bluuebunny/crossref_metadata_2025 huggingface dataset:
Tissue‐type plasminogen activator mutants imitating urokinase in the peptide link between kringle and protease domains and at selected sites with the protease domain
Authors: HINZMANN Bernd , WERNICKE Dirk , PFEIFER Monika , ZACHARIAS Ute , FISCHER Bernhard , EISENMENGER Frank , WILL Horst
Published: March 2005

Tissue‐type plasminogen activator (tPA) mutants which, at selected amino acid positions, mimic urokinase‐type plasminogen activator (uPA) were expressed in Chinese hamster ovary cells and examined for their catalytic properties.

In one series of mutants, the dipeptide Ser262 Thr263 between kringle 2 and the protease domain of tPA was (a) replaced by an Ala residue, (b) lengthened by additional Ser and Ala residues, (c) exchanged for the 16‐amino‐acid link between kringle and protease domains of uPA and an additional Ala residue. The activities of the latter two mutants toward plasminogen were, in the absence of fibrin, 3–5‐fold higher and, in the presence of fibrin, comparable to or lower than the activity of tPA. The kinetic data suggest a short interdomain peptide in tPA as most favorable for high fibrin stimulation of tPA activity.

In a second series of mutants, selected amino acid residues of the tPA protease domain were replaced by residues of the homologous uPA domain. Positins chosen for exchange are either close to the active site or are part of a tPA‐specific insertion in the variable region preceding the activesite Ser residue. Compared to authentic tPA, protease‐domain mutants exhibited 7.3–424‐fold lower activities toward plasminogen, mainly due to lower kcat values. Km values differed only moderately. A mutant containing an additional hydroxyl group at the S1 site, tPA A473S, has lost the preference of tPA for Arg over Lys as the P1 residue in peptide substrates.
Comparative proteolysis of sorbitol and alcohol dehydrogenases
Authors: ROUIMI Patrick , LOOMES Kerry , JÖRNVALL Hans
Published: March 2005

Mammalian alcohol and sorbitol dehydrogenases, with distantly related subunits but different substrates, quaternary structures and zinc contents, were evaluated by comparison of their sensitivities to proteases. Sorbitol dehydrogenase is more sensitive to proteolysis than alcohol dehydrogenase, but both enzymes show limited cleavage with Lys‐specific and Glu‐specific proteases. With the former, the major cleavage in both proteins involves Lys‐Lys‐Pro segments, at positions 247–248 in alcohol dehydrogenase, surface‐positioned after the most distal β‐strand in the coenzyme‐binding domain, and at 61–62 in sorbitol dehydrogenase, at another surface in the catalytic domain. Further cleavages affect these two and a third surface. A non‐surface cleavage was obtained with the Gluspecific protease and sorbitol dehydrogenase, after insufficient protease inhibition before analysis by SDS/PAGE. It probably reflects non‐native conditions, and the fact that this protease is active in strong SDS, necessitating pre‐analytical use of a specific inhibitor.

Differences in cleavage patterns between the two proteins do not involve areas corresponding to the dimer interactions in alcohol dehydrogenase. Hence, these areas are likely to be the same in sorbitol dehydrogenase. However, major differences involve regions that flank the surface which has a 20‐residue loop segment in alcohol dehydrogenase that is missing in sorbitol dehydrogenase. This segment, previously highlighted from comparisons, is therefore also emphasized by experimental results with proteolysis. The surface exposed by the missing segment is likely to correlate with the separate quaternary structures and to indicate possible sites for the additional subunit interactions in the sorbitol dehydrogenase tetramer versus the alcohol dehydrogenase dimer.
Crystallographic studies of azide binding to human carbonic anhydrase II
Authors: NAIR Satish K. , CHRISTIANSON David W.
Published: March 2005

The crystal structures of human carbonic anhydrase II (CAII) at pH 5.7 and 8.0 have been determined at 0.21‐nm resolution in the presence of 20 mM azide, which is a noncompetitive inhibitor of the CAII‐catalyzed CO2 hydration reaction. Although azide often facilitates the crystallization of CAII and its variants, this small anion does not cause any significant structural changes in the enzyme active site or in the overall protein structure, and zinc coordination remains tetrahedral over the pH range 5.7–8.0. Importantly, the binding of azide at pH 8.0 has implications for the zinc‐binding mode of the catalytic product, bicarbonate ion. Since azide is a competitive inhibitor of the reverse reaction of bicarbonate dehydration, and since the zinc‐bound azide nitrogen makes a non‐hydrogen‐bonded van der Waals contact with the hydroxyl group of Thr199, it is possible that a zinc‐bound bicarbonate oxyanion could likewise make a non‐hydrogen‐bonded, van der Waals contact with the hydroxyl group of Thr199. Therefore, the donation of a hydrogen bond to Thr199 is not absolutely required for anion binding to tetracoordinate zinc.
Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A2
Authors: LUGTIGHEID Richard B. , OTTEN‐KUIPERS Maaike A. , VERHEIJ Hubertus M. , DE HAAS Gerard H.
Published: March 2005

The X‐ray structure of a mutant porcine pancreatic phospholipase A2 inhibitor complex [Thunnissen et al. (1990) Nature 347, 689–691] has been determined. This structure shows several interactions between the sn‐2‐acyl chain and the phosphate moiety of the inhibitor at sn‐3 and the protein. The interactions of the remaining part of the polar head group are less clear. Because Arg53 is in close proximity to the head group, we tested the importance of charge at position 53 on enzymatic activity and specificity. Arg53 has been replaced by a glutamine and a glutamic acid in mutants R53Q and R53E, respectively. The effects of the mutations were tested with both zwitterionic and anionic substrates. With monomeric, zwitterionic, (R,S)‐1,2‐dihexanoyldithiopropyl‐3‐phosphocholine as substrate, the mutants R53Q and R53E display twofold and sevenfold, respectively, increased kcat/Km values, composed of increased kcat and decreased Km values. Tested on micelles of zwitterionic (R)‐1,2‐dioctanoylglycero‐3‐phosphocholine the mutants R53Q and R53E are more active than the native enzyme, whereas these mutations have an opposite effect on the activity on anionic (R)‐1,2‐dioctanoylglycero‐3‐phosphoglycol. Thus, whereas the native enzyme is 0.3 times as active on zwitterionic as on the anionic substrate, these ratios are 1.0 (R53Q) and 1.7 (R53E) for the mutants. No changes in activity were observed with the anionic substrate (R)‐1,2‐dioctanoylglycero‐3‐sulfate. Binding studies with substrate‐derived inhibitors confirmed the increased affinity for zwitterionic phospholipids and the reduced affinity for anionic phospholipids. The kinetic and binding data indicate the involvement of the charge of residue 53 in head‐group specificity and suggest a position of residue 53 closer to the choline or glycol than to the phosphate.
Haemonectin, a granulocytic‐cell‐binding protein, is related to the plasma glycoprotein fetuin
Authors: WHITE Harry , TOTTY Nicholas , PANAYOTOU George
Published: March 2005

Haemonectin, a protein present in rabbit bone marrow extracellular matrix extracts, has been reported to bind granulocytes in a developmentally regulated manner. We have purified haemonectin from such extracts and determined the partial amino‐acid sequence. The sequence obtained shows 60–70% similarity with the sequence of the plasma glycoprotein fetuin from other mammal species. This difference is consistent with the difference between fetuins from different species. We conclude that the rabbit haemonectin molecule is related to fetuin. The similarity between haemonectin and fetuin is reinforced by analysis with Western blots of one‐ and two‐dimensional gels. These show that haemonectin, like fetuin, is present in serum and that migration of haemonectin from serum and extracellular matrix extracts, on two‐dimensional gels, co‐incides with that of human fetuin (α2HS‐glycoprotein) from serum, extracellular matrix extracts and in purified form. Also, antihaemonectin antibodies cross react with human fetuin. These data imply that the rabbit haemonectin molecule is closely related to fetuin, but do not rule out the possibility that these molecules are functionally distinct.
Purification and properties of N5‐methyltetrahydromethanopterin: coenzyme M methyltransferase from Methanobacterium thermoautotrophicum
Authors: GÄRTNER Peter , ECKER Axel , FISCHER Reinhard , LINDER Dietmar , FUCHS Georg , THAUER Rudolf K.
Published: March 2005

N
5‐Methyltetrahydromethanopterin: coenzyme M meth‐yltransferase is an integral membrane protein found in methanogenic archaea. It catalyzes an energy‐conserving step in methane formation from CO2 and from acetate. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) has been purified 30‐fold to apparent homogeneity. The purified enzyme had an apparent molecular mass of 670 kDa and was composed of seven different polypeptides of 34 kDa, 28 kDa, 24 kDa, 23 kDa, 21 kDa, 13 kDa, and 12 kDa. The N‐terminal amino acid sequences of these polypeptides were determined. The native 670‐kDa enzyme was found to contain 7.6 mol 5‐hydroxybenzimidazolyl cobamide/mol, 37 mol non‐heme iron/mol and 34 mol acid‐labile sulfur/mol. Cobalt analyses after sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that the corrinoid was bound to the 23‐kDa polypeptide.

The apparent molecualr masses of the polypeptides given above were determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis without boiling the samples prior to analysis. When the samples were boiled, as is usally done, the 23‐kDa polypeptide changed its apparent molecular mass to 33 kDa and the 21‐kDa, 24‐kDa, and 28‐kDa polypeptides formed aggregates.

The specific activity (apparent Vmax) of the purified methyltransferase preparation was 11.6 μmol · min−1· mg protein−1. The apparent Km for N5‐methyltetrahydromethanopterin was 260 μM and that for coenzyme M was 60 μM. The preparation was absolutely dependent on the presence of Ti(III) for activity. ATP enhanced the activity 1.5–2‐fold.
tRNA fluorescent labeling at 3′ end inducing an aminoacyl‐tRNA‐like behavior
Authors: SERVILLO Luigi , BALESTRIERI Ciro , QUAGLIUOLO Lucio , IORIO Eugenio L. , GIOVANE Alfonso
Published: March 2005

A fluorescent tRNA derivative labeled at 3′‐O position of the ultimate adenosine residue by reaction, under mild conditions, of tRNA with isatoic anhydride [3,1‐benzoxazine‐ 2,4(1H)‐dione] was obtained. The labeling selectivity was determined by several criteria: digestion with RNase, followed by HPLC of the digest, produces only one labeled nucleoside, identified as 3′‐O‐anthraniloyladenosine; the ratio of the absorbance at 260 nm to 332 nm also suggests a 1:1 molar ratio between the nucleic acid and the fluorophore; finally, the incapacity of the labeled tRNA to be charged by the specific aminoacyltransferase further demonstrates the engagement of the 3′‐O position.

Although the 3′‐O‐anthraniloyl‐labeled tRNA does not seem to be functionally active, as far as the aminoacyl charging activity is concerned, surprisingly we found that it is able to form the ternary complex with elongation factor Tu (EF‐Tu) and GTP with an affinity consistently higher than uncharged tRNA. From fluorescence anisotropy measurements the ternary complex dissociation constant was estimated as 73 nM for Escherichia coli and 140 nM for yeast anthraniloyl‐tRNAPhe. These results may be interpreted in terms of the particular structure of the anthraniloyl group that makes the labeled tRNA similar to an aminoacyl‐tRNA.
Troponin‐C mutants with increased calcium affinity
Authors: da SILVA Ana Claudia R. , de ARAUJO Alexandre H. B. , HERZBERG Osnat , MOULT John , SORENSON Martha , REINACH Fernando C.
Published: March 2005

Binding of two Ca2+ to the regulatory sites I and II of troponin C (TnC) induces a conformational transition believed to be responsible for the activation of muscle contraction. Based on the known crystal structure (2Ca2+ state), a model for the transition to the 4Ca2+ state has been proposed [Herzberg, O., Moult, J. & James, M. N. G. (1986) J. Biol. Chem. 261, 2638–2644]. The proposed conformational transition predicts that during Ca2+ binding a number of nonpolar residues become exposed to the solvent, creating a hydrophobic patch. Such a model implies that mutation of the hydrophobic to polar residues should increase the Ca2+ affinity at the regulatory sites and reduce the Ca2+ concentration necessary for muscle activation. To test this prediction, we have constructed and functionally characterized two troponin‐C mutants (V45T and M48A mutations).

Direct calcium‐binding measurements in the mutants demonstrate an increase in the Ca2+ affinity for two low‐affinity sites. Replacement of endogenous troponin C in skinned muscle fibers by TnC with mutations V45T or M48A increased the Ca2+ sensitivity of their tension development. These results show that the model can be used to construct mutants that regulate muscle contraction at lower Ca2+ concentrations. They provide further experimental support for the proposed calcium‐induced conformational change of troponin C and suggest that the predicted transition plays a central role in the activation of the thin filament.
Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase
Authors: BAUNSGAARD Lone , DALBØGE Henrik , HOUEN Gunnar , RASMUSSEN E. Michael , WELINDER Karen G.
Published: March 2005

Sequence analysis and cDNA cloning of Coprinus peroxidase (CIP) were undertaken to expand the understanding of the relationships of structure, function and molecular genetics of the secretory heme peroxidases from fungi and plants. Amino acid sequencing of Coprinus macrorhizus peroxidase, and cDNA sequencing of Coprinus cinereus peroxidase showed that the mature proteins are identical in amino acid sequence, 343 residues in size and preceded by a 20‐residue signal peptide. Their likely identity to peroxidase from Arthromyces ramosus is discussed. CIP has an 8‐residue, glycine‐rich N‐terminal extension blocked with a pyroglutamate residue which is absent in other fungal peroxidases. The presence of pyroglutamate, formed by cyclization of glutamine, and the finding of a minor fraction of a variant form lacking the N‐terminal residue, indicate that signal peptidase cleavage is followed by further enzymic processing. CIP is 40–45% identical in amino‐acid sequence to 11 lignin peroxidases from four fungal species, and 42–43% identical to the two known Mn‐peroxidases. Like these white‐rot fungal peroxidases, CIP has an additional segment of approximately 40 residues at the C‐terminus which is absent in plant peroxidases. Although CIP is much more similar to horseradish peroxidase (HRP C) in substrate specificity, specific activity and pH optimum than to white‐rot fungal peroxidases, the sequences of CIP and HRP C showed only 18% identity. Hence, CIP qualifies as the first member of a new family of fungal peroxidases. The nine invariant residues present in all plant, fungal and bacterial heme peroxidases are also found in CIP. The present data support the hypothesis that only one chromosomal CIP gene exists. In contrast, a large number of secretory plant and fungal peroxidases are expressed from several peroxidase gene clusters. Analyses of three batches of CIP protein and of 49 CIP clones revealed the existence of only two highly similar alleles indicating less peroxidase polymorphism in C. cinereus strains than observed in plants and white‐rot fungi.
Identification of novel phosphorylation sites in murine A‐type lamins
Authors: EGGERT Martin , RADOMSKI Norbert , LINDER Dietmar , TRIPIER Dominique , TRAUB Peter , JOST Erich
Published: March 2005

We report the distribution of phosphorylation sites in murine lamins A and C (A‐type lamins) in vitro and in vivo followed by reverse‐phase high‐performance liquid chromatography and micro‐sequencing of peptides spanning the almost complete lamin sequence. We show that two distinct protein kinases, cell‐division‐cycle‐2 kinase (cdc2 kinase) and protein kinase C (PKC), phosphorylate murine A‐type lamins at the non‐α‐helical amino‐ and carboxy‐terminal domains in vitro and in vivo. Cdc2 kinase, but not PKC, is capable of inducing depolymerization of the nuclear lamina in permeabilized cells. Accordingly, lamins were proposed to be direct in vivo substrates of cdc2 kinase and PKC with different effects on the lamina dynamics. Analysis of the original A‐type lamins revealed phosphorylation of residues Ser5 and Ser392. Residue Ser392 was substoichiometrically phosphorylated in the substrate and by cdc2 kinase in vitro. PKC phosphorylated peptides with its kinase‐specific motifs surrounding Ser5, Thr199, Thr416, Thr480 and Ser625. In vivo, a mitosis‐specific phosphorylation at the cdc2‐kinase‐specific phosphoacceptor site Ser392 and of the N‐terminal peptide was identified. An interphase‐specific phosphorylation at Ser525 matching the PKC consensus sequence and of peptides phosphorylated by unknown kinases was determined. The results lead us to propose that different cyclin‐dependent kinase activities act as lamin kinases in mitosis and in interphase. Other kinases may cooperate with cdc2 kinase during reversible disassembly in mitosis and may modulate the supramolecular assembly of lamin filaments.
Orthorhombic crystal structure of the A‐DNA octamer d(GTACGTAC)
Authors: d'ESTAINTOT Béatrice LANGLOIS , DAUTANT Alain , COURSEILLE Christian , PRECIGOUX Gilles
Published: March 2005

The X‐ray crystal structure of the double‐helical A‐DNA octanucleotide d(GTACGTAC) has been solved by molecular replacement and refined to a resolution of 0.219 nm. The final R‐factor is equal to 16.1% for 1516 observed reflections with F > 4σ(F). The sequence crystallizes as an A‐DNA‐type double helix in the orthorhombic space group P21212, with one duplex molecule solvated by 66 water molecules in the asymmetric unit. Cell parameters are a= 3.860 nm, b= 5.082 nm, c= 2.174 nm. It is the first time that such a crystal form has been observed. This orthorhombic structure has been compared with the tetragonal structure of the same oligonucleotide. It adopts a bent structure with an unusual packing between symmetry‐related molecules.
Functional domains in the Escherichia coli release factors
Authors: MOFFAT John G. , DONLY B. Cameron , McCAUGHAN Kim K. , TATE Warren P.
Published: March 2005

Chimeras between Escherichia coli release factors RF‐1 and RF‐2 have been constructed to study the role of the release factors in termination, in particular whether each possesses specific domains for recognition of the stop codon, and for facilitating peptidyl‐tRNA hydrolysis. One hybrid factor showed normal codon‐recognition activity but was defective in its ability to facilitate hydrolysis. Overexpression of this protein was toxic to the cell. Conversely, another hybrid factor showed complete loss of codon recognition but retained some hydrolysis activity. These two functional activities of the release factors were not localised in domains within either the amino‐terminal or carboxy‐terminal halves of the primary sequence as previously predicted. Evidence from the activities of the hybrid proteins and from earlier studies suggests that a combination of residues from the beginning and middle of the sequence, including a region of very high sequence conservation, contribute to the hydrolysis domain, whereas residues from both the amino‐terminal and carboxy‐terminal halves of the molecule are important for the codon recognition domain.
CaBP2 is a rat homolog of ERp72 with proteindisulfide isomerase activity
Authors: VAN Phuc Nguyen , RUPP Katrin , LAMPEN Alfons , SÖLING Hans‐Dieter
Published: March 2005

Ca‐binding protein 2 (CaBP2) has been described previously as an intracisternal calcium‐binding microsomal glycoprotein [1]. We report now the primary sequence of this protein as deduced from the corresponding cDNA. The protein possesses a C‐terminal ‐KEEL retention sequence and three repeats of the thioredoxin‐like motive ‐EFYAPNCGHCK‐, and represents the rat homolog of ERp72 [2]. In contrast to earlier reports on ERp72, CaBP2 possesses significant proteindisulfide isomerase activity. Furthermore, in contrast to ERp72, CaBP2 is a glycoporotein containing O‐linked glycans. The amount of CaBP2 in H‐35 Reuber hepatoma cells increases in parallel with that of immunoglobin heavy‐chain‐binding protein under conditions which lead to impaired glycosylation, while the amount of calreticulin, another KDEL‐containing glycoprotein, remains almost unchanged.
Human β2‐adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP‐binding protein to adenylyl cyclase
Authors: KLEYMANN Gerald , BOEGE Fritz , HAHN Mathias , HAMPE Wolfgang , VASUDEVAN Subhash , REILÄNDER Helmut
Published: March 2005

Spodoptera frugiperda insect cells (Sf9) containing the stably integrated human β2‐adrenergic receptor gene under the control of the baculovirus IE1 promoter expressed up to 350000 human receptors/cell. The number of receptors did not change with cell density or age of culture. The adrenergic receptors overexpressed in the insect cells were functional with respect to their ligand binding and signalling properties. Coupling of the receptors to endogenous GTP‐binding proteins is demonstrated by hormone‐dependent stimulation of GTPase and adenylyl cyclase activity in the transformed insect cells. Western‐blot analysis revealed that the endogenous GTP‐binding protein appears to be of the heterotrimeric type. Antibodies raised against the mammalian α subunit of stimulatory GTP‐binding proteins cross‐react with the insect α subunit of GTP‐binding proteins, which also exhibits the same apparent molecular mass as its mammalian counterpart. The β subunit of GTP‐binding proteins from insect cells reacts with anti‐peptide serum directed against the C‐terminal amino acids of the mammalian β subunit of GTP‐binding proteins, but is about ∼2 kDa larger than that of the β subunit of GTP‐binding proteins from bovine brain. Exposure of the transformed insect cells to L‐isoproterenol rapidly induces uncoupling and internalization of 30% of the heterologously expressed receptors. In contrast to the situation in mammalian cells, prolonged exposure of the agonist (24 h) does not result in down regulation of the remanining 70% of the receptors.
Crude liver membrane fractions and extracellular matrix components as substrata regulate differentially the preservation and inducibility of cytochrome P‐450 isoenzymes in cultured rat hepatocytes
Authors: SAAD Bashar , SCHOLL Florence A. , THOMAS Helmut , SCHAWALDER Hanspeter , STREIT Veronika , WAECHTER Felix , MAIER Peter
Published: March 2005

The influence of cell‐substrata interactions on the preservation of basal or in‐vivo‐induced micro‐somal cytochrome P‐450 isoenzyme contents in cultured rat hepatocytes and on the adaptive responses after exposure to phenobarbital or 3‐methylcholanthrene in vitro, was investigated. Hepatocytes from untreated or phenobarbital‐treated rats were cultured in serum‐free, aprotinin‐supplemented culture medium in 96‐well microtiter plates coated with collagen type I (COL), laminin, fibronectin or crude liver membrane fractions/collagen type I (CMF/COL). Basal cell functions were characterized by measuring the total protein content and lactate dehydrogenase release. The relative contributions of CYP1A1/2, CYP2B1/2, CYP2C6, CYP2C11, CYP3A and CYP4A isoenzymes were determined with ELISA using monoclonal antibodies raised against purified cytochromes P‐450 from rat liver microsomes.

The characterization of the CMF revealed that contaminations with mitochondria, nuclei and lysosomes are relatively low. Among these, membranes derived from the endoplasmic reticulum appeared to be the major organelle contaminant of the CMF. The matrix components laminin, fibronectin and collagen type IV were found in appreciable amounts.

Hepatocytes from untreated rats, cultured for up to nine days on CMF/COL‐coated plates, retained their relative cytochrome P‐450 contents at 1.5–3‐fold higher levels when compared to cells cultured on COL, fibronectin or laminin. Similarly, hepatocytes from phenobarbital‐treated rats preserved the contents of barbiturate‐inducible CYP2B1/2 and CYP3A proteins best when cultured on CMF/COL. After exposure of hepatocytes cultured on CMF/COL to phenobarbital from days 3–6, CYP3A proteins were enhanced more than twofold and CYP2B1/2, depending on the exposure level, increased 1.3–6‐fold. After exposure to 3‐methylcholanthrene, a threefold increase of CYP1A proteins was found in CMF/COL and laminin cultures. These result indicate that CMF/COL, as a substratum in rat hepatocyte cultures, regulates gene expression of cytochromes P‐450 isoenzymes for up to 9 days and provides a matrix which enables the cells to respond qualitatively similar to the response observed in different zones of the liver. This activity cannot be replaced by single matrix components.
The contraction of hepatic stellate (Ito) cells stimulated with vasoactive substances
Authors: KAWADA Norifumi , TRAN‐THI Thuy‐Anh , KLEIN Heike , DECKER Karl
Published: March 2005

We have studied the contractility of liver sinusoidal stellate (Ito) cells stimulated with endothelin 1, nitric‐oxide donors and eicosanoids. Contraction and relaxation of stellate cells were detected by the use of a silicone‐rubber method that revealed the traction forces exerted by these cells. Endothelin 1 was a strong elicitor for stellate‐cell contraction. 78, 55, 59 and 56% of stellate cells were contracted 2.5, 5, 10 and 20 min, respectively, after exposure to 10 nM endothelin 1. The effect of endothelin 1 was dose dependent and still detectable at an endothelin 1 concentration of 100 pM. Concomitantly, an endothelin‐dependent formation of inositol phosphates was apparent; values of Insp, InsP2, and InsP3 were 881±99%, 1965±368%, and 791±120% of control, respectively, 20 min after addition of 10 nM endothelin 1. In addition, endothelin 1 caused a transient increase of [Ca2+]i in stellate cells from a basal value of 121±9 nM to maximal 1015±86nM. These endothelin‐1 effects were much stronger than those of the thromboxane‐A2 analogue U46619 and of prostaglandin F2a. In contrast, IIoprost, prostaglandin E2, and sodium nitroprusside promoted stellate‐cell relaxation; for example, 82, 83 and 71% of stellate cells relaxed 5, 10, and 20 min, respectively, after addition of 500μM sodium nitroprusside to contracted cells. Prostaglandin E2 and Iloprost led to elevation of cAMP levels in stellate cells from a basal value of 9.2±0.8 pmol/well to 55.1±8.0 and 122.2±12.2 pmol/well 10 min after addition of prostaglandin E2 (5μM), and Iloprost (5μM), respectively, in the presence of 3‐isobutyl‐1‐methylxanthine (0.5 mM). However, sodium nitroprusside was a trigger for cGMP accumulation. Intracellular cGMP increased from a basal value of 0.9±0.007 pmol/well 13.4±6.7 pmol/well 10min after addition of 500 μM sodium nitroprusside into the medium. It is interesting that Iloprost and sodium nitroprusside also induced the disappearance of actin stress fibers in contracted cells; F‐actin stress fibers became less numerous and de‐aggregated; more than 90% of stellate cells were void of stress fibers after 10 μM Iloprost treatment for 30 min. Thus, endothelin 1, eicosanoids and sodium nitroprusside are able to modulate the contractility of stellate cells. The effect of endothelin 1 and prostaglandin F2a seems to be mediated by inositol‐phosphate formation and increase of the intracellular Ca2+ level, that of Iloprost and prostaglandin E2 by cAMP. cGMP may be a second messenger for the action of sodium nitroprusside.
High‐mobility‐group (HMG) proteins and histone H1 subtypes expression in normal and tumor tissues of mouse
Authors: GIANCOTTI Vincenzo , BANDIERA Antonella , CIANI Lorenza , SANTORO Daniela , CRANE‐ROBINSON Colyn , GOODWIN Graham H. , BOIOCCHI Mauro , DOLCETTI Riccardo , CASETTA Bruno
Published: March 2005

Exhaustive extraction of mouse tissues with perchloric acid has been used together with reverse‐phase HPLC and electrophoresis to quantify the amounts of chromosomal proteins HMG17, HMG14 and HMGI, relative to histone H1. Normal lung and thymus contain ∼3% HMG17/HMG14 but only ∼2% HMGI. In tumor tissues (Lewis lung carcinoma and lymphoma NQ35), the amount of HMG17/HMG14 is not greatly alterated but HMGI levels rise considerably, reaching 10% in Lewis lung carcinoma. HMGI synthesis does not replace HMG17/HMG14 proteins, suggesting that HMGI proteins contribute to the structure of chromatin regions in a manner distinct from those of HMG17/HMG14. Ion‐spray mass spectrometry has been used to determine the molecular masses of H1 subtypes from the same four mouse tissues. In addition to the six known species H1°, H1a, H1b, H1c, H1d and H1e, a newly defined subtype of mass 21756 Da from Lewis lung carcinoma, named H1L was identified. Several phosphorylated H1 subtypes have also been defined by mass spectrometry. The combined use of reverse‐phase HPLC and electrophoresis permitted quantification of these seven histone H1 subtype in the four mouse tissues. Increased phosphorylation of H1 subtypes in tumors parallels the phosphorylation of HMGI proteins which are present in great amounts, showing that both are involved as post‐translational‐modified forms in the structure of the chromatin of neoplastic systems.
Natural cycloheximide resistance in yeast
Authors: DEHOUX Pierre , DAVIES Julian , CANNON Michael
Published: March 2005

The yest Kluyveromyces lactis is resistant to high concentrations (1 mg/ml) of the antibiotic cycloheximide. Using in vitro translation studies it was confirmed that this extreme resistance is a property of ribosomes. The resistance determinant from K. lactis was cloned into Saccharomyces cerevisiae. Nucleotide sequence analysis of the determinant demonstrated that resistance was conferred by the K. Lactis ribosomal protein L41. K. lactis was shown to contain only one copy of the gene that encodes this protein and the gene was located to chromosome III. In contrast, S. cerevisiae was found to contain multiple copies of the gene for the corresponding ribosomal protein L41 which mapped to two of the three chromosomes V, XIV and VIII. Since the cycloheximide‐resistance gene of K. lactis causes essentially complete protection against inhibition by the drug, it is likely to be particularly useful as a selective marker in eukaryotic gene transfer studies.
LymnaDFamides, a new family of neuropeptides from the pond snail, Lymnaea stagnalis
Authors: JOHNSEN Anders H. , REHFELD Jens F.
Published: March 2005

Five tridecapeptides have been identified from the central nervous system of the pond snail, Lymnaea stagnalis. The sequences are Pro‐Xaa‐Asp‐Arg‐Ile‐Ser‐Yaa‐Ser‐Ala‐Phe‐Ser‐Asp‐Phe · NH2, where Xaa is either Tyr or Phe and Yaa either Asn, Ser or Gly. The peptides are named lymnaDFamides to acknowledge identity with the C‐terminal dipeptide of the mammalian neuropeptides, cholecystokinin (CCK) and gastrin. They were detected by an antiserum that recognizes the biologically active C‐termini of cholecystokinin and gastrin. LymnaDFamide‐1 (Xaa = Tyr and Yaa = Asn) had no effect on trout gallbladder, which responds equally to CCK and gastrin. We propose that the lymnaDFamides belong to an Asp‐Phe‐amide superfamily, which includes CCK and gastrin, and suggest that the widespread CCK/gastrin immunoreactivity in invertebrates is due to peptides belonging to such a superfamily.
Translational repression of EF‐1α mRNA in vitro
Authors: SLOBIN Lawrence I. , RAO Manjunath N.
Published: March 2005

In this report we show that when 10000 ×g supernatant extracts of growth arrested murine erythroleukemia (MEL) cells are incubated there is a rapid conversion of essentially all mRNAs to non‐translating messenger ribonucleoprotein (RNP) particles. Most of these RNPs are readily translated in an initiation‐dependent manner when added to a nuclease‐treated rabbit reticulocyte lysate. A notable exception is the RNP containing eucaryotic elongation factor 1α (EF‐1α) mRNA. The mRNA for poly(A)‐binding protein behaved similarly to EF‐1α. Previous work has demonstrated that the translation of both these mRNAs are repressed in vivo when the growth of a number of different mammalian cells is arrested [Slobin L. I. and Jordan, P. (1984) Eur J. Biochem. 145, 1984; Thomas, G. and Thomas, G. (1986) J. Cell Biol. 103, 1986]. Translational activity of EF‐1α mRNA could be restored by treating RNP particles with 0.5 M KCl, provided that the RNPs were separated from salt wash by chromatography on oligo(dT)‐cellulose. Addition of the salt wash to total MEL cell mRNA significantly and selectively inhibited EF‐ 1α mRNA translation, suggesting that a component of the salt wash acts as a trans‐acting translational repressor of EF‐1α mRNA.
Primary structures of the N‐linked carbohydrate chains from honeybee venom phospholipase A2
Authors: KUBELKA Viktoria , ALTMANN Friedrich , STAUDACHER Erika , TRETTER Verena , MÄRZ Leopold , HÅRD Karl , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G.
Published: March 2005

The N‐linked carbohydrate chains of phospholipase A2 from honeybee (Apis mellifera) were released from glycopeptides with peptide‐N‐glycanase A and reductively aminated with 2‐aminopyridine. The fluorescent derivatives were separated by size‐fractionation and reverse‐phase HPLC, yielding 14 fractions. Structural analysis was accomplished by compositional and methylation analyses, by comparison of the HPLC elution patterns with reference oligosaccharides, by stepwise exoglycosidase digestions which were monitored by HPLC, and, where necessary, by 500‐MHz 1H‐NMR spectroscopy.

Ten oligosaccharides consisted of mannose, N‐acetylglucosamine and fucose α1–6 and/or α1–3 linked to the innermost N‐acetylglucosamine. Four compounds, which comprised 10% of the oligosaccharide pool from phospholipase A2, contained a rarely found terminal element with N‐acetylgalactosamine. The structures of the 14 N‐glycans from honeybee phospholipase A2 can be arranged into the following three series:

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Creatinase in its collapsed A state shows properties of a molten globule with dimeric quaternary structure
Authors: SCHUMANN Judith , JAENICKE Rainer
Published: March 2005

In the past, the molten globule state at acidic pH (A state) has mainly been observed for small single‐domain proteins. For more complex proteins such as immunoglobulin, alternatively folded states, with certain characteristics of the molten globule but different thermodynamic properties, were observed. In the present work, the acid‐induced structural characteristics of a homodimer, creatinase from Pseudomonas putida, are described. The 91‐kDa protein at pH 2 shows molten globule behavior in that there is (a) a high content of native‐like secondary structure (monitored by far‐ultraviolet circular dichroism), (b) changes in the solvent accessibility of intrinsic fluorophores (acrylamide quenching of protein fluorescence), (c) increased hydrophobic surface area (indicated by anomalous dye binding) and (d) a slight expansion of the hydrodynamic volume (calculated from S20,solv, obtained from analytical ultracentrifugation). The enzyme at pH 2 shows reversible cooperative transitions in guanidinium chloride or urea (at elevated ionic strength). Its quaternary structure remains unaltered, indicating that native‐like subunit interactions are involved in the stabilization of the A state of the enzyme. Anions stabilize the compact conformation due to reduced intramolecular charge repulsion; for the same reason, the enzyme in its A state shows a strong tendency to aggregate at >0.3 M NaCl.
Photochemical cross‐linking of the skeletal myosin head heavy chain to actin subdomain‐1 at Arg95 and Arg28
Authors: BONAFÉ Nathalie , CHAUSSEPIED Patrick , CAPONY Jean Paul , DERANCOURT Jean , KASSAB Ridha
Published: March 2005

F‐actin specifically substituted with the photocross‐linker, p‐azidophenylglyoxal, at Arg95 and Arg28 was isolated and characterized. Upon complexation with myosin subfragment‐1 (S1) and photolysis at 365 nm, it was readily cross‐linked to the S1 heavy chain with a yield of about 13–25%, generating four major actin–heavy‐chain adducts with molecular masses in the range 165–240 kDa. The elevated Mg2+‐ATPase of the covalent complexes displayed a turnover rate of 33±8 s−1 which is similar to the values reported earlier for other acto‐S1 conjugates. The cross‐linking between various proteolytic S1 and actin derivatives, combined with the fluorescent and immunochemical detection of the photocross‐linked products, indicated that the arylnitrene group on Arg95 was inserted predominantly in the central 50‐kDa region, whereas that attached to Arg28 mediated the selective cross‐linking of the COOH‐terminal 22–21‐kDa fragments of the heavy chain, most probably by reacting at or near the connector segment between the 50‐kDa and 20‐kDa fragments. The rapid photoactivation and cross‐linking to S1 of the substituted F‐actin, which can be accomplished on a millisecond time scale, may serve to probe the structural dynamics of the interaction of the S1 heavy chain with subdomain‐1 of actin during the ATPase cycle.
Collagen II from articular cartilage and annulus fibrosus
Authors: YANG Chunlin L. , RUI Haifeng , MOSLER Stephan , NOTBOHM Holger , SAWARYN Andrzej , MÜLLER Peter K.
Published: March 2005

Collagen II was isolated and characterized from hyaline cartilage (articular cartilage) and fibrocartilage (annulus fibrosus). Collagen II from the latter tissue has a substantially higher degree of hydroxylation and glycosylation than that isolated from articular cartilage. The higher degree of posttranslational modification was associated with a slower electrophoretic mobility, a greater resistance to mammalian collagenase digestion and a higher thermal stability. An increase of glycosylation accelerates the initial steps in fibril formation of collagen molecules but slows down the following lateral growth. The newly formed aggregates of collagen II from anulus fibrosus consisted of fibrils with a smaller diameter.
Expression of the Xenopus D2 dopamine receptor
Authors: MARTENS Gerard J. M. , GROENEN Peter M. A. , GRÖNEVELD Diet , VAN RIEL Maarten C. H. M.
Published: March 2005

In the amphibian Xenopus laevis the D2 dopamine receptor is involved in the regulation of the melanotrope cells of the intermediate pituitary during background adaptation of the animal. The Xenopus D2 receptor has been found to be pharmacologically different from the mammalian D2 receptor. In a number of mammalian species alternative splicing generates two molecular forms of the D2 receptor. These isoforms differ by the presence or absence of 29 amino acids in the third cytoplasmic loop which is thought to be involved in guanine‐nucleotide‐binding‐regulatory‐protein (G‐protein) binding of the receptor. We previously described a cDNA encoding the large isoform of the Xenopus D2 receptor. Here we report on the isolation of a brain cDNA encoding a second, structurally different Xenopus D2 dopamine receptor. Both Xenopus receptors correspond to the large isoform of the D2 receptor and they display a high degree of sequence identity with their mammalian counterparts. Their occurence reflects the expression of two Xenopus D2 receptor genes and they are expressed to approximately the same level. In contrast to mammals, PCR analysis gave no evidence for alternative splicing during D2 receptor expression in Xenopus brain and pituitary. Tissue‐specific expression of the Xenopus D2 receptor was observed in the pituitary during background adaptation. The low level of receptor mRNA in melanotrope cells of white animals compared to that of black animals may be caused by chronic dopamine stimulation of melanotrope cells in white animals with consequent cellular desensitization and down regulation of the D2 receptor gene.
Genomic organization, splice variants and expression of CGM1, a CD66‐related member of the carcinoembryonic antigen gene family
Authors: NAGEL Gerhard , GRUNERT Fritz , KUIJPERS Taco W. , WATT Suzanne M. , THOMPSON John , ZIMMERMANN Wolfgang
Published: March 2005

The tumor marker carcinoembryonic antigen (CEA) belongs to a family of proteins which are composed of one immunoglobulin variable domain and a varying number of immunoglobulin constant‐like domains. Most of the membrane‐bound members, which are anchored either by a glycosylphosphatidylinositol moiety or a transmembrane domain, have been shown to convey cell adhesion in vitro. Here we describe two splice variants of CGM1, a transmembrane member of the CEA family without immunoglobulin constant‐like domains. CGM1a and CGM1c contain cytoplasmic domains of 71 and 31 amino acids, respectively. The cytoplasmic region of CGM1a is encoded by four exons (Cyt1‐Cyt4). Differential splicing of the Cyt1 exon (53 bp) leads to the formation of CGM1c. The presence or absence of potential protein kinase phosphorylation sites in the cytoplasmic domains and a sequence consensus motif involved in signal transduction in multichain immune recognition receptors indicates that this splice event is of functional importance. CGM1a mRNA, the predominant CGM1 transcript, was found in the granulocytic lineage, but not in monocytes, lymphocytes nor in a number of tumors derived from all three germ layers. Weak staining using monoclonal antibodies Tu2 and 73 in fluorescence‐activated cell scan analyses indicate low concentrations of CGM1 protein on the surface of granulocytes. The CGM1 protein is also recognized by CD66 antibodies. Therefore, the granulocyte‐specific CD66 epitope is present on at least four CEA family members: CGM1, CEA, NCA‐50/90 and NCA‐160.
Does Escherichia coli possess a second citrate synthase gene?
Authors: PATTON Amanda J. , HOUGH David W. , TOWNER Paul , DANSON Michael J.
Published: March 2005

Escherichia coli possesses a hexameric citrate synthase that exhibits allosteric kinetics and regulatory sensitivity, and for which the gene (gltA) has previously been cloned and sequenced. A citrate‐synthase‐deficient strain of E. coli (K114) has been mutated to generate a revertant (K114r4) that produces a dimeric citrate synthase with altered kinetic and regulatory properties. On cloning and sequencing the gltA gene from both K114 and K114r4, a single mutation was found that caused the replacement of Asp362 with Asn. Asp362 has been previously shown to be a catalytically essential residue in E. coli citrate synthase, and we demonstrate that the hexameric enzyme produced on expression of the gltA gene from K114 and K114r4 is inactive. The dimeric citrate synthase from K114r4 has been purified and shown to be immunologically distinct from the wild‐type hexameric enzyme. Determination of its N‐terminal amino acid sequence demonstrates that the mutant citrate synthase is encoded by a gene distinct from the E. coli gltA gene. The N‐terminal sequence is compared with those of other eukaryotic, eubacterial and archaebacterial citrate synthases.
Characterization of macrophage proteins bearing the functional leukotriene D4‐binding site of an anti‐cysteinylleukotriene monoclonal antibody
Authors: Van HILTEN Joost A. , BRUNE Kay , BICKEL Daniela , MOLLENHAUER Jürgen
Published: March 2005

Protein conformations of the putative cysteinylleukotriene (LT) receptor of macrophages were characterized using anti‐idiotypic IgG (AIAb) against an anti‐LT monoclonal Ab (LTmAb). The AIAb nature of two rabbit antisera were demonstrated with titers of up to 1: 1000 against F(ab')2 from the LTmAb (in an enzyme‐linked immunoassay) which also inhibit LTD4 binding to the LTmAb (in a radioimmunoassay), whereas non‐immunized rabbit serum was not reactive. The specific reactivity of Fc‐purified AIAb towards LTmAb was measured by two fractions obtained after passage over columns of Sepharose either coupled with LTmAb (fraction A, representing immunoglobulins not absorbed to LTmAb) or coupled with homologous immunoglobins (fraction B, representing immunoglobulins not absorbed to homologous IgG). The difference in immunoreactivity between both fractions showed that fraction B contains AIAb against a LT‐recognizing domain of the LTmAb (in enzyme‐linked immunoassays coated with LTmAb and homologous IgG) and AIAb against the functional LT‐binding site of LTmAb (in radioimmunoassay).

Using the antisera, Western‐blot analysis with peritoneal cell proteins detected signals at 236, 198, 118, 99, 75, 25 and 18kDa. Dithiothreitol‐reduced proteins were detected at 25kDa and 18 kDa. In general, this suggested recognition of a 236‐kDa oligomeric protein composed of subunits with molecular masses of 25kDa and 18 kDa, including intramolecular disulfide bridges all bearing an epitope similar to the LTmAb. From these conformations, an overlay assay with [3H]LTD4 favoured a 75‐kDa protein. Immunohistochemical analysis demonstrated that the recognized proteins may be located at cell membranes, because (a) in an ELISA, enriched plasma membrane preparations from peritoneal cells showed a threefold increase in reactivity to the AIAb, compared to the original cell homogenate; (b) after Western‐blot analysis, the membrane‐enriched protein fraction exhibited stronger protein signals than the microsomal fraction and the original cell homogenate; (c) regions of AIAb binding on the surface of cultured mouse peritoneal macrophages were detected by indirect immunofluorescence. Taken together, this study demonstrated AIAb binding to macrophage membrane‐associated proteins bearing the LTD4‐binding site of LTmAb, which may include identification of the putative LT receptor.
Molecular characterization of Limulus Polyphemus C‐reactive protein
Authors: AMATAYAKUL‐CHANTLER Supavadee , DWEK Raymond A. , TENNENT Glenys A. , PEPYS Mark B. , RADEMACHER Thomas W.
Published: March 2005

The N‐linked oligosaccharides of C‐reactive protein (CRP) from the arachnid Limulus polyphemus, the horsehoe crab, were characterized after their release by hydrazinolysis, re‐N‐acetylation, and reduction with NaB3H4. High‐voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel‐permeation chromatography on Bio‐Gel P4 yielded five fractions. The oligosaccharide fractions were further fractionated using high‐voltage borate paper electrophoresis and Dionex BioLC ion‐exchange chromatography. The oligosaccharides were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis and methylation analysis. Three major structures were found, of which two were the biantennary oligomannose type with compositions Man5GlcNAc2 (B‐1), Man4GlcNAc2 (C‐3) and one was the monoantennary structure Man3GlcNAc2 (d‐1). The biantennary oligomannose structures B‐1 and C‐3 contained the structural unit Manα6Manα6R. This unusual arrangement of mannose linkages suggests a biosynthetic pathway in Limulus which differs from that reported in mammals, plants and the parasitic protozoa.
Purification and partial amino acid sequence of the actin‐fragmin kinase from Physarum polycephalum
Authors: GETTEMANS Jan , DE VILLE Yvette , VANDEKERCKHOVE Joel , WAELKENS Etienne
Published: March 2005

The 80‐kDa actin‐fragmin kinase (AFK) was purified from Physarum polycephalum microplasmodia to apparent homogeneity through a procedure involving six chromatographic steps. Taking the activity at the first purification step as 100%, the kinase was purified more than 1500‐fold, with an overall yield of 8%. The specific activity of the purified enzyme was 700 U/mg. The total amount of AFK present could be estimated as 34 ng/mg extracted protein. One of the polyclonal antibodies raised against four tryptic peptides of the purified 80‐kDa AFK recognized the 80‐kDa band in an immunoblot and could inhibit the AFK activity up to 100%. A minor 78‐kDa protein, also displaying AFK activity, appeared to be derived from the 80‐kDa AFK. A partial amino acid sequence analysis, covering up to 20% of the protein (150 amino acids), was perfomed and confirmed the lack of similarity with any of the sequenced kinases. These data are in agreement with the unique physical and enzymatic properties of the AFK.