bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of pH and KCl on the Conformations of Creatine Kinase from Rabbit Muscle
Authors: Raimbault Cyrille , Couthon Fabienne , Vial Christian , Buchet René
Published: September 2004

The activity loss of creatine kinase (CK), observed at low pH (midpoint was 4.8) corresponded to the monomerization of the dimeric protein and was correlated with structural changes. The acid‐induced unfolding was not complete at this pH, as probed by circular dichroic (CD) and fluorescence methods. Further decrease of pH, led to a second transition (midpoint was pH 3.5). The loss of activity was irreversible at pH 4.8 (<20% native activity was recovered) while it was almost fully reversible (>90% of native activity was recovered) for the enzyme incubated at pH 0.9–2.5. The amount of intermolecular β‐sheets (monitored with the 1620 cm−1 infrared component band) was maximal when the enzyme was incubated at pH 4.8, as a consequence of protein aggregation, while it was minimal at extremes of pH and at low ionic strength. Acid‐induced and alkaline‐induced denaturations promoted different structural changes, leading to distinct partially unfolded conformational states. The addition of KCl (from 0.05 M to 0.5 M) to an acidic solution of monomeric creatine kinase (pH 1.6) resulted in a highly cooperative transition from the partially unfolded conformation (UA) to the more compact conformation (A) with the properties of a molten globule, as probed by CD spectra and by fluorescence. The formation of intermolecular β‐sheets in the compact conformation was observed by infrared spectroscopy, indicating formation of unstable aggregates.
Muscarinic Toxins from the Black Mamba Dendroaspis polylepis
Authors: Jolkkonen Mikael , Van Giersbergen Paul L. M. , Hellman Ulf , Wernstedt Christer , Oras Aldo , Satyapan Nisamanee , Adem Abdu , Karlsson Evert
Published: September 2004

Three new toxins acting on muscarinic receptors were isolated from the venom of the black mamba Dendroaspis polylepis. They were called muscarinic toxins α, β, and γ (MTα, MTβ, and MTγ), All of the toxins have four disulphide bonds and 65 or 66 amino acids. The sequences of MTα and MTβ were determined. The muscarinic toxins, of which about 12 have been isolated from venoms of green and black mambas, have 60–98% sequence identity with each other, and are similar to many (about 180) other snake venom components, such as α‐neurotoxins, cardiotoxins, and fasciculins. In contrast to the α‐neurotoxins, muscarinic toxins do not bind to nicotinic acetylcholine receptors.

The binding constants of MTα and MTβ were determined for human muscarinic receptors of subtypes m1–m5 stably expressed in Chinese hamster ovary cells. The toxins are less selective than the earlier discovered muscarinic toxins from the green mamba Dendroaspis angusticeps.

MTα and the muscarinic toxin MT4 from D. angusticeps differ only in a region of three amino acids (residues 31–33), which are Leu‐Asn‐His in MTα and Ile‐Val‐Pro in MT4. This difference causes a pronounced shift in subtype selectivity. MTα has high affinity to all subtypes, with Ki(inhibition constant) values of 23 nM (m1; pKi= 7.64±0.10), 44 nM (m2; pKi= 7.36±0.06), 3 nM (m3: pKi= 8.46±0.14), 5 nM (m4; pKi= 8.32±0.07), and 8 nM (m5; pKi= 8.09±0.07). MT4 has high affinity only to m1 (Ki= 62 nM) and m4 (87 nM) receptors, and low (Ki>1 μM) affinity to m2, m3, and m5. The region at positions 31–33 evidently plays an important role in the toxin‐receptor interaction.

MTβ has low affinity for ml and m2 receptors Ki>1 μM) and intermediate affinity for m3 (140 nM; pKi= 6.85±0.03), m4 (120 nM; pKi= 6.90±0.06), and m5 (350 nM; pKi= 6.46±0.01). The low affinity of MTβ may reflect a tendency for spontaneous inactivation.
Characterization of Apolipoproteins B‐100, AI and C from Plasma Lipoprotein in the Goose, Anser anser
Authors: Hermier Dominique , Sellier Nadine , Rousselot‐Pailley Daniel , Forgez Patricia
Published: September 2004

In this study we have characterized four of the principle goose apolipoproteins and compared their physicochemical properties with human and avian counterparts. Goose ApoB‐100 and ApoAI amino acid compositions were very similar to their chicken and human homologous proteins. The partial N‐terminal sequence from goose ApoAI was 91% and 82% similar to the corresponding duck and chicken proteins, respectively. Most of the observed amino acid changes detected between the ApoAI sequences were amino acid replacements having the same characteristics and could be the result of a single base mutation.

The N‐terminal portion of two ApoC‐like apolipoproteins were also studied. Goose ApoCa had an electrophoretic mobility of 0.31 and exhibited a nine‐residue motif that was well conserved between ApoCIII sequences from different species. We therefore suggest that ApoCa is the equivalent of mammalian ApoCIII. The N‐terminal portion of goose ApoCb, the second major ApoC in high‐density apolipoprotein, showed no similarity to proteins previously described in the literature. This protein displayed two isomorphs in alkaline urea gel electrophoresis called ApoCb1 and ApoCb2 with Rf values of 0.36 and 0.39, respectively. A genetic polymorphism was detected in the population whereby 25% of the animals carried only one isomorph and 50% exhibited both ApoCb isomorphs. These frequencies were similar in females and males. The transmission mode of these ApoCb isomorphs was consistent with two segregating alleles from a single codominantly expressed gene.
Purification and Properties of Coenzyme F390 Hydrolase from Methanobacterium thermoautotrophicum (strain Marburg)
Authors: Vermeij Paul , Vinke Esther , Keltjens Jan T. , Van Der Drift Chris
Published: September 2004

8‐Hydroxyadenylylated coenzyme F420 (coenzyme F390‐A) is formed in methanogenic bacteria upon oxidative stress. After reinstatement of anaerobic conditions, coenzyme F390 is degraded into coenzyme F420 and AMP. The enzyme catalyzing the latter reaction, coenzyme F390 hydrolase, was purified to homogeneity from Methanobacterium thermoautotrophicum strain Marburg 355‐fold to a specific activity of 12.1 μmol · min1· mg protein−1. The enzyme consisted of one polypeptide of approximately 27 kDa. Coenzyme F390 hydrolase displayed an apparent Km for coenzyme F390 of 40 μM. The enzyme required the presence of a reducing agent like dithiothreitol to become active. Activity could be manipulated by applying various ratios of reduced and oxidized dithiothreitol. Activation proceeded by a two‐electron reduction, which indicates that one S‐S bridge is involved the activation/inactivation of the enzyme. Dithiothreitol could be replaced by the methanogenic C1‐carrier 2‐mercaptoethanesulfonate (H‐S‐CoM), but not by N7‐mercaptoheptanoyl‐l‐threonine phosphate (H‐S‐HTP) or other naturally occurring thiol‐containing compounds. The addition of the heterodisulfide of H‐S‐CoM and H‐S‐HTP (CoM‐S‐S‐HTP) diminished the stimulatory effect of H‐S‐CoM.
Production of Recombinant Human Brain type I Inositol‐1,4,5‐trisphosphate 5‐phosphatase in Escherichia coli
Authors: Erneux Christophe , De Smedt Florence , Moreau Colette , Rider Mark , Communi David
Published: September 2004

The dephosphorylation of inositol 1,4,5‐trisphosphate (InsP3) to inositol 1,4‐bisphosphate is catalyzed by InsP3 5‐phosphatase. The coding region of human brain type I InsP3 5‐phosphatase was expressed as a fusion protein with the maltose‐binding protein (MBP) in Escherichia coli, using the pMAL‐cR1 vector. The relative molecular mass of the purified fusion protein (MBP‐InsP3‐5‐phosphatase) was approximately Mr 85000 as analysed by SDS/PAGE. The yield was about 10 mg fusion protein/l lysate. After cleavage from MBP with factor Xa, the specific activity of recombinant 5‐phosphatase was 120–250 μmol · mg−1· min−1. The molecular mass of purified protein by SDS/PAGE was Mr 43000. The activity was inactivated by p‐hydroxymercuribenzoate. The possibility that protein kinase C might phosphorylate InsP3 5‐phosphatase was tested on the purified 43000 Mrprotein. In this study, we show that recombinant 5‐phosphatase is not a substrate of protein kinase C.
The Mechanism of Substrate and Coenzyme Binding to Clostridial Glutamate Dehydrogenase During Reductive Amination
Authors: Basso Luiz A. , Engel Paul C. , Walmsley Adrian R.
Published: September 2004

The binding of NADH and 2‐oxoglutarate to glutamate dehydrogenase (GDH) from Clostridium symbiosum has been studied by fluorescence spectroscopy. The Kd values for the binding of these ligands have been measured by titration of either the nucleotide or protein fluorescence. During ternary complex formation, the substrate and coenzyme binding sites interact in a positive cooperative manner, but steady‐state studies reveal a decrease in affinity of the catalytic complex indicative of negative cooperativity. It was possible to determine the kinetics of formation of the glutamate‐dehydrogenase–NADH complex by stopped‐flow fluorescence spectroscopy but formation of the glutamate‐dehydrogenase–2‐oxoglutarate complex was optically silent. Ternary complex formation was characterized by a large quench in protein fluorescence. The binding of NADH to the glutamate‐dehydrogenase–2‐oxoglutarate binary complex is characterised by a linear increase in the association rate constant, consistent with a one‐step binding process. However, the binding of 2‐oxoglutarate to the glutamate‐dehydrogenase–NADH binary complex is characterised by a decrease in the rate for the observed transient. This suggests that 2‐oxoglutarate binds to a different conformation of the enzyme to that stabilized by NADH, and that the transition between these different conformational forms is rate limiting for ternary complex formation. NADH and 2‐oxoglutarate can therefore stabilize different conformational states of the enzyme. Collectively, these studies are suggestive of a kinetic model for ternary complex formation that involves the oscillation of the free, binary, and ternary glutamate dehydrogenase complexes between two different conformational states, termed E1 and E2. The equilibrium constants for ternary complex formation via the predominant pathway have been determined. The cooperativity betwen the substrate and coenzyme binding sites can be accounted for by the displacement of the equilibria between the E1 and E2 states because of their difference in affinities for NADH and 2‐oxoglutarate.
Synthesis of a Divalent Sialyl Lewis x O‐glycan, a Potent Inhibitor of Lymphocyte‐Endothelium Adhesion
Authors: Maaheimo Hannu , Renkonen Risto , Turunen Juha Pekka , Penttilä Leena , Renkonen Ossi
Published: September 2004

The recognition of cell‐surface l‐selectin by its carbohydrate ligands causes lymphocytes to roll on capillary endothelium at sites of inflammation. As this primary contact is a prerequisite for extravasation of the leukocytes to the tissue, its inhibition by free oligosaccharides capable of competing with the natural l‐selectin ligands is an attractive therapeutic possibility.

The exact structures of the biological ligands of l‐selectin are not yet known, but the principal carbohydrate epitopes share some structural features: they are O‐glycosidically linked mucin‐type oligosaccharides with N‐acetyllactosamine backbone, which is 3′‐sialylated or 3′‐sulfated, 3‐fucosylated and sometimes 6‐ or 6′‐sulfated at the distal N‐acetyllactosamine termini. Multivalency of the ligand, which is believed to enhance the binding, is achieved by a branched polylactosamine backbone or by a clustered array of O‐glycans.

We report here enzymic synthesis of a large oligosaccharide fulfilling several of the features characteristic to the l‐selectin ligands: it is a dodecameric O‐glycosidic core‐2‐type oligosaccharide alditol with a branched polylactosamine backbone carrying two distal α‐2,3′‐sialylated and α‐1,3‐fucosylated N‐acetyllactosamine groups (sialyl Lewis x, sialyl Lex). The structure of each saccharide on the synthesis route from disaccharide Galβ1–3GalNAc to the dodecasaccharide alditol was established by several methods including one‐ and two‐dimensional 1H‐NMR spectroscopy. The last step of the synthesis, the α‐1,3‐fucosylation of the 6‐linked arm proceeded sluggishly, and was associated with a noticeable shift in H1 resonance of the GlcNAc residue of the branch‐bearing N‐acetyllactosamine unit.

The final synthesis product and its analogs lacking one or both of the fucose residues were tested as inhibitors of l‐selectin‐mediated lymphocyte‐endothelium interaction in vitro in rejecting rat kidney transplant. While the non‐fucosylated O‐glycosidic oligosaccharide alditol did not possess any inhibitory activity, the mono‐fucosylated one (i.e. monovalent sialyl Lex) prevented the binding significantly and the difucosylated dodecasaccharide alditol (i.e. divalent sialyl Lex) was a very potent inhibitor (IC50, inhibitory concentration preventing 50% of binding = 0.15 μM). Besides the multivalency, also the Galβ1–3GalNAc‐ol sequence of the O‐glycosidic core appeared to increase the affinity of the glycan to l‐selectin. This was indicated by parallel inhibition experiments, where a disialylated and difucosylated branched polylactosamine decasaccharide, similar to the divalent dodecasaccharide alditol, but lacking the reduced O‐glycosidic core, was a less effective inhibitor (IC50= 0.5 μM) than the O‐glycosidic dodecasaccharide alditol.
Effect of the Aminosteroid, U73122, on Ca2+ Uptake and Release Properties of Rat Liver Microsomes
Authors: De Moel Mariëlle P. , Van De Put Frans H. M. M. , Vermegen Tino M. J. A. , De Pont JanJoep H. H. M. , Willems Peter H. G. M.
Published: September 2004

The putative phospholipase C inhibitor, U73122, transiently increases the cytosolic free Ca2+ concentration in rabbit pancreatic acinar cells by stimulating the release of Ca2+ from intracellular stores [Willems, Van de Put, Engbersen, Bosch, Van Hoof & De Pont (1994) Pflügers Arch, 427, 233–243]. In order to elucidate the exact mechanism of action of U73122 we studied its effects on both Ca2+‐stimulated Mg2+‐dependent ATPase activity and Ca2+‐stimulated ATP‐dependent Ca2+ uptake in rat liver microsomes. In addition, we studied its effects on Ca2+ release from steady‐state loaded microsomes. The effects of U73122 were compared with those of thimerosal, described in the literature as inhibiting Ca2+‐ATPases and sensitizing inositol 1,4,5‐trisphosphate‐operated Ca2+ release channels, and thapsigargin, a specific inhibitor of sarcoplasmic and endoplasmic reticulum Ca2+‐ATPases. Both U73122 (IC50= 9 μM) and thimerosal (IC50= 11 μM) dose‐dependently inhibited Ca2+‐stimulated Mg2+‐dependent ATPase activity, without significantly affecting Mg2+‐stimulated ATPase activity. Similarly, both U73122 (IC50= 9 μM) and thimerosal (IC50= 14 μM) dose‐dependently inhibited ATP‐dependent Ca2+ uptake. At concentrations beyond 20 μM, U73122 stimulated Ca2+ release from steady‐state loaded microsomes at a rate considerably higher than obtained with a maximally inhibitory concentration of thapsigargin (1 μM). This observation, which was not reached with equally inhibitory concentrations of thimerosal, demonstrates that higher U73122 concentrations cause an additional increase of the passive Ca2+ leak. The data presented demonstrate that U73122 stimulates the release of actively stored Ca2+ primarily through inhibition of the internal Ca2+ pump.
The Genomic Response of Tumor Cells to Hypoxia and Reoxygenation
Authors: Rupec Rudolf A. , Baeuerle Patrick A.
Published: September 2004

Hypoxia and reoxygenation are important pathophysiological conditions that occur during injury, ischemia, reperfusion and stroke. In tumors, hypoxia and oxidative stress are regarded as triggers for enhanced proliferation and metastasis. Hypoxia and reoxygenation exert part of their biological effects by inducing the expression of novel genes but very little is known about the transcription factors involved. Here, we have compared the behaviour of two redox‐controlled factors, AP‐1 and NF‐κB, during hypoxia and reoxygenation. We report that the DNA‐binding and transcriptional activity of transcription factor AP‐1 is very strongly induced in a biphasic response when HeLa cells are exposed to reduced oxygen pressure. This induction required new AP‐1 protein synthesis. Different members of the Jun/Fos family of transcription factors were found in the first and second maxima of activation. The pathogen‐responsive, pre‐existing transcription factor NF‐κB was not activated under hypoxic conditions. However, a p50‐p65 heterodimer of NF‐κB was rapidly and strongly activated when HeLa cells were re‐exposed to normal oxygen pressure. This explains the induction of NF‐κB‐controlled inflammatory cytokine genes during reperfusion of ischemic tissue. Our data suggest that the genomic response to hypoxia is primarily mediated by AP‐1 while the inflammatory response to reoxygenation is mediated by NF‐κB.
Complexes of Tissue Kallikrein with Protein C Inhibitor in Human Semen and Urine
Authors: España Francisco , Fink Edwin , Sanchez‐Cuenca Jaime , Gilabert Juan , Estellés Amparo , Witzgall Karlheinz
Published: September 2004

An ELISA was developed for quantifying the complex between tissue kallikrein (tKK) and protein C inhibitor (PCI) (tKK:PCI) in seminal plasma and urine. The ELISA used purified tKK:PCI complex as a standard and was specific for this complex with a detection limit of about 1.1 pM. Purified tKK:PCI complex was obtained from human urine and was 95% homogeneous as judged by SDS/PAGE. The 90‐kDa band corresponding to the purified tKK:PCI complex reacted with anti‐tKK and anti‐PCI antibodies as judged by immunoblotting. Seminal plasma collected in the absence of extrinsic inhibitors contained 1.8±0.6 nM tKK:PCI complex and 4.7±2.8 nM immunoreactive tKK (mean±SD, n= 10), which indicates that about 28% of the total tKK immunoreactivity is forming complexes with PCI. When semen was collected in the presence of tKK inhibitors it had only about 6% of the tKK complexed to PCI. In vitro studies showed that the tKK:PCI complex formation in semen was accomplished in about 1 h and that heparin stimulated both the rate and the extent of complexation of tKK with PCI. Native urine showed low levels of tKK:PCI complex, but after dialysis urine had 0.17±0.05 nM complex. Formation of tKK:PCI complex in urine and semen was also demonstrated by immunoblotting. These results suggest that PCI is a physiological inhibitor of tKK and provide additional evidence of the involvement of PCI in human reproduction.
The Replacement of Trp392 by Alanine Influences the Decarboxylase/Carboligase Activity and Stability of Pyruvate Decarboxylase from Zymomonas mobilis
Authors: Bruhn Heike , Pohl Martina , Grötzinger Joachim , Kula Maria‐Regina
Published: September 2004

The bulky tryptophan residue 392 located in the deep cleft leading to the active center of pyruvate decarboxylase (PDC) from Zymomonas mobilis was changed to alanine which is found in the equivalent position of PDC from yeast. The mutation reduced the decarboxylase activity towards pyruvate by a factor of two (60–70 U/mg), whereas the Km (1.1 mM in Mes/KOH buffer) remains unchanged compared with the wild‐type enzyme. The apparent Km for thiamine diphosphate (thiamin–P2) in the presence of 5 mM MgSO4 was increased by a factor of 10 (84 μM in Mes/KOH buffer) and the tetrameric mutant protein was less stable, as indicated by urea denaturation experiments. The mutation enhanced the carboligase activity of the enzyme towards benzaldehyde by a factor of four. The resulting α‐hydroxyketone was identified as (R)‐phenylacetylcarbinol.
Involvement of Janus Kinases in the Insulin Signaling Pathway
Authors: Giorgetti‐Peraldi Sophie , Peyrade Frédéric , Baron Véronique , Van Obberghen Emmanuel
Published: September 2004

The adaptor molecule growth‐factor‐receptor‐bound protein‐2 (Grb2) plays a role in insulin action since it links tyrosine phosphorylated IRS–1 and Shc to the guanine‐nucleotide‐exchange factor, Sos, which initiates the mitogen‐activated‐protein (MAP) kinase cascade by producing Ras‐GTP. Both IRS‐1 and Shc are phosphorylated by the insulin‐receptor tyrosine kinase. In the present study, we have investigated whether the tyrosine kinases of the Janus kinase family (JAK) could be involved in insulin signaling by acting on Grb2. In fibroblasts over‐expressing insulin receptors we observed that two tyrosine‐phosphorylated proteins interact with Grb2 and with a mutant of Grb2, which lacks the Src homology 2 (SH2) domain, indicating that these proteins associate with the SH3 domains of Grb2. Further, we found that both JAK1 and JAK2 constitutively associate with Grb2, through interaction with the SH3 domains of Grb2. Finally, insulin appears to induce the tyrosine phosphorylation of JAK1, but does not modify the tyrosine phosphorylation state of JAK2. In conclusion, our results suggest that the JAK proteins could participate in insulin signal transduction, and could therefore constitute an alternative pathway for mediating some of the pleiotropic responses induced by insulin.
Soluble Human Interleukin‐6 Receptor
Authors: Weiergräber Oliver , Hemmann Ulrike , Küster Andrea , Müller‐Newen Gerhard , Schneider Johannes , Rose‐John Stefan , Kurschat Peter , Brakenhoff Just P. J. , Hart Margreet H. L. , Stabel Silvia , Heinrich Peter C.
Published: September 2004

The extracellular domain of the human interleukin‐6 (IL‐6) receptor, comprising 339 amino acids following the signal peptide, has been expressed in baculovirus‐infected insect cells (Sf158). When the soluble receptor secreted into the culture medium was purified by affinity chromatography, using IL‐6 immobilized on Sepharose, 6 mg soluble receptor was isolated from 1 l conditioned medium of Sf158 suspension cultures. A molar absorption coefficient of 9.3 × 104l · mol−1· cm−1 was calculated from the ultraviolet spectrum of the soluble IL‐6 receptor. After SDS/PAGE and silver staining, an apparent molecular mass of 48 kDa was estimated for the purified protein. Deglycosylation with peptide N‐glycosidase F resulted in an increase in electrophoretic mobility and a decrease in the apparent molecular mass from 48 kDa to about 41–44 kDa. As expected, the soluble human IL‐6 receptor bound human 125I‐labeled IL‐6 with low affinity (Kd= 500 pM). Furthermore, the binding of soluble human IL‐6 receptor to immobilized IL‐6 was studied using real‐time interaction analysis. The recombinant soluble receptor showed biological activity on HepG2 cells stably transfected with a cDNA coding for IL‐6 (HepG2‐IL‐6 cells). Haptoglobin mRNA synthesis was induced by the soluble IL‐6 receptor at concentrations as low as 10 ng/ml. Five monoclonal antibodies were generated. Two groups of antibodies were identified mapping to amino acids 1–67 and 68–143 of the soluble IL‐6 receptor, respectively. The plasma clearance of soluble 125I‐labeled IL‐6 receptor in the absence and presence of IL‐6 was studied in rats as a model system. The kinetics was biphasic. Soluble IL‐6 receptor/IL‐6 complexes were cleared more rapidly than the soluble receptor alone. Intravenously injected soluble 125I‐labeled IL‐6 receptor, as well as complexes with IL‐6, rapidly accumulated in liver and to a lesser extent in skeletal muscle, skin and kidneys, Subsequently, the radioactivity appeared in the gut content.
Characterization of the Ovine‐Lens Plasma‐Membrane Protein‐Kinase Substrates
Authors: Arneson Mark L. , Cheng Hsueh‐Ling , Louis Charles F.
Published: September 2004

The cAMP‐dependent protein‐kinase‐catalyzed phosphorylation of the two major intrinsic lens fiber cell plasma membrane proteins, MP20 and MP26, is likely restricted to the inner cortical and nuclear regions of the lens in vivo. The ovine‐lens‐specific connexin, MP70, that has been identified as Cx50 in mice and Cx45.6 in the chick, is also a protein kinase substrate although it does not appear to be phosphorylated by a number of protein kinases including cAMP‐dependent protein kinase, calmodulin‐dependent protein kinase or protein kinase C. Rather, an extrinsic lens membrane fraction was isolated which contained protein kinase activity that catalyzed the phosphorylation of MP70; this protein kinase activity was cAMP‐independent, Ca2+‐independent, Mg2+‐dependent, phosphorylated MP70 on a serine residue(s) and migrated with a molecular mass of 35 kDa on a gel filtration column. Both MP70 phosphorylation and the endogenous protein kinase activity were restricted to the lens outer cortical region. This membrane‐associated protein kinase activity represents the first reported partial characterization of an endogenous lens fiber cell protein kinase activity that catalyzes the phosphorylation of a lens connexin protein. The phosphatase‐induced shift in the electrophoretic mobility of MP70 is not reversed by this protein kinase, indicating that MP70 is likely phosphorylated on different residues by two or more protein kinases.
Diethylumbelliferyl Phosphate Inhibits Steroidogenesis by Interfering with a Long‐Lived Factor Acting Between Protein Kinase A Activation and Induction of the Steroidogenic Acute Regulatory Protein (StAR)
Authors: Choi Yong‐Soon , Stocco Douglas M. , Freeman Dale A.
Published: September 2004

Diethylumbelliferyl phosphate (DEUP) is an organophosphate cholesteryl ester hydrolase inhibitor that blocks steroidogenesis mainly by preventing cholesterol transport into the mitochondria of steroidogenic cells. In the present study, we show that DEUP blocks the cAMP‐stimulated mitochondrial accumulation of the 30‐kDa mitochondrial proteins (recently named steroidogenic acute regulatory StAR proteins) that are believed to be the cycloheximide‐sensitive factors induced by trophic hormones and cAMP. Inhibition of mitochondrial StAR accumulation by DEUP is dose dependent and closely parallels inhibition of progesterone synthesis. Stimulated lactate production, another cAMP‐dependent process in MA‐10 cells, is also inhibited by DEUP. Inhibition of protein kinase A (PKA) action would explain the inhibition of these two unrelated processes. However, the cytosolic PKA activity of DEUP‐treated MA‐10 cells was normal. Moreover, the activity of purified PKA was unaffected by DEUP. The inhibition of StAR synthesis was not caused by a direct effect of DEUP on the labile proteins since DEUP‐treated cells required more than 24 h to recover steroidogenic capacity after DEUP treatment. Further evidence that the synthesis of StAR was not directly affected was obtained using the constitutively active R2C cells. Progesterone synthesis by these cells also involves StAR, but neither StAR synthesis or steroid synthesis is sensitive to DEUP. Lactate formation in dibutyryl‐cAMP‐stimulated R2C cells is, however, sensitive to inhibition by DEUP. These data can be best explained by DEUP acting on a long‐lived factor involved in the cAMP/PKA response pathway, but not involved in constitutive steroidogenesis.
Perturbation of the CuA Site in Cytochrome‐c Oxidase of Paracoccus denitrificans by Replacement of Met227 with Isoleucine
Authors: Zickermann Volker , Verkhovsky Michael , Morgan Joel , Wikström Mårten , Anemüller Stefan , Bill Eckhard , Steffens Guy C. M. , Ludwig Bernd
Published: September 2004

Subunit II of cytochrome‐c oxidase contains a redox centre, CuA, with unusual spectroscopic properties; this site consists of two copper atoms and acts as the entry point for electrons from cytochrome c. We have constructed a site–directed mutant of cytochrome‐c oxidase from Paracoccus denitrificans in which the CuA site has been disturbed by replacement of Met227 with isoleucine. The purified, fully assembled enzyme complex has been investigated with various techniques including metal analysis, EPR and visible spectroscopies, steady‐state and fast kinetics. The stoichiometry of the metals in the enzyme remains unchanged but a clear perturbation of the CuA site can be observed in the EPR and near‐infrared optical spectra. It is concluded that in the mutant CuA is still binuclear but that the two nuclei are no longer equivalent, converting the delocalized [Cu(1.5).…Cu(1.5)] centre of the wild type into a localized [Cu(I).…Cu(II)] system. Changes in the overall kinetics of the mutant are correlated with a diminished electron transfer rate between CuA and heme a.
High‐Affinity Interaction of Human Immunodeficiency Virus Type‐1 Reverse Transcriptase with Partially Complementary Primers
Authors: Zakharova Ol'ga D. , Tarrago‐Litvak Laura , Maksakova Galija , Andréola Marie‐Line , Dufour Emmanuelle , Litvak Simon , Nevinsky Georgy A.
Published: September 2004

The comparison of Km, and Vmax values for various primers in the reaction of polymerization catalyzed by the human immunodeficiency virus type‐1 (HIV‐1) reverse transcriptase was carried out. The primers were: (a) complementary to the template, (b) partially complementary with mismatched nucleotides at different positions from the 3′ end or (c) non‐complementary. Non‐complementary primers were not elongated by HIV‐1 reverse transcriptase. However, if they contained only one residue complementary to the template or an abasic unit at the 3′ end, they could serve as primers. The most effective discrimination between matched and mismatched primers, due to a decrease in the affinity and Vmax, was found in the case of oligonucleotides containing non‐complementary bases at the second or third position from the 3′ end of the primer. The efficiency of discrimination by HIV‐1 reverse transcriptase between matched and mismatched base‐paired primers was about 1–1.5 orders of magnitude lower than that of procaryotic, eucaryotic and archaebacterial DNA polymerases and avian myeloblastosis virus reverse transcriptase. Oligonucleotides such as (dT)4(dCdG)k(dT)4 showed higher affinity for the enzyme than (dT)4 or (dT)8 primers. These data suggest that HIV‐1 reverse transcriptase, in contrast to procaryotic, eucaryotic and archaebacterial DNA polymerases, forms additional contacts with the 5′‐end region of the non‐complementary primer. In addition, using tRNA3Lys, the natural primer of HIV‐1, it was shown that the p66 subunit of reverse transcriptase can be crosslinked, in the presence of a platinum derivative, to the 5′ end of tRNA. Thus. besides the normal binding site for the 3′ end of tRNA, which is crucial for the initiation of cDNA synthesis, the 5′ end of the tRNA also interacts with a specific site on the enzyme.
Phosphorylation of the N‐Terminal Intracellular Tail of Sucrase–Isomaltase by cAMP‐Dependent Protein Kinase
Authors: Keller Patrick , Semenza Giorgio , Shaltiel Shmuel
Published: September 2004

This paper reports the phosphorylation of the intracellular N‐terminal tail of sucrase‐isomaltase by protein kinase A and shows that this phosphorylation is targeted to Ser6 within a sequence Arg/Lys/Lys‐Phe‐Ser, which is conserved in all sucrase–isomaltase sequences known so far. By dephosphorylation of native sucrase–isomaltase with an immobilized acid phosphatase and rephosphorylation with protein kinase A, it is shown that Ser6 may be partially phosphorylatedin vivo, raising the possibility that the tail itself and its phosphorylation by protein kinase A may be physiologically significant.
The basic isoform of profilin in pathogenic Entamoeba histolytica
Authors: Binder Marina , Ortner Stephan , Erben Hertha , Scheiner Otto , Wiedermann Gerhard , Valenta Rudolf , Duchêne Michael
Published: September 2004

In the human parasite Entamoeba histolytica, components of the cytoskeleton are involved in the pathogenicity by their contribution to immune evasion by antibody capping and shedding. In this study, we focus on profilin as a central regulatory component of the cytoskeleton. Profilin was isolated from trophozoites of the pathogenic E. histolytica strain SFL‐3, and partial amino acid sequences were used to devise a probe for isolating a profilin cDNA. The deduced complete primary structure was divergent: plant profilins with amino acid sequence identities in the range 33–38% were more closely related than the mammalian profilins with sequence identities 21–28%. The cDNA was expressed as a nonfusion protein in Escherichia coli. Isoelectric focussing of the natural profilin isolated from E. histolytica showed two isoforms with different isoelectric points; the recombinant profilin migrated with the basic isoform. In a blot overlay experiment, purified 125I‐labeled recombinant profilin bound not only to plant actin, but also to mammalian actin, demonstrating that cytoskeletal components from distantly related organisms with divergent primary structures can be compatible.
Ras oncogene induces β‐galactoside α2,6‐sialyltransferase (ST6Gal I) via a RalGEF‐mediated signal to its housekeeping promoter
Authors: Dalziel Martin , Dall'Olio Fabio , Mungul Arron , Piller Véronique , Piller Friedrich
Published: September 2004

Several oncogenic proteins are known to influence cellular glycosylation. In particular, transfection of codon 12 point mutated H‐Ras increases CMP‐Neu5Ac: Galβ1,4GlcNAc α2,6‐sialyltransferase I (ST6Gal I) activity in rodent fibroblasts. Given that Ras mediates its effects through at least three secondary effector pathways (Raf, RalGEFs and PI3K) and that transcriptional control of mouse ST6Gal I is achieved by the selective use of multiple promoters, we attempted to identify which of these parameters are involved in linking the Ras signal to ST6Gal I gene transcription in mouse fibroblasts. Transformation by human K‐Ras or H‐Ras (S12 and V12 point mutations, respectively) results in a 10‐fold increase in ST6Gal I mRNA, but no alteration in the expression of related sialyltransferases. Using an inducible H‐RasV12 expression system, a direct causal link between activated H‐Ras expression and elevated ST6Gal I mRNA was demonstrated. The accumulation of the ST6Gal I transcript in response to activated Ras was accompanied by an increase of α2,6‐sialyltransferase activity and of Neu5Acα2,6Gal at the cell surface. Results obtained with H‐RasV12 partial loss of function mutants H‐RasV12S35 (Raf signal only), H‐RasV12C40 (PI3‐kinase signal only) and H‐RasV12G37 (RalGEFs signal only) suggest that the H‐Ras induction of the mouse ST6Gal I gene (Siat1) transcription is primarily routed through RalGEFs. 5′‐Rapid amplification of cDNA ends analysis demonstrated that the increase in ST6Gal I mRNA upon H‐RasV12 or K‐RasS12 transfection is mediated by the Siat1 housekeeping promoter P3‐associated 5′ untranslated exons.
Characterization of recombinant forms of the yeast Gas1 protein and identification of residues essential for glucanosyltransferase activity and folding
Authors: Carotti Cristina , Ragni Enrico , Palomares Oscar , Fontaine Thierry , Tedeschi Gabriella , Rodríguez Rosalía , Latgé Jean Paul , Vai Marina , Popolo Laura
Published: September 2004

Gas1p is a glycosylphosphatidylinositol‐anchored plasma membrane glycoprotein of Saccharomyces cerevisiae and is a representative of Family GH72 of glycosidases/transglycosidases, which also includes proteins from human fungal pathogens. Gas1p, Phr1‐2p from Candida albicans and Gel1p from Aspergillus fumigatus have been shown to be β‐(1,3)‐glucanosyltransferases required for proper cell wall assembly and morphogenesis. Gas1p is organized into three modules: a catalytic domain; a cys‐rich domain; and a highly O‐glycosylated serine‐rich region. In order to provide an experimental system for the biochemical and structural analysis of Gas1p, we expressed soluble forms in the methylotrophic yeast Pichia pastoris. Here we report that 48 h after induction with methanol, soluble Gas1p was produced at a yield of ≈ 10 mg·L−1 of medium, and this value was unaffected by the further removal of the serine‐rich region or by fusion to a 6 × His tag. Purified soluble Gas1 protein showed β‐(1,3)‐glucanosyltransferase activity that was abolished by replacement of the putative catalytic residues, E161 and E262, with glutamine. Spectral studies confirmed that the recombinant soluble Gas1 protein assumed a stable conformation in P. pastoris. Interestingly, thermal denaturation studies demonstrated that Gas1p is highly resistant to heat denaturation, and a complete refolding of the protein following heat treatment was observed. We also showed that Gas1p contains five intrachain disulphide bonds. The effects of the C74S, C103S and C265S substitutions in the membrane‐bound Gas1p were analyzed in S. cerevisiae. The Gas1‐C74S protein was totally unable to complement the phenotype of the gas1 null mutant. We found that C74 is an essential residue for the proper folding and maturation of Gas1p.
Antioxidant defences and homeostasis of reactive oxygen species in different human mitochondrial DNA‐depleted cell lines
Authors: Vergani Lodovica , Floreani Maura , Russell Aaron , Ceccon Mara , Napoli Eleonora , Cabrelle Anna , Valente Lucia , Bragantini Federica , Leger Bertrand , Dabbeni‐Sala Federica
Published: September 2004

Three pairs of parental (ρ+) and established mitochondrial DNA depleted (ρ0) cells, derived from bone, lung and muscle were used to verify the influence of the nuclear background and the lack of efficient mitochondrial respiratory chain on antioxidant defences and homeostasis of intracellular reactive oxygen species (ROS). Mitochondrial DNA depletion significantly lowered glutathione reductase activity, glutathione (GSH) content, and consistently altered the GSH2 : oxidized glutathione ratio in all of the ρ0 cell lines, albeit to differing extents, indicating the most oxidized redox state in bone ρ0 cells. Activity, as well as gene expression and protein content, of superoxide dismutase showed a decrease in bone and muscle ρ0 cell lines but not in lung ρ0 cells. GSH peroxidase activity was four times higher in all three ρ0 cell lines in comparison to the parental ρ+, suggesting that this may be a necessary adaptation for survival without a functional respiratory chain. Taken together, these data suggest that the lack of respiratory chain prompts the cells to reduce their need for antioxidant defences in a tissue‐specific manner, exposing them to a major risk of oxidative injury. In fact bone‐derived ρ0 cells displayed the highest steady‐state level of intracellular ROS (measured directly by 2′,7′‐dichlorofluorescin, or indirectly by aconitase activity) compared to all the other ρ+ and ρ0 cells, both in the presence or absence of glucose. Analysis of mitochondrial and cytosolic/iron regulatory protein‐1 aconitase indicated that most ROS of bone ρ0 cells originate from sources other than mitochondria.
Evidence for the presence of ferritin in plant mitochondria
Authors: Zancani Marco , Peresson Carlo , Biroccio Antonino , Federici Giorgio , Urbani Andrea , Murgia Irene , Soave Carlo , Micali Fulvio , Vianello Angelo , Macrì Francesco
Published: September 2004

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25–26 kDa (corresponding to that of ferritin), was cross‐reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI‐TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross‐sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.
Determination of the reopening temperature of a DNA hairpin structure in vitro
Authors: Pan Xuefeng
Published: September 2004

A novel method, based upon primer extension, has been developed for measuring the reopening temperature of a single type of DNA hairpin structure. Two DNA oligonucleotides have been utilized and designated as primers 1 and 2. Primer 1, with its 5‐ and 3′‐termini fully complementary to the hairpin flanking sequences, was used to evaluate primer extension conditions, and primer 2, with its 3′‐end competing with the DNA hairpin stem, was used to detect the DNA hairpin reopening temperature. A single DNA hairpin structure was formed on the DNA template by thermal denaturation and renaturation, and this hairpin structure was predicted to prevent the annealing of the 3′‐end of primer 2 with the template DNA, which leads to no primer extension. By incubating at different temperatures, the DNA hairpin structure can be reopened at a particular temperature where the primer extension can be carried out. This resulted in the appearance of double‐stranded DNA that was detected on an agarose gel. This temperature is defined here as the hairpin reopening temperature.
Biosynthesis of platelet glycoprotein V expressed as a single subunit or in association with GPIb‐IX
Authors: Strassel Catherine , Moog Sylvie , Baas Marie‐Jeanne , Cazenave Jean‐Pierre , Lanza François
Published: September 2004

Glycoprotein (GP) V is noncovalently linked to GPIbα, GPIbβ and GPIX within the platelet GPIb–V–IX complex, a receptor for von Willebrand factor and thrombin. Two functions have been ascribed to GPV, namely, the modulation of thrombin‐ and collagen‐dependent platelet responses. The biosynthesis of this molecule was investigated in pulse–chase metabolic labelling experiments performed in CHO cell lines transfected with GPV, alone or in the presence of GPIb–IX. GPV could not be detected at the surface of cells expressing the single subunit but was found instead as a soluble form in the culture medium. In pulse–chase studies, an immature 70 kDa protein was detected in cell lysates, whereas a fully processed 80–82 kDa form was only observed in the culture supernatants at later chase times. Immature GPV was N‐glycosylated and retained before the medial Golgi while the secreted molecule contained complex sialylated sugars. The mature soluble form of GPV was produced by an enzymatic cleavage which was not affected by inhibitors of proteasome, calpain or metalloproteinases. When GPV was cotransfected with GPIb–IX, the former was no longer found in the culture supernatant but was retained in the cell membrane as shown by fluorescence‐activated cell sorting and confocal microscopy analyses. Surface expressed GPV was processed from an immature 70 kDa form to produce a mature 80 kDa protein, processing similar to the intracellular trafficking of GPIbα. These results indicate that correct biosynthesis and surface expression of GPV in platelets requires the presence of the other subunits of the GPIb–V–IX complex.
l‐3,4‐Dihydroxyphenyl alanine‐extradiol cleavage is followed by intramolecular cyclization in lincomycin biosynthesis
Authors: Novotná Jitka , Honzátko Aleš , Bednář Petr , Kopecký Jan , Janata Jiří , Spížek Jaroslav
Published: September 2004

The LmbB1 protein, participating in the biosynthesis of lincomycin, was heterologously expressed in Escherichia coli, purified in its active form, and characterized as a dimer of identical subunits. Methods for purification and analysis of the LmbB1 reaction product were developed. Molecular mass and fragmentation pattern of the product revealed by capillary electrophoresis‐mass spectrometry were in agreement with its proposed structure, 4‐(3‐carboxy‐3‐oxo‐propenyl)‐2,3‐dihydro‐1H‐pyrrole‐2‐carboxylic acid. The LmbB1 is therefore a dioxygenase catalysing the 2,3‐extradiol cleavage of the l‐3,4‐dihydroxyphenyl alanine aromatic ring. The final LmbB1 reaction product, a unique compound found in biosynthesis of lincomycin and expected in anthramycins, arises through subsequent cyclization of the primary cleavage product, 2,3‐secodopa. A possible role of LmbB1 in 2,3‐secodopa cyclization and alternative ways of the cyclization in the formation of biosynthetically related compounds, muscaflavin and stizolobinic acid, are discussed.
Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF‐Tu
Authors: Rao Damodara , Momcilovic Ivana , Kobayashi Satoru , Callegari Eduardo , Ristic Zoran
Published: September 2004

The protein synthesis elongation factor, EF‐Tu, is a protein that carries aminoacyl‐tRNA to the A‐site of the ribosome during the elongation phase of protein synthesis. In maize (Zea mays L) this protein has been implicated in heat tolerance, and it has been hypothesized that EF‐Tu confers heat tolerance by acting as a molecular chaperone and protecting heat‐labile proteins from thermal aggregation and inactivation. In this study we investigated the effect of the recombinant precursor of maize EF‐Tu (pre‐EF‐Tu) on thermal aggregation and inactivation of the heat‐labile proteins, citrate synthase and malate dehydrogenase. The recombinant pre‐EF‐Tu was purified from Escherichia coli expressing this protein, and mass spectrometry confirmed that the isolated protein was indeed maize EF‐Tu. The purified protein was capable of binding GDP (indicative of protein activity) and was stable at 45 °C, the highest temperature used in this study to test this protein for possible chaperone activity. Importantly, the recombinant maize pre‐EF‐Tu displayed chaperone activity. It protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation. To our knowledge, this is the first observation of chaperone activity by a plant/eukaryotic pre‐EF‐Tu protein. The results of this study support the hypothesis that maize EF‐Tu plays a role in heat tolerance by acting as a molecular chaperone and protecting chloroplast proteins from thermal aggregation and inactivation.
Mechanism for transcriptional synergy between interferon regulatory factor (IRF)‐3 and IRF‐7 in activation of the interferon‐β gene promoter
Authors: Yang Hongmei , Ma Gang , Lin Charles H. , Orr Melissa , Wathelet Marc G.
Published: September 2004

The interferon‐β promoter has been studied extensively as a model system for combinatorial transcriptional regulation. In virus‐infected cells the transcription factors ATF‐2, c‐Jun, interferon regulatory factor (IRF)‐3, IRF‐7 and NF‐κB, and the coactivators p300/CBP play critical roles in the activation of this and other promoters. It remains unclear, however, why most other combinations of AP‐1, IRF and Rel proteins fail to activate the interferon‐β gene. Here we have explored how different IRFs may cooperate with other factors to activate transcription. First we showed in undifferentiated embryonic carcinoma cells that ectopic expression of either IRF‐3 or IRF‐7, but not IRF‐1, was sufficient to allow virus‐dependent activation of the interferon‐β promoter. Moreover, the activity of IRF‐3 and IRF‐7 was strongly affected by promoter context, with IRF‐7 preferentially being recruited to the natural interferon‐β promoter. We fully reconstituted activation of this promoter in insect cells. Maximal synergy required IRF‐3 and IRF‐7 but not IRF‐1, and was strongly dependent on the presence of p300/CBP, even when these coactivators only modestly affected the activity of each factor by itself. These results suggest that specificity in activation of the interferon‐β gene depends on a unique promoter context and on the role played by coactivators as architectural factors.