation of multinucleated cells. In the presence of vanadate, the H1 kinase activity dropped to near normal levels and the amount of the inactive form of p34cdc2 protein phosphorylated at a tyrosine residue was increased. H1 and H3 were also extensively de‐ phosphorylated, the latter being tightly associated with chromosome decondensation. Serine/threonine‐protein phosphatase in late mitosis of the mutant works normally at 39°C. The results indicate that (a) the genetic defect in the mutant may be involved in the control mechanism of the p34cdc/H1 kinase activity in the late M phase rather than the phosphatase, (b) normalization of the defect of the mutant by vanadate results from inactivation of H1 kinase, and (c) late mitosis‐specific events (p34cdc2/H1 kinase inactivation, mitosis‐specific dephosphorylation of histone H1 and H3) are closely operating with chromosome decondensation.
Expression of DNA‐Dependent Protein Kinase Holoenzyme Upon Induction of Lymphocyte Differentiation and V(D)J Recombination
Authors: Grawunder Ulf , Finnie Nicholas , Jackson Stephen P. , Riwar Brigitte , Jessberger Rolf
Published: July 2004

Murine preB lymphocytes grow in tissue culture in the presence of stromal cells and interleukin 7 (IL‐7), and can be induced to differentiate to surface‐immunoglobulin‐positive B cells in vitro by withdrawal of IL‐7. Upon differentiation, proliferation ceases, and upregulation of Rag‐1 and Rag‐2 expression, and induction of V(D)J immunoglobulin‐gene rearrangements occur. DNA‐dependent protein kinase (DNA‐PK) is required for effective V(D)J recombination and repair of DNA double‐strand breaks. The holoenzyme comprises a catalytic subunit (DNA‐PKcs) and the Ku heterodimer (Ku70/Ku80). We have analyzed expression of Ku70, Ku80 and DNA‐PKcs upon induction of differentiation in preB cells derived from wild‐type, severe combined immunodeficiency (SCID) and Rag‐2−/− mice. Protein levels of Ku80 and Ku70 moderately decrease after induction in all three cell types. A distinct polypeptide that cross‐reacts with anti‐Ku Ig appears in the cytoplasm of wild‐type and Rag‐2−/− cells, but not of SCID cells. In mouse preB cells, Ku70 and Ku80 are present in the nuclei and cytoplasm before and after onset of differentiation. In vivo, Ku70 is predominantly expressed in V(D)J‐recombination‐active, early‐preB and CD4−/CD8− thymocyte cell populations. Upon differentiation, protein levels of DNA‐PKcs are unaltered. DNA‐PK activity, which is not detectable in SCID cells, increases in wild‐type and Rag‐2−/− cells more than twofold shortly after induction of differentiation, then falls back to about 50% of starting levels.
Si‐Face Stereospecificity at C5 of Coenzyme F420 for F420‐Dependent Glucose‐6‐Phosphate Dehydrogenase from Mycobacterium smegmatis and F420‐Dependent Alcohol Dehydrogenase from Methanoculleus thermophilicus
Authors: Klein Andreas R. , Berk Holger , Purwantini Endang , Daniels Lacy , Thauer Rudolf K.
Published: July 2004

Coenzyme F420 is a 5‐deazaflavin. Upon reduction, 1,5‐dihydro‐coenzyme F420 is formed with a prochiral center at C5. In this study we report that the F420‐dependent glucose‐6‐phosphate dehydrogenase from Mycobacterium smegmatis and the F420‐dependent alcohol dehydrogenase from Methanoculleus thermophilicus are Si ‐face stereospecific with respect to C5 of the 5‐deazaflavin. These results were obtained by following the stereochemical course of the reversible incorporation of 3H into F420 from tritium‐labeled substrates. Our findings bring to eight the number of coenzyme‐F420‐dependent enzymes shown to be Si ‐face stereospecific. No F420‐dependent enzyme with Re ‐face stereospecificity is known. This is noteworthy since coenzyme F420 is functionally similar to pyridine nucleotides for which both Si ‐face and Re ‐face specific enzymes have been found.
Purification of Active Calpain by Affinity Chromatography on an Immobilized Peptide Inhibitor
Authors: Anagli John , Vilei Edy M. , Molinari Maurizio , Calderara Silvio , Carafoli Ernesto
Published: July 2004

Most purification schemes of calpain (CANP) involve a number of chromatographic steps. The final preparations often contain impurities, including degradation fragments. Two peptide‐affinity columns were developed, using peptides of 27 amino acids and 30 amino acids, corresponding to the products of exons 1B and 1C, respectively, of the natural inhibitor (calpastatin) gene, coupled to CNBr‐activated Sepharose® 4B. Crude preparations of calpain, isolated by anion‐exchange chromatography on a DEAE‐Sepharose® column, were incubated with a reversible or an irreversible synthetic inhibitor which blocks the catalytic subunit of the enzyme in the inactive 80‐kDa form. The crude preparation was then loaded onto the peptide column in the presence of calcium. Calpain was eluted with an EGTA‐containing buffer. Using the two peptide‐affinity columns connected in tandem, calpain was isolated with a high degree of purity, suitable for structural and mechanistic studies, i.e. as an 80/30‐kDa heterodimer or in the form of dissociated monomers.
Three α‐Galactosidase Genes of Trichoderma reesei Cloned by Expression in Yeast
Authors: Margolles‐Clark Emilio , Tenkanen Maija , Luonteri Elina , Penttilä Merja
Published: July 2004

Three α‐galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC‐30 constructed in the yeast Saccharomyces cerevislae by screening the library on plates containing the substrate 5‐bromo‐4‐chloro‐3‐indolyl‐α‐D‐galactopyranoside. The genes agll, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the α‐galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLIII showed similarity with the bacterial α‐galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p‐nitrophenyl‐α‐d‐galactopyran‐oside and the small galactose‐containing oligosaccharides, melibiose and raffinose. They liberated galac‐tose from polymeric galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo‐l,4‐β‐mannanase of T. reesei. AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo‐1,4‐β‐mannanase of T. reesei and a β‐mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the α‐galactosidase previously purified from T. reesei.
Isolation, Expression and Characterization of the Gene for an ADP‐Ribosylation Factor from the Human Malaria Parasite, Plasmodium Falciparum
Authors: Stafford William H. L. , Stockley Richard W. , Ludbrook Steven B. , Holder Anthony A.
Published: July 2004

We have isolated an ADP‐ribosylation factor (ARF) gene from the human malarial parasite, Plasmodium falciparum. The gene (P. falciparum arf 1) has four introns and the exons encode a protein of 181 amino acids with high similarity to the mammalian class I ARF proteins 1–3 (?74% amino acid identity). Southern hybridization suggests there is at least one additional arf in the P. falciparum genome. Northern analysis identified a single P. falciparum arf1 mRNA of 1.8 kb in the asexual blood stage form of the parasite. The P. falciparum arf1 mRNA levels are developmentally regulated, reaching a maximum during nuclear division towards the end of the intraerythrocytic cycle. P. falciparum arfl cDNA was isolated by reverse‐transcriptase polymerase chain reaction and used to express a recombinant protein in Escherichia coli. Recombinant P. falciparum ARF1 protein was purified with stoichiometric amounts of bound GDP, although intrinsic guanose triphosphatase activity of the protein could not be detected. The protein stimulated cholera‐toxin‐catalyzed ADP‐ribosyltransferase activity in a reaction that was dependent upon the addition of both dimyristoylglycerophosphocholine and cholate. The protein bound GTP with first‐order kinetics with an apparent rate constant, k', of 0.0145 (±0.0019) min‐1. These results suggest that P. falciparum ARF1 is a member of the class 1 ARF family and provide additional evidence for the existence of a classical secretory pathway in P. falciparum.
Expression of Tyrosine‐Sulfated Secretory Proteins in Xenopus laevis Oocytes
Authors: Vannier Christian , Huttner Wieland B.
Published: July 2004

Xenopus laevis oocytes were used to study the tyrosine sulfation and secretion of exogenous proteins. Secretogranin II (SgII), a tyrosine‐sulfated protein found in secretory granules of a wide variety of endocrine cells and neurons, became tyrosine‐sulfated by the oocytes when expressed by injection of poly(A)‐rich RNA isolated from the neuroendocrine cell line PC12. The same result was observed when SgII was expressed from cloned SgII cRNA, showing that its tyrosine sulfation did not require the coexpression of exogenous tyrosylprotein sulfotransferase (TPST) but occurred by means of the endogenous oocyte TPST. Sulfophilin, an artificial protein consisting of 12 repeats of a heptapeptide tyrosine‐sulfation site, was highly sulfated upon injection of its RNA, indicating the presence of TPST levels sufficient for stoichiometric sulfation of appropriate reporter proteins. Comparison of the secretion of [35S]sulfate‐labelled SgII with that of sulfophilin and an exogenous heparan sulfate proteoglycan (HSPG), two proteins delivered to the cell surface by the constitutive pathway of secretion, revealed striking differences. The majority of sulfophilin and the HSPG was found in the medium, whereas that of SgII was found intracellularly. Prolactin, another secretory granule protein, showed the same secretion behaviour as SgII. These results show that oocytes express TPST and that these cells secrete constitutive and regulated secretory proteins in a differential manner.