bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and orientation of the surfactant‐associated protein C in a lipid bilayer
Authors: VANDENBUSSCHE Guy , CLERCX Anne , CURSTEDT Tore , JOHANSSON Jan , JÖRNVALL Hans , RUYSSCHAERT Jean‐Marie
Published: March 2005

The secondary structure of native and depalmitoylated porcine surfactant‐associated protein C (SP‐C) was studied by attenuated total reflection Fourier‐transform infrared spectroscopy. Both forms of procine SP‐C adopt mainly an α‐helical conformation. These two forms of the protein were reconstituted in a lipid bilayer. The insertion of the protein in a membrane is associated with an increase of the α‐helical content. Dichroic measurements show that, in both cases, the long axis of the α‐helix is oriented parallel to the lipid acyl chains.
Differential rates of glycoprotein secretion by isolated rat hepatocytes studied in terms of concanavalin A binding
Authors: POÜS Christian , DRECHOU Anne , ROUZEAU Jean‐Denis , GUIBOURDENCHE Jean , EL MOUJAHED Abderahman , DURAND Geneviève
Published: March 2005

Using a concanavalin‐A‐based method which respects cell function, we have shown that the kinetics of glycoprotein secretion appear to depend on the nature of the oligosaccharide moiety. In 37°C pulse/chase experiments using freshly isolated normal rat hepatocytes, we found that except for transferrin, whose rate of secretion was independent of its concanavalin A reactivity, the secretion of the concanavalin‐A‐retained forms of α1 acid glycoprotein, T‐kininogen, α1 protease inhibitor and α1 inhibitor III was slower than that of the concanavalin‐A‐non‐retained forms. When hepatocytes were incubated at 20°C, secretion was blocked with the accumulation of mainly endoglycosidase‐H‐sensitive forms. The secretion kinetics of the concanavalin‐A‐differentiated forms were still different when the temperature was shifted back to 37°C. The divergence between the secretion rates of the concanavalin‐A‐differentiated forms would appear to be due to a late event in intracellular protein trafficking, which may depend on the sugar content and/or the number of carbohydrate chains of the glycoproteins.
Hormonal regulation of malic enzyme and glucose‐6‐phosphate‐dehydrogenase expression in fetal brown‐adipocyte primary cultures under non‐proliferative conditions
Authors: VALVERDE Angela M. , BENITO Manuel , LORENZO Margarita
Published: March 2005

The expression of malic enzyme and glucose‐6‐phosphate (Glc6P) dehydrogenase was investigated in primary cultures of fetal brown adipocytes after the prolonged presence (6 d or 10 d) of various hormones under non‐proliferative conditions.

The presence of triiodothyronine for 6 d and 10 d resulted in maturation of the triiodothyronine regulatory mechanism of malic‐enzyme expression at the mRNA level. However, triiodothyronine had no effect on Glc6P dehydrogenase expression. Insulin increased malic‐enzyme and Glc6P dehydrogenase expression at the mRNA and protein level after 6 d and 10 d of culture. The joint presence of triiodothyronine and insulin produced an additive effect on malic‐enzyme expression at the mRNA and protein level after 6 d and 10 d of culture, by two independent mechanisms. Noradrenaline prevented the effect at the protein level after 6 d, but not after 10 d, probably due to loss of the β‐adrenergic response of brown adipocytes after prolonged culture. Triiodothyronine overexpressed the Glc6P dehydrogenase mRNA induced by the presence of insulin at 6 d and 10 d of culture. There was no adrenergic regulation of Glc6P dehydrogenase expression in cultured fetal brown adipocytes, regardless of the time of culture.
Effect of protein kinase C activators on the phosphorylation and the surface expression of the CDw50 leukocyte antigen
Authors: LOZANO Francisco , ALBEROLA‐ILA José , PLACES Lourdes , VIVES Jordi
Published: March 2005

The CDw50 antigen is a constitutively non‐phosphorylated leukocyte surface molecule which becomes highly phosphorylated in all the normal and lymphoblastoid cells analyzed (peripheral blood mononuclear cells, Molt 4, CEM, 8402, Namalwa), after stimulation with tumor promoter agents (phorbol 12‐myristate 13‐acetate, phorbol 12,13‐dibutyrate, mezerein). This phosphorylation is rapid (within 1–5 min), dose‐dependent and results in the incorporation of PO43‐ groups on serine residues. Furthermore, the level of CDw50 phosphorylation induced by tumor promoter agents is decreased by the protein kinase C inhibitors staurosporine and 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine. Activation of peripheral lymphocytes with concanavalin A, phytohemagglutinin and cross‐linking of CD3 molecules also induces CDw50 phosphorylation, but the response is delayed and less intense than when tumor promoting agents are used. Treatment with any of the aforementioned agents is not accompanied by quantitative changes in the CDw50 surface expression. We therefore conclude that protein‐kinase‐C‐mediated mechanisms are involved in phosphorylation, but not in regulation of the surface expression of the CDw50 leukocyte antigen.
Interaction of Hoechst 33258 with the minor groove of the A + T‐rich DNA duplex d(GGTAATTACC)2 studied in solution by NMR spectroscopy
Authors: EMBREY Kevin J. , SEARLE Mark S. , CRAIK David J.
Published: March 2005

The interaction of Hoechst 33258 with the minor groove of the A + T‐rich DNA duplex d(GGTAATTACC)2 has been investigated in solution by two‐dimensional 1H‐NMR spectroscopy. Many intermolecular NOEs define the position and orientation of the Hoechst molecule at the centre of the duplex, binding to a single site located within the minor groove of the AATT sequence. This is despite the opportunity for Hoechst 33258 to bind to as many as five unique sites containing the minimum requirement of three consecutive A · T base pairs. All intermolecular NOEs are detected between protons on the concave edge of Hoechst, many with the adenine H2s on the floor of the minor groove. The NOEs are consistent with interproton distances measured within a partially restrained energy‐minimised structure of the complex. The AATT sequence provides the key recognition features for tight binding, including a particularly narrow minor groove, with no evidence for the partial G · C specificity previously proposed to be necessary to accommodate the bulky N‐methylpiperazine ring. While chemical exchange cross‐peaks between symmetry‐related adenine H2s on opposite strands of the duplex identify a very slow rate of dissociation of the drug from the complex at 298 K (exchange rate ∼ 1.2 s−1). Averaging of chemical shifts in the phenyl ring of Hoechst 33258 are indicative of rapid ring‐flipping motions in the bound state.
Characterization of the nuclear proteins binding the CACCC element of a glucocorticoid‐responsive enhancer in the tyrosine aminotransferase gene
Authors: DeVACK Carol , LUPP Barbara , NICHOLS Mark , KOWENZ‐LEUTZ Elisabeth , SCHMID Wolfgang , SCHÜTZ Günther
Published: March 2005

The nuclear proteins which act synergistically with the glucocorticoid receptor to induce transcription of the tyrosine aminotransferase gene include factors recognizing the CACCC element. We have purified and characterized the proteins from rat liver nuclei which bind to the CACCC motif in the glucocorticoid‐inducible enhancer of the gene. Three protein‐DNA complexes (C1, C2, and C3) were detected in band‐shift assays. The protein component of complex C1 also binds a GC motif (a Sp1 binding site) and is recognized by anti‐Sp1 antiserum. The proteins forming complexes C2 and C3 have been purified by DNA‐affinity chromatography and their molecular masses (75–80 kDa and 35–40 kDa, respectively) have been determined by ultraviolet cross‐linking to radiolabelled DNA and SDS/PAGE. The DNA‐affinity‐purified C2 and C3 activities do not bind significantly to the GC motif and are not recognized by anti‐Sp1 antiserum. Methylation interference analysis indicates that the nucleotides of the CACCC element bound by the C2 and C3 proteins correspond to those of the glucocorticoid‐responsive enhancer which are contacted in vivo following glucocorticoid administration. Our data suggest that these proteins contribute to glucocorticoid‐induced transcription of the tyrosine aminotransferase gene.
On the mechanism of energy transfer to Tb3+ ions in proteins
Authors: MARTINI Jean‐Louis , TETREAU Catherine , POCHON François , TOURBEZ Hervé , LENTZ Jean‐Michel , LAVALETTE Daniel
Published: March 2005

Intraprotein energy transfer to terbium ions is widely used for probing distances of calcium sites in proteins. In this work we have performed a time‐resolved study of the sensitized luminescence in elastase using a pulsed laser excitation at 265 nm. Terbium‐sensitized luminescence was found to build‐up within about 150 μs, which indicates that the protein transfers energy at a rate several orders of magnitude slower than expected for a singlet state donor. From the rise time of the signal and from its variation with the oxygen concentration, it can be deduced that 80% of the transfer originates from the first triplet excited state of one unique aromatic residue. From the comparison of protein fluorescence and sensitized terbium luminescence excitation spectra the sensitizer was identified as a tryptophan, presumably Trp141, which is situated only 0.7–0.9 nm away from the Tb site. The results are at variance with the usual assumption that energy is transferred from the first excited singlet state of aromatic residues according to a long‐range dipole–dipole interaction and are more consistent with a short‐distance exchange mechanism.
Expression of the γ‐subunit gene of desulfoviridin‐type dissimilatory sulfite reductase and of the α‐ and β‐subunit genes is not coordinately regulated
Authors: KARKHOFF‐SCHWEIZER RoxAnn R. , BRUSCHI Mireille , VOORDOUW Gerrit
Published: March 2005

It has been shown [Pierik, A. J., Duyvis, M. G., van Helvoort, J. M. L. M., Wolbert, R. B. G. & Hagen, W. R. (1992) Eur. J. Biochem. 205, 111–115] that desulfoviridin, the dissimilatory sulfite reductase of sulfate‐reducing bacteria of the genus Desulfovibrio, contains a third, γ, subunit (11 kDa), in addition to the well‐established α (50 kDa) and β (40 kDa) subunits, and an α2β2γ2 subunit structure has been proposed. Cloning and sequencing of the dsvC gene indicated it to encode a protein of 105 amino acids (11.9 kDa; γ subunit). The finding that the dsvC gene, located on a 3.5‐kb SacII fragment, is transcribed in both Escherichia coli and Desulfovibrio vulgaris as an mRNA of only 400–600 nucleotides, and that both the dsvA and dsvB genes are present on a 7.2‐kb SacII fragment, indicates that dsvC forms a separate transcriptional unit. The steady‐state level of α and β subunits expressed in D. vulgaris Hildenborough cells is rather constant, while that of the γ subunit increased strongly in the stationary growth phase. Biochemical analysis of the purified protein, expressed in E. coli, and library comparison of its sequence, have so far failed to establish the function of γ.
Immunochemical characterisation of a dehydroepiandrosterone sulfotransferase in rats and humans
Authors: SHARP Sheila , BARKER Emma V. , COUGHTRIE Michael W. H. , LOWENSTEIN Pedro R. , HUME Robert
Published: March 2005

A member of the rat liver hydroxysteroid sulfotransferase (ST) enzyme family metabolising dehydroepiandrosterone (DHEA) was purified from female rats and used to raise rabbit polyclonal antibodies. Characterisation of this antibody preparation demonstrated that it was specific for DHEA ST, and recognised a single 30‐kDa protein on immunohistochemical localisation of the protein in female rat liver determined that DHEA ST was distributed homogeneously in the cytoplasm of hepatocytes. Examination of the extrahepatic expression of this protein showed it to be located predominantly in the liver, although a small amount of enzyme activity was found in the kidney which was not apparently subject to the same sex difference as the hepatic activity. Immunological analysis suggested that this activity was not due to the action of DHEA ST, but to another, unidentified ST isozyme The antibody cross‐reacted strongly with adult human liver DHEA ST, recognising a protein of 35 kDa on immunoblotting. Using this antibody preparation, the distribution of DHEA ST in mid‐trimester human fetal tissues was examined, and it was shown that the enzyme is expressed in the adrenal and liver, but not to any significant extent in the kidney or lung. This antibody therefore provides a powerful tool for investigating the function of DHEA ST.
Cloning, sequencing and expression of the gene encoding glucose dehydrogenase from the thermophilic archaeon Thermoplasma acidophilum
Authors: BRIGHT Jeremy R. , BYROM David , DANSON Michael J. , HOUGH David W. , TOWNER Paul
Published: March 2005

The gene encoding glucose dehydrogenase has been identified by Southern analysis of doubly restricted genomic Thermoplasma acidophilum DNA, using two redundant 17‐residue oligonucleotide probes reverse translated from protein N‐terminal sequence data. A 1670‐bp BamH1–EcoR1 restriction fragment was ligated into pUC19 and pUC18 (constructs pTaGDH1 and pTaGDH2, respectively) and cloned in Escherichia coli. The sequence of the whole fragment was determined, and a 1059‐bp open reading frame identified as the gene encoding glucose dehydrogenase. Cellfree extracts from E. coli carrying construct pTaGDH1 displayed glucose dehydrogenase activity indistinguishable from controls, but extracts from cells carrying pTaGDH2 displayed a 600‐fold increase in glucose dehydrogenase activity. For high‐level expression and purification of native protein, the glucose dehydrogenase coding sequence was subcloned into pMEX8. Glucose dehydrogenase purified from E.coli expressing the pMEX8 construct was indistinguishable by SDS/PAGE, N‐terminal amino‐acid sequence and kinetic analysis from the native enzyme purified from Tp. acidophilum. The derived 352‐amino‐acid sequence shows less than 20% identity with the glucose dehydrogenases of Bacillus subtilis and Bacillus megaterium but, by comparison with other eubacterial and eukaryotic dehydrogenase sequences, a protion of its sequence has been tentatively identified as a cofactor‐binding region.
1H‐ and 13C‐NMR investigation of redox‐state‐dependent and temperature‐dependent conformation changes in horse cytochrome c
Authors: TURNER David L. , WILLIAMS Robert J. P.
Published: March 2005

The redox‐state dependent changes in chemical shift, which have been measured for almost 100 CHn groups in the 13C‐NMR spectra of horse cytochrome c [Santos, H., and Turner, D. L. (1992) Eur. J. Biochem. 206, 721–728], have been used to investigate the nature of the redox‐related change in conformation. Apart from the haem and its axial ligands, the shifts are found to be dominated by the electron–nuclear dipolar coupling in the oxidised form, as was the case in 1H‐NMR studies. These pseudocontact shifts are well described by using an empirically determined magnetic susceptibility tensor in conjunction with atomic coordinates for the horse cytochrome c. The groups which fit least well are located in the vicinity of Trp59. Comparison between 1H and 13C shifts and their temperature dependence shows that the differences from expectation based on a single structure for both oxidation states are caused largely by changes in the diamagnetic contribution to the chemical shifts. Since these are different for 1H and 13C resonances they indicate, independently from crystal structure data, some redox‐related movement of the protein under the haem. The significance of these results for understanding electron transfer pathways is discussed. Finally, the temperature dependence of the pseudocontact shifts in the range 30–50°C is shown to be anomalous. Approximately half of the anomalous effect may be attributed to Zeeman mixing of the electronic wavefunctions with a spin‐orbit coupling constant γ– 241 cm−1, while the other half is attributed to thermal expansion of the protein.
Interleukin‐1β‐specific partial agonists defined by site‐directed mutagenesis studies
Authors: GUINET Françoise , GUITTON Jean‐Dominique , GAULT Nathalie , FOLLIARD Françoise , TOUCHET Nathalie , CHEREL Jean‐Michel , CRESPO André , DESTOURBE Armelle , BERTRAND Philippe , DENEFLE Patrice , MAYAUX Jean‐François , BOUSSEAU Anne , DUCHESNE Marc , TERLAIN Bernard , CARTWRIGHT Terence
Published: March 2005

Monocyte‐derived interleukin 1 (IL‐1) mediates a wide range of biological effects including destruction of the cartilage matrix in articular diseases such as rheumatoid and osteoarthritis. To elucidate further the relationships between protein structure and biological activities, we have analyzed the sequence of several IL‐1 polypeptides using the algorithm of Parker, the hydrophobic cluster analysis method and published structural data. This led us to identify several residues that seemed to be strictly topologically conserved, with respect to identifiable secondary structures features, although this was not readily apparent from sequence alignments. We performed site‐directed mutagenesis on some of these conserved residues, as well as on those predicted to occur in external loops of the polypeptide. Human IL‐1β mutant polypeptides were expressed in Escherichia coli in soluble form and purified to homogeneity by anion‐exchange and gel‐filtration chromatography. Their biological effects (binding to EL4–6.1 murine thymocytes, Raji human B cells and rabbit chondrocytes cells, lymphocyte activation, neutral protease induction, proteoglycan degradation and synthesis) have been determined. Among the 20 IL‐1β mutant polypeptides we present here, four showed a markedly reduced activity in cartilage matrix assays without any significant change in their binding to the cartilage matrix cells (chondrocytes). Furthermore, some of these mutants were specific partial agonists of the effects of IL‐1 on connective tissue since they have a low affinity for thymocytes.
Comparison of stability properties of lactate dehydrogenase B4/ɛ‐crystallin from different species
Authors: VOORTER Christina E. M. , WINTJES Liesbeth T. M. , HEINSTRA Pieter W. H. , BLOEMENDAL Hans , DE JONG Wilfried W.
Published: March 2005

ɛ‐Crystallin occurs as an abundant lens protein in many birds and in crocodiles and has been identified as heart‐type lactate dehydrogenase (LDH‐B4). Lens proteins have, due to their longevity and environmental conditions, extraordinary requirements for structural stability. to study lensprotein stability, we compared various parameters of LDH‐B4/ɛ‐crystallin from lens and/or heart of duck, which has abundant amounts of this enzyme in its lenses, and of chicken and pig, which have no ɛ‐crystallin. measuring the thermostability of LDH‐B4 from the different sources, the t50 values (temperature at which 50% of the nezyme activity remains after a 20‐min period) for LDH‐B4 from duck heart, duck lens and chicken heart were all found to be around 76°C, whereas pig heart LDH‐B4 was less thermostable, having a t50 value of 62.5°C. A similar tendency was fond with urea inactivation studies. Plotting the first‐order rate constants obtained from inactivation kinetic plots against ruea concentration, it was clear the LDH‐B4 from pig heart was less stable in urea than the homologous enzymes from duck heart, chicken heart and duck lens. the duck and chicken enzymes were also much more resistant against proteolysis than the porcine enyzme. Therefore, it is concluded that avian LDH‐B4 is structurely more stable than the homologous enzyme in mammals. this greater stability might make it suitable to function as a crystallin, as in duck, but is not necessarily associated with high lens expression, as in chicken.
Neurotrophin‐3 receptors in the developing chicken retina
Authors: RODRÍGUEZ‐TÉBAR Alfredo , de la ROSA Enrique J. , ARRIBAS Angeles
Published: March 2005

Neurotrophin 3 (NT‐3) had specific high‐affinity receptors (HNT‐3R) in the developing chick retina at all ages between embryonic day (E) 4 and E14. The affinity of HNT‐3R for 125I‐NT‐3 did not change with the developmental state. A dissociation constant (kd) of 13 pM was obtained. However, the amount of HNT‐3R appeared to be developmentally regulated; the number of receptors/cell increased from E4 up to E6–7 (coinciding with the main onset of neuronal differentiation), then decreased until E9 and increased again by E12, when all retinal cells were differentiated. Kinetic and cross‐linking experiments showed that HNT‐3R from two prototypical developmental ages, E7 and E14, were different. E7 and E14 HNT‐3R could be distinguished from each other on the basis of different inhibition patterns of 125I‐NT‐3 binding in the presence of nerve growth factor or brain‐derived neurotrophic factor. Chemical cross‐linking of increasing concentrations of 125I‐NT‐3 to its receptors showed (a) one 100‐kDa band corresponding to neurotrophin low‐affinity receptors in both E7 and E14 cells; (b) one 130‐kDa band also present in both E7 and E14 cells. Densitometric measurements showed that this 130‐kDa band behaved as HNT‐3R in E14 cells (kd∼10 pM) but not in E7 cells (kd≥0.2 nM). Furthermore, the 130‐kDa band in both E7 and E14 retinal cells displayed a trk‐like immunoreactivity. Our data show that, in neurons, one particular neurotrophin may induce different actions mediated through distinct and specific receptors.
Isolation and characterization of the rat gene encoding glutamate dehydrogenase
Authors: DAS Atze T. , ARNBERG Annika C. , MALINGRÉ Helga , MOERER Petra , CHARLES Rob , MOORMAN Antoon F. M. , LAMERS Wouter H.
Published: March 2005

The concentration of glutamate dehydrogenase (GDH) varies strongly between different organs and between different regions within organs. To permit further studies on the regulation of GDH expression, we isolated and characterized the rat gene encoding the GDH protein. This gene contains 13 exons and spans approximately 34 kbp. The GDH gene is present as a single, autosomally located copy in the Wistar rat genome, but shows an extensive restriction‐fragment‐length polymorphism for several enzymes. Promoter activity of the 5′‐flanking sequence is shown by transient transfection experiments. The 5′‐flanking sequence contains a TTAAAA sequence at position – 29, instead of a consensus TATA box and, like many other TATA‐less promoters, is characterized by a very high G+C content. In addition, consensus sequences for the binding sites of the transcription factors Sp1 and Zif268 are present in the G+C‐rich upstream region.
Site‐directed mutagenesis and chemical modification of histidine residues on an α‐class chick liver glutathione S‐transferase CL 3‐3
Authors: CHANG Li‐Hsueh , TAM Ming F.
Published: March 2005

Each chick liver glutathione S‐transferase CL 3 subunit contains three histidine residues: His 142, His 158 and His228. CL 3‐3 can be inactivated by treating with diethylpyrocarbonate. The inactivation process is pH dependent and the pKa of the modified residue is 6.4. The second‐order inhibition rate constant is 741 M−1min−1 at pH 7.0. Based on difference‐spectrum and kinetic analysis, inactivation coincides with the modification of one histidine residue. However, hydroxylamine treatment of the diethylpyrocarbonate‐modified enzyme only partially restored the activity (30–50%) of CL 3‐3. By tryptic mapping and amino acid sequence analysis, His228 and Lys14 have been identified as the modified residues.

Mutants with histidine to serine replacement (H142S and H158S) or C‐terminal histidine deletion (des‐H228) were constructed and over‐expressed in Spodoptera frugiperda cells using a baculovirus system. The mutants are enzymically active. Furthermore, the des‐H228 mutant can be inactivated by diethylpyrocarbonate. These results support the conclusion that histidines are not involved in the enzymic mechanism of CL 3‐3.
Importance of the conserved active‐site residues Try7, Glu106 and Thr199 for the catalytic function of human carbonic anhydrase II
Authors: LIANG Zhiwei , XUE Yafeng , BEHRAVAN Gity , JONSSON Bengt‐Harald , LINDSKOG Sven
Published: March 2005

The catalytic mechanism of carbonic anhydrase includes the reaction of zinc‐bound hydroxide ion with the CO2 substrate. This hydroxide ion is part of a hydrogen‐bonded network involving the conserved amino acid residues Thr199, Glu106 and Tyr7. To investigate the functional importance of these residues, a number of site‐specific mutants have been made. Thus, Thr199 has been changed to Ala, Glu106 to Ala, Gla and Asp, and Try7 to Phe. The effects of these mutations on catalyzed CO2 hydration and ester hydrolysis have been measured, as well as the binding of some inhibitors. The results show that the CO2 hydration activity of the mutant with Phe7 is only marginally reduced, whereas the esterase activity is larger than that of unmodified enzyme. It is concluded that Tyr7 is not a functionally required element of the hydrogen‐bonded network. The CO2 hydration activity (kcat as well as kcatlKm) and the esterase activity of the mutant with Ala199 are reduced about 100‐fold. The affinity for the sulfonamide inhibitor dansylamide, is only slightly reduced while the mutant has an enhanced affinity for bicarbonate and the anionic inhibitor, SCN‐. The activities of the mutants with Ala106 and Gln106 are also reduced. The reduction of the esterase activity is about 100‐fold, while kcat for CO2 hydration has decreased by a factor of 1000. The parameter kcatlKm is only about one order of magnitude smaller for these mutants than for the unmodified enzyme. The binding of dansylamide and another sulfonamide inhibitor, acetazolamide, are about 20‐times weaker to the mutant with Gln106 than to unmodified enzyme. These results suggest important roles for Thr199 and Glu106 in carbonic anhydrase catalysis. The mutant with Asp106 is almost fully active suggesting that the structure has undergone a compensatory change to maintain the interaction between residue 106 and Thr199.
Identification of a distinct human high‐density lipoprotein subspecies defined by a lipoprotein‐associated protein, K‐45
Authors: BLATTER Marie‐Claude , JAMES Richard W. , MESSMER Sylvia , BARJA Francisco , POMETTA Daniel
Published: March 2005

In an attempt to provide immunological tools for subfractionation of high‐density lipoproteins (HDL), monoclonal antibodies were raised against HDL complexes. Two clones identified a peptide, provisionally named K‐45 (pI 4.5–4.9; molecular mass 45 kDa, range 42–48 kDa), whose plasma distribution and lipoprotein association were fully characterised. Gel filtration localised the peptide to the HDL region of human plasma where it co‐eluted with apolipoprotein (apo) A‐I, the structural protein of HDL. Complementary studies employing immunoabsorption with anti‐(apo A‐I) antibodies removed 90% of K‐45 from plasma: conversely, anti‐(apo A‐II) antibodies eliminated only 10% of K‐45. Immunoaffinity chromatography on an anti‐(K‐45) column revealed that the peptide was present in a distinct. HDL subspecies containing three major proteins: K‐45, apo A‐I and clusterin or apo J. The lipoprotein nature of the bound fraction was indicated by electron microscopy (diameter 9.6 ± 3.3 nm) and quantification of lipids, the latter showing an unusually high triacyglycerol concentration. Plasma concentrations of K‐45 were positively correlated with apo A‐I and HDL‐cholesterol and negatively correlated with apo B and total cholesterol. Thus, the peptide appears to be linked, directly or indirectly, to processes which give rise to an anti‐atherogenic lipid profile. After completion of the present studies, an N‐terminal sequence identical to that of K‐45 was reported in recently isolated cDNA clones. These clones encode paraxonasc.
In vivo biological activity of retinoids partially correlates to their affinity to recombinant retinoic‐acid receptor α and recombinant‐cellular retinoic‐acid‐binding protein I
Authors: KEIDEL Siegfried , SZARDENINGS Michael , MUELLER Walter H.
Published: March 2005

Several known and some new retinoids were synthesized and their in vivo activity was investigated by an assay, based on induction of alkaline phosphatase in P19 teratocarcinoma cells, human prostate carcinoma cells and primary cultures of neonatal rat heart cells. The assay used in this study was found to be reproducible and useful for rapid screening of retinoids for biological activity. Two newly synthesized compounds exhibit high biological activity. The biological potency of the compounds was compared to their ability to bind to recombinant retinoic‐acid receptor α and to cellular retinoic‐acid‐binding protein I determined by Charsorb‐binding assay. mRNA of both retinoic‐acid‐binding proteins could be detected in the three cell lines investigated. As expected from the number of different retinoic‐acid receptors, the results suggest that retinoids do not need to bind retinoic‐acid receptor α nor cellular retinoic‐acid‐binding protein I in order to exhibit biological activity, but most retinoids investigated show a clear correlation between binding to these proteins and their biological activity.
Isolation of a ripening and wound‐induced cDNA from Cucumis melo L. encoding a protein with homology to the ethylene‐forming enzyme
Authors: BALAGUÉ Claudine , WATSON Colin F. , TURNER Andrew J. , ROUGE Pierre , PICTON Steve , PECH Jean‐Claude , GRIERSON Don
Published: March 2005

A cDNA clone (pMEL1) was isolated from a climacteric melon fruit cDNA library using the tomato ethylene‐forming‐enzyme (EFE) cDNA, pTOM13, as a probe. Northern analysis of RNA isolated from wounded leaf and fruit tissue and from preclimacteric and climacteric pericarp tissue was used to determine the pattern of pMEL1 RNA expression. pMEL1 hybridized to a 1.3‐kb transcript in climacteric fruit and wounded leaf tissue but was undetectable in preclimacteric fruit and unwounded leaves. Maximal expression of pMEL1 RNA occurred in wounded ripe fruit. pMEL1 contained a 1230‐bp insert containing a predicted open reading frame of 318 amino acids and molecular mass of 35.3 kDa. The predicted amino acid sequence of pMEL1 was 73–81% identical to the deduced amino acid sequences of tomato (pTOM13) EFE and EFE‐related genes isolated from tomato and avocado fruit and senescent carnation petals. Genomic Southern analysis indicated that pMEL1 hybridized to a number of genomic fragments consistent with the presence of more than one pMEL1‐related gene in melon. On Western analysis of total protein extracts from ripe tomato and melon fruit, using an antibody raised against tomato EFE (pTOM13) expressed in Escherichia coli, a single 35.5‐kDa protein was detected. A 35‐kDa product translated from in‐vitro transcribed pMEL1 and immunoadsorbed by anti‐EFE serum was very similar in size to the predicted 35.3‐kDa pMEL1 cDNA protein product. These results indicate that pMEL1 may encode an EFE gene involved in ethylene biosynthesis during fruit ripening and may be identical to or share extensive sequence similarity with an EFE expressed in response to tissue wounding.
Glucose‐6‐phosphate dehydrogenase
Authors: JEFFERY Jonathan , PERSSON Bengt , WOOD Irene , BERGMAN Tomas , JEFFERY Rachel , JÖRNVALL Hans
Published: March 2005

The primary structure of glucose‐6‐phosphate dehydrogenase from the yeast Pichia jadinii (formerly Candida utilis) has been determined. It consists of a 495‐residue, N‐terminally acetylated protein chain. The structure shows extensive differences from those of the corresponding mammalian, fruit fly, and bacterial enzymes (52–68% residue non‐identities), but also from that of another yeast, Saccharomyces cerevisiae (38%).

A eubacterial type and a yeast type of glucose‐6‐phosphate dehydrogenase are discerned, in addition to the known mammalian type. They are distinguished from each other, from the mammalian type, and the insect enzyme, on the basis of both specific residues and pattern differences.

The distribution of residues conserved in all forms locates short segments in which identities are closely grouped. Approximately 50% of these segments correspond to predicted turns and appear to mark the principal folds characteristic of the enzyme's tertiary structure. A region in the N‐terminal part of the protein chain has characteristics suggestive of a coenzyme‐binding site, while, in the middle third, another functionally important segment may be related to glucose‐6‐phosphate binding and catalysis.
1H‐NMR investigation of oxidized and reduced high‐potential iron‐sulfur protein from Rhodopseudomonas globiformis
Authors: BERTINI Ivano , CAPOZZI Francesco , LUCHINAT Claudio , PICCIOLI Mario
Published: March 2005

1H one‐dimensional and two‐dimensional NMR spectra have been recorded for the oxidized and reduced forms of the high‐potential iron‐sulfur protein (HiPIP) from Rhodopseudomonas globiformis which has the highest known reduction potential. The spectrum of the oxidized protein is similar to that of Chromatium vinosum and Rhodocyclus gelatinosus HiPIP but different from that of the HiPIP II from Ectothiorhodospira halophila. Surprisingly, site‐specific assignment has shown that in the oxidized protein the distribution of oxidation numbers within the cluster is very similar to that found for E. halophila HiPIP II and different from that of the other two proteins. The spectrum of the reduced species is very similar to that of all other HiPIPs known to date, indicating very similar electronic and geometric structures for the reduced forms. These findings are discussed in terms of cluster structure in HiPIPs and of redox potentials.
An N‐terminal peptide from link protein is rapidly degraded by chondrocytes, monocytes and B cells
Authors: MARTIN Harry , DEAN Michael
Published: March 2005

A peptide cleaved from the link‐protein component of human and pig proteoglycan aggregates by trypsin and stromelysin was taken up and degraded further by human monocytes, B cells, chondrocytes and by mouse peritoneal macrophages. Monocytes were able to process the peptide twice as rapidly as peritoneal macrophages and some 16 times more rapidly than articular chondrocytes. The B cell line Priess, which unlike the monocytes and macrophages could not take up or degrade whole proteoglycan aggregates, was able to degrade the peptide at a rapid rate. Synthetic, unglycosylated peptides consisting of the first 16 and 13 N‐terminal amino acids of human link protein, corresponding to its stromelysin‐cleavage and trypsin‐cleavage products, were also taken up and degraded in a similar manner to the natural products and, in addition, were able to block uptake of the 125I‐labelled natural peptides. The isoelectric points of the re‐secreted breakdown fragment from both the synthetic and natural peptides were identical and each peptide was processed by the cells to produce a single radiolabelled fragment. Each of these fragments was eluted with the same retention time during HPLC, indicating that the natural peptides were derived from the N‐terminal region of the link. Since a proportion of the link protein extracted from human and pig cartilage has already undergone proteolysis to remove peptides from its N‐terminal region, these peptides may be produced in articular cartilage during the normal process of turnover and ageing. Although a physiological function for this protein has not been established, it may have a homeostatic role in the regulation of proteoglycan synthesis.
Purification and characterization of recombinant human β1–4 galactosyltransferase expressed in Saccharomyces cerevisiae
Authors: KREZDORN Christian H. , WATZELE Gabriele , KLEENE Ralf B. , IVANOV Svetoslav X. , BERGER Eric G.
Published: March 2005

A protease‐defective strain of Saccharomyces cerevisiae (BT 150) was used to express full‐length cDNA of HeLa cell β‐D‐N‐acetylglucosaminide‐β‐1,4‐galactosyltransferase (gal‐T). To ascertain import of the recombinant gal‐T into the secretory pathway of yeast cells, metabolically labeled enzyme was immunoprecipitated from extracts of yeast transformants, analysed by SDS/PAGE/fluorography and tested for sensitivity to treatment with endoglycosidase‐H. Untreated recombinant gal‐T had an apparent molecular mass of 48 kDa, which was reduced to 47 kDa after treatment, indicating that the recombinant enzyme was N‐glycosylated and, therefore, competent for translocation across the membranes of the endoplasmic reticulum.

Using specific gal‐T assays employing N‐acetylglucosamine or glucose in combination with α‐lactalbumin as exogenous acceptor substrates, recombinant gal‐T enzyme activity could readily be detected in crude homogenates. Analysis of the disaccharide products by 1H‐NMR spectroscopy demonstrated that only β‐1–4 linkages were formed by the recombinant gal‐T. The recombinant gal‐T was detergent solubilized and subsequently purified by affinity chromatography on N‐acetylglucosamine‐derivatized Sepharose followed by α‐lactalbumin–Sepharose. The purified enzyme preparation had a specific activity comparable to that of the soluble gal‐T isolated from human milk. Furthermore, kinetic parameters determined for both acceptor and donor substrates of both enzymes differed only slightly.

This work shows that yeast provides an appropriate host system for the heterologous expression of mammalian glycosyltransferases.
Purification and characterization of a coenzyme‐A‐dependent succinate‐semialdehyde dehydrogenase from Clostridium kluyveri
Authors: SÖHLING Brigitte , GOTTSCHALK Gerhard
Published: March 2005

Cell extracts of Clostridium kluyveri, grown on ethanol plus succinate contained a succinyl‐CoA:CoA transferase (0.28 U/mg), a coenzyme‐A‐dependent succinate‐semialdehyde dehydrogenase (0.73 U/mg) and a NAD+‐dependent 4‐hydroxybutyrate dehydrogenase (0.25 U/mg). The semialdehyde dehydrogenase, which catalyzed the NADPH‐dependent reduction of succinyl‐CoA to succinate semialdehyde, was purified 59‐fold to homogeneity. A molecular mass of 115000 Da was determined for the native enzyme; SDS/PAGE revealed one protein band at 55000, indicating that the active form is a dimer. The enzyme was highly specific for succinyl‐CoA and succinate semialdehyde. The pH optium was 7.0 for the reduction of succinyl‐CoA, and 8.5 for the reverse reaction Km values were determined for both the forward and reverse directions. The kinetic data suggest a ping‐pong mechanism.
Asymmetry of orientation and voltage gating of the Acidovorax delafieldii porin Omp34 in lipid bilayers
Authors: BRUNEN Manfred , ENGELHARDT Harald
Published: March 2005

The functional properties of the major outer‐membrane protein of Acidovorax delafieldii, the anion‐selective porin Omp34, were investigated in artificial membranes. Detergent‐solubilized porin incorporates into the membrane in a undirectional orientation solely determined by protein features. This enabled us to characterize the vectorial properties of the porin channels. Omp34 is electrostatically asymmetric regarding both the ion conductivity of a single trimer and the macroscopic ion conductance of multiple porin molecules. Voltage‐dependent closing occured at negative potentials; 50% of the channels were already closed at −10 mV (switching voltage). Our experimental results suggest that protein charges situated on flexible parts inside the channel are involved in the gating mechanism. A simple model is proposed illustrating the mechanism of voltage‐dependent opening and closing of the porin channels. This model explains the functional characteristics of Omp34 and the dependence of the switching voltage on the electrolyte concentration in particular. Further factors influencing voltage gating include the buffer concentration as well as the technique used for membrane formation. Altogether these factors may explain the relatively high voltages needed to obtain voltage gating with other porins.
Effects of subsite alterations on substrate‐binding mode in the active site of hen egg‐white lysozyme
Authors: KUMAGAI Izumi , MAENAKA Katsumi , SUNADA Futoshi , TAKEDA Shigeki , MIURA Kin‐ichiro
Published: March 2005

The subsite structures in the active site of hen egg‐white lysozyme were altered by site‐directed mutagenesis. Replacement of Trp62, which is involved in apolar interaction with a sugar ring, and Asp101, which is hydrogen bonded to the same sugar ring in subsite B, led to a shift of the oligosaccharide‐binding mode in the active‐site cleft. Consequently, the double‐mutant lysozyme (Trp62His, Asp101Gly) exhibited a drastic change of substrate‐binding without any significant loss of enzymic activity. Conversion of Asn37, which is postulated to be involved in interaction with a sugar ring in subsite F, had a reverse effect on substrate binding. Nuclear magnetic resonance analysis of mutant lysozymes, in which Trp62 was replaced with Phe or His, suggested that these replacements not only altered the structure of the amino acid side chain at position 62 of the lysozyme, but also induced local structural changes around the residue at position 62.
Cooperative effects on filament stability in actin modified at the C‐terminus by substitution or truncation
Authors: Drewes Gerard , Faulstich Heinz
Published: March 2005

We have studied the contribution of the C‐terminus of actin to filament stability by chemical modification and limited proteolysis. Formation of mixed disulfides of the penultimate C‐terminal cysteine residue 374 with various low‐molecular‐mass thiols resulted in filament destabilization, as reflected by an increase in critical concentration and steady‐state ATPase activity. These effects were fully reversed by the addition of phalloidin. Both the destabilization by glutathionylation and the reversal of it by phalloidin exhibited a high degree of cooperativity; half‐maximal destabilization required the modification of four out of five actin subunits, and half‐maximal restabilization by phalloidin was already reached when only one out of 20 actin subunits was complexed. C‐terminal truncation by limited trypsinolysis of filamentous actin resulted in a similar destabilization of the polymer, as shown by a 2–3‐fold increase in the steady‐state ATPase activity. This effect was likewise cooperative and could be reversed by phalloidin. Since truncation of the C‐terminus of actin has an effect on stability similar to that of chemical modification with bulky substituents, the possibility can be excluded that, in the latter case, destabilization was caused by steric hindrance. Rather, it seems that the highly conserved C‐terminal part of actin plays an active role in establishing a tight contact between neighbouring subunits.
H2‐forming N5,N10‐methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum
Authors: SCHWÖRER Beatrix , FERNANDEZ Victor M. , ZIRNGIBL Carmen , THAUER Rudolf K.
Published: March 2005

H2‐forming N5,N10‐methylenetetrahydromethanopterin dehydrogenase is a novel hydrogenase found in most methanogenic archaea. It catalyzes the reversible conversion of N5,N10‐methylenetetrahydromethanopterin (CH2= H4MPT) to N5,N10‐methenyltetrahydromethanopterin (CH ≡ H4MPT+) and dihydrogen; CH2= H4MPT + H+⇌ CH ≡ H4MPT++ H2; ΔG°′=+ 5.5 kJ/mol. In the following investigation, the formation of H2, HD and D2 was studied in experiments in which either the methylene group of CH2= H4MPT or water were deuterium labelled. In the case of CD2= H4MPT and H2O, the dihydrogen formed immediately after the start of the reaction was composed of approximately 50% HD and 50% of H2 at all pH tested. In the case of CH2= H4MPT and D2O, the dihydrogen generated was composed of approximately 50% HD and 50% D2 at pD 5.7 and of approximately 85% HD and 15% D2 at pD 7.0. Evidence is presented that the enzyme catalyzes a CH ≡ H4MPT+‐dependent isotopic exchange between HD and H2O and between HD and D2O, yielding H2 and D2, respectively. A catalytic mechanism aimed to explain these findings is discussed.
Glucose‐6‐phosphate phosphohydrolase of detergent‐treated liver microsomal membranes exhibits a specific kinetic behaviour towards glucose 6‐phosphate
Authors: AJZANNAY Ahmed , MINASSIAN Carol , RIOU Jean‐Paul , MITHIEUX Gilles
Published: March 2005

With the aim of questioning the apparent loss of specificity of the microsomal glucose‐6‐phosphate phosphohydrolase after detergent‐treatment, we performed competitive inhibition experiments among the four best substrates of the enzyme, i. e. the 6‐phosphates of glucose (Glc6P), mannose‐6 (Man6P), glucosamine (GlcN6P) and 2‐deoxyglucose (dGlc6P). The Km and Vmax of glucose‐6‐phosphatase (Glc6Pase) and mannose‐6‐phosphatase (Man6Pase), assayed either by complex formation determination of Pi produced or by radiometric determination of [U‐14C]Glc or [U‐14C]Man, were very close to 1 mM and 0.64 μmol · min−1· mg−1 microsomal protein, respectively. The Km of the enzyme for GlcN6P and for dGlc6P, determined by colorimetric assay of Pi, were equal to 1.53 ± 0.07 mM and 2.35 ± 0.15 mM, respectively, whilst the Vmax was not different from that of Glc6Pase and Man6Pase. Unexpectedly, the Ki of Man6P (1.61 ± 0.22 mM), GlcN6P (2.24 ± 0.17 mM) and dGlc6P (3.40 ± 0.07 mM) for Glc6Pase, assayed by liberation of [U‐14C]Glc, were significantly (50%) higher than their Km previously determined. The Ki of Glc6P (0.66 ± 0.05 mM) for Man6Pase, assayed by liberation of [U‐14C]Man, was significantly lower than its Km previously determined. In contrast, the Ki of GlcN6P (1.55 ± 0.05 mM) for Man6Pase, assayed by the radiometric assay, was not different from its Km previously determined. It can be inferred from these data that Glc6P phosphohydrolase exhibits specific behaviour towards Glc6P after the detergent‐treatment of the microsomal membrane.
The metal‐dependent conformational changes in Factor IX associated with phospholipid binding.
Authors: LIEBMAN Howard A.
Published: March 2005

Factor IX undergoes two sequential metal‐ion‐dependent conformational transitions [Liebman, H. A. (1987) J. Biol. Chem. 262, 7605–7612]. The first transition is metal ion dependent, but cation nonselective. The second transition is metal ion selective for Ca(II) or Sr(II) and associated with the expression of conformational determinants necessary for phospholipid membrane binding. Using antibodies raised against a synthetic peptide containing Factor IX amino acid residues 28–46, it was demonstrated that the aromatic‐amino‐acid‐stack domain (amino acid residues 41–46) of Factor IX is surface exposed in the metal‐free conformer and internalized with the initial metal‐ion‐dependent conformational transition. The metal‐ion‐induced internalization of this region of Factor IX protects the tryptophan at position 42 from oxidation by N‐bromosuccinimide. The oxidation of Factor IX tryptophan residues is associated with the rapid loss of coagulant activity, but protection of Trp42 allows for the continued expression of the Ca(II)‐specific antigenic determinants on Factor IX and the retention of phospholipid binding. These results suggest that the Trp42 residue located in the aromatic amino acid stack domain of human Factor IX is internalized with the metal‐ion‐dependent conformational transition and is essential for the expression of the phospholipid membrane binding site on Factor IX.