bluuebunny/crossref_metadata_2025 huggingface dataset:
Energised (entatic) States of Groups and of Secondary Structures in Proteins and Metalloproteins
Authors: Williams Robert J. P.
Published: September 2004

In this review I have examined the functional value of selected states of isolated groups in proteins energised away from their expected ground states whether they are observed with or without energy perturbation of larger parts of a protein structure. These energisations, found in the absence of substrates, are called ‘entatic states of groups’ [Vallee, B. L. & Williams, R. J. P. (1968) Proc. Natl Acad. Sci. USA 59, 498–505]. A group can be part of an amino acid or any bound metal ion or cofactor. In some particular cases the apoprotein, where the energised metal ion or cofactor has been removed, or the protein in which the energised amino acid has been replaced, has the same back‐bone structure as the holoprotein and even side‐chains are only slightly adjusted. This case is quite different from a condition of a group simultaneously energised with a protein fold, due to their combination, and which therefore involves conformational change in the protein and the group and which may adjust the group while the protein tightens. The final condition is again stable but removal of the group now must result in a reversed protein conformation change. This simultaneous energisation of both the adjustable protein and the group can be local, as in an induced fit or more extensive when it can be likened in some cases to a stretching by rack action, or may involve a change from an almost random to a structured protein when the group is energised in a very limited way. The various energisations must not be confused since they differ functionally. The first can give rise to optimal heightened catalytic (or other functional) potential of the local group but cannot be connected either to excitation of other parts of a protein as in induced fitting, or to a relay of energy (larger conformational change) in the protein. Clearly it restricts the rate of exchange of a group. Induced fit can also give rise to group activation, though to a somewhat reduced degree, while increasing exchange rate. A device such as a rack may rather give rise to a mechanical activity (message transmission), which is relayed a large distance into the protein, and can only give considerably lower activation of individual groups but exchange may now be fast. The final case involves very modest energisation of the group with gross rearrangement and energisation of the protein and may be associated with storage or carrier functions. The groups upon which I concentrate are metal ions since detailed electronic and structural knowledge of their ground states are well known, allowing energised states to be easily detected, but the ideas apply equally to organic side‐chains of proteins as will be shown. A further energisation can arise from the addition of a substrate to each kind of protein. In fact all the ideas of energisation applicable to groups having cyclic activity in permanent features of protein structure are equally well applied to substrate binding or conversion of substrates through excited stales to products.
Thermostability of a Nuclear‐Targeted Luciferase Expressed in Mammalian Cells
Authors: Michels Annemieke A. , Nguyen Van Trung , Konings Antonius W. T. , Kampinga Harm H. , Bensaude Olivier
Published: September 2004

Protein denaturation and aggregation are most likely the cause for the noxious effects of heat shock. There are some indications that the nucleus is one of the most sensitive cellular compartments. To test the possibility that the intranuclear microenvironment might be detrimental to the heat stability of proteins, we compared the in situ thermal stability of a reporter protein localized in the nucleus or in the cytoplasm. A recombinant firefly (Photynus pyralis) luciferase carrying a point mutation in the C‐terminal domain remains in the cytoplasm (cyt‐luciferase). A nuclear localization sequence was fused to the N‐terminal domain of cyt‐luciferase; the resulting nuc‐luciferase was efficiently targeted to the cell nucleus.

In both cases, decreased luciferase activity and solubility were found in lysates from heat‐shocked cells. These characteristics were taken as an indication of thermal denaturation in situ. The heat‐inactivated luciferases were partially reactivated during recovery after stress, indicating the capacity of both the cytoplasmic and nuclear compartments to reassemble proteins from an aggregated state.

Although both the nuc‐ and the cyt‐luciferases were heat inactivated at similar rates in vitro, nucluciferase was more susceptible to thermal denaturation in situ compared to cyt‐luciferase. This observation suggests that the microenvironment of an intracellular compartment may modulate the thermal stability of proteins. The local concentration might be one element of this microenvironment affecting the heat‐stability of proteins.

In cells made thermotolerant by a priming shock, the thermal inactivation of the recombinant luciferases occurred at a slower rate during a second challenging stress. However, this decreased thermal sensitivity was less pronounced for the nuc‐luciferase (threefold) than for the cyt‐luciferase (sevenfold). The nuclear luciferase might become a useful tool to investigate the action of molecular chaperones in the nucleus.
Fatty Acids and Fibrates are Potent Inducers of Transcription of the Phosphenol pyruvate Carboxykinase Gene in Adipocytes
Authors: Antras‐Ferry Jocelyne , Robin Pierre , Robin Danielle , Forest Claude
Published: September 2004

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) plays a critical role in adipose tissue glyceroneogenesis. We have previously shown that transcription of the PEPCK gene was stimulated by isoprenaline and retinoic acid in 3T3‐F442A adipocytes. We also showed that oleate increased PEPCK mRNA. Here, we analysed the effect that fatty acids of various chain lengths and unsaturation degrees exerted on PEPCK gene expression in 3T3‐F442A adipocytes. When maintained in serum‐free, glucose‐free medium, differentiated cells responded to unsaturated long‐chain fatty acids by a large increase in PEPCK mRNA whereas saturated fatty acids were inefficient. A maximum fivefold stimulation by oleate was attained at 4h of treatment with 1 mM fatty acid bound to albumin in a 6:1 ratio. The poly‐unsaturated very long‐chain fatty acid all‐cis‐4,7,10,13,16,19‐docosahexaenoic acid (C22:6) was even more potent and produced a tenfold increase. The expression of the genes encoding glycerol‐3‐phosphate dehydrogenase, hormone‐sensitive lipase or actin remained unaffected by oleate exposure. A 4‐h treatment by the hypolipidemic drug clofibrate, 0.5–2 mM, also produced a large (3–9‐fold) increase in PEPCK mRNA. When used at non‐saturating concentrations, oleate and clofibrate acted in an additive manner. At maximally effective concentrations, additivity was lost, suggesting that fatty acids and fibrates might act through similar mechanisms. Nuclear transcription experiments showed that oleate and clofibrate stimulated the transcription rate of the gene. 3T3‐F442A cells were stably transfected with a plasmid containing the base pairs–2100 to +69 of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase gene. These differentiated stable transfectants responded to oleate and clofibrate by a specific increase in chloramphenicol acetyltransferase activity. Adipocytes express various isoforms of peroxisome‐proliferator‐activated receptors that can be activated by fibrates and fatty acids. Potential recognition sequences for peroxisome‐proliferator‐activated receptors are present in the–2100 to +69 fragment of the PEPCK gene promoter. Thus, this gene represents an ideal molecular target for understanding the complex transcriptional control exerted by fatty acids and peroxisome proliferators.
Human Monoclonal Antibodies to Cytomegalovirus
Authors: Böldicke Thomas , Haase Bernd , Böcher Michael , Lindenmaier Werner
Published: September 2004

Human monoclonal antibodies (mAb) to human cytomegalovirus (HCMV) were established from spleen cells of a HCMV‐positive donor. The antibodies (γ3, λ) secreted from a stable heterohybridoma cell line were further characterized by immunoprecipitation and immune‐fluorescence microscopy using HCMV infected cells and recombinant cell lines expressing HCMV glycoprotein B. The antibody reacted with the entire glycoprotein B or the extracellular domain expressed as glycoprotein‐B‐β‐galactosidase fusion protein in the native state, but the antibody was not neutralizing HCMV. Denatured and reduced forms of glycoprotein B were not recognized by this antibody, however, native glycoprotein B on the surface of infected cells was detected efficiently. The genes encoding the Fab part of the antibody were cloned and expressed in Escherichia coli, Recombinant Fab fragments specifically binding the extracellular domain of glycoprotein B could easily be isolated from the periplasmic space. Recombinant antibodies provide the opportunity to modify effector functions and to add tags to diagnostic antibodies for more efficient detection of CMV‐infected cells.
Characterization of the cDNA and Pattern of Expression of a New Gene Over‐Expressed in Human Hepatomas and Colonic Tumors
Authors: Charrasse Sophie , Mazel Martine , Taviaux Sylvie , Berta Philippe , Chow Terry , Larroque Christian
Published: September 2004

A cDNA clone of 6.449 kb ch‐TOG (for colonic and hepatic tumor over‐expressed gene) initially selected from various human libraries and completed by 5′ rapid amplification of cDNA ends (RACE) PCR is described. The original cDNA clone was extracted from an expression library constructed from a human tumoral brain. This library was screened with an antibody raised against the cytochrome P 450tu that was shown to be over‐expressed in chemically induced mouse hepatic tumors. Using this cDNA as a probe, a full‐length cDNA was characterized. Its nucleotide sequence shows no significant similarity with any of the gene sequences collected in the various DNA data bases. The translation of the larger open reading frame leads to a putative protein of 1972 amino acids (molecular mass = 218453 Da). Hybridization analyses on Southern blot and on metaphase chromosomes indicate that this gene is present as a single copy in the genome and is localized on the short arm of chromosome 11. ch‐TOG transcripts are present in several human tissues. Over‐expression of ch‐TOG in neoplastic liver and colon compared with the corresponding normal corresponding tissues is demonstrated. The level of the expression of ch‐TOG transcripts was also studied in the various differentiation stages of the human colonic adenocarcinoma cell line Caco‐2.
Post‐Translational Heterocyclic Backbone Modifications in the 43‐Peptide Antibiotic Microcin B17
Authors: Bayer Anja , Freund Stefan , Jung Günther
Published: September 2004

Microcin B17 (McB17), the first known gyrase inhibitor of peptidic nature, is produced by ribosomal synthesis and post‐translational modification of the 69‐residue precursor protein by an Escherichia coli strain. To elucidate the chemical structure of the mature 43‐residue peptide antibiotic, fermentation and purification protocols were established and optimized which allowed the isolation and purification of substantial amounts of highly pure McB17 (non‐labelled, 15N‐labelled and 13C/15N‐labelled peptide. By ultraviolet‐absorption spectroscopy, HPLC‐electrospray mass spectrometry and GC‐mass spectrometry, amino acid analysis, protein sequencing, and, in particular, multidimensional NMR, we could demonstrate and unequivocally prove that the enzymic modification of the precursor backbone at Gly‐Cys and Gly‐Ser segments leads to the formation of 2‐aminomethylthiazole‐4‐carboxylic acid and 2‐aminomethyl‐oxazole‐4‐carboxylic acid, respectively. In addition, two bicyclic modifications 2‐(2‐aminomethyl‐oxazolyl)thiazole‐4‐carboxylic acid and 2‐(2‐aminomethylthiazolyl)oxazole‐4‐carboxylic acid were found that consist of directly linked thiazole and oxazole rings derived from one Gly‐Ser‐Cys and one Gly‐Cys‐Ser segment. Analogous to the thiazole and oxazole rings found in antitumor peptides of microbial and marine origin, these heteroaromatic ring systems of McB17 presumably play an important role in its gyrase‐inhibiting activity, e.g. interacting with the DNA to trap the covalent protein‐DNA intermediate of the breakage‐reunion reaction of the gyrase.
The Handedness of the Subunit Arrangement of the Nicotinic Acetylcholine Receptor from Torpedo californica
Authors: Machold Jan , Weise Christoph , Utkin Yuri , Tsetlin Victor , Hucho Ferdinand
Published: September 2004

Cross‐linking an α‐neurotoxin with a known three‐dimensional structure and with photoactivatable groups in known positions to native membrane‐bound acetylcholine receptor reveals its quaternary structure, including the handedness of its circular subunit arrangement. Photolabelling with α‐neurotoxin carrying the photoactivatable group at position Lys46 is inhibited by the competitive antagonist (+)‐tubocurarine in a biphasic manner, indicating that it reacts with both α‐subunits that were shown to have different affinities for this antagonist [Neubig, R. R. & Cohen, J. B. (1979) Biochemistry 18, 5464–5475]. Lys46 is located on loop III of the neurotoxin. The other information necessary for the elucidation of the handedness was provided by the recent finding that the central loop of the toxin (loop II) is orientated towards the central pore of the receptor, securing the overall orientation of the bound toxin [Machold, J., Utkin, Y. N., Kirsch, D., Kaufmann, R., Tsetlin, V. & Hucho, F. (1995b) Proc. Natl Acad. Sci. USA 92, 7282–7286].

Looking at the receptor from the synaptic side of the postsynaptic membrane, it was concluded that the clockwise subunit arrangement is αH‐γ‐αL‐δ‐β (αH and αL, are the α‐subunits binding (+)‐tubocurarine with high and low affinity, respectively). Its mirror image αL‐γ‐αH‐β‐δ‐could thus be excluded.
Structure/Function Relationships in the Hemoglobin Components from Moray (Muraena Helena)
Authors: Pellegrini Mariagiuseppina , Giardina Bruno , Olianas Alessandra , Sanna Maria Teresa , Deiana Anna Maria , Salvadori Susanna , Di Prisco Guido , Tamburrini Maurizio , Corda Marcella
Published: September 2004

Concerning the number and type of the hemoglobin components, the moray Muraena helena is characterized by three different phenotypes whose frequencies are nearly identical. Thus, the cathodal component is present in all individuals, whereas one or both of two anodal components may be present in the same phenotype. These components have been separated by chromatography.

The oxygen binding properties of the purified hemoglobin components have been studied in the absence and presence of saturating concentrations of ATP or GTP and as a function of pH. The cathodal component shows an intrinsic O2 affinity four times higher than that of both anodal components, a very small Bohr effect and a significant decrease in O2 affinity upon addition of ATP and GTP (three and four times respectively with respect to stripped conditions), the latter being more effective than the former over the entire pH range examined. The anodal components do not appear functionally distinguishable and show the presence of an enhanced Bohr effect (Root effect) that is under the strict control of nucleotide triphosphates ATP, GTP, which, unlike in the cathodic component, exert the same effect on oxygen affinity. The complete sequence of the β chains of the cathodal and of one of the anodal components have been determined.

The possible molecular basis of these different functional characteristics are discussed in the light of the globin sequence and of those amino acid residues which are known to be responsible of hemoglobin functional behaviour.
The Sequence of Arrestins from Rod and Cone Photoreceptors in the Frogs Rana catesbeiana and Rana pipiens
Authors: Abdulaeva Galina , Hargrave Paul A. , Smith W. Clay
Published: September 2004

Members of the arrestin protein family are known to participate in the inactivation of rhodopsin and other heptahelical receptors. Arrestins bind to the activated and phosphorylated state of these receptors, consequently blocking the ability of the receptors to activate the guanine‐nucleotide‐binding protein (G protein). We have determined the sequences of four retinal arrestins from two species of frog, Rana catesbeiana and Rana pipiens. Using polymerase chain reaction on reverse‐transcribed mRNA isolated from single photoreceptor cells, we show that two of these arrestins are from rod photoreceptors and two are from cone photoreceptors. Comparison of these arrestins with the twenty known arrestin sequences identifies three regions of the protein that are well conserved across all phylogenetic groups. These regions may function in the binding of the arrestin to the heptahelical receptors. In addition, the Rana arrestins contain a uniquely acidic C‐terminal sequence.
Purification and Two‐Dimensional Crystallization of Bacterial Cytochrome Oxidases
Authors: Warne Antony , Wang Da Neng , Saraste Matti
Published: September 2004

A novel strategy which employes chromatography on an immobilized metal ion has been developed for the purification of bacterial cytochrome c and quinol oxidases. Many bacterial oxidase complexes appear to have a natural affinity to bind to the chelated copper ion. A combination of three different chromatographic principles (anion exchange, metal‐affinity and gel filtration) makes an effective tool chest for the preparation of homogeneous and protein‐chemically pure bacterial oxidases. These preparations have been used for two‐dimensional crystallization. Until now, crystals have been obtained using the Paracoccus denitrificans and Rhodobacter sphaeroides cytochrome aa3, and the Escherichia coli cytochrome bo. The crystals diffract to approximately 2.5 nm in negative stain and have potential for further structural studies.
Purification and Properties of NAD(P)H:(quinone‐acceptor) Oxidoreductase of Sugarbeet Cells
Authors: Trost Paolo , Bonora Patrizia , Scagliarini Sandra , Pupillo Paolo
Published: September 2004

NAD(P)H:(quinone‐acceptor) oxidoreductase [NAD(P)H‐QR], a plant, cytosolic protein, was purified from cultured sugarbeet cells by a combination of ammonium sulfate fractionation, FPLC Superdex 200 gel filtration, Q‐Sepharose anion‐exchange chromatography, and a final Blue Sepharose CL‐6B affinity chromatography with an NADPH gradient. The subunit molecular mass is 24 kDa and the active protein (94 kDa) is a tetramer. The isoelectric point is 4.9. The enzyme was characterized by ping‐pong kinetics and extremely elevated catalytic capacity. It prefers NADPH over NADH as electron donor (Kcat/Km ratios of 1.7×108 M−1 s−1 and 8.3×107M−1 s−1 for NADPH and NADH, respectively, with benzoquinone as electron acceptor). The acridone derivative 7‐iodo‐acridone‐4‐carboxylic acid is an efficient inhibitor (I0.5= 5×10−5M), dicumarol is weakly inhibitory. The best acceptor substrates are hydrophilic, short‐chain quinones such as ubiquinone‐0 (Q‐0), benzoquinone and menadione, followed by duroquinone and ferricyanide, whereas hydrophobic quinones, cytochrome c and oxygen are reduced at negligible rates at best. Quinone acceptors are reduced by a two‐electron reaction with no apparent release of free semiquinonic intermediates. This and the above properties suggest some relationship of NAD(P)H‐QR to DT‐diaphorase, an animal flavoprotein which, however, has distinct structural properties and is strongly inhibited by dicumarol. It is proposed that NAD(P)H‐QR by scavenging unreduced quinones and making them prone to conjugation may act in plant tissues as a functional equivalent of DT‐diaphorase.
Pigment‐Protein Complexes from the Photosynthetic Membrane of the Cyanobacterium Synechocystis sp. PCC 6803
Authors: Barbato Roberto , De Laureto Patrizia Polverino , Rigoni Fernanda , De Martini Elena , Giacometti Giorgio M.
Published: September 2004

Photosystem I and II core complexes were resolved in a single step from the thylakoid membrane of Synechocystis sp. PCC 6803 by using a mild solubilization procedure in dodecyl β‐d‐maltoside and Deriphat/PAGE. For each photosystem, two green bands were obtained containing oligomeric and monomeric forms of the core complexes of either photosystem. The oligomers are likely to be trimers in the case of photosystem I and dimers for photosystem II. The absorption spectra, polypeptide and pigment composition of green bands corresponding to either photosystem I or photosystem II were identical for monomeric and oligomeric forms. The cytochrome b‐559 content of photosystem II was evaluated to be one cytochrome b‐559/reaction centre both in the monomeric and dimeric forms. Two new 15‐kDa and 22‐kDa carotenoid‐binding proteins were isolated and their polypeptides purified to homogeneity.
The Mechanism of Action of Hepatic Sympathetic Nerves on Ketone‐Body Output from Perfused Rat Liver
Authors: Yamamoto Tetsuya , Iwai Masaru , Kimura Shigeru , Shimazu Takashi
Published: September 2004

The regulatory mechanism of ketone‐body output by the hepatic sympathetic nerves was studied in rat liver perfused in situ. Enrichment of the perfusion medium with 1 mM octanoate increased the basal ketone‐body output from the liver up to 1.5 μmol · min−1· g liver−1. Under these conditions, electrical stimulation of the hepatic nerves (20 V, 20 Hz, 2 ms) decreased the output of both acetoacetate and β‐hydroxybutyrate, and was accompanied by an accumulation of β‐hydroxybutyrate in the liver. The effects of nerve stimulation were inhibited by the α1‐antagonist bunazosin (10 μM). However, noradrenaline, a typical sympathetic neurotransmitter, at a concentration of 1 μM decreased the output of acetoacetate but did not affect β‐hydroxybutyrate output. Prostaglandin F2α, at a concentration of 10 μM produced an effect similar to treatment with noradrenaline, without a decrease in β‐hydroxybutyrate output. ATP at 50 μM, however, decreased the output of both acetoacetate and β‐hydroxybutyrate and increased the tissue concentration of β‐hydroxybutyrate, mimicking the effect of nerve stimulation. Moreover, in the presence of 0.2 μM ATP, a concentration that produced neither metabolic nor hemodynamic changes, noradrenaline (1 μM) was shown to decrease the β‐hydroxybutyrate output. These results indicate the possible involvement of ATP in the action of hepatic sympathetic nerves on β‐hydroxybutyrate output from the liver, presumably through its interaction with noradrenaline.
Inhibition of Human Glutathione Reductase by S‐nitrosoglutathione
Authors: Becker Katja , Gui Ming , Schirmer R. Heiner
Published: September 2004

S‐Nitrosoglutathione (GSNO) represents a major transport form of nitric oxide (NO) in biological systems. Since NO and GSNO have been shown to modulate the function of various proteins, we studied the influence of GSNO and other NO donors on human glutathione reductase (GR). Catalyzing the reaction NADPH + GSSG + H+→ NADP++ 2 GSH, the dimeric flavoprotein GR is the central enzyme of the glutathione redox metabolism.

GSNO was found to inhibit crystalline erythrocyte GR in two ways: (a) as a reversible inhibitor GSNO is competitive with glutathione disulfide (GSSG), the Ki being appr. 0.5 mM; (b) as an irreversible inhibitor; after 1 h (3 h) incubation with 1 mM GSNO, GR (2.5 U/ml, representing intraerythrocytic concentrations) was inhibited by 70% (90%). This inhibition depended on the presence of NADPH and could not be reversed by dilution nor by reducing agents. Absorption spectra indicate that the charge‐transfer interaction between Cys63 and the flavin is abolished by this modification. In a GR sample inhibited by 90% with GSNO, the Km values for the substrates GSSG and NADPH were not significantly changed nor did the modification induce oxidase activity of the enzyme.

GSNO was found not to be a substrate in the forward reaction of GR. This implies that GSNO is not accounted for by methods which employ GR for determining total glutathione.

Incubating isolated GR for 60 min with other NO donors, namely 1 mM sodium nitroprusside or 1 mM S‐nitroso‐N‐acetyl‐dl‐penicillamine (SNAP), resulted in only 25% and 10% inhibition, respectively. This attests to a specific affinity of GSNO to the enzyme.

GSNO inhibition patterns comparable to purified authentic GR were obtained for purified recombinant GR, a GR mutant lacking the 15 N‐terminal amino acids including Cys2, and for the enzyme present in diluted fresh haemolysates (0.02 U/ml); in concentrated haemolysates the inhibition was less pronounced. GR of intact erythrocytes was not affected when exposed to GSNO in the medium.

Our results suggest that the irreversible inhibition of GR by GSNO involves nitrosylation of Cys63 and/or Cys58 at the catalytic site of the enzyme. To further investigate the mechanism of inactivation we have crystallized GSNO‐modified GR for X‐ray diffraction analysis.
Basic Fibroblast Growth Factor Controls the Expression and Molecular Structure of Heparan Sulfate in Corneal Endothelial Cells
Authors: Schmidt Annette , Skaletz‐Rorowski Adriane , Buddecke Eckhart
Published: September 2004

Cultured bovine corneal endothelial cells express 5–8 ng basic fibroblast growth factor (bFGF)/mg cell protein and distribute it between the intracellular and pericellular compartment. Confluent cultures retain approximately 80% of the total bFGF intracellularly, whereas 20% is present in the pericellular (trypsin‐releasable) compartment. No bFGF can be detected in the culture medium.

The presence of 1–2 ng/ml medium of endogenous or exogenous (human recombinant) bFGF is sufficient to support cell growth. Simultaneously, cells incorporate [35S]sulfate and [3H]glucosamine into the sulfated proteoglycans associated with the cell layer at a rate that is three times higher than in the absence of bFGF.

The enhanced proteoglycan synthesis is accompanied by a shift in proteoglycan distribution. In control cells, cell‐associated heparan sulfate accounts for about 30% of the total glycosaminoglycans, whereas under the influence of bFGF the amount of heparan sulfate increases to approximately 60%. At the same time, the molecular structure of the heparan sulfate molecule undergoes bFGF‐specific changes as indicated by the [35S]oligosaccharide pattern generated by heparitinase I degradation. The proportion of [35S]oligosaccharides with greater than six monosaccharides decreases on account of disaccharides and tetrasaccharides under the influence of bFGF. Pretreatment of bFGF with neutralizing antibodies against bFGF abolishes its biological activity.

The results suggest a bFGF‐dependent change in the rate of synthesis and structural features of the membrane‐associated heparan sulfate in corneal endothelial cells. The modification of the heparan sulfate structure could influence its bFGF‐binding and antiproliferative activity.
Expression of a Biologically Active Murine Tissue Inhibitor of Metalloproteinases‐1 (TIMP‐1) in Baculovirus‐Infected Insect Cells
Authors: Kurschat Peter , Graeve Lutz , Erren Andrea , Gatsios Petros , Rose‐John Stefan , Roeb Elke , Tschesche Harald , Koj Aleksander , Heinrich Peter C.
Published: September 2004

Murine tissue inhibitor of metalloproteinases‐1 (mTIMP‐1) was expressed in baculovirus‐infected insect cells (Sf9). The protein secreted into the culture medium was purified to homogeneity by means of heparin‐Sepharose CL‐6B and FPLC. The purified protein showed metalloproteinase‐inhibitory activity in two independent assays: reverse zymography and inhibition of collagenase activity. Digestion of the recombinant TIMP‐1 with peptide‐N‐glycanaseF revealed that both N‐glycosylation sites are used. 125I‐mTIMP‐1 intraveneously injected into a male Sprague Dawley rat disappeared within 2 min from the circulation. 5 min after injection more than 50% of the 125I‐mTIMP‐1 were found in the liver and 20% in the kidneys. At later times, trichloroacetic‐acid‐soluble material accumulated in the intestinal tract.
Complete Nucleotide Sequence, Expression, and Chromosomal Localisation of Human Mixed‐Lineage Kinase 2
Authors: Dorow Donna S. , Devereux Lisa , Tu Guo‐fen , Price Gareth , Nicholl Jillian K. , Sutherland Grant R. , Simpson Richard J.
Published: September 2004

Protein kinases play pivotal roles in the control of many cellular processes. In a search for protein kinases expressed in human epithelial tumour cells, we discovered two members of a novel protein kinase family [Dorow, D. S., Devereux, L., Dietzsch, E. & de Kretser, T. A. (1993) Eur. J. Biochem. 213, 701–710]. Due to the unique mixture of structural domains within their amino acid sequences, we named the family mixed‐lineage kinases (MLK). We initially isolated clones encoding partial cDNAs of MLK1 and 2 from a human colonic cDNA library. The MLK2 cDNA was subsequently used to screen a human brain cDNA library and we have now cloned and sequenced a 3454‐bp cDNA encoding the full‐length MLK2 protein. The predicted MLK2 polypeptide has 954 amino acids and contains a src homology 3 (SH3) domain, a kinase catalytic domain, a double leucine zipper and basic domain, and a large C‐terminal domain. The 22‐amino‐acid N‐terminal region has four glutamic acid residues immediately following the initiator methionine. Beginning at amino acid 23, the 55‐amino‐acid SH3 domain contains a 5‐amino‐acid insert in a position corresponding to inserts of 6 and 15 residues in the SH3 domains of n‐src and the phosphatidylinositol 3′‐kinase. Adjacent to the SH3 domain is a kinase catalytic domain with conserved motifs associated with both serine/threonine and tyrosine specificity. Beginning nine residues C‐terminal to the catalytic domain, there are two leucine/isoleucine zippers separated by a 13‐amino‐acid spacer sequence and followed by a stretch of basic residues. The polybasic sequence contains a motif that is similar to nuclear localisation signals from several proteins. The C‐terminal domain is composed of 491 amino acids of which 17% are serine or threonine and 16% are proline. This domain also has a biased ratio of basic to acidic amino acids with a calculated pI of 9.38. When used as a probe to examine mRNA expression in human tissues, a MLK2 cDNA hybridised to a species of 3.8 kb that was expressed at highest levels in RNA from brain and skeletal muscle. The 3454‐bp cDNA was also used for fluorescence in situ hybridisation to localise the MLK2 gene to human chromosome 19 q13.2.
The Effect of pH on the Folding and Stability of the Myosin Rod
Authors: Ozog Anne , Béchet Jean‐Jacques
Published: September 2004

The far‐ultraviolet circular dichroism and fluorescence emission intensities of the myosin rod were studied at pH 2–11, in the absence and presence of guanidine hydrochloride. The protein kept its helicity in this pH range. Its stability in the denaturant was higher at acidic pH than at pH 7. This may be due to favorable interactions involving protonated glulamic acid residues at the interface of the two α‐helical chains of the molecule. At alkaline pH, the fluorescence of the myosin rod was quenched, and the tryptophan region of the protein became less stable in the presence of guanidine hydrochloride, due to ionization of tyrosine residues or other amino acids close to tryptophans in the double‐helix arrangement.
A Comparative Study of the Inactivation of Wild‐Type, Recombinant and Two Mutant Horseradish Peroxidase Isoenzymes C by Hydrogen Peroxide and m‐chloroperoxybenzoic Acid
Authors: Hiner Alexander N. P. , Hernández‐Ruíz Josefa , García‐Cánovas Francisco , Smith Andrew T. , Arnao Marino B. , Acosta Manuel
Published: September 2004

The mechanism‐based inactivation of four horseradish peroxidase (HRP‐C) enzyme variants has been studied kinetically with either hydrogen peroxide or the xenobiotic m‐chloroperoxybenzoic acid (m ClO2‐BzOH) as sole substrate. The concentration and time dependence of inactivation was investigated for the wild‐type plant enzyme (HRP‐C), the unglycosylated recombinant enzyme (HRP‐C*), and two site‐directed mutants with Phe143 replaced by Ala ([F143A]HRP‐C*) or Arg38 replaced by Lys ([R38K]HRP‐C*). The number of turnovers (r) of H2O2 required to completely inactivate the enzymes was found to vary between the different enzymes with HRP‐C being most resistant to inactivation (r= 625), HRP‐C* and [F143A]HRP‐C* being approximately twice as sensitive (r= 335 and 385, respectively) in comparison, and [R38K]HRP‐C* being inactivated much more easily (r= 20). In the cases of HRP‐C* and [F143A]HRP‐C*, compared to HRP‐C the differences were due to the absence of glycosylation on the exterior of the proteins, whilst the [R38K]HRP‐C* variant exhibited a distinct mechanistic difference. When m ClO2BzOH was used as the substrate the differences in sensitivity to inactivation disappeared. The values of r were all around 3 reflecting the strong affinity of m ClO2BzOH for the active site. The apparent rate constant for inactivation by H2O2 was found to be about twofold higher in [R38K]HRP‐C* than the other enzymes and the catalytic constant for turnover of H2O2 was approximately ten times lower. The affinity of compound I for H2O2 leading to the formation of a transitory intermediate implicated in the inactivation of peroxidase decreased in the order HRP‐C, HRP‐C*, [F143A]HRP‐C*, [R38K]HRP‐C*.
Receptor‐Mediated Regulation of Leukotriene C4 Synthase Activity in Human Platelets
Authors: Tornhamre Susanne , Edenius Charlotte , Lindgren Jan Åke
Published: September 2004

Human platelets possess a specific membrane‐bound leukotriene (LT) C4 synthase, which catalyzes the conversion of LTA4 to LTC4. Stimulation of the receptors for thrombin, collagen or thromboxane A2 provoked inhibition of this enzyme, as judged by suppressed transformation of exogenous LTA4 to LTC4. Similarly, direct activation of protein kinase (PK) C with nanomolar concentrations of 4β‐phorbol 12‐myristate 13‐acetate (PMA) inhibited the production of LTC4. Kinetic studies demonstrated that the inhibition induced by thrombin and PMA was non‐competitive. Elevation of intracellular cAMP levels with carbacyclin did not affect basal LTC4 formation, but abolished the attenuation of platelet LTC4 synthase activity induced by the thromboxane receptor agonist U‐46619. The unselective protein kinase inhibitor staurosporine prevented both receptor‐mediated and PMA‐induced suppression of LTC4 formation. In contrast, two selective PKC inhibitors, Ro 31–8220 and GF 109203X, reversed the inhibitory effect provoked by PMA, but failed to prevent thrombin‐induced inhibition. Furthermore, the protein tyrosine phosphatase inhibitor, sodium orthovanadate, induced dose‐dependent inhibition of LTC4 production in platelet sonicates. In conclusion, receptor‐mediated activation of human platelets leads to decreased LTC4 synthase activity via phosphoregulation. Although the present results demonstrate that platelet LTC4 synthase can be regulated via PKC‐dependent events, alternative mechanisms appears to be involved in the physiological regulation of this enzyme. The findings suggest the possible importance of protein tyrosine phophorylations in this process.
Ligand Specificity of Pheromone‐Binding Proteins of the Processionary Moth
Authors: Feixas Joan , Prestwich Glenn D. , Guerrero Angel
Published: September 2004

Photoaffinity labeling of proteins extracted from sensory hairs and antennal branches of the processionary moth Thaumetopoea pityocampa with a tritium‐labeled diazoacetate analogue of the sex pheromone (Z)‐13‐hexadecen‐11‐ynyl acetate revealed a 15‐kDa pheromone‐binding protein in male moth sensory hairs (SH‐15). A different 15‐kDa protein in male antennal branches (B‐15) was not photolabeled. All extracts except male sensory hairs showed a photolabeled 20‐kDa protein; a photolabeled male 30‐kDa protein in the branches (B‐30) was also observed. The 20‐kDa proteins in the sensory hairs (SH‐20) and branches (B‐20) showed differing affinities for the photoaffinity analogues; moreover, SH‐15 exhibits higher affinity for the natural pheromone, (Z)‐13‐hexadecen‐11‐ynyl acetate, than for its alcohol metabolite and other analogues in competitive displacement experiments. The affinity shown by the pheromone‐binding protein for the metabolic product suggests that the alcohol may be also transported by the binding protein. Interestingly, a shift in labeling from SH‐15 to SH‐20 was produced in the presence of an excess of the natural pheromone, its alcohol and other analogues. The binding showed little discrimination among structurally similar analogues of the pheromone, while saturated and aromatic molecules showed little affinity for the proteins of either sensory hairs or antennal branches.
A Short Isoform of Carcinoembryonic‐Antigen‐Related Rat Liver Cell‐Cell Adhesion Molecule (C‐CAM/gp110) Mediates Intercellular Adhesion
Authors: Lucka Lothar , Cichocka Iwona , Bäumler Karin , Bechler Karin , Reutter Werner
Published: September 2004

Rat liver cell‐cell adhesion molecule (C‐CAM) is a type I transmembrane glycoprotein belonging to the immunoglobulin (Ig)‐superfamily. Within this family it is related to the carcinoembryonic antigen (CEA) proteins. C‐CAM, previously known as gp110, cell‐CAM 105, HA4/pp120 or ecto‐ATPase, is a highly glycosylated protein with an apparent Mr of 100000–115000 and an isoelectric point of 3–3.5. It was analysed as a molecule that stimulates reaggregation of isolated hepatocytes. So far three different isoforms have been cloned. Only the isoform with a long intracellular tail (71 amino acids), C‐CAM1, was shown to be involved in intercellular adhesion. C‐CAM2, an isoform with only 10 cytoplasmic amino acids and a slightly different N‐terminal Ig‐like loop did not function as an adhesion molecule. In this study we show the existence of another short. C‐CAM isoform (C‐CAM2a), which is an alternatively spliced product of the C‐CAM1 gene. Like C‐CAM2, it has a short cytoplasmic tail, but in the, extracellular region it is identical to C‐CAM1. To investigate whether C‐CAM2a can function as an adhesion molecule, we stably expressed the corresponding cDNA in Chinese hamster ovary (CHO) cells. In these cells, we detected a specific increase of intercellular adhesion, indicating that, in contrast to the other short isoform, C‐CAM2a can induce adhesion. This adhesion is homophilic and Ca2+ independent.
The Role of Membrane Proximal Threonine Residues Conserved among Guanine‐Nucleotide‐Binding‐Protein‐Coupled Receptors in Internalization of the m4 Muscarinic Acetylcholine Receptor
Authors: Van Koppen Chris J. , Lenz Wolfgang , Nunes J. Pedro L. , Zhang Chunyi , Schmidt Martina , Jakobs Karl H.
Published: September 2004

Many guanine‐nucleotide‐binding‐protein‐coupled receptors contain consensus sequences for phosphorylation by cAMP‐dependent protein kinase (PKA), often located in the membrane proximal regions critically important for receptor signalling. In the present study, we have evaluated by site‐directed mutagenesis the role of the putative PKA phosphorylation sites in the m4 muscarinic acetylcholine receptor (mAChR), i.e. Thr145 in the second cytoplasmic loop and Thr399 in the third cytoplasmic loop, and the influence of PKA on m4 mAChR function and internalization. Antagonist binding was unaltered by any of the mutations studied, while the agonist‐binding affinity was either not affected (Thr145 alanine), increased (Thr399 alanine) or decreased (Thr399 serine or aspartic acid). m4 mAChR‐mediated inhibition of adenylyl cyclase was unaltered by the mutations, except for an approximately tenfold reduced agonist potency of the Thr399 aspartic acid mutated receptor. Agonist‐induced receptor internalization was unaltered with Thr399 serine or aspartic acid mutations of the receptors, but was strongly decreased in its rate and extent upon replacement of Thr399, Thr145 or both of these residues with alanine. These mutational effects could not be reproduced by treatment of wild‐type receptor‐expressing cells with the PKA inhibitor H‐8. Furthermore, maximal stimulation of cellular PKA neither affected receptor internalization nor signalling measured as receptor‐mediated Ca2+ mobilization. We conclude that the membrane proximal threonine residues of the m4 mAChR are not required for receptor signalling, but replacement by alanine residues can significantly affect receptor internalization, independently of PKA phosphorylation. Sequence comparisons suggest that threonine residues at corresponding positions may be relevant to internalization of other guanine‐nucleotide‐binding‐protein‐coupled receptors.
Characterization of the Human α1(VI) Collagen Promoter and its Comparison with Human α2(VI) Promoters
Authors: Saitta Biagio , Chu Mon‐Li
Published: September 2004

From a human cosmid library, we isolated a clone (5B) with an insert of 32 kb, encoding the amino‐terminal and the 5′‐end flanking region of the α1(VI) collagen gene. Exon 1 was found to be 194 bp and contain the 5′ untranslated region plus 97 bp coding sequence. Exon 2 consists of 130bp, a size that is conserved across the chicken and mouse species. S1‐nuclease‐protection assays and primer‐extension analysis, using mRNA from human dermal fibroblasts, show the presence of multiple transcription start sites located in a region of ≈20 nucleotides. Canonical TATA and CAAT boxes, as found in the chicken and mouse α1 promoters, were absent in the human α1(VI) promoter. The promoter region from positions–1 to–190, is a polypyrimidine/polypurine‐rich region containing 12 CCCTCCCC (CT element consensus) sequences and has multiple potential binding sites for the Sp1 and AP2 transcription factors. These regulatory proteins bind to the α2(VI) promoters [Saitta, B. & Chu, M.‐L. (1994) Eur. J. Biochem. 223, 675–682]. To test the transcriptional activity of the α1 promoter, transient transfection experiments of the DNA constructs were performed in human dermal fibroblasts and in human fibrosarcoma (HT1080) cell lines. The DNA constructs drive the expression of the chloramphenicol acetyl transferase (CAT) gene. The results show strong CAT activity for the constructs at positions–1700,–298 and–257, while low activity was found for the constructs at positions–4400,–142 and–5 when transfected in fibroblasts. The experiments also identified positive and negative regulatory regions in the α1(VI) promoter CAT constructs when transfected in fibroblasts, but did not identify them in the fibrosarcoma cells.
Phosphorylation of Elongation Factor 1 (EF‐1) by Protein Kinase C Stimulates GDP/GTP‐Exchange Activity
Authors: Peters Holme I. , Chang Yu‐Wen Edith , Traugh Jolinda A.
Published: September 2004

Phosphorylation of the α, β and δ subunits of elongation factor (EF) 1 by protein kinase C results in stimulation of elongation activity up to threefold both in vivo and in vitro [Venema, R. C., Peters, H. I. & Traugh, J. A. (1991) J. Biol. Chem. 266, 11993–11998; Venema, F., C., Peters, H. I. & Traugh, J. A. (1991) J. Biol. Chem. 266, 12574–12580]. The α subunit catalyzes the GTP‐dependent binding of amino‐acyl‐tRNA to the ribosome, while the βγ and δ subunits of EF‐1 catalyze exchange of the residual GDP on EF‐1α for GTP. To determine whether the change in elongation rate following phosphorylation by protein kinase C is due to stimulation of GDP/GTP exchange activity, EF‐1 and EF‐1 · valyl‐tRNA‐synthetase have been purified from rabbit reticulocytes, phosphorylated in vitro by protein kinase C and the effect of phosphorylation on nucleotide‐exchange activity analyzed. The α, β and δ subunits are phosphorylated only on serine, and phosphopeptide maps show distinct phosphopeptides for each subunit. Following quantitative phosphorylation of EF‐1 by protein kinase C on the α, β, and δ subunits, a twofold enhancement of the rate of nucleotide exchange over the non‐phosphorylated controls is observed with EF‐1 and EF‐1 · valyl‐tRNA synthetase. Stimulation of nucleotide exchange results in a two‐fold increase in the formation of EF‐1α· GTP · Phe‐tRNA, leading to an increased rate of binding of Phe‐tRNA to ribosomes. The magnitude of stimulation of the exchange rate is similar to that reported previously for the rate of elongation following phosphorylation of EF‐1 by protein kinase C. Thus, the enhancement of EF‐1 activity in response to 4β‐phorbol 12‐myristate 13‐acetate appears to be due to stimulation of the rate of GDP/GTP exchange following phosphorylation of EF‐1 by protein kinase C.
Cell‐Density‐Dependent Modulation of the Rat Insulin‐Like‐Growth‐Factor‐Binding Protein 2 and its Gene
Authors: Kutoh Eiji , Schwander Jürg , Margot Jean B.
Published: September 2004

The steady‐state level of the rat insulin‐like‐growth‐factor‐binding protein 2 (IGFBP‐2) and insulin‐like‐growth‐factor‐II (IGF‐II) mRNA increased approximately 20‐fold when BRL‐3A cells were cultured at increasingly higher cell densities. This increase could not be accounted for by paracrine or autocrine factors, or by the addition of insulin, IGF‐I, transforming growth factor β (TGF‐β), cAMP or IGFBP‐2 to the culture medium. A reporter gene assay carrying the promoter domain of the IGFBP‐2 gene indicated that the promoter‐dependent IGFBP‐2 transcription is tenfold higher in high‐density cells. The increase in the IGFBP‐2 message was accompanied by an increase in the level of protein in the medium. When confluent BRL‐3A cells were reseeded at low cell density, the IGFBP‐2 mRNA disappeared at a rate significantly faster than in normal conditions. A protein synthesis inhibitor, cycloheximide, was able to prevent the decay of the message observed after the switch from high to low densities. In summary, these findings suggest a regulatory link between cell density and IGFBP‐2.
Crystal Structure of the Transthyretin–Retinoic‐Acid Complex
Authors: Zanotti Giuseppe , D'acunto Maria Rosa , Malpeli Giorgio , Folli Claudia , Berni Rodolfo
Published: September 2004

Retinoids are quite insoluble and chemically unstable compounds in the aqueous medium, such that natural retinoids need to be bound to specific retinoid‐binding proteins to be protected, solubilized and transported in body fluids. All‐trans retinoic acid exhibits a relatively high affinity for thyroxine‐binding transthyretin in vitro and this protein is a good candidate for the transport of retinoic acid administered as pharmacological or antitumor agent. To define structural features essential for the recognition by transthyretin of a ligand which is structurally unrelated to thyroxine, we have cocrystallized human transthyretin with retinoic acid and determined its structure at 0.18‐nm resolution. The retinoid fits into the two chemically identical thyroxine‐binding sites, which are located in the central channel that runs through the tetrameric transthyretin. The cyclohexene ring of the bound retinoid is innermost, occupying the same position of the phenolic ring of the bound 3,3′‐diiodo‐l‐thyronine, whereas the carboxylate group, like the same group of the thyroid hormone, participates in an ionic interaction with the Lys15 side chain at the entrance of the channel. Despite the fact that transthyretin was cocrystallized with all‐trans‐retinoic acid, the isoprene chain of the bound retinoid has been found in a non‐extended conformation. This feature, that allows the carboxylate to orient in a manner suitable for ion‐pair association with the Lys15 side chain, is attributable to the conversion of all‐trans‐retinoic acid into cis‐misomers or folded conformers. It is concluded that the presence, in an essentially hydrophobic molecular core of the appropriate size, of a negatively charged group at the correct position is a crucial requirement for ligand‐transthyretin recognition. Whereas the binding of the ligand has no remarkable consequences for the protein structure, all‐trans‐retinoic acid undergoes structural changes such as to interact favorably with residues present in the thyroxine‐binding sites, resembling roughly the natural ligand.