bluuebunny/crossref_metadata_2025 huggingface dataset:
Methylation state of the human HLA‐DRA gene in T‐lymphocytes and B‐lymphocytes of transgenic mice
Authors: FERIOTTO Giordana , POZZI Laura , CIUCCI Alessandra , CAMARDA Grazia , MISCHIATI Carlo , D'AGNANO Igea , GAMBARI Roberto , GIACOMINI Patrizio
Published: March 2005

A consistent lack of DNA methylation at one or both of two GCGC (Hha I) restriction sites in the 5′ region of the HLA‐DRA gene has been previously documented by the use of methyl‐sensitive restriction enzymes in human cells and tissues, irrespectively of their expression of DRα products. Evidence presently available, however, does not exclude that a lack of methylation in this region, although not sufficient, might be necessary for gene expression. In this report, we show that only one of the 5′‐GCGC sites is protected, although less efficiently than in man, from CG→mCG modifications in tissues and cells of transgenic mice carrying an expressed single copy of the HLA‐DRA gene/diploid genome. We demonstrate that the two 5′GCGC sites of the HLA‐DRA transgene are fully methylated in DRα− splenocytes (more than 80% T‐lymphocytes), while one of them (the most 5′ site) is not methylated in a fraction of DRα+ splenocytes (more than 95% B‐lymphocytes). These results provide evidence that absence of DNA methylation in the 5′ region is not necessary for, but might be associated with and possibly secondary to the expression of the DRA gene.
Ser2 is the autophosphorylation site in the β subunit from bicistronically expressed human casein kinase‐2 and from native rat liver casein kinase‐2β
Authors: BOLDYREFF Brigitte , JAMES Peter , STAUDENMANN Werner , ISSINGER Olaf‐Georg
Published: March 2005

Human casein kinase‐2 (CK‐2) subunits α and β were bicistronically expressed in bacteria. The recombinant holoenzyme shared all investigated properties with the native CK‐2 from mammalian sources (rat liver, Krebs II mouse ascites tumour cells). Contrary to recombinant human CK‐2 produced by self‐assembly in vitro, the bicistronically expressed β subunit was autophosphorylated during formation of the holoenzyme in bacteria. Electrospray ionisation mass spectrometry (ESI) revealed Ser2 (second amino acid, first serine) as the only phosphate acceptor site. Kinetic data obtained with either the phosphorylated or the unphosphorylated form of CK‐2 did not differ significantly, suggesting that the autophosphorylation had no influence on the kinetic parameters Km and Vmax. In parallel, native rat liver CK‐2β subunit was shown to incorporate 0.1 mol phosphate/mol holoenzyme, which suggests that the enzyme is already heavily phosphorylated. ESI analysis also revealed Ser2 as the only phosphorylated residue at the amino terminus. In the case of both recombinant human CK‐2 and native rat liver CK‐2, treatment with alkaline phosphatase readily reversed the phosphorylated form of the β subunit to the faster migrating dephosphorylated polypeptide.
General occurrence of binding synergism in zinc proteases and its possible significance
Authors: CHAN William W.‐C. , PFUETZNER Richard A.
Published: March 2005

The observation of binding synergism has been successfully extended to include carboxypeptidases A and B. The behaviour of these two enzymes follows the same pattern previously found for three other Zn‐proteases. Thus in all cases examined, the affinity of a suitable Zn‐ligand is increased in the presence of a compound (specificity probe) which contains the key structural features of specific substrates. A bifunctional ligand such as phosphonoacetate is particularly useful for generating synergism in both carboxypeptidases. Presumably the carboxylate moiety binds to the C‐terminal recognition site while the other functional group interacts with the metal ion. Several basic compounds (e.g. methyl guanidine) act as effective specificity probes for carboxypeptidase B while phenol and other hydrophobic substances serve this purpose in carboxypeptidase A.

The above phenomenon appears to be a mechanism designed to enhance catalytic efficiency through a substrate‐induced conformational change. We postulate that there is a requirement for at least one ionizable group at the active site. The proposed mechanism keeps this group in the correct ionization state in the presence of water and increases its reactivity after exclusion of water by substrate binding. We suggest the term xerophilic shift for this process. Since proton transfer is a common process in enzyme reactions, the xerophilic‐shift mechanism may play a similar role in many instances. It should therefore be possibe to detect binding synergism in a wide variety of enzymes.
Influence of the supramolecular structure of free lipid A on its biological activity
Authors: BRANDENBURG Klaus , MAYER Hubert , KOCH Michel H. J. , WECKESSER Jürgen , RIETSCHEL Ernst Th. , SEYDEL Ulrich
Published: March 2005

The three‐dimensional supramolecular structures and the states of order of the acyl chains of lipid A from different Gram‐negative species were investigated at 40°C, high water content (80–90%), and different [lipid A]/[Mg2+] molar ratios using synchrotron radiation X‐ray diffraction and Fourier‐transform infrared spectroscopy. Measurements were made on free lipid A from Salmonella minnesota R595, mono‐ and bi‐phosphoryl, as well as those from the non‐enterobacterial strains Rhodobacter capsulatus 37 b4, Rhodopseudomonas viridis F, and Rhodocyclus gelatinosus 29/1. Parallel to differences in their chemical primary structure, the structural polymorphisms and states of order at 37°C of the non‐enterobacterial lipid A were found to be different from those of enterobacterial lipid A. A clear correlation between the supramolecular structure and previously determined biological activities was found. Lipid A with a strong preference for lamellar structures (Rb. capsulatus and Rp. viridis) are endotoxically inactive and lack cytokine‐inducing capacity; the compounds assuming a mixed lamellar/nonlamellar structure (monophosphoryl lipid A from S. minnesota) are of lower toxicity in vivo, but may induce cytokines in vitro; those lipid A with a strong tendency to form non‐lamellar inverted structures (lipid A from S. minnesota and Rc. gelatinosus) exhibit full endotoxicity in vitro and in vivo. In contrast, anti‐complementary activity is most pronounced for compounds with lamellar and least expressed for those with inverted structures. The states of order at 37°C vary non‐systematically, exhibiting the highest values for lipid A of S. minnesota and the lowest for that of Rc. gelatinosus.

We propose to extend the term ‘endotoxic conformation’, which is used to describe the conformation of a single lipid A molecule required for optimal triggering of biological effects, to ‘endotoxic supramolecular conformation’ which denotes the particular organization of lipid A aggregates in physiological fluids causing biological activity.
A drug‐resistant variant of topoisomerase IIα in human HL‐60 cells exhibits alterations in catalytic pH optimum, DNA binding and sub‐nuclear distribution
Authors: BOEGE Fritz , KJELDSEN Eigil , GIESELER Frank , ALSNER Jan , BIERSACK Harald
Published: March 2005

Anion‐exchange chromatography of partially purified human HL‐60 topoisomerase II resolves the known α (170 kDa) and β (180 kDa) isoenzymes at 150 mM NaCl and 230 mM NaCl, respectively. An additional topoisomerase II fraction was eluted by > 300 mM NaCl. It could be identified by Western blotting as a late‐eluting variant of topoisomerase IIα, which is functionally altered as compared to the early‐eluting form, having the following properties: a shift in the catalytic optimum to pH 9; increased stability in DNA complex formation; approximately 100‐fold resistance to orthovanadate; approximately 1000‐fold resistance to the cytostatic substances N‐[4‐(9‐acridinylamino)‐3‐methoxyphenyl]‐methanesulphonamide (amsacrine) and the podophyllotoxin etoposide (VP 16). 80% of the late‐eluting topoisomerase IIα could be captured by SDS on calf thymus DNA without further enhancement by drugs. In contrast, the early‐eluting topoisomerase IIα exhibits 10% complex formation with SDS alone, and an increase to 90% complex formation in the presence of drugs. A HL‐60 subline (HL‐60/R), approximately 1000‐fold resistant to etoposide and amsacrine, has equivalent proportions of topoisomerase IIα and topoisomerase IIβ and similar levels of both isoenzymes, as compared to the drug‐sensitive HL‐60/WT cells. However, determination of the cellular levels of the early‐eluting and late‐eluting forms of topoisomerase IIα revealed that the HL‐60/R cell line contains approximately 80% of the late‐eluting topoisomerase IIα, whereas the sensitive HL‐60/WT cell line contains only 15–20% of this form. The nuclear distribution of the two forms also differs. Sensitive HL‐60/WT cells show a diffuse nuclear distribution but in resistant cells the distribution is localized in the nucleoli. Apparently two functionally distinct subforms of topoisomerase IIα coexist in drug‐sensitive and drug‐resistant HL‐60 cells and changes in their relative levels affect the cellular sensitivity to topoisomerase‐II‐targeting drugs.
Gene structure of CYP3A4, an adult‐specific form of cytochrome P450 in human livers, and its transcriptional control
Authors: HASHIMOTO Hisashi , TOIDE Kenji , KITAMURA Ryuji , FUJITA Masako , TAGAWA Sanae , ITOH Susumu , KAMATAKI Tetsuya
Published: March 2005

CYP3A4 is the adult‐specific form of cytochrome P450 in human livers [Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. & Kamataki, T. (1990) Biochemistry 29, 4430–4433]. The sequences of three genomic clones for CYP3A4 were analyzed for all exons, exon‐intron junctions and the 5′‐flanking region from the major transcription site to nucleotide position ‐1105, and compared with those of the CYP3A7 gene, a fetal‐specific form of cytochrome P450 in humans. The results showed that the identity of 5′‐flanking sequences between CYP3A4 and CYP3A7 genes was 91%, and that each 5′‐flanking region had characteristic sequences termed as NFSE (P450NF‐specific element) and HFLaSE (P450HFLa specific element), respectively. A basic transcription element (BTE) also lay in the 5′‐flanking region of the CYP3A4 gene as seen in many CYP genes [Yanagida, A., Sogawa, K., Yasumoto, K. & Fujii‐Kuriyama, Y. (1990) Mol. Cell. Biol. 10, 1470–1475]. The BTE binding factor (BTEB) was present in both adult and fetal human livers.

To examine the transcriptional activity of the CYP3A4 gene, DNA fragments in the 5′‐flanking region of the gene were inserted in front of the simian virus 40 promoter and the chloramphenicol acetyltransferase structural gene, and the constructs were transfected in HepG2 cells. The analysis of the chloramphenicol acetyltransferase activity indicated that (a) specific element(s) which could bind with a factor(s) in livers was present in the 5′‐flanking region of the CYP3A4 gene to show the transcriptional activity.
Evidence for a glycolipid anchor of gp64, a putative cell‐cell adhesion protein of Polysphondylium pallidum
Authors: SAITO Tamao , OCHIAI Hiroshi
Published: March 2005

The membrane‐bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell‐cell adhesion protein identified by adhesion‐blocking antibody fragments. Since gp64 can be purified in a few days and in substantial yields, it is a good candidate for clarifying the structure of a cell‐cell adhesion protein. This study reveals that gp64 possesses a glycolipid anchor which is sensitive to deamination but resistant to phosphatidylinositol‐specific phospholipase C from Bacillus thuringiensis. Although the anchor resistance to phosphatidylinositol‐specific phospholipase C can be ascribed to the presence of an additional acyl chain on the inositol ring in the glycosyl phosphatidylinositol anchors, this was not the case. After a mild‐base treatment that released an additional acyl chain on the inositol ring, only a trace amount of fatty acid was detected but, after strong acid hydrolysis, we detected both amide‐linked fatty acids and a long‐chain base. The long‐chain base was further analysed by gas‐chromatography/mass spectrometry and was found to be phytosphingosine. Both fatty acids and myo‐inositol were also analysed by gas‐chromatography/mass spectrometry. These data suggest that gp64 possesses a glycolipid anchor which contains ceramide and myo‐inositol.
Probing different conformational states of pregnancy‐zone protein
Authors: ARBELAEZ Luis F. , JENSEN Poul Erik H. , SHANBHAG Vithaldas P. , STIGBRAND Torgny
Published: March 2005

The binding of the fluorescence probe 4,4′‐bis(8‐anilino‐1‐naphthalenesulphonate) (bis‐ANS) to the human proteinase inhibitor pregnancy‐zone protein (PZP) and its complexes with methylamine and chymotrypsin were investigated. The existence of dimeric PZP‐chymotrypsin complex was demonstrated and both the dimeric and the tetrameric PZP‐chymotrypsin complexes could be studied separately. The fluorescence data indicate that bis‐ANS binds to two different sites on PZP and its complexes. The values of the dissociation constant, Kd1, for the binding to the high‐affinity site were determined to be 231 ± 14, 220 ± 28, 114 ± 15 and 49 ± 1 nM, for the binding to native PZP, PZP‐methylamine and dimeric and tetrameric PZP‐chymotrypsin, respectively. An 11–30‐fold decrease was observed in the affinity for the second site, the corresponding values of the dissociation constant, Kd2, being 1.5 − 2.8 ± 1.0 μM, which are not significantly different for PZP and its derivatives. The results suggest that the probe bis‐ANS discriminates between the different conformational states of PZP and that while the conformation of the complex with methylamine does not differ much from that of the native protein, there is a significant change in conformation when chymotrypsin cleaves the bait region. This is substantiated by a 30%–45% decrease in the maximum enhancement of fluorescence intensity when PZP is treated with chymotrypsin. Although the dimeric and tetrameric forms of PZP‐chymotrypsin complexes differ in Kd1 values, the difference in the maximum enhancement of the fluorescence of bis‐ANS by the two forms is not significant. This indicates that dimer‐dimer interaction in the tetrameric form does not involve hydrophobic sites. The necessity of bait‐region cleavage for extensive conformational changes in PZP distinguishes it from α2‐macroglobulin, the other α‐macroglobulin in human plasma.
Purification and characterization of a macromolecular‐translocation inhibitor III of activated glucocorticoid‐receptor‐complex binding to nuclei from rat liver
Authors: LIU Gang , OKAMOTO Kazuki , ISOHASHI Fumihide
Published: March 2005

Macromolecular‐translocation inhibitors (MTI) of binding of the activated glucocorticoid‐receptor complex (GRC) to nuclei from rat liver are separated into at least three components (MTI‐I–III) by DEAE‐cellulose column chromatography [Okamoto, K., Isohashi, F., Horiuchi, M. & Sakamoto, Y. (1982) Biochem. Biophys. Res. Commun. 108, 1655–1660]. In this study, we have purified MTI‐III from the livers of adrenalectomized rats to apparent homogeneity, as determined by SDS/PAGE. The purification procedure consisted of DEAE‐cellulose chromatography, acid treatment and sequential chromatographies using gel filtration, S‐Sepharose and Mono S columns. The purified protein had a molecular mass of approximately 69 kDa, as estimated by SDS/PAGE, and the molecular mass of the inhibitor was approximately 68 kDa, as estimated by gel filtration. Thus, MTI‐III exerts its inhibitory activity as a monomer. The sedimentation coefficient of MTI‐III was approximately 3.7 S. Purified MTI‐III was fairly stable at 4°C but at higher temperatures, especially above 25°C, it was rapidly inactivated. Under low‐salt conditions, MTI‐III was associated with activated GRC (4.2 S) and the resulting complex was detected on sucrose density gradients as a larger species (6.8 S). Initial treatment of nuclei or DNA‐cellulose with MTI‐III did not alter their abilities to bind activated GRC. These results indicate that MTI‐III acts through an interaction with GRC.
Kinetic characterization of the EcaI methyltransferase
Authors: SZILÁK László , DÉR András , DEÁK Ferenc , VENETIANER Pál
Published: March 2005

A kinetic analysis of the EcaI adenine‐N6‐specific methyltransferase (MTase) is presented. The enzyme catalyzes the transfer of a methyl group from S‐adenosyl‐L‐methionine (AdoMet) to the adenine of the GGTNACC sequence with a random rapid‐equilibrium mechanism. Experiments with a synthetic, 14‐bp DNA substrate suggest that recognition of the specific site of DNA occurs after the binding of AdoMet. Proton concentration does not affect the dissociation constant of AdoMet while Vm and the dissociation constant of DNA show a maximum around pH 8. Increasing the amount of S‐adenosyl‐L‐homocysteine decreases the inhibitory effect of methylated DNA which proves the active role of AdoMet in site recognition. Experiments with hemimethylated DNA show that the methylase binds the double‐stranded DNA asymmetrically.
Effect of proglycosyn and other phenolic compounds on glycogen metabolism in isolated hepatocytes
Authors: VAN SCHAFTINGEN Emile , DE HOFFMANN Edmond
Published: March 2005

The mechanism by which proglycosyn (LY 177 507) stimulates glycogen synthesis in isolated hepatocytes [Harris, R. A., Yamanouchi, K., Roach, P. J., Yen, T. T., Dominiani, S. J. & Stephens, T. W. (1989) J. Biol. Chem. 264, 13674–13680] has been investigated. When incubated in the presence of hepatocytes, proglycosyn was metabolized to an O‐demethylated glucuronidated derivative, as determined by fast‐atom‐bombardment mass spectrometry and enzymic analysis. This metabolite accumulated almost linearly inside the cells to reach a concentration of approximately 3 μmol/g protein after 50 min, without apparent release into the medium. In confirmation of previous work, proglycosyn decreased the level of phosphorylase a and increased that of synthase a in hepatocytes. Washing of cells incubated with proglycosyn for 30 min considerably decreased the concentration of the drug without significantly modifying the intracellular concentration of the metabolite and the activation state of glycogen synthase.

Several compounds bearing structural analogy with proglycosyn were also tested for their effect on glycogen metabolism. At millimolar or submillimolar concentrations, resorcinol, m‐anisidine, phenol, 3‐hydroxyacetophenone, and 3‐acetamidophenol, although not 4‐acetamidophenol, stimulated the incorporation of [14C]glucose into glycogen, decreased the level of phosphorylase a and increased the level of synthase a. In the case of phenol, the effect on the glycogen enzymes paralleled the intracellular accumulation of phenylglucuronide. Furthermore, ethanol and D‐galactosamine, which decreased the conversion of phenol to phenylglucuronide and the intracellular concentration of phenylglucuronide, counteracted the effect of phenol on the synthase and on the phosphorylase.

From these results, it is suggested that the effect of proglycosyn and of simpler phenol derivatives is mediated by glucuronidated metabolites, which act on an intracellular target.
Formation of diacylglycerol, inositol phosphates, arachidonic acid and its metabolites in macrophages
Authors: DIETER Peter , FITZKE Edith
Published: March 2005

Treatment of macrophages with zymosan, 4β‐phorbol 12‐myristate 13‐acetate (PMA) and fluoride but not with A 23187 or arachidonic acid (ΔAch) leads to a generation of diacylglycerol (acyl2Gro). Formation of inositol phosphates is achieved with zymosan, only. An elevation of intracellular calcium is obtained with zymosan and A 23187 but not with PMA, fluoride or ΔAch. Prior treatment of the cells with phorbol ester for 3 h which has been shown recently to result in a down regulation of protein kinase (PK) C‐β but not PKC‐δ [Duyster, J., Schwende, H., Fitzke, E., Hidaka H. & Dieter P. (1993) Biochem. J. 292, 203–207] has no effect on the zymosan‐induced formation of acyl2Gro or inositol phosphates but inhibits the PMA‐induced generation of acyl2Gro. Down regulation of PKC‐δ by prior phorbol ester treatment for 24 h augments the zymosan‐induced generation of acyl2Gro and inositol phosphates. The acyl2Gro lipase inhibitor RG 80267 inhibits the PMA‐induced and fluoride‐induced generation of prostaglandin (PG) E2, reduces the zymosan‐induced release of PGE2 by 50% but has no effect on PGE2 formation of unstimulated, A 23187 treated or ΔAch‐treated cells. Furthermore, RG 80267 enhances accumulation of ΔAch‐labeled acyl2Gro in response to zymosan, PMA and fluoride. These data indicate that zymosan activates a phosphatidylinositol 4,5‐bisphosphate‐specific phospholipase (PL) C, that generation of acyl2Gro by PMA and fluoride occurs via hydrolysis of other phospholipids, that PKC‐β is involved in the PMA‐induced generation of acyl2Gro and PKC‐δ negatively modulates the zymosan‐induced activation of PLC and PMA and fluoride induce a liberation of ΔAch from acyl2Gro, A 23187 activates the PLA2 pathway and zymosan stimulates both, the acyl2Gro‐ and PLA2‐pathway.
Structures of the N‐linked oligosaccharides on porcine plasma vitronectin
Authors: YONEDA Atsuko , OGAWA Haruko , MATSUMOTO Isamu , ISHIZUKA Ineo , HASE Sumihiro , SENO Nobuko
Published: March 2005

The structures of N‐linked oligosaccharides, especially the distribution of sialic acid species, present on porcine plasma vitronectin were elucidated. Oligosaccharides were released from the vitronectin by N‐glycosidase F digestion and tagged with 2‐aminopyridine, and the pyridylamino‐oligosaccharides were fractionated by anion‐exchange and reverse‐phase HPLC. Nine major pyridylamino‐oligosaccharides were isolated. The linkages and locations of sialic acids were determined by a novel approach involving desialylation with Salmonella sialidase in combination with acid desialylation. After desialylation, the asialo‐forms were analyzed by two‐dimensional sugar mapping, component sugar analysis and 400‐MHz 1H‐NMR spectroscopy.

The major oligosaccharides of porcine vitronectin were of the fucosylated biantennary type, with a small amount of the triantennary N‐acetyllactosamine type, to which 1–3 mol sialic acids was linked. Sialic acids were linked predominantly through α2–6 linkages, although α2–3 linkages were also present, and fucose was linked to the innermost N‐acetylglucosamine through an α1–6 linkage. It was found that every pyridylamino‐oligosaccharide population contained N‐glycolylneuraminic acid and N‐acetylneuraminic acid in a molar ratio of 1:2–9, and that N‐glycolylneuraminic acids were located predominantly on the Man α1–6 arm.
Insulin and tumor‐promoting agent regulate an inhibitor of the 44‐kDa mitogen‐activated protein kinase/extracellular signal‐regulated protein kinase 1 in fibroblasts
Authors: PERALDI Pascal , VAN OBBERGHEN Emmanuel
Published: March 2005

We have examined the negative regulation of the 44‐kDa mitogen‐activated protein kinase (MAP kinase), also known as extracellular signal‐regulated protein kinase 1 (ERK1), in NIH3T3 cells transfected with an expression plasmid encoding the human insulin receptor (NHIR cells). In these cells ERK1 activation is induced by two distinct stimuli, insulin and tumor‐promoting agent (TPA). While insulin was found to be more potent than TPA for ERK1 activation, both stimuli produced the same transient activation pattern with a rapid peak (reached within 5 min) followed by a fast decrease within 20 min. By performing reconstitution experiments with immunoprecipitated ERK1 and lysates from NHIR cells, we showed that extracts from untreated cells exhibit an ERK1 inhibitory activity. Interestingly, this inhibitor was found to be regulated by insulin and TPA with a profile that is the mirror image of ERK1 activity. This repressing activity was sensitive to tyrosine phosphatase inhibitors, such as sodium orthovanadate and zinc acetate, but it was not affected by serine/threonine phosphatase inhibitors, such as sodium fluoride and okadaic acid. Moreover, it was possible to observe in extracts of NHIR cells an activity dephosphorylating ERK1. The time course of this phosphatase activity was comparable to that of the ERK1 inhibition, suggesting that the repressing activity could reflect a dephosphorylating action. Interestingly, phosphatase 2A treatment of extracts from 5‐min TPA‐treated cells (where the ERK1 inhibitor was weak) was able to induce an increase in the ERK1 repressing activity. This suggests that serine/threonine dephosphorylation of ERK1 inhibitor leads to an increase in its activity. In summary, we have shown that NHIR cells contain a regulatable ERK1 inhibitor, which is likely to be due to tyrosine phosphatase(s). We would like to suggest that such activities are key components in the fine‐tuning of the MAP kinase cascade.
Investigation of intra‐domain and inter‐domain interactions of glutathione transferase P1–1 by limited chymotryptic cleavage
Authors: MARTINI Filippo , ACETO Antonio , SACCHETTA Paolo , BUCCIARELLI Tonino , DRAGANI Beatrice , DI ILIO Carmine
Published: March 2005

Limited proteolysis of glutathione transferase P1–1 (GSTP1–1) by chymotrypsin performed at 20°C and 30°C mainly generates two complementary peptides of 17 kDa and 6 kDa molecular mass with concomitant loss of catalytic capacity. Sequence analysis of these peptides showed that the peptide bond between Tyr47 and Gly48 was cleaved. The analysis of the recently resolved three‐dimensional structure of GSTP1–1 [Reinemer, P., Dirr, H. W., Ladenstein, R., Huber, R., Lo Bello, M., Federici, G. & Parker, M. W. (1992) J. Mol. Biol. 227. 214–226] suggests that the proteolytically cleaved bond results located in a portion of the polypeptide chain lining the G‐site which has been demonstrated to be part of an exposed and flexible region of the N‐terminal domain (structural elements αB1 and αB2) [Aceto, A., Caccuri, A. M., Sacchetta, P., Bucciarelli, T., Dragani, B., Rosato, N., Federici, G. & Di Ilio, C. (1992) Biochem. J. 285, 241–245]. The fragments which are generated by proteolysis at 20°C, remain linked by noncovalent interaction in a complex (nicked GSTP1–1) which is dissociated by incubation at higher temperatures. As shown by circular dichroic analysis, although inactive, nicked GSTP1–1 retains an overall secondary structure closely resembling that of the parent enzyme. However, the fluorescence data of the nicked GSTP1–1 indicate that the Trp38, which is near the chymotrypsin‐cleavable bond, becomes exposed in a more polar environment. This indicates that, in the nicked enzyme, the polypeptide portion containing the structural elements αB1 and αB2 has more freedom of fluctuation. The fact that this polypeptide chain portion contains two essential amino acid residues of the G‐site (Trp38 and Lys42) might account for the loss of ability to bind glutathione by the nicked enzyme which is consequently catalytically inactive. Proteolysis performed at 30°C generated a homodimeric 17–kDa fragment. The structural analysis of this fragment suggests that the GSTP1–1 αC helix, which is located in the domain I and is thought to be involved in the inter‐domain interaction, could exert a critical role in maintaining the native folding of domain II.
The mature form of imported mitochondrial proteins undergoes conformational changes upon binding to isolated mitochondria
Authors: HARTMANN Claudia M. , GEHRING Heinz , CHRISTEN Philipp
Published: March 2005

Mature mitochondrial proteins (aspartate aminotransferase, malate dehydrogenase, hydroxyacyl coenzyme A dehydrogenase, creatine kinase) and cytosolic proteins (aldolase, glyceraldehyde‐3‐phosphate dehydrogenase) with a basic pI were found to bind to isolated mitochondria, electrostatic interactions being mainly responsible for their binding. Mitochondrial aspartate aminotransferase bound with a Kd' of 30 nM in 0.6 M sorbitol, 20 mM Hepes/KOH, pH 7.4, at 25°C. Cytosolic aspartate aminotransferase and glutamate dehydrogenase (a protein located in the mitochondrial matrix) both with an acidic pI, did not bind to mitochondria. Treatment of mitochondria with proteinases did not affect the subsequent binding of imported mitochondrial proteins. Their association with both intact and proteinase‐treated mitochondria resulted in a marked increase in their susceptibility toward proteinase K. In contrast, the basic cytosolic proteins tested bound only to intact mitochondria and thereby did not become more susceptible toward proteolytic attack. Treatment of mitochondria with adriamycin, a drug binding to acidic phospholipids, prevented the subsequent association of mitochondrial aspartate aminotransferase with mitochondria and the ensuing conformational labilization. Apparently, the mature moiety of imported mitochondrial proteins is partially unfolded upon interaction with the lipid component of the mitochondrial envelope. Both the binding of the mitochondrial proteins and their conformational labilization is independent of ATP and the electrochemical potential across the inner membrane.
Formation of a functionally active sodium‐translocating hybrid F1F0 ATPase in Escherichia coli by homologous recombination
Authors: KAIM Georg , DIMROTH Peter
Published: March 2005

A deletion mutant of Escherichia coli lacking the genes for ATPase subunits a, c, b, δ and part of the α subunit was transformed with a plasmid containing the corresponding genes of the sodium‐translocating ATPase of Propionigenium modestum. The respective DNA fragment of P. modestum was integrated into the genome of the E. coli deletion mutant by site‐specific homologous recombination. The sites of this recombination event were identified by cloning and DNA sequencing. As a consequence of the recombination event, a functionally active hybrid ATPase was obtained by in vivo complementation. The biochemical characterization of this hybrid ATPase revealed high sensitivity to dicyclohexylcarbodiimide as well as strong activation by the addition of sodium ions. After reconstitution into liposomes, the hybrid ATPase catalysed the transport of Na+ upon ATP addition. In the absence of Na+, the ATPase hybrid was able to pump protons, as was shown by the ATP‐dependent fluorescence quenching of 9‐amino‐6‐chloro‐2‐methoxyacridine.
Basal Mg2+‐dependent ATPase activity of rat liver microsomes is not influenced by ambient free Ca2+
Authors: VAN DE PUT Frans H. M. M. , VISSER Gea J. , DONKERS Elizabeth A. M. , THEUVENET Alexander P. R. , WILLEMS Peter H. G. M.
Published: March 2005

The potent inhibitor of Ca2+, Mg2+‐ATPase activity, thapsigargin, has been used to investigate the effect of ambient free Ca2+ on basal Mg2+‐dependent ATPase activity in rat liver microsomes. Thapsigargin non‐competitively inhibited both Ca2+ ‐stimulated ATP‐dependent Ca2+ uptake and Ca2+‐stimulated Mg2+‐dependent ATPase activity. At a concentration of 1 μM, thapsigargin completely inhibited the Ca2+‐pump activity, without affecting Mg2+ ‐dependent ATPase activity measured in the absence of Ca2+. Increasing the ambient free Ca2+ concentration did not alter the basal Mg2+‐dependent ATPase activity. The data presented indicate that ATPase activity measured in the absence of Ca2+ is a reliable measure for the basal Mg2+‐dependent ATPase activity and that, consequently, the Ca2+‐stimulated Mg2+‐dependent ATPase activity can indeed be defined as the difference between the ATPase activity measured in the presence and the absence of Ca2+.
Aromatization of 4‐oxocyclohexanecarboxylic acid to 4‐hydroxybenzoic acid by two distinctive desaturases from Corynebacterium cyclohexanicum
Authors: KANEDA Toshi , OBATA Hitoshi , TOKUMOTO Masakazu
Published: March 2005

We have previously demonstrated that Corynebacterium cyclohexanicum degrades cyclohexanecarboxylic acid, a bacteriocide, through a pathway including the aromatization of 4‐oxocyclohexanecarboxylic acid to 4‐hydroxybenzoic acid [Kaneda, T. (1974) Biochem. Biophys. Res. Commun. 58, 140–144]. Aromatization has now been shown to be catalysed by two desaturase enzymes. Under the action of desaturase I, 4‐oxocyclohexanecarboxylic acid is converted to (+)‐4‐oxocyclohex‐2‐enecarboxylic acid which is then aromatized by desaturase II to 4‐hydroxybenzoic acid. The latter reaction is presumed to occur via the unstable intermediate, 4‐oxocyclohex‐2,5‐dienecarboxylic acid, which is spontaneously isomerized to 4‐hydroxybenzoic acid. Desaturase I has been purified in an electrophoretically homogeneous form. It is monomeric with a molecular mass of 67 kDa and contains one tryptophan, one histidine and two cysteine residues per enzyme molecule. The enzyme produces an equivalent amount of 4‐oxocyclohex‐2‐enecarboxylic acid and hydrogen peroxide from 4‐oxocyclohexanecarboxylic acid. The properties of desaturase I have been studied in detail. Desaturase II is unstable and has been partially purified. Its characterization is therefore limited. However, the molecular mass of desaturase II was estimated to be 43 kDa by gel filtration chromatography. The characterization of both desaturase enzymes is described in this paper. The possible environmental importance of microbial aromatization in the biodegradation of compounds with the cyclohexane structure is discussed.
Characterisation of high‐affinity and low‐affinity receptors for ciliary neurotrophic factor
Authors: HUBER Johann , DITTRICH Falk , PHELAN Pauline
Published: March 2005

Ciliary neurotrophic factor (CNTF) supports the survival of a wide variety of neuronal cells in culture. To characterise the receptor(s) mediating the biological response of CNTF we measured the binding of radiolabelled CNTF to chick sympathetic neurons and human neuroblastoma cells. Two distinct CNTF‐binding sites with high and low affinity for the ligand were identified by steadystate binding experiments. Furthermore, two low‐affinity binding sites could be discriminated on the basis of the dissociation rates. Cross‐linking experiments showed that CNTF interacts with two proteins, one of 80 kDa and one of 140kDa. The identity of the 80‐kDa protein was determined by transient transfection experiments with the rat CNTF‐binding protein CNTFRα while the properties of the 140‐kDa protein correspond to those of gp130. Antisense experiments confirmed that CNTFRα is necessary for high affinity binding of 125I‐CNTF and therefore a necessary subunit of the high‐affinity receptor.
Intracellular degradation of prohormone‐chloramphenicol‐acetyl‐transferase chimeras in a pre‐lysosomal compartment
Authors: DANOFF Ann , MAI Xiao Ping , SHIELDS Dennis
Published: March 2005

Small peptide hormones (less than 50 amino acids) are synthesized as larger inactive precursors. Work from several laboratories, including our own, has implicated the propeptide of various precursors in mediating intracellular transport and targeting to secretory granules. We previously demonstrated that the proregion of prosomatostatin, one of the simplest peptide hormone precursors, when fused to α‐globin, enabled the globin polypeptide to be transported to the regulated secretory pathway. To identify sorting motifs in this propeptide, we have now constructed a chimera comprising the somatostatin signal peptide and proregion fused to chloramphenicol acetyl transferase (CAT) and a control protein consisting of the signal peptide fused to CAT, both of which were expressed in rat anterior‐pituitary GH3 cells. Both molecules were translocated into the endoplasmic reticulum (ER) efficiently and core‐glycosylated on the single cryptic N‐linked glycosylation site present in CAT. Surprisingly, the glycosylated propeptide‐CAT and signal without CAT were degraded intracellularly with half‐lives of 30 min and 90 min, respectively. Based on the kinetics of degradation, temperature sensitivity, and resistance to lysosomotrophic agents, we suggest that degradation occurred in the ER. Our data imply that the pro‐region is not an a priori universal sorter, but only directs heterologous peptides to the secretory pathway when the passenger peptide assumes a secretion‐competent conformation.
Flavin dynamics in reduced flavodoxins
Authors: LEENDERS Rik , KOOIJMAN Martin , van HOEK Arie , VEEGER Cees , VISSER Antonie J. W. G.
Published: March 2005

The time‐resolved fluorescence and fluorescence anisotropy characteristics of reduced flavin mononucleotide in solution as well as bound in flavodoxins isolated from the bacteria Desulfovibrio gigas, Desulfovibrio vulgaris, Clostridium beijerinckii MP and Megasphaera elsdenii have been examined. All fluorescence and fluorescence anisotropy decays were analyzed by two different methods: (a) least‐squares fitting with a sum of exponentials and (b) the maximum entropy method to yield distributed lifetimes and correlation times. The results of both approaches are in excellent agreement. The fluorescence decay of the free as well as protein‐bound reduced flavin chromophore is made up of three components. The shortest component proves to be relatively sensitive to the environment and can therefore be used as a diagnostic tool to probe the microenvironment of the reduced isoalloxazine ring system. The other two longer fluorescence lifetime components are insensitive to the chromophore environment and seem therefore to be related to intrinsic, photophysical properties of the reduced chromophore. Fluorescence anisotropy decays show that the flavin mononucleotide in all four reduced flavodoxins is immobilized within the protein matrix, as indicated by the recovery of a single rotational correlation time, reflecting the rotation of the whole protein. No indications are found that rapid structural fluctuations occur in reduced flavodoxins, and the mechanism of electron transfer from flavodoxin to other redox proteins seems to involve immobilized reduced flavin.
Comparison of the dynamic structure of α‐chymotrypsin in aqueous solution and in reversed micelles by fluorescent active‐site probing
Authors: DOROVSKA‐TARAN Victoria N. , VEEGER Cees , VISSER Antonie J. W. G.
Published: March 2005

A highly fluorescent anthraniloyl (Ant) group was covalently attached to the active site of α‐chymotrypsin (CT), probably at Ser195. Ant‐CT is stable at neutral pH for months, allowing a detailed fluorescence study of Ant‐CT as a model protein to investigate its physical properties in 0.1 M Tris/HCl, pH 8.2, and in reversed micelles of n‐octane, 0.1 M Tris/HCl, pH 8.2, and sodium bis(2‐ethylhexyl)sulfosuccinate (AOT).

Steady‐state fluorescence measurements of the progressive red‐shift of the center of gravity of the emission band as function of degree of hydration, wo, defined as [H2O]/[AOT], indicate that the average polarity in the vicinity of the probe is approaching that of bulk water at wo > 12. Time‐resolved fluorescence measurements of Ant‐CT in water and in reversed micelles showed that the active site has different properties in reversed micelles compared to those in water. Some specific changes at very low water content (0.6 < wo < 5) can be observed, which correlate with enzyme activity measurements in the same wo region (unpublished results). These effects are, for instance, significant changes in the average fluorescence lifetime and the internal flexibility of the probe. The overall rotational‐correlation time of the enzyme in AOT reversed micelles seems to be independent on wo (5 < wo < 29), which suggests that the enzyme creates its own micelle.
Engineered yeast cells as model to study coupling between human xenobiotic metabolizing enzymes
Authors: GAUTIER Jean‐Charles , URBAN Philippe , BEAUNE Philippe , POMPON Denis
Published: March 2005

Human microsomal epoxide hydrolase and cytochrome P450 (P450) 1A1 were coexpressed in Saccharomyces cerevisiae from expression cassettes integrated respectively into the host chromosomal DNA and on a multicopy plasmid in a strain already overexpressing yeast NADPH‐cytochrome P450 reductase (P450 reductase). A styrene‐oxide‐hydrolase activity (2 nmol · min−1· mg microsomal protein−1) and a 7‐ethoxyresorufin‐O‐deethylase activity (320 pmol · min−1· mg microsomal protein−1) characteristic respectively of microsomal epoxide hydrolase and P450 1A1 were detected. The conversion of benzo[a]pyrene (B[a]P) to B[a]P‐7,8‐dihydrodiol both in microsomal preparations and in growing yeast cells was observed, demonstrating an efficient coupling between the two human enzymes. Kinetic analysis indicated that the B[a]P‐7,8‐oxide produced by the P450‐1A1‐dependent reaction does not accumulate before hydrolysis by microsomal epoxide hydrolase. This system was also used as a control to evaluate the coupling efficiency of a mixture of microsomes or of yeast cells containing separately the individual enzymes (i.e., human P450 1A1 and microsomal epoxide hydrolase). B[a]P‐7,8‐oxide was well converted to the corresponding dihydrodiol with a mixture of microsomes. In contrast, when the same experiment was repeated with a mixture of cells expressing independently the two activities, dihydrodiol formation was not observed. Coexpression of human phase I and phase II enzymes in a single yeast cell and microsome mixture thus appear to be complementary tools for the simulation of human‐drug‐metabolism or carcinogen‐metabolism pathways.
Action spectrum of light‐harvesting‐II precursor apoprotein transcript accumulation and coordinated translation
Authors: TRIANTAPHYLLOPOULOS Kostas A. , ARGYROUDI‐AKOYUNOGLOU Joan H.
Published: March 2005

In an attempt to ascertain that the phytochrome chromoproteins do indeed constitute the photorecptor involved in light‐harvesting‐II‐apoprotein‐transcript accumulation and the synchronization of the endogenous circadian clock [Tavladoraki, P., Kloppstech, K. & Argyroudi‐Akoyunoglou, J. H. (1989)Plant Physiol. (Bethesda) 90, 665–672], we studied the action spectrum of Cab transcription in 9–10‐day‐old etiolatedPhaseolus vulgarisleaves. Transcript accumulation was detected by hybridization experiments or immunoprecipitation of thein‐vitrotranslation products, obtained from isolated leaf poly(A)‐rich mRNA species. It was found that the accumulation of the precursor‐light‐harvesting‐II‐apoprotein mRNA depends highly on the wavelength of light exposure, the action spectrum of its accumulation being closely correlated with the absorption spectrum of the red‐absorbing phytochrome form (Pr); the reversal by far‐red light of the light‐induced Cab‐mRNA accumulation depends also on wavelength of light exposure, used prior to far red for activation of transcription; the action spectrum thus obtained resembles the difference absorption spectrum of the purified phytochrome protein (ΔAPr–Pfr). In addition, immunoblot analysis of total lithium‐dodecyl‐sulphate‐solubilized protein extracts of leaves showed that the appearance of the mature lightharvesting‐II apoprotein in etiolated leaves previously exposed to a light pulse of equal dose but varying wavelength, closely follows the pattern of its transcript accumulation and the action spectrum of protochlorophyllide photoconversion. Thus, translation of the proteinin vivo, as monitored by the steady level of the protein appearing in Western blots, is closely coordinated with the accumulation of its transcript, the action spectrum of both processes closely resembling the absorption spectrum of phytochrome. The results suggest that phytochrome is involved in photoreception mediating Cabgene transcription in 9–10‐day‐old etiolated bean leaves and that Cab transcription, appearance of the mature protein and stabilization of the apoprotein in thylakoids by the chlorophyll formed during the light pulse are in close coordination.
A purification method and N‐glycosylation sites of a 36‐cysteine‐containing, putative cell/cell adhesion glycoprotein gp64 of the cellular slime mold, Polysphondylium pallidum
Authors: SAITO Tamao , KUMAZAKI Takashi , OCHIAI Hiroshi
Published: March 2005

A 64‐kDa membrane‐bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell/cell adhesion protein identified by adhesion‐blocking antibody fragments (Fab). gp64 is expressed on the cell surface of growth‐phase cells and seems to mediate cell/cell adhesion. This paper describes an improved purification method based on the lipophilic nature of this protein. A critical step in the purification method is to collect an insoluble top layer appearing during ammonium sulfate precipitation. The sequence of cDNA encoding gp64 and its deduced amino acid sequence have been determined previously. Based on cDNA sequence data, the structure of gp64 protein was analyzed: almost all amino acid compositions and partial amino acid sequences of lysylendopeptidase‐digested peptides of gp64 were determined by protein analysis; all six asparagine‐linked glycosylation sites (Asn‐Xaa‐Ser/Thr) in fact contain carbohydrates, and all 36 cysteine residues were involved in forming disulfide bridges. From these data, gp64 seems to be a unique protein among cell/cell adhesion proteins.
The immediate activator of the NADPH oxidase is arachidonate not phosphorylation
Authors: HENDERSON Lydia M. , MOULE S. Kelly , CHAPPELL J. Brian
Published: March 2005

Superoxide generation is rapidly triggered following the addition of a stimulus to neutrophils. The signal‐transduction pathway culminates in the activation of protein kinase C, whose phosphorylation of a protein component is considered to activate the oxidase. Arachidonate stimulated the oxidase in a concentration‐dependent manner but, unlike phorbol‐12‐myristate‐13‐acetate (PMA), was not inhibited by staurosporine, a protein kinase inhibitor. Increase protein phosphorylation, apparent with PMA, was not observed when superoxide generation was triggered by arachidonate. Inhibitors of phospholipase A2 inhibit the PMA activation of the oxidase. Therefore, we propose that arachidonate and not phosphorylation is the immediate stimulus for superoxide generation.
Purification and characterization of ribonucleoproteins from pea chloroplasts
Authors: SUBBAIAH Chalivendra Chenchu , TEWARI Krishna K.
Published: March 2005

RNA‐binding proteins are known to mediate the post‐transcriptional regulation of genes in many organisms. Recently they have been found to be important in the expression of plastid genes.

We have purified a group of three single‐stranded nucleic‐acid‐specific acidic proteins (33, 30 and 28 kDa) from chloroplast extracts of pea (Pisum sativum L.), using single‐stranded DNA affinity chromatography. All of them have acidic amino termini but the amino acid sequences are unique to each polypeptide, with partial similarities to the recently reported ribonucleoproteins from tobacco chloroplasts. The pea proteins are also antigenically distinct, as shown by Western blot analysis using polyclonal antisera for purified proteins. Further, from their large nucleic‐acid‐binding domains and the polynucleotide substrate affinities, they are predicted to belong to a family of pea plastid ribonucleoproteins. In vivo radiolabeling of proteins in the presence of translational inhibitors as well as in vitro translation of leaf tissue RNA suggest that these proteins are encoded in the nucleus. Antibody cross‐reactivity experiments reveal that their genes are conserved during plastid evolution.
NMR solution structure of the [Ala26]parathyroid‐hormone‐related protein(1 – 34) expressed in humoral hypercalcemia of malignancy
Authors: RAY Fiona R. , BARDEN Julian A. , KEMP Bruce E.
Published: March 2005

The structure of the biologically active N‐terminal domain of human parathyroid‐hormone‐related protein (residues 1–34) containing an Ala substituted for a His in position 26 was studied by two‐dimensional proton NMR spectroscopy. Unambiguous NMR assignments of all backbone and side‐chain hydrogens were made with the aid of totally correlated spectroscopy experiments, which provided through‐bond 1H‐1H connectivities, and NOE spectroscopy, which provided through‐space and sequential backbone connectivities. The NMR data were utilized in distance‐geometry algorithms to generate a family of structures. The major structural features include two segments of α‐helix extending from Glu4 to Lys13 and from Leu27 to Thr33, with two turns from Gln16 to Arg19 and Phe22 to His25. A salt‐bridge appears likely between Arg20 and Glu30 which may be critical for holding the receptor‐binding domain together.