bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation and substrate specificity of the human protein kinase C α and ζ isoenzymes
Authors: KOCHS Georg , HUMMEL Richard , MEYER Diana , HUG Hubert , MARMÉ Dieter , SARRE Thomas F. 
Published: March 2005

Protein kinase C (PKC), a class of serine/threonine kinases activated by Ca2+ and/or phospholipids, is involved in a variety of cellular processes such as proliferation, differentiation and secretion. Nine members of the PKC gene family are known; these are differentially expressed in eukaryotic cells and can be divided into two sub‐groups: the Ca2+ ‐dependent (classical) PKC isoenzymes α, βI, βII and γ, and the Ca2+ ‐independent neoPKC isoenzymes δ, ε, ζ, ν and θ. A detailed biochemical characterisation of these PKC isoenzymes is one prerequisite for the elucidation of their distinct roles within cellular signal transduction.In this study, we report the cloning of a human PKC‐ζ cDNA, its expression in recombinant baculovirus‐infected insect cells and the partial purification of the PKC‐ζ isoenzyme. In comparison to higly purified human PKC α, a representative of the classical PKC subgroup, purified PKC ζ was characterised with respect to activator requirement, substrate specificity, proteolytic activation and sensitivity towards PKC inhibitors. In contrast to PKC α, PKC ζ exhibits a constitutive kinase activity which is independent of Ca2+, phosphatidylserine and diacylglycerol. Arachidonic acid alone or a combination of γ‐linolenic acid and phosphatidylserine slightly enhance PKC ζ activity. In the presence of the classical PKC activators phosphatidylserine/diacylglycerol, PKC α phosphorylates a PKC‐α pseudosubstrate‐derived peptide, an epidermal‐growth‐factor‐receptor‐derived peptide, histone III‐S and myelin basic protein to an equal extent, whilst PKC ζ phosphorylates only the PKC‐α‐derived peptide. However, arachidonic acid greatly diminishes PKC‐α activity towards the epidermal‐growth‐factor‐receptor‐derived peptide, histone III‐S and myelin basic protein, but enhances PKC‐ζ activity towards the PKC‐α‐derived peptide. These results indicate a possible modulation of substrate specificity of these two PKC isoenzymes by (the binding of) different activators (to their regulatory domains). In the case of PKC ζ, this finding is strengthened by the fact that the epidermal growth factor receptor‐derived peptide, which is not a substrate for the holoenzyme, is significantly phosphorylated by a protein fragment generated by limited proteolysis and comprising only the kinase domain. Furthermore, PKC ζ, in contrast to PKC α, is insensitive to PKC inhibitors known to interfere either with the regulatory or the catalytic domain and cannot be activated by phorbol ester treatment of NIH 3T3 cells or insect cells, overexpressing the respective PKC isoenzyme. The potential implications of these findings on the mechanism(s) activation and the substrate specificity of PKC ζ are discussed.
The l‐lactate dehydrogenase gene of the hyperthermophilic bacterium Thermotoga maritima cloned by complementation in Escherichia coli
Authors: OSTENDORP Ralf , LIEBL Wolfgang , SCHURIG Hartmut , JAENICKE Rainer 
Published: March 2005

The gene for a l(+)‐lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was cloned by complementation of an Escherichia coli pfl. ldh mutant. The gene is part of a 4.5 kb SauIIIA fragment obtained by partial digestion of the Thermotoga genome. The DNA fragment was physically mapped and the putative Shine‐Dalgarno sequence within the non‐coding region determined. The gene contains 960 bp, including the stop codon, corresponding to 319 amino acids/subunit of the homotetrameric enzyme.Part of the amino acid sequence was confirmed by Edman degradation of peptides obtained from nanomolar quantities of the purified enzyme by tryptic digestion. A comparison of the amino acid sequenc̀e with those of known prokaryotic l‐lactate dehydrogenases reveals a high similarity, especially with the enzyme from thermophilic sources, where up to 48% identity is found.The gene was expressed as an active enzyme in a heterologous host.
Purification and primary structure of snail metallothionein
Authors: DALLINGER Reinhard , BERGER Burkhard , HUNZIKER Peter E. , BIRCHLER Neil , HAUER Charles R. , KÄAGI Jeremias H. R. 
Published: March 2005

A cadmium‐binding metallothionein has been purified from metal‐exposed Roman snails (Helix pomatia) using gel‐permeation, ion‐exchange and reverse‐phase high‐performance liquid chromatorgraphy. The S‐methylated protein was digested with trypsin and the endoproteinases Asp‐N, Glu‐C and Arg‐C. While most of the resulting peptides could be sequenced by Edman degradation, the intact protein, as well as the N‐terminal peptide, proved to be blocked. Analysis by mass spectrometry showed that the N‐terminal amino acid was an acetylated serine residue.Snail metallothionein, which is suggested to be involved in the detoxification of cadmium, contains 66 amino acid residues, 18 of which are cysteine residues arranged in seven Cys‐Xaa‐Cys motifs. The calculated molecular mass of the protein is 6.62 kDa. The primary structure of snail metallothionein reveals a clear relationship with molluscan and vertebrate metallothioneins, but lower similarity with metallothioneins of other invertebrate species. The N‐terminal region of the isolated protein proved to be unique among the metallothionein sequences determined so far, showing high degrees of similarity with the N‐terminal sequences of histones H2A and H4 which may be important for regulatory functions.
Studies on O‐glycans of Plasmodium‐falciparum‐infected human erythrocytes Evidence for O‐GlcNAc and O‐GlcNAc‐transferase in malaria parasites
Authors: DIECKMANN‐SCHUPPERT Angela , BAUSE Ernst , SCHWARZ Ralph T. 
Published: March 2005

O‐Glycosylation is the major form of protein glycosylation in human erythrocytes infected with the asexual intraerythrocytic stage of the malaria parasite, Plasmodium falciparum. This study compares aspects of O‐glycosylation in P. falciparum‐infected and uninfected erythrocytes.Non‐labeled and metabolically glucosamine‐labeled O‐glycans were obtained from the protein fraction of infected or uninfected erythrocytes by β elimination. Additional label was introduced by reduction with sodium borohydride, or by the attachment of radioactive Gal to peripheral GlcNAc using galactosyltransferase. 2–4‐times more labeled O‐glycans were obtained from infected erythrocytes compared to the same number of uninfected ones, consistent with additional biosynthesis by the parasite. Our analysis of these O‐glycans showed no significant qualitative divergence between the O‐glycans of the infected and those of the uninfected red cell.According to preliminary alditol analyses, the O‐glycans of P. falciparum‐infected red cells do not contain GalNAc at their reducing terminus. Moreover, GalNAc was not synthesized by P. falciparum from either Glc, Gal, GlcN or GalN. At least one O‐glycan found in P. falciparum‐infected erythrocytes contains GlcNAc at its reducing terminus.Gel‐filtration results had suggested the presence of O‐GlcNAc on proteins in the infected erythrocyte. Probing with a synthetic pentapeptide, we could show that P. falciparum expresses its own O‐GlcNAc transferase during intracrythrocytic development. Using this peptide, the enzyme was characterized to some degree. The localization and function of O‐GlcNAc in P. falciparum remains to be elucidated.
Conformational study of a short Pertussis toxin T cell epitope incorporated in a multiple antigen peptide template by CD and two‐dimensional NMR
Authors: ESPOSITO Gennaro , FOGOLARI Federico , VIGLINO Paolo , CATTARINUSSI Serge , DE MAGISTRIS Maria Teresa , CHIAPPINELLI Lorella , PESSI Antonello 
Published: March 2005

The immunogenic efficacy of multiple antigen peptides, MAPs, i.e. branched molecules in which multiple copies of a given immunogenic peptide are attached on a scaffold of lysine residues via both α and ε linkages, has been repeatedly demonstrated, but little is known about the structural arrangement of these peptide constructs. A conformational characterization was therefore performed for a known T cell epitope of the S1 subunit of Pertussis toxin, whose sequence is predicted to form α‐helix.The peptide DNVLDHLTGR, its N‐acetylated and C‐amidated analogue and a tetrabranched MAP based on the N‐acetylated peptide were prepared and studied by CD and two‐dimensional 1H‐NMR. No evidence of helical structure was obtained in water for the isolated peptides. In contrast, in trifluoroethanol, the isolated epitopes fold into a helical structure spanning the segment Val3–Thr8 in the uncapped molecule and encompassing also the N‐terminal region in the capped analogue. The mobile C‐terminal region tends to adopt a distorted turn arrangement in both peptides due to the folding of Arg10 guanidinium over the backbone. No distortion of the helix structure was observed for the single‐copy epitope in the four‐branched MAP molecule in trifluoroethanol: each peptide chain is equivalent within the MAP and shows an even more regular helical pattern than the isolated end‐blocked sequence. A slight difference was located at the junction with the lysine scaffold: the peptide bond to εNH was found in a much more extended conformation than the corresponding link to αNH.These structural results correlate with in vitro T cell stimulatory activity of the three compounds examined and provide arguments supporting the previous suggestion that MAP tetramers are unlikely to elicit an immune response specific for the synthetic template assembly, a feature necessary to retain the advantage of the polymeric epitope presentation.
Inhibition by the protein kinase C activator 4β‐phorbol 12‐myristate 13‐acetate of the prostaglandin F2α‐mediated and noradrenaline‐mediated but not glucagon‐mediated activation of glycogenolysis in rat liver
Authors: PÜSCHEL Gerhard P. , MIURA Hisayuki , NEUSCHÄFER‐RUBE Frank , JUNGERMANN Kurt 
Published: March 2005

In perfused rat livers, infusion of prostaglandin F2α (PGF2α) or noradrenaline increased glucose and lactate output and reduced flow. Glucagon increased glucose output and decreased lactate output without influence on flow. Infusion of phorbol 13‐myristate 14‐acetate (PMA) for 20 min prior to these stimuli strongly inhibited the metabolic and hemodynamic effects of noradrenaline, reduced the metabolic actions of PGF2α but did not alter the effects of glucagon.In isolated rat hepatocytes PGF2α noradrenaline and glucagon activated glycogen phosphorylase but only PGF2α and noradrenaline increased intracellular inositol 1,4,5‐trisphosphate (InsP3). The noradrenaline‐ or PGF2α‐elicited activation of glycogen phosphorylase and increase in InsP3 were largely reduced after preincubation of the cells for 10 min with PMA, whereas the glucagon‐mediated enzyme activation was not affected. In contrast to PMA, the phorbol ester 4α‐phorbol 13,14‐didecanoate, which does not activate protein kinase C, did not attenuate the PGF2α‐ and noradrena‐line‐elicited stimulation of glucose output, glycogen phosphorylase and InsP3 formation. Stimulation of InsP3 formation by AlF4−, which activates phospholipase C independently of the receptor, was not attenuated by prior incubation with PMA.Plasma membranes purified from isolated hepatocytes had both a high‐capacity, low‐affinity and a low‐capacity, high‐affinity binding site for PGF2α. The Kd of the high‐capacity, low‐affinity binding site was close to the concentration of PGF2α that increased glycogen phosphorylase activity half‐maximally. Binding to the high‐capacity, low‐affinity binding site was enhanced by guanosine 5′‐O‐(3‐thio)triphosphate (GTP[S]). This high‐capacity, low‐affinity site might thus represent the receptor. The Bmax and Kd of the high‐capacity site, as well as the enhancement by GTP[S] of PGF2α binding to this site, remained unaffected by PMA pretreatment.It is concluded that, in hepatocytes, activation of protein kinase C by PMA interrupted the InsP3‐mediated signal pathway from PGF2α via a PGF2α receptor and phospholipase C to glycogen phosphorylase at a point distal of the receptor prior to phospholipase C.
Structural and functional properties of a multi‐enzyme complex from spinach chloroplasts
Authors: GONTERO Brigitte , MULLIERT Guillermo , RAULT Magali , GIUDICI‐ORTICONI Marie‐Thérèse , RICARD Jacques 
Published: March 2005

The carboxylase activity of free ribulose 1,5‐bisphosphate carboxylase‐oxygenase has been compared to that of the five‐enzyme complex present in chloroplasts. Kinetic results have shown that the V/active site is lower for the free enzyme than for the complex. Conversely the Km is smaller for the complex than for the free enzyme. This implies that the catalytic activity of the enzyme is enhanced when it is embedded in the complex.Under reducing conditions and in the presence of reduced thioredoxin, inactive oxidized phosphoribulokinase, free in solution or inserted in the multi‐enzyme complex, becomes active. The kinetics of this activation process has been studied and shown to be exponential. The time constant of this exponential decreases, for the free enzyme, as thioredoxin concentration is increased. Alternatively, for the enzyme embedded in the complex, this time constant increases with thioredoxin concentration almost in a linear fashion. This implies that the complex is much more rapidly activated by reduced thioredoxin than is the free phosphoribulokinase.The variation of the amplitude of this activation process as a function of thioredoxin concentration is a hyperbola. The concentration of thioredoxin which results in half the asymptotic value of this hyperbola is smaller for the complex than for the free enzyme.A kinetic model has been proposed and the dynamic equations resulting from this model have been derived. They fit the experimental results exactly. From the variation of the amplitude of the activation process one may derive the binding constants of thioredoxin on either the oxidized enzyme or on a partly dithiothreitol‐reduced enzyme (both of them free or inserted in the complex). In either case, the affinity of reduced thioredoxin is larger for the complex than for the free enzyme. The individual values of some of the rate constants have also been estimated from the variation of the time constants as a function of thioredoxin concentration.Taken together, these results show that at least two enzymes, ribulose 1,5‐bisphosphate carboxylase‐oxygenase and phosphoribulokinase, have quite different kinetic properties depending on whether they are in free solution or embedded in the multi‐enzyme complex.
Increased phosphorylation of eukaryotic initiation factor 4α during early activation of T lymphocytes correlates with increased initiation factor 4F complex formation
Authors: Morley Simon J. , RAU Michael , KAY John E. , PAIN Virginia M. 
Published: March 2005

Mature porcine peripheral blood mononuclear cells (PPBMCs) exist in a resting state both in vivo and when maintained in culture, with low translation rates consistent with their non‐proliferative state. When cultured in the presence of the appropriate mitogen, there is a 2–4‐fold increase in the rate of protein synthesis per ribosome within 4 h of stimulation [Kay, J. E., Ahern, T. and Atkins, M. (1971) Biochim. Biophys. Acta 247, 322–334]. Studies on extracts prepared from unstimulated cells have suggested lesions in initiation factor activity, primarily affecting the binding of mRNA to ribosomes [Ahern, T., Sampson, J. and Kay, J. E. (1974) Nature 248, 519–521].In these studies, we have demonstrated that activation of quiescent PPBMCs with the phorbol ester phorbol 12‐myristate 13‐acetate or concanavalin A leads to a rapid 2–4‐fold increase in the rate of protein synthesis within 1 h or 4 h, respectively, which is insensitive to the transcriptional inhibitor, 5,6‐dichlorobenzimidazole riboside. Relative to control cells, both phorbol ester and concanavalin A induce a 2–4‐fold increase in labelling of the eukaryotic initiation factor eIF‐4α with phosphate in vivo, which primarily reflects a small net increase in phosphorylation rather than phosphate turnover on eIF‐4α. Similarly, with the human leukaemic T cell line JURKAT, stimulation of the T cell receptor with the monoclonal antibody, OKT‐3, or treatment with phorbol ester induces a 2–3‐fold increase in eIF‐4α phosphorylation within 30 min. Analysis of phosphorylation by two‐dimensional gel electrophoresis and measurement of kinase activity towards synthetic peptides, indicate that this increased labelling also reflects increased eIF‐4α kinase activity rather than phosphate turnover on eIF‐4α.Of central importance is the finding that, concomitant with increased rates of protein synthesis following stimulation of PPBMCs with either phorbol ester or concanavalin A, there is a significant increase in the level of eIF‐4α recovered in high‐molecular‐mass complexes. These data suggest that, in quiescent PPBMCs, eIF‐4F may be limiting and that the association of eIF‐4α and eIF‐4γ into high‐molecular‐mass complexes is regulated by phosphorylation and may play a pivotal role in translational control.
Partial purification and characterization of a circulating hypertensive factor in spontaneously hypertensive rats
Authors: SCHLÜTER Hartmut , KLUTH Brigitte , BÖRJESSON‐STOLL Regina , NORDHOFF Eckhard , ZIDEK Walter 
Published: March 2005

Parabiosis and cross‐circulation experiments with spontaneously hypertensive and normotensive rats gave indications for a previously unidentified circulating hypertensive agent. In this study, plasma from normotensive and hypertensive rats was fractionated and the vasopressor action of the corresponding fractions was measured in the isolated perfused rat kidney. One of three vasoactive fractions obtained by gel filtration (Biol‐Gel P2) from hypertensive rats showed a significantly higher activity (increase in perfusion pressure by 1502.9 ± 438.9 Pa) than that from normotensive rats (increase in perfusion pressure by 505.4 ± 186.2 Pa, P<0.01). Further chromatographic separations of this fraction revealed that the hypertensive factor is hydrophilic and has no ionic groups or vicinal diol groups. The molecular mass was estimated by dialysis and the matrix‐assisted laser desorption/ionization mass spectrometry to be in the range of 1 kDa. The vasopressor is heat resistant and not degradable with trypsin or carboxypeptidase Y. The vasopressor action was not inhibited with the angiotensin‐II‐receptor antagonist saralasin, the α‐receptor antagonist phentolamine, the thromboxane‐receptor antagonist carbocyclic thromboxane A2 or the serotonin antagonist ketanserine.The results confirm the existence of a vasopressor factor in the plasma of hypertensive rats and, in a lower concentration, of normotensive rats, which is possibly related to the pathogenesis of essential hypertension. The chromatographic behaviour suggests that this factor is different from the parathyroid hypertensive factor described recently.
Aminoacyl chloromethanes as tools to study the requirements of NADPH oxidase activation in human neutrophils
Authors: CHOLLET‐PRZEDNOWED Emmanuelle , LEDERER Florence 
Published: March 2005

Previous studies from this laboratory described the kinetic characteristics of the inhibition by tosylphenylalanine chloromethane (TosPheCH2Cl) on superoxide anion production by human neutrophils (PMN) stimulated with a phorbol ester (PMA). In this study we present further evidence concerning the potential role of the chloromethane target in the normal cellular activation of NADPH oxidase. When PMN are treated with TosPheCH2Cl and subsequently PMA, or with the two reagents in the reverse order, the inhibition of superoxide production by the intact cells is still present in a particulate NADPH oxidase fraction prepared from these cells. Nevertheless, when cells incubated only with the chloromethane and not with PMA are disrupted, both their cytosolic and membrane fractions are fully competent in the cell‐free activation assay. Thus, the chloromethane target has a role in NADPH oxidase activation exclusively at the cellular level. This observation constitutes additional evidence in favour of the idea that activation in the cell‐free system reflects only partially the events which occur in the cells.When cells are activated with PMA, their cytosol displays a loss of activating capacity in the cell‐free activation assay in the presence of arachidonate, as was shown before with SDS as activator [Ambruso, D. R., Bolsher, B. G. J. M., Stockman, P. M., Verhoeven, A. J. & Roos, D. (1990) J. Biol. Chem. 265, 924–930]. This phenomenon was shown to arise most probably from the translocation of cytosolic factors to the membrane, resulting in a depleted cytosol. When superoxide production was inhibited by cell treatment with TosPheCH2Cl, either before or after activation with PMA, the cytosol from inhibited cells showed a recovery of activation capacity in the cell‐free system. This effect probably results from TosPheCH2Cl inhibiting the translocation of the cytosolic factors when added before PMA. This results in an insufficient activation at the membrane level, which was previously considered as an inhibition. The effect of TosPheCH2Cl, when added after PMA, can best be explained again as an inhibition of translocation in the frame of the continuous replenishment‐deactivation hypothesis proposed by Akard et al. [Akard, L. P., English, D. & Gabig, T. G. (1988) Blood 72, 322–327]. Thus, TosPheCH2Cl is apparently a promising new tool for studying the activation of NADPH oxidase at the cellular level.In this study, we have also found that [3H]TosPheCH2Cl‐labeled cells show a prominent radiola‐beled band of Mr 15000 after SDS/PAGE analysis of the cytosolic proteins from inhibited cells; this species has the same migration as the labeled membrane protein detected previously, but appears much more abundant on a protein/cell basis. The potential role of the TosPheCH2Cl protein target is discussed.
Nucleotide sequence and promoter‐specific effect of a negative regulatory region located upstream of the mouse proliferating cell nuclear antigen gene
Authors: MATSUOKA Shuhei , YAMAGUCHI Masamitsu , HAYASHI Yuko , MATSUKAGE Akio 
Published: March 2005

Different portions of the 5′‐upstream region of the mouse proliferating cell‐nuclear‐antigen (PCNA) gene were combined with the bacterial chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in mouse neuroblastoma N18TG2 cells transfected with these recombinant plasmids and RNase protection analysis have revealed the existence of a negative regulatory region between nucleotides –1231 and –624 (+1 denotes the transcription initiation site). The CAT expression levels were gradually increased, depending on the extent of deletion from the 5′‐terminus in this region, suggesting that the negative regulatory region consists of multiple elements with rather weak repressing activities.Significant sequence similarity was found between the negative regulatory region of the PCNA gene and those of the several reported genes. A 752‐bp segment containing this negative regulatory region repressed the function of the PCNA gene promoter in an orientation‐independent and position‐independent manner. However, the negative regulatory region showed almost no repressing effect on the functions of the heterologous gene promoters such as the simian virus 40 enhancer promoter, the enhancer promoter in the Rous sarcoma virus long‐terminal repeat and the mouse DNA polymerase β gene promoter. These results suggest that the negative regulatory region of the mouse PCNA gene functions specifically to its own promoter. This unique property is discussed in comparison with that of the negative regulatory elements of the mouse DNA polymerase β gene.