bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of yeast pGKL 128‐kDa killer‐toxin secretion signal sequence
Authors: TOKUNAGA Masao , KAWAMURA Akiko , OMORI Akira , HISHINUMA Fumio
Published: March 2005

The linear double‐stranded DNA plasmid pGKL1 in yeast encodes a killer toxin consisting of 97‐kDa 31‐kDa and 28‐kDa subunits. A 128‐kDa protein precursor of the 97‐kDa and 31‐kDa subunits, was first synthesized with a 29‐amino‐acid extension at its NH2‐terminus as a secretion signal sequence. In the present study, the property of this signal sequence was studied by the analysis of a fusion protein with mouse α‐amylase. Using the secretion signal sequence of the killer protein, the mouse α‐amylase was successfully secreted into the culture medium. An intracellular precursor form of α‐amylase was identified and purified. Analysis of the NH2‐terminal sequence of this precursor molecule indicated that it corresponded to the secretory intermediate (pro form) of α‐amylase with the removal of the hydrophobic segment (Met1–Gly16) of the secretion signal. Both the secretion of α‐amylase into the culture medium and the detection of the pro‐α‐amylase species in the cells were prohibited by a sec11 mutation, or by the conversion of Gly to Val at the 16th position of the secretion signal. These results strongly suggest that the cleavage occurs between Gly16 and Leu17 by a signal peptidase, and that this cleavage is required for the secretion of α‐amylase into the medium. Based on the data from the NH2‐terminal amino acid sequences of secreted α‐amylases, we conclude that the 29‐amino‐acid secretion signal present in the 128‐kDa killer toxin precursor protein is a prepro structure.
The differential transcriptional activity of two amphibian U1 small‐nuclear RNA genes correlates with structural differences in the proximal sequence element
Authors: MURGO Sylvie , KROL Alain , CARBON Philippe
Published: March 2005

We previously analyzed the transcription of an axolotl U1 small‐nuclear RNA (snRNA) gene (AmU1) by microinjection into Xenopus laevis oocytes. In such an assay, AmU1 showed a low template activity compared to that of an X. laevis U1 snRNA gene (XlU1B2). Swapping the proximal sequence element (PSE) with that of XlU1B2 was required for AmU1 to acquire a transcription level equal to that of XlU1B2. In the present work, we examine the functional importance of the nucleotides that are common or different in both PSEs with the aim of identifying which nucleotides within the Xenopus U1 PSE are critical for this enhancement of Ambystoma mexicanum U1 snRNA transcription. The PSE mutation analysis showed that the central, phylogenetically conserved C‐58/C‐57 doublet is absolutely required for U1 promoter activity. In the 3′ portion of this element, a CGC to ATG change (positions ‐54/‐52) which partially restores the XlU1B2 PSE sequence, enables the AmU1 gene to gain the same transcriptional activity as XlU1B2. Remarkably, in this clustered point mutation, the sole C‐54 to A‐54 change is sufficient to obtain this increased level. Therefore, the activity of the AmU1 gene in injected Xenopus oocytes is strongly affected by a single sequence difference between AmU1 and XlU1B2 PSEs. This finding underscores the crucial importance of the nucleotide identity at position ‐54 to the function of the Xenopus U1 PSE.
Functional study of the human thyroid peroxidase gene promoter
Authors: ABRAMOWICZ Mare J. , VASSART Gilbert , CHRISTOPHE Daniel
Published: March 2005

Structure/function relationships in the human thyroid peroxidase gene promoter have been studied by deletion and mutation analyses and confronted with footprint patterns obtained with thyroid nuclear extracts and the purified thyroid transcription factor TTF‐1. Crude nuclear extracts from dog thyroid primary cultures were shown to contain a binding activity recognizing the –119 to –105 segment of the promoter (coordinates relative to the transcriptional start site). Deletion, or site‐directed mutagenesis of this segment dramatically reduced transcriptional activity in transient expression experiments on gene fusions of the thyroid peroxidase promoter and the growth hormone reporter. This binding activity was increased in nuclear extracts from thyrocytes cultured in the presence of the cAMP‐agonist forskolin. A mutation that decreased the promoter function in forskolin‐stimulated thyrocytes resulted in weakening of the corresponding footprint. The binding site displays no significant sequence similarities with known cAMP‐responsive elements. Mutagenesis of another region of the promoter (–99 to –94) induced the binding of an additional factor, resulting in a dramatically enhanced promoter activity.

We show that the thyroid‐specific transcriptional factor TTF‐1 is not directly involved in the above‐mentioned interactions and provide evidence suggesting that, in spite of displaying a similar binding pattern to thyroperoxidase and thyroglobulin promoters in vitro, TTF‐1 plays a less important role in the former.

Altogether, our data delineate the minimal thyroid peroxidase gene promoter in the human and identify the binding sites of two trans‐activating factors, one of them being potentially the mediator of a non‐conventional cAMP control, independent of the cAMP‐responsive element and factor AP‐2.
Photoaffinity labelling of cholinesterases
Authors: EHRET‐SABATIER Laurence , SCHALK Isabelle , GOELDNER Maurice , HIRTH Christian
Published: March 2005

Two para‐dialkylaminobenzenediazonium salts, the dimethylamino (A) and dibutylamino (B) derivatives, are presented as structural probes for acetylcholinesterase and butyrylcholinesterase. While being reversible competitive inhibitors in the dark, A and B behave, upon irradiation and through the formation of arylcation species, as irreversible labels of ammonium‐binding sites of both enzymes. The observed variations of the different inactivation rate constants point to a different structural environment for acetylcholinesterase‐binding and butyrylcholinesterase‐binding sites. Moreover, in the case of acetylcholinesterase, protection experiments with specific ligands (edrophonium and propidium) showed that the dimethylamino salt A exclusively labels the hydrolytic anionic site, whereas the dibutylamino salt B also labels the peripheral site. Specificities and stoechiometries of the incorporations were determined and, in the case of acetylcholinesterase, the irradiated protein was submitted to chemical degradation. Peptide maps were obtained by gelpermeation chromatography and HPLC, giving access to labelled peptides which belong either to the active or to the peripheral site.
Purification and characterization of F420H2‐dehydrogenase from Methanolobus tindarius
Authors: HAASE Peter , DEPPENMEIER Uwe , BLAUT Michael , GOTTSCHALK Gerhard
Published: March 2005

The oxidation of F420H2 (reduced coenzyme F420) is a key reaction in the final step of methanogenesis. This step is catalyzed in Methanolobus tindarius by the membrane‐bound F420H2‐dehydrogenase which was purified 31‐fold to apparent homogeneity. The apparent molecular mass of the native enzyme was 120 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of five different subunits of apparent molecular masses of 45 kDa, 40 kDa, 22 kDa, 18 kDa and 17 kDa. The purified F420H2‐dehydrogenase, which was yellowish, contained 16 ± 2 mol iron and 16 ± 3 mol acid‐labile sulfur/mol enzyme. No flavin could be detected. The oxygen‐stable enzyme catalyzed the oxidation of F420H2 (apparent Km= 5.4 μM) with methylviologen and metronidazole as electron acceptors at a specific rate of 13 μmol · min−1± min−1± mg−1 (Kcat= 25.5 s−1). The isoelectric point was at pH 5.0. The temperature optimum was at 37°C and the pH optimum at 6.8.
Mechanism of racemization of amino acids by aspartate aminotransferase
Authors: KOCHHAR Sunil , CHRISTEN Philipp
Published: March 2005

Aspartate aminotransferase (mitochondrial isoenzyme from chicken) has been found to racemize very slowly dicarboxylic amino acid substrates in the presence of their cognate oxo acids [Kochhar, S. & Christen, P. (1988) Eur. J. Biochem. 175, 433–438]. Tyrosine, phenylalanine and alanine are racemized at the same rate although they undergo the transamination reaction 3–5 orders of magnitude more slowly than the dicarboxylic substrates. Similarly, the truncated enzyme aspartate aminotransferase‐(27/32–410) catalyzes the racemization at the same rate as the native enzyme, while its rate of transamination is decreased to 3% of that of the native enzyme. Apparently, the rate‐limiting step in racemization is not immediately linked to the transamination cycle. Decreasing the water concentration in the reaction medium by adding methanol at 0°C drastically reduces the rate of racemization without affecting the rate of transamination. On the basis of these and additional kinetic data and the model of the three‐dimensional structure of the active site, we conclude that a water molecule is responsible for the protonation of Cα of the coenzyme‐substrate intermediate from the wrong side. The diffusion of the water molecule into the interior of the enzyme appears to be the rate‐limiting step in aspartate‐aminotransferase‐catalyzed racemization.
Purification and characterization of proteinase yscJ, a new yeast peptidase
Authors: WAGNER Jean‐Claude , WOLF Dieter H.
Published: March 2005

A newly recognized peptidase, designated proteinase yscJ, was purified from the yeast Saccharo‐myces cerevisiae. The enzyme is of non‐vacuolar origin and cleaves the Tyr‐Lys bond of the synthetic peptide substrate Cbz‐Tyr‐Lys‐Arg‐NH‐Ph (Cbz, benzyloxycarbonyl; NH‐Ph, 4‐nitroanilide) and the Glu‐Lys bond of the substrate Boc‐Glu‐Lys‐Lys‐NH‐Mec (Boc, butoxycarbonyl; Mec, 4‐methylcoumarinyl) with high efficiency. Optimum pH for cleavage of Cbz‐Tyr‐Lys‐Arg‐NH‐Ph is in the range 7.0–7.5. The purified enzyme has a molecular mass of approximately 58 kDa, as judged by gel filtration on a Superose 12 FPLC column. Mercury compounds and EDTA were found to be potent inhibitors of proteinase yscJ activity.
Differential proteolytic sensitivity of yeast fatty acid synthetase subunits α and β contributing to a balanced ratio of both fatty acid synthetase components
Authors: SCHÜLLER Hans‐Joachim , FÖRTSCH Brigitte , RAUTENSTRAUSS Bernd , WOLF Dieter H. , SCHWEIZER Eckhart
Published: March 2005

The Saccharomyces cerevisiae genes FAS1 and FAS2 encoding the β and α subunit of yeast fatty acid synthetase (FAS), respectively, were individually deleted by one‐step gene disruption. Northern blot analysis of RNA from the resulting fas null allele mutants indicated that deletion of FAS2 did not influence the transcription of FAS1, while FAS2 transcription was significantly reduced in the Δfas1 strain. These data suggest an activating role of subunit β on FAS2 gene expression or, alternatively, a repression of FAS2 by an excess of its own gene product. Compared to the intact α6β6 complex, the individual FAS subunits synthesized in the Δfas1 or Δfas2 strains exhibit a considerably increased sensitivity towards the proteinases present in the yeast cell homogenate. Using yeast mutants specifically defective in the vacuolar proteinases yscA (PRA1/PEP4 gene product) and/or yscB (PRB1 gene product), it was shown that in vitro, subunit α is efficiently degraded by proteinase yscA while for degradation of subunit β, the combined action of proteinases yscA and yscB is necessary. In vivo, besides the vacuolar proteinases, an additional proteolytic activity specifically affecting free FAS subunit α becomes increasingly apparent in cells entering the stationary growth phase. In contrast, under similar conditions uncomplexed FAS subunit β is stable in strains lacking the vacuolar proteinases yscA and yscB. The reduced FAS subunit levels, at the stationary phase, were independent of the corresponding FAS transcript concentrations. Thus, differential degradation pathways are obviously removing an excess of either FAS subunit, at least under starvation conditions. A combination of both regulation of FAS gene expression and proteolysis of free FAS polypeptides may therefore explain the equimolar amounts of both FAS subunits observed in yeast wild‐type cells.
Diurnal rhythm of phosphorylation of rat liver acetyl – CoA carboxylase by the AMP‐activated protein kinase, demonstrated using freeze‐clamping
Authors: DAVIES Stephen P. , CARLING David , MUNDAY Michael R. , HARDIE D. Grahame
Published: March 2005

Acetyl‐CoA carboxylase was purified to homogeneity, in the presence of protein phosphatase inhibitors, from rat liver sampled without freeze‐clamping. The enzyme was in a highly phosphorylated state (4.8 mol/subunit) of low specific activity, and could be dramatically reactivated by treatment with protein phosphatase‐2A. Amino acid sequencing and fast‐atom‐bombardment mass spectrometry showed that the enzyme was phosphorylated in Ser79, Ser1200 and Ser1215, the three sites known to be phosphorylated in cell‐free assays by the AMP‐activated protein kinase.

The inactive enzyme could also be completely reactivated using a limited treatment with trypsin, which removes the N‐terminal segment containing Ser79 and reduces the phosphate content to 3.5 mol/subunit. These results strengthen previous findings that it is phosphorylation at Ser79 by the AMP‐activated protein kinase that is responsible for the inactivation, and not the phosphorylation of the 220‐kDa core fragment (which contains Ser1200 and Ser1215).

Analysis of the phosphorylation state of Ser79 in acetyl‐CoA carboxylase from rat liver showed that phosphorylation occurs post mortem if freeze‐clamping is not used. The higher phosphorylation observed in extracts made without freeze‐clamping correlates with a large increase in AMP and decrease in ATP (presumably caused by hypoxia during removal of the liver), and with increased activity of the AMP‐activated protein kinase. These results provide a rational explanation for the post mortem phosphorylation events, and re‐emphasize the point that rapid cooling of cells and tissues is essential when measuring the expressed activity of acetyl‐CoA carboxylase (as well as 3‐hydroxy‐3‐methylglutaryl‐CoA reductase).

Using the freeze‐clamping procedure, the ratio of‘expressed’ activity (measured in the presence of protein phosphatase inhibitors) to ‘total’ activity (measured after complete dephosphorylation) of rat liver acetyl‐CoA carboxylase showed a marked diurnal rhythm, changing from 50% in the active form in the middle of the dark period to less than 10% active in the middle of the light period. The very low activity in the light period was associated with a high level of phosphorylation in Ser79. This diurnal rhythm is very similar to that previously described for the phosphorylation of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase, another substrate for the AMP‐activated protein kinase. Neither the activity of the AMP‐activated protein kinase nor the content of AMP, ADP or ATP changed between the dark or light periods.

Feeding of high‐fat diets (beef tallow or sunflower oil) prevented the dephosphorylation of liver acetyl‐CoA carboxylase that normally occurs during the dark period in rats on chow diet, as well as dramatically reducing the total amount of enzyme.
Molecular cloning of the murine substance K and substance P receptor genes
Authors: SUNDELIN Johan B. , PROVVEDINI Diego M. , WAHLESTEDT Claes R. , LAURELL Henrik , POHL Jens S. , PETERSON Per A.
Published: March 2005

The peptides substance K and substance P evoke a variety of biological responses via distinct, guanosine‐nucleotide‐binding‐regulatory‐protein‐coupled receptors. We have screened a murine genomic cosmid library using oligonucleotide probes and have isolated, cloned and characterized the substance K receptor and the substance P receptor genes. The coding portion of the substance K receptor gene consists of five exons distributed over 13 kbp. The substance P receptor gene is considerably larger than that of substance K (more than 30 kbp), however, the bundaries of the four exons that have been characterized in the substance P receptor gene correspond exactly to the homologous exons in the substance K receptor gene. To verify the identity of the isolated genes, we have cloned the corresponding cDNA by means of the polymerase chain reaction and we have expressed these cDNA species in Xenopus laevis oocytes. The ligand binding characteristics determined in this system pharmacologically confirm the identity of the two receptors. The deduced amino acid sequence of the mouse substance K receptor is 94% identical to the rat sequence and 85% identical to the bovine and human sequences. The mouse substance P receptor amino acid sequence is 99% identical to the rat sequence. The cloning of the murine substance K and substance P receptor genes should contribute substantially to the generation of in vivo models for the detailed analysis of the functional significance of these receptors.
Inducible expression of human phospholipase C‐γ2 and its activation by platelet‐derived growth factor B‐chain homodimer and platelet‐derived growth factor A‐chain homodimer in transfected NIH 3T3 fibroblasts
Authors: TOTZKE Frank , MARMÉ Dieter , HUG Hubert
Published: March 2005

Growth factors such as platelet‐derived growth factor (PDGF) have been shown to activate phospholipase C‐γ1 (PLC‐γ1). We have overexpressed the human PLC‐γ2 (hPLC‐γ2) cDNA in murine NIH 3T3 fibroblasts using the interferon‐type‐I inducible murine Mx promoter. Northern blot analysis revealed an induction of hPLC‐γ2 mRNA by interferon (IFN) α of about 25‐fold as compared to the uninduced transcript level. Western blot analysis with anti(bovine PLC‐γ2) antiserum showed increased hPLC‐γ2 protein levels in hPLC‐γ2 transfected cells. Induction with IFN α resulted only in a slight further increase. After labelling the cells with [35S]methionine an increase of radioactive label in a protein migrating at 148 kDa could be detected in IFN‐α‐stimulated, hPLC‐γ2 overexpressing cells. PLC activity in homogenates from hPLC‐γ2 overexpressing cells was increased as compared to control cells transfected with the vector lacking the hPLC‐γ2 cDNA insert. There was no difference between in vitro PLC activity in homogenates from PDGF B‐chain homodimer (BB) treated and untreated cells. PLC activity was mainly present in the soluble fraction. After incubation of hPLC‐γ2 overexpressing cells with IFN α, the in vitro activity of PLC increased significantly in the soluble fraction. Stimulation with PDGF BB increased inositol phosphate production about 3.5‐fold in control cells and about 10‐fold in hPLC‐γ2 overexpressing cells. PDGF A‐chain homodimer (AA) showed slightly smaller effects. These results demonstrate that human PLC‐γ2 can be expressed functionally in murine NIH 3T3 fibroblasts and can be activated by both murine PDGF receptors, α and β type.
Modifications of hepatic alpha‐1‐acid glycoprotein and albumin gene expression in rats treated with phenobarbital
Authors: BERTAUX Odile , FOURNIER Thierry , CHAUVELOT‐MOACHON Laurence , PORQUET Dominique , VALENCIA Richard , DURAND Geneviéve
Published: March 2005

The serum level of α1‐acid glycoprotein (α1‐AGP) is significantly increased in various animal species by treatment with cytokines, glucocorticoids and phenobarbital. The mechanisms responsible for the cytokine‐induced and glucocorticoid‐induced increases are now well documented, but not so in the case of phenobarbital. The main purpose of this study was to assess whether phenobarbital acts on α1‐AGP synthesis in the liver at the transcriptional or translational level. Male Dark Agouti rats received 70 mg phenobarbital/kg daily for 7 days. The analysis of total hepatic RNA showed that a single injection of phenobarbital induced an 11‐fold increase in phenobarbital‐dependent cytochrome P450IIB mRNA, whereas seven injections of phenobarbital were required to induce a maximum 5.5‐fold increase in α1‐AGP mRNA. Concurrently, the transcription rate of the α1‐AGP gene rose 3.5‐fold. Hepatocytes isolated after the seventh injection of phenobarbital showed a threefold increased capacity to secrete α1‐AGP, corresponding to a 3.2‐fold increased α1‐AGP mRNA content in the liver. In conditions in which its effect on the induction of α1‐AGP synthesis was maximum, phenobarbital caused a 30 % reduction in liver albumin mRNA and in albumin secretion by isolated hepatocytes, resulting from a 60–70 % reduction in the rate of transcription of the albumin gene measured in isolated nuclei. We conclude that the effect of phenobarbital on α1‐AGP and albumin gene expression occurs at the transcriptional rather than the translational level.
Site‐specific O‐glycosylation of human granulocyte/macrophage colony‐stimulating factor secreted by yeast and animal cells
Authors: ERNST Joachim F. , MERMOD Jean‐Jacques , RICHMAN Larry H.
Published: March 2005

To compare the site specificity of O‐glycosylation in lower and higher eukaryotes, we expressed human granulocyte/macrophage colony‐stimulating factor (hGM‐CSF) in the yeast Saccharomyces cerevisiae and in COS‐1 cells. Analyses of specific hGM‐CSF mutants secreted by yeast led to the conclusion that efficient O‐glycosylation in yeast requires residues S9 and T10. However, only S9 is used as an attachment point for an extended O‐glycosyl chain in a 15.5‐kDa hGM‐CSF form. A 14.5‐kDa hGM‐CSF form, secreted by yeast, appears substituted by single mannosyl residues at both positions S9 and T10, indicating that O‐glycosylation at T10 inhibits extension of the O‐glycosyl chain attached to S9. As in yeast cells, the addition of O‐glycosyl chains to hGM‐CSF secreted by COS‐1 cells requires the presence of S9 and T10 residues. These results demonstrate that, inspite of different biosynthetic routes, the selection of O‐glycosylation sites is similar between lower and higher eukaryotes.
Structure of the Clostridium thermocellum gene licB and the encoded β‐1,3‐1,4‐glucanase
Authors: SCHIMMING Silke , SCHWARZ Wolfgang H. , STAUDENBAUER Walter L.
Published: March 2005

The nucleotide sequence of the Clostridium thermocellum gene licB, coding for a thermoactive β‐1,3‐1,4‐glucanase, has been determined. The gene is located downstream, but in opposite orientation to the β‐glucosidase gene bglA. A coding region of 1002 bp is flanked by canonical promoter and transcription terminator sequences. The primary translation product of the licB gene has a predicted molecular mass of 37896 Da. The protein sequence can be divided into several discrete segments: an N‐terminal signal peptide, a catalytic region, a segment rich in Pro and Thr residues and a C‐terminal reiterated domain. The catalytic region shows close similarity to lichenases of bacilli (52 – 58% identity) and Fibrobacter succinogenes (35% identity), but is unrelated to barley β‐1,3‐1,4‐glucanases. It consists of two domains, which in the case of the F. succinogenes lichenase are arranged in reversed order to that of C. thermocellum and Bacillus lichenases. The C‐terminal reiterated domain of C. thermocellum lichenase is homologous to the duplicated non‐catalytic domain of endo‐β‐1,4‐glucanases and xylanase Z from the same organism. This domain is considered a characteristic feature of clostridial cellulases organized as multienzyme complex (cellulosome). The β‐1,3‐1,4‐glucanase encoded by the licB gene might therefore be an additional enzyme component of the C. thermocellum cellulosome.
Hybrid myosin light chains containing a calcium‐specific site from troponin C
Authors: da SILVA Ana Claudia Rasera , KENDRICK‐JONES John , REINACH Fernando C.
Published: March 2005

Recombinant DNA approaches have allowed us to probe the mechanisms by which the regulatory light chains (RLCs) regulate myosin function by identifying the functional importance of specific regions of the RLC molecule. For example, we have demonstrated that the presence of a high‐affinity Ca2+/Mg2+‐binding site in the N‐terminal domain of the RLC is essential for the regulation of myosin‐actin interaction [Reinach, F. C., Nagai, K. & Kendrick‐Jones, J. (1986) Nature 322, 80–83]. To explore further the role of this metal‐binding site in the RLC and generate an RLC with a Ca2+‐specific site, we constructed four chicken skeletal muscle myosin regulatory light chain hybrid ‘genes’. In these, the first domain containing the high‐affinity, Ca2+/Mg2+‐binding site in the RLC was replaced with that containing the lower‐affinity, Ca2+‐specific, regulatory site from troponin C (TnC). In two of these hybrids, we replaced only the Ca2+‐binding EF hand, while in the other two the EF hand and the N‐terminal helix of TnC were transplanted. These hybrids were expressed in Escherichia coli in high yields and the purified proteins were used in calcium‐binding experiments to assay the affinity and specificity of the sites and incorporated into scallop myosin to assay their regulatory behaviour. The results obtained show that the calcium‐binding site from TnC, when transplanted into the RLC backbone, had a low affinity although most of its specificity appeared to be retained. As a result, although the TnC/RLC hybrids bound to scallop myosin and were able to activate the MgATPase activity of scallop acto‐myosin, they were unable to regulate it. These results are in agreement with our previous findings that occupancy of the Ca2+/Mg2+ site in the RLC is essential for regulation. Our results suggest that the specificity and affinity of the calcium‐binding site in troponin C is dependent on both intra‐ and inter‐domain interactions within troponin C and that these latter interactions appear to be missing when this binding site is transplanted into the light chain backbone.
NOE and two‐dimensional correlated 1H‐NMR spectroscopy of cytochrome c′ from Chromatium vinosum
Authors: BANCI Lucia , BERTINI Ivano , TURANO Paola , VICENS OLIVER Margarita
Published: March 2005

1H two‐dimensional (nuclear Overhauser effect spectroscopy (NOESY) and two‐dimensional correlated spectroscopy (COSY) spectra of cytochrome c′ from Chromatium vinosum have been obtained. The protein is of medium size (Mr 28000), essentially high spin (S= 5/2) although some quantum mechanical spin admixing with S= 3/2 may be present. Under these circumstances NOESY cross peaks have been revealed between geminal protons (α‐CH2 propionate and β‐CH2 protons of the bound histidine) and between α‐CH2 propionate protons and the heme methyl groups. COSY maps have confirmed the geminal nature of the proton pairs, even with a linewidth as large as 900 Hz; the J value is about 12 Hz. This assignment has rationalized on a sound basis the biochemical behavior of this protein with pH and has showed the utility of this kind of spectroscopy for the other cytochromes c′ structures and analogous systems.
Action pattern of xylo‐oligosaccharide hydrolysis by Schizophyllum commune xylanase A
Authors: BRAY Mark R. , CLARKE Anthony J.
Published: March 2005

The endo‐1,4‐β‐xylanase of the basidiomycete Schizophyllum commune, designated xylanase A, was studied to determine its action pattern, rates of reaction and bond‐cleavage frequencies on xylo‐oligomer and xylo‐alditol substrates ranging in degree of polymerization (Dp) from xylotriose (X3) to xyloheptaose (X7). An HPLC method using a Dionex HPLC and Carbopac PA1 ion‐exchange column with pulsed amperometric detection was developed to quantify both substrate loss and increase of products. Xylanase A had no detectable activity on xylobiose (X2) and low activity on xylotriose and xylotetraose (X4) but cleaved X5–X7 rapidly with X2 and X3 as major products. Initial rate data from hydrolyses of individual oligomers at 25°C and pH 5.81 indicated that the Michaelis constant (Km) decreased with increasing chain length (n) of oligomer. Turnover number (kcat) increased with chain length up to n= 7 suggesting that the specificity region of xylanase A spans about seven xylose units. Bond‐cleavage frequencies obtained from xylanase A hydrolysis of xylo‐alditols indicated a strong preference for internal linkages of the xylose chain. The action pattern of xylanase A on reduced substrates suggests that the catalytic site is located assymetrically within the binding cleft of the enzyme.
Role of the β subunit of casein kinase‐2 on the stability and specificity of the recombinant reconstituted holoenzyme
Authors: MEGGIO Flavio , BOLDYREFF Brigitte , MARIN Oriano , PINNA Lorenzo A. , ISSINGER Olaf‐Georg
Published: March 2005

Recombinant human α subunit from casein kinase‐2 (CK‐2) was subjected, either alone or in combination with recombinant human β subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the β subunit could drastically reduce the loss of kinase activity, strongly suggesting a protective function for the β subunit. Assaying different peptides for specificity toward the recombinant α subunit and the recombinant reconstituted enzyme, showed that the presence of the β subunit could modify the specificity of the catalytic α subunit. Therefore, a dual function for the β subunit is proposed which confers both specificity and stability to the catalytic α subunit within the CK‐2 holoenzyme complex. The peptide DLEPDEELEDNPNQSDL, reproducing the highly acidic amino acid 55–71 segment of the human β subunit, counteracts the stimulatory effect of the β subunit on the α subunit activity and partially substitutes the β subunit in conferring thermal stability to the α subunit. No such effect is induced by the peptide MSSSEEVSW, reproducing the N‐terminal segment of the β subunit including the autophosphorylation site. It is suggested that the acidic domain of the β subunit, encompassing residues 55–71, plays a role in the interactions between the β and α subunits.
A supramolecular light‐harvesting complex from chloroplast photosystem‐II membranes
Authors: BASSI Roberto , DAINESE Paola
Published: March 2005

In this study, we report on the composition of a photosystem‐II antenna preparation which contains three chlorophyll‐a/b proteins (CP), CP29, CP24 and light‐harvesting complex (LHC) II obtained from Zea mays grana membranes as previously described [Dainese, P. & Bassi, R. (1991) J. Biol. Chem. 266, 8136–8142]. We demonstrate that the three chlorophyll proteins are present in the preparation with a 3:3:9 molar ratio and that they form a supramolecular antenna complex which represents one third of the photosystem‐II antenna system.

Phosphorylation experiments show that this complex is involved in the mechanism of regulation of excitation‐energy distribution between photosystems: phosphorylation of the membranes induces dissociation of the LHCII moiety from the CP29‐CP24 moiety and changes in the aggregation state of LHCII components of the CP29‐CP24‐LHCII complex. The LHCII subpopulations of the complex are shown to be distinct from the total LHCII population by isoelectrofocusing analysis. On the basis of these data and in the light of the stoichiometry of photosystem‐II chlorophyll‐binding proteins, we propose a model for the organization of photosystem‐II antenna system.
Electron – proton coupling in cytochrome c studied using protein variants
Authors: GAO Yuan , McLENDON George , PIELAK Gary J. , WILLIAMS Robert J. P.
Published: March 2005

An NMR study of the cytochrome c variant Asn52Ile is used to show how the redox state change in native cytochrome c is coupled to a rearrangement of a proton network which runs through the cytochrome c molecule. The substitution breaks the H‐bond network and removes the coupling. The uncovering of this putative proton channel and the connection of changes to it with redox state changes of the iron centre of the protein allows a possible description of the way in which redox energy state changes can be coupled to energisation and gating of protons in membranes.
Purification and characterization of cathepsin J from rat liver
Authors: NIKAWA Takeshi , TOWATARI Takae , KATUNUMA Nobuhiko
Published: March 2005

Cathepsin J has been partially purified [Liao, J. C. R. & Lenney, J. F. (1984) Biochem. Biophys. Res. Commun. 124, 909–916], but its detailed properties are still unknown. In this study, we have purified cathepsin J completely and characterized it. It was purified to homogeneity from the mitochondrial‐lysosomal fraction of rat liver by acid treatment, followed by ammonium sulfate precipitation (20–65%), and chromatographies on S‐Sepharose, ConA‐Sepharose, Affi‐gel 501, HPLC DEAE‐5PW and HPLC TSK G3000SW. Cathepsin J was found to be a lysosomal high‐molecular‐mass cysteine protease of about 160 kDa consisted of two different subunits. One subunit (α subunit) was a glycoprotein with a molecular mass of 19–24 kDa which was reduced to 19 kDa by treatment with endoglycosidase F. It has the amino acid sequence LPESWDWRNVR at its N‐terminus, which was very similar to those at the N‐termini of rat cathepsins B, H and L. The other subunit (β subunit) was a glycoprotein with a molecular mass of 17 kDa, which was reduced to 14 kDa by treatment with endoglycosidase F. It had DTPANETYPDLLG at its N‐terminus, which had no similarity with the N‐terminal sequences of other cathepsins. Cathepsin J showed strong affinity for synthetic substrates such as N‐benzyloxycarbonyl‐phenylalanyl‐arginine 4‐methyl‐coumaryl‐7‐amide and glycyl‐arginine β‐naphthylamide. It was activated by thiol reagents and chloride ion and was inhibited by cysteine protease inhibitors. However, its initial inhibition constant Ki(initial) by N‐(L‐3‐trans‐carboxyoxirane‐2‐carbonyl)‐L‐leucine‐3‐methylbutylamide (E‐64‐c) was 1800 nM, which was 100–500 times those of cathepsins B and L. Many properties of cathepsin J were similar to those of cathepsin C (dipeptidylaminopeptidase I) reported as a lysosomal cysteine protease with dipeptidyl‐aminopeptidase activity [McDonald, J. K., Reilly, T. J. & Ellis, S. (1964) Biochem. Biophys. Res. Commun. 16, 135–140]. Furthermore, antiserum against rat liver cathepsin C reacted with rat liver cathepsin J. These findings suggested that cathepsin J is identical with cathepsin C.
The thyroliberin receptor interacts directly with a stimulatory guanine‐nucleotide‐binding protein in the activation of adenylyl cyclase in GH3 rat pituitary tumour cells
Authors: PAULSSEN Ruth H. , PAULSSEN Eyvind J. , GAUTVIK Kaare M. , GORDELADZE Jan O.
Published: March 2005

The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine‐nucleotide‐binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gsα‐subunit cDNA clone (Gsα) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3′ non‐translated region. Transient expression of Gsα antisense RNA in GH3 cells resulted in the specific decrease of Gsα mRNA levels, followed by decreased Gsα protein levels. Thyroliberin‐elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30–40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gsα and Gi‐1α/Gi‐2α support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs‐like protein in addition to the coupling to phospholipase C.
Natural protein proteinase inhibitors and their interaction with proteinases
Authors: BODE Wolfram , HUBER Robert
Published: March 2005

The substrate‐like ‘canonical' inhibition by the ‘small’ serine proteinase inhibitors and the product‐like inhibition by the carboxypeptidase inhibitor have provided the only atomic models of protein inhibitor–proteinase interactions for about 15 years. The recently published structures of cystatin/stefin – papain complexes and of hirudin – thrombin complexes reveal novel non‐substrate ‐ like interactions. In addition, the structure of pro‐carboxypeptidase showes a mode of inactivation which bears resemblance to proteinase/protein inhibitor systems. Considerable progress in understanding the transition between native and cleaved states of the serpins has also been made by several recent structural studies.
Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium
Authors: RAGONE Raffaele , FACCHIANO Francesco , CACCIAPUOTI Giovanna , PORCELLI Marina , COLONNA Giovanni
Published: March 2005

The thermal stability of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium, has been characterized thermodynamically by a Van't Hoff analysis. Conformational transitions induced by guanidine hydrochloride, as well as by temperature, have been linked together in a scheme involving six equilibria, which arise from both dissociation and unfolding. The mechanism by which the protein achieves thermal stabilization is quite unusual. It is driven by a conformational equilibrium between two forms of different stability. The stability of each form towards denaturation is characterized by a specific temperature dependence. The low‐temperature form, indicated as ‘form A’, is stable over 12–89°C. Its stability maximum is 36.8 kJ/mol at 50°C. ‘Form B’, which is populated at higher temperature, spans the interval 28–146°C. Its stability maximum is 71.6 kJ/mol at 87°C. A possible explanation for the mechanism underlying this behaviour is discussed assuming that two major terms contribute to stability, i.e. hydrophobic interactions arising from burying of the accessible surface residues as well as conformational entropy. The thermal stabilization of the enzyme seems to depend on effects related to both an overall increase of flexibility and a concomitant decrease of the area buried upon folding. In this regard proteins from extreme thermophilic organisms appear to be a useful model to shed new light on the general problem of protein stability.
Characterization by mass spectrometry of a recombinant hepatitis δ virus antigen and its proteolytic products
Authors: TECCE Mario F. , PETRACCA Roberto , GIULIANI Marzia M. , RUOPPOLO Margherita , MARINO Gennaro , MALORNI Antonio , PUCCI Pietro
Published: March 2005

The recombinant hepatitis δ virus antigen was obtained as a chimaeric protein fused to the C‐terminus of the phage MS2 RNA polymerase. Following induction of the temperature‐sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34‐kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast‐atom‐bombardment mass spectrometry. The most represented degradation product, i.e. the 29‐kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of δ antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis δ antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35–40 amino acids at the C‐terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti‐(hepatitis δ virus) antisera.
Arginine catabolism in the phototrophic bacterium Rhodobacter capsulatus E1F1
Authors: MORENO‐VIVIÁN Conrado , SOLER Germán , CASTILLO Francisco
Published: March 2005

The phototrophic bacterium Rhodobacter capsulatus E1F1 grew with L‐arginine or L‐homoarginine as nitrogen source under light/anaerobiosis. However, when L‐arginine was used as the only source of both carbon and nitrogen, the bacterium exhibited weak growth levels and the excess of nitrogen was excreted to the medium as ammonia. By contrast, L‐ornithine was used under phototrophic conditions as either nitrogen or carbon source. Other compounds of the arginine catabolic pathways, such as putrescine or proline, also supported phototrophic growth of this bacterium. Under heterotrophic/dark conditions, R. capsulatus always showed a low growth rate with those nitrogen compounds. Cells growing on media containing L‐arginine, L‐homoarginine or L‐ornithine induced an Mn2+‐dependent arginase activity regardless of the presence of ammonium ions and other readily utilizable nitrogen sources. Arginase activity was strongly inhibited by Zn2+, Cu2+, borate, L‐cysteine, L‐ornithine and γ‐guanidinobutyrate. Mercurials also inactivated arginase, the activity being partially restored by the presence of thiols. Arginase was purified to electrophoretic homogeneity and found to consist of four identical subunits of 31 kDa. The molecular parameters and kinetic constants of arginase from R. capsulatus E1F1 resembled those previously described for the Saccharomyces cerevisiae enzyme rather than those of bacterial arginases.
Enzymatic synthesis and isolation of thymidine diphosphate‐6‐deoxy‐D‐xylo‐4‐hexulose and thymidine diphosphate‐L‐rhamnose
Authors: MARUMO Kenji , LINDQVIST Lennart , VERMA Naresh , WEINTRAUB Andrej , REEVES Peter R. , LINDBERG Alf A.
Published: March 2005

A two‐step enzymatic synthesis of dTDP‐l‐rhamnose is developed using enzymes from sonicated extracts of cultures of Escherichia coli K12 strains harboring plasmids containing different parts of the rfb gene cluster of Salmonella enterica LT2. The intermediate dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose was isolated after a 1‐h reaction, using only dTDP‐d‐glucose and dTDP‐d‐glucose 4,6‐dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 89%). In a two‐step reaction using dTDP‐d‐glucose and dTDP‐d‐glucose 4,6‐dehydratase in the first step, and with NADPH, dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose 3,5‐epimerase and NADPH:dTDP‐6‐deoxy‐l‐lyxo‐4‐hexulose‐4‐reductase in the second hour of incubation, the dTDP‐d‐glucose was fully converted to dTDP‐l‐rhamnose. The hexoses of both products were identified by mass spectroscopy. The molar yield of dTDP‐l‐rhamnose, after protein precipitation, anion‐exchange chromatography and desalting by gel chromatography, was 62%, corresponding to more than 150 mg, starting from 250 mg of dTDP‐d‐glucose. When stored lyophilysed under nitrogen, these products were found to be stable for several months.

Both dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose and dTDP‐l‐rhamnose have light absorption maxima at 267 nm, with molar absorption coefficients close to that of dTMP. However, the absorption coefficient of dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose at the absorption maximum of 320 nm (specific for sugars containing keto groups) was found to be approximately 20% higher than values presented earlier.

Furthermore, an HPLC technique is presented for determining the net activity of dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose 3,5‐epimerase and NADPH:dTDP‐6‐deoxy‐l‐lyxo‐4‐hexulose‐4‐reductase, based on separation of dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose and dTDP‐l‐rhamnose. The HPLC technique is also suitable for determination of all the nucleotide components involved in the synthesis.
Structural analysis of three prokaryotic 5S rRNA species and selected 5S rRNA–ribosomal‐protein complexes by means of Pb(II)‐induced hydrolysis
Authors: CIESIOLKA Jerzy , LORENZ Siegfried , ERDMANN Volker A.
Published: March 2005

Lead ions have been applied to the structural analysis of 5S rRNA from Thermus thermophilus, Bacillus stearothermophilus and Escherichia coli. Based on the distribution of Pb(II)‐induced cleavages, some minor modifications of the consensus secondary structure model of 5S rRNA are proposed. They include the possible base pairing between nucleotides at positions 11 and 109, as well as changes in secondary interactions within the helix B region. The ‘prokaryotic arm' region is completely resistant to hydrolysis in the three RNA species, suggesting that it is a relatively stable, highly ordered structure. Hydrolysis of E. coli 5S rRNA complexed with ribosomal protein L18 shows, besides the shielding effect of the bound protein, a highly enhanced cleavage between A108 and A109. It supports the concept that the major L18‐induced conformational change involves the junction of helices A, B and D.
Phenylalanine ammonia‐lyase in potato (Solanum tuberosum L.)
Authors: JOOS Hans‐Jürgen , HAHLBROCK Klaus
Published: March 2005

Potato (Solanum tuberosum L. cv. Datura) contains approximately 40 – 50 phenylalanine ammonia‐lyase (PAL) genes/haploid genome. Considerable cDNA heterogeneity indicates that at least about 10, and probably more, of these genes are potentially active. One subfamily, represented by one selected member (PAL‐1), was analyzed with respect to genomic complexity, nucleotide and deduced amino acid sequence, and mode of constitutive or induced expression. For comparison, a second gene (PAL‐2), representing several subfamilies that are easily distinguished from PAL‐1, was included in these studies. Extensive structural similarities were observed both between the TATA‐proximal portions of the PAL‐1 and PAL‐2 promoters, particularly in the areas containing putative cis‐acting elements, and among all presently known PAL proteins from various higher and lower plants.

The relative abundance of PAL mRNA varied greatly in several major potato organs. However, the patterns obtained with probes detecting either total PAL mRNA or more specifically, PAL‐1‐related or PAL‐2‐related mRNA species, were the same within experimental error. Mature leaves contained particularly low levels of PAL mRNA. Infection of these leaves with the pathogenic fungus, Phytophthora infestans, resulted in a large, transient induction of PAL mRNA. The relative timing of PAL‐1 and PAL‐2 mRNA expression, however, differed in compatible (fungus virulent, plant susceptible) but not in incompatible interactions (fungus avirulent, plant resistant). Wounding of leaves caused an extremely rapid and transient induction of both PAL mRNA species.
Total synthesis of horse heart apocytochrome c by conformation‐assisted condensation of two chemically synthesized fragments
Authors: VITA Claudio , GOZZINI Luigia , Di BELLO Carlo
Published: March 2005

A fully synthetic peptide, corresponding to the entire 104‐residue sequence of horse heart apocytochrome c with Met65 replaced by homoserine, has been obtained by an original conformation‐assisted three‐fragment condensation procedure. The method involves the selective joining of two synthetic fragments, namely residues 1–65 of the apopeptide with Met65 replaced by homoserine lactone and residues 66–104 of the protein in the presence of fragment 1–25 of the native heme‐containing peptide. The joining conditions have been optimized with regard to solvent, pH and possible influence of additives. The presence of radical scavengers and the complete exclusion of oxygen were found essential in order to prevent oxidative side reactions. A sensitive method based on reverse‐phase HPLC has been used to monitor the course of the reaction. Condensation yields up to 80% were obtained. The data obtained by this new three‐fragment rejoining approach are discussed and compared to those of a similar two‐fragment condensation procedure. Our data demonstrate how the folding properties of large synthetic peptide fragments, organized in a complex, can be utilized to extend the presently improved solid‐phase peptide methods to the synthesis of a functioning protein with more than 100 residues.