bluuebunny/crossref_metadata_2025 huggingface dataset:
The protein kinase C family
Authors: AZZI Angelo , BOSCOBOINIK Daniel , HENSEY Carmel
Published: March 2005

Protein kinase C represents a structurally homologous group of proteins similar in size, structure and mechanism of activation. They can modulate the biological function of proteins in a rapid and reversible manner. Protein kinase C participates in one of the major signal transduction systems triggered by the external stimulation of cells by various ligands including hormones, neurotransmitters and growth factors. Hydrolysis of membrane inositol phospholipids by phospholipase C or of phosphatidylcholine, generates sn‐1,2‐diacylglycerol, considered the physiological activator of this kinase. Other agents, such as arachidonic acid, participate in the activation of some of these proteins. Activation of protein kinase C by phorbol esters and related compounds is not physiological and may be responsible, at least in part, for their tumor‐promoting activity. The cellular localization of the different calcium‐activated protein kinases, their substrate and activator specificity are dissimilar and thus their role in signal transduction is unlike. A better understanding of the exact cellular function of the different protein kinase C isoenzymes requires the identification and characterization of their physiological substrates.
In vivo and in vitro modulation of the mRNA‐binding activity of iron‐regulatory factor
Authors: MÜLLNER Ernst W. , ROTHENBERGER Sylvia , MÜLLER Anna Maria , KÜHN Lukas C.
Published: March 2005

The mRNA‐binding protein, iron‐regulatory factor (IRF) has a central role in iron metabolism. It coordinately increases transferrin‐receptor mRNA stability and inhibits translation of ferritin and erythroid δ‐aminolevulinate synthase mRNA by binding to specific mRNA structures, the iron‐responsive elements (IRE): In gel‐retardation assays, IRF had a broad tissue distribution, showing activity in cytosolic extracts from 12 mouse organs tested. In all these extracts, IRF could be further activated in vitro by 2‐mercaptoethanol. In cultured mouse 3T6 fibroblasts, growth stimulation after low serum arrest increased IRF activity 10‐fold, mainly through activation of existing inactive IRF. No change was observed during progresion of 3T6 cells through the cell cycle.

IRF activation by iron chelators has been postulated to result in the reduction of an intramolecular sulfhydryl group. In a search for redox conditions that regulate IRE binding of IRF, we studied several compounds in vitro or in vivo. Hemin, known to inactivate IRF in vivo, showed a similar, reversible effect in vitro, presumably by oxidizing IRF. However, this did not appear to be relevant for the mode of IRF regulation in vivo. Addition of protoporphyrin IX to intact cells induced IRF activity almost to the same extent as desferrioxamine. This effect was inhibited by iron salts, indicating that IRF is activated in vivo through depletion of a chelatable iron pool. In vitro activation by reductants other than 2‐mercaptoethanol suggested some selectivity in their access to relevant sulfhydryl groups, but did not reveal which natural redox‐sensitive compound might regulate IRF in vivo. However, in cultured cells, inactivation of free IRF by the sulfhydryl‐specific oxidizing agent diamide was much more rapidly reversed than inactivation by iron salts. This indicates the direct involvement of a cellular reductant in setting IRF activity and suggests a rate‐limiting IRF conformation that is reached only in the presence of iron, but not after diamide oxidation.
Studies on ribonucleoside‐diphosphate reductase from Escherichia coli
Authors: SHEN Binghua , ALLARD Peter , KUPRIN Sergei , EHRENBERG Anders
Published: March 2005

Ribonucleoside‐diphosphate reductase (EC 1.17.4.1) from Escherichia coli consists of two protein subunits, R1 of 171.5 kDa and R2 of 86.8 kDa, and catalyzes the reduction of all four common ribonucleoside diphosphates. In a search for ligands that bind weakly to the enzyme active site and may be in fast exchange suitable for NMR studies, we have found that the product dCDP is a competitive inhibitor. Kinetics with CDP as substrate shows Km= 4.8 × 10−5 M and dCDP inhibits with Ki= 1.6 × 10−4 M. With an assumed diffusion limited binding rate ≲ 109 M−1s−1, the dissociation rate of dCDP would be ≲ 105 s−1. In 1H‐NMR experiments studying linewidths, i. e. spin–spin relaxation, dCDP is indeed demonstrated to be in fast exchange. Enzyme subunit R1 causes a line broadening of dCDP resonances. Unexpectedly less broadening was observed when subunit R2 combined with R1. No paramagnetic interaction from the tyrosyl radical of R2 could be detected. It is concluded that dCDP is a promising NMR probe for studies of active‐site properties of the enzyme.
Mechanistic studies on rhodopsin kinase
Authors: BROWN Neil G. , FOWLES Charles , SHARMA Ram , AKHTAR Muhammad
Published: March 2005

The phosphorylation of a synthetic peptide, corresponding to the C‐terminal 11 amino acids of bovine rhodopsin (VII, residues 338–348), was studied under different conditions. The peptide was only phosphorylated in the presence of photoactivated rhodopsin. Using the same protocol, 12 other peptides, mapping in the rhodopsin C‐terminal, were screened for their effectiveness as substrates for rhodopsin kinase. It was found that the peptides became poorer substrates with increasing length, and the best substrates comprised the most C‐terminal 9–12 amino acids as opposed to other parts of the C‐terminus. It was noted that the absence of the two‐terminal residues Pro347 and Ala348 impaired peptide phosphorylation.

The effect of the decay of metarhodopsin II on the phosphorylation of rhodopsin and the peptides was determined, and it was found that the rhodopsin and peptide phosphorylations decayed with half times of approximately 33 min and 28 min, respectively.

The sites of phosphorylation on the peptides were determined and in all cases the phosphorylation was found to be predominantly on serine residues. Only the 11‐residue peptide (VII, residues 338–348) contained significant threonine phosphorylation, which was about 25% that on serine residues. Cumulatively, the results suggest that Ser343 is the preferred site of phosphorylation in vitro.

The reason for the poor substrate effectiveness of the larger peptides was examined by competitive experiments in which it was shown that a poorly phosphorylated larger peptide successfully inhibited the phosphorylation of a ‘good’ peptide substrate. The studies above support a mechanism for rhodopsin kinase that we have termed the ‘kinase‐activation hypothesis’. This requires that the kinase exists in an inactive form and is activated only after binding to photoactivated rhodopsin.
Occurrence of α1‐2‐fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver
Authors: NUCK Rolf , ORTHEN Bruno , REUTTER Werner
Published: March 2005

A comparative study was undertaken to characterize the linkages of l‐fucose in N‐glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum.

After in‐vivo radiolabelling of rats with l‐[6‐3H]fucose, the asparagine‐linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride.

Two specific α‐l‐fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of l‐fucose in the oligosaccharide chains.

Fucose α1‐2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total l‐[3H]fucose in N‐glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose α1‐3/4 linked to N‐acetyl‐d‐glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained α‐fucosyl residues linked at the C6 position of the proximal N‐acetyl‐d‐glucosamine.
Determination of the redox properties of the Rieske [2Fe‐2S] cluster of bovine heart bc1 complex by direct electrochemistry of a water‐soluble fragment
Authors: LINK Thomas A. , HAGEN Wilfred R. , PIERIK Antonio J. , ASSMANN Claus , von JAGOW Gebhard
Published: March 2005

The redox potential of the Rieske [2Fe‐2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water‐soluble fragment of the iron/sulfur protein. At the nitric‐acid‐treated bare glassy‐carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25°C and I of 0.01 M was Em=+ 312 ± 3 mV. This value corresponds within 20 mV to results of an EPR‐monitored dye‐mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with up to I= 2.5 M. From the cathodic‐to‐anodic peak separation, the heterogeneous rate constant, K°, was calculated to be approximately 2 × 10−3 cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as S°=−155 J · K−1· mol−1. The pH dependence of the midpoint potential was followed over pH 5.5–10. Above pH 7, redox‐state‐dependent pK changes were observed. The slope of the curve, −120 mV/pH above pH 9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox‐dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex.
Precursor of mitochondrial aspartate aminotransferase synthesized in Escherichia coli is complexed with heat‐shock protein DnaK
Authors: SCHMID Daniel , JAUSSI Rolf , CHRISTEN Philipp
Published: March 2005

On expression of the cDNA encoding the precursor of chicken mitochondrial aspartate aminotransferase (pmAspAT) in Escherichia coli, the bulk of pmAspAT was found to be associated with the 70‐kDa heat‐shock protein DnaK which is closely related to mitochondrial 70‐kDa heat‐shock protein (HSP70). Purification protocols for the DnaK/pmAspAT complex and its individual components were elaborated. The complex dissociated on treatment with MgATP or at pH 5.5. Like the mature enzyme, pmAspAT is a dimer (2 × 47 kDa) and exhibits about a third of its enzyme activity. In the DnaK/pmAspAT complex, one DnaK molecule is bound to each subunit of pmAspAT; this tetramer may further aggregate to an octamer. The complex is catalytically almost as active as free pmAspAT. It could be reconstituted from isolated DnaK and pmAspAT. No complex was formed with mAspAT. Apparently, DnaK binds to the solvent‐exposed presequence of folded pmAspAT without significantly changing the structure and functional properties of its mature moiety.
A high‐resolution solid‐state 13C‐NMR study on crystalline bovine heart cytochrome‐c oxidase and lysozyme
Authors: TUZI Satoru , SHINZAWA‐ITOH Kyoko , ERATA Tomoki , NAITO Akira , YOSHIKAWA Shinya , SAITÔ Hazime
Published: March 2005

We have recorded 100.6‐MHz high‐resolution solid‐state 13C‐NMR spectra of crystalline cytochrome‐c oxidase from bovine heart muscle and hen egg‐white lysozyme, to compare conformation and dynamics of a typical membrane‐protein complex with those of lysozyme. The absence of severe interference with the solid‐state 13C‐NMR spectra, from both the line broadenings from paramagnetic centers and overlapping of intense detergent signals, provided spectral resolution of 13C‐NMR feature of cytochrome‐c oxidase crystals comparable to that of lysozyme crystal and better than that of dissolved or lyophilized samples. In fact, the observed peak intensities of the polar heads of the detergents BL8SY and Brij 35 were only about 10% and 3% of the anticipated values, respectively.

The dynamic behavior of the backbone and side chains of cytochrome‐c oxidase was compared with that of lysozyme on the basis of the 13C spin‐lattice relaxation times (T1): the backbone of the cytochrome‐c oxidase turned out to be more flexible than that of lysozyme. Molecular motions of the detergent molecules attached to the proteins are found to be highly heterogeneous. Detergent molecules undergo rapid tumbling motions in the crystals in about 10 ns as detected by T1. In addition to rapid motions, slow motions were detected by 1H spin‐lattice relaxation time in the rotating frame (TH1Q) and cross‐polarization time (TCH), together with data from static spectra, indicating that the aliphatic portion of the detergent interacts more strongly with hydrophobic protein surfaces than do the polar heads.
Characterization of Nω‐phosphoarginine hydrolase from rat liver
Authors: KUBA Masako , OHMORI Hitoshi , KUMON Akira
Published: March 2005

N
ω‐Phosphoarginine hydrolase from rat liver hydrolyzed Nω‐phosphoarginine into arginine and inorganic phosphate, whereas it did not release inorganic phosphate from 19 other phosphorylated compounds containing a N‐P bond, an O‐P bond or a C‐P bond. In addition, it was not able to transfer the phosphoryl moiety from Nω‐phosphoarginine to ADP. These results indicated that this enzyme was distinct from both phosphoamidase and arginine kinase. Its properties were as follows: thiol compounds were essential for its activity; it was stimulated by 1.5–2‐fold in the presence of 0.001% Lubrol, Tween 20, poly(oxyethylene) 9‐lauryl ether and Nonidet P‐40, while 0.004% sodium lauryl sulfate inhibited the activity completely; concentrations of sodium molybdate and sodium vanadate necessary for 50% inhibition were 7 μM respectively; some proteins stimulated the activity, while lysophosphatidic acid, lysophosphatidylinositol, and phosphatidic acid suppressed the activity even in the presence of poly(oxyethylene) 9‐lauryl ether.
Transient activation of hepatic glycogenolysis by thrombin in perfused rat livers
Authors: YAMANAKA Hirofumi , NUKINA Sadayuki , HANDLER Jeffrey A. , CURRIN Robert T. , LEMASTERS John J. , THURMAN Ronald G.
Published: March 2005

Thrombin, a peptide with native protease activity, caused a rapid (<1 min) increase in glycogenolysis of about 30% assessed from rates of production of glucose + lactate + pyruvate, and in oxygen uptake in perfused rat liver. These increases were followed by a rapid return to basal values within 5 min. The effect of thrombin on glycogenolysis was dose‐dependent and was maximal at perfusate concentrations around 1 U/ml. Interestingly, the effect of thrombin on glycogenolysis could be elicited only once in any given liver. The activation of glycogenolysis by thrombin was diminished nearly 50% by prior infusion of the protease inhibitor, diisopropyl fluorophosphate (10 μM), and over 90% when thrombin was treated with diisopropyl fluorophosphate prior to infusion. The stimulation of glycogenolysis by thrombin could not be detected in isolated hepatocytes or in livers stored for 24 h in cold Euro‐Collins solution, a treatment which destroys endothelial cells. Further, thrombin stimulated production of prostaglandin D2 from arachidonic acid in cultured hepatic endothelial but not Kupffer cells. The effect of thrombin on carbohydrate output was also blocked by a phospholipase A2 inhibitor (quinacrine, 50 μM) and by an inhibitor of the cyclooxygenase (indomethacin, 20 μM), suggesting the involvement of cyclooxygenase in the mechanism of action of thrombin. In support of this idea, the transient kinetics of stimulation of glycogenolysis by thrombin and arachidonic acid was nearly identical to release of thromboxane B2 (80–420 pg/ml) and prostaglandin D2 (300–900 pg/ml) from the perfused liver. Further, a second addition of thrombin failed to increase thromboxane and prostaglandin D2 release as well as carbohydrate production, supporting a causal link between these phenomena. Taken together, these data support the hypothesis that thrombin interacts with receptors in the liver, possibly on endothelial cells, leading to activation of phospholipase A2 and subsequent transient production of prostaglandins and thromboxanes. These mediators subsequently interact with receptors on parenchymal cells, leading to a transient stimulation of glycogenolysis.
Production of Fenton's reagent by cellobiose oxidase from cellulolytic cultures of Phanerochaete chrysosporium
Authors: KREMER Simon M. , WOOD Paul M.
Published: March 2005

The reduction of dioxygen by cellobiose oxidase leads to accumulation of H2O2, with either cellobiose or microcrystalline cellulose as electron donor. Cellobiose oxidase will also reduce many Fe(III) complexes, including Fe(III) acetate. Many Fe(II) complexes react with H2O2 to produce hydroxyl radicals or a similarly reactive species in the Fenton reaction as shown: H2O2+ Fe2+→ HO•+ HO−+ Fe3+. The hydroxylation of salicylic acid to 2,3‐dihydroxybenzoic acid and 2,5‐dihydroxybenzoic acid is a standard test for hydroxyl radicals. Hydroxylation was observed in acetate buffer (pH 4.0), both with Fe(II) plus H2O2 and with cellobiose oxidase plus cellobiose, O2 and Fe(III). The hydroxylation was suppressed by addition of catalase or the absence of iron [Fe(II) or Fe(III) as appropriate]. Another test for hydroxyl radicals is the conversion of deoxyribose to malondialdehyde; this gave positive results under similar conditions. Further experiments used an O2 electrode. Addition of H2O2 to Fe(II) acetate (pH 4.0) or Fe(II) phosphate (pH 2.8) in the absence of enzyme led to a pulse of O2 uptake, as expected from production of hydroxyl radicals as shown: RH + HO•→ R•+ H2O;R•+ O2→ RO•2→ products. With phosphate (pH 2.8) or 10 mM acetate (pH 4.0), the O2 uptake pulse was increased by Avicel, suggesting that the Avicel was being damaged. Oxygen uptake was monitored for mixtures of Avicel (5 g · 1−1), cellobiose oxidase, O2 and Fe(III) (30 μM). An addition of catalase after 20–30 min indicated very little accumulation of H2O2, but caused a 70% inhibition of the O2 uptake rate. This was observed with either phosphate (pH 2.8) or 10 mM acetate (pH 4.0) as buffer, and is further evidence that oxidative damage had been taking place, until the Fenton reaction was suppressed by catalase. A separate binding study established that with 10 mM acetate as buffer, almost all (98%) of the Fe(III) would have been bound to the Avicel. In the presence of Fe(III), cellobiose oxidase could provide a biological method for disrupting the crystalline structure of cellulose.
Cloning and molecular analysis of the poly(3‐hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1
Authors: TIMM Arnulf , STEINBÜCHEL Alexander
Published: March 2005

From genomic libraries, the polyhydroxyalkanoate gene locus of Pseudomonas aeruginosa PAO1 was cloned and characterised at the molecular level. Two genes coding for polyhydroxyalkanoate synthases, phaC1Pa and phaC2Pa, a polyhydroxyalkanoate depolymerase gene, phaDPa, and four adjacent open reading frames (ORF1, ORF2, ORF3 and ORF4) were identified from the nucleotide sequence. Two transcriptional start sites, which were preceded by sequences resembling the Escherichia coli consensus sequences for σ54 and σ70 promoters, were identified experimentally upstream of phaC1Pa, which was shown by Northern blot analysis to constitute an operon together with phaDPa. A third putative promoter resembling the E. coli consensus sequence for σ70‐dependent promoters was proposed upstream of phaC2Pa, which is in a bicistronic operon with ORF3. Investigations of rpoN‐negative mutants of related strains revealed that polyhydroxyalkanoate accumulation from gluconate required an intact rpoN locus in P. aeruginosa. Complementation experiments revealed multiple evidence that either polyhydroxyalkanoate synthase is involved in polyhydroylkanoate accumulation from gluconate as well as from octanoate. The P. aeruginosa PAO1 polyhydroxyalkanoate gene locus was expressed in the polyhydroxyalkanoate‐negative mutant Alcaligenes eutrophus PHB−4 and in the poly(3‐hydroxybutyrate)‐accumulating strain P. oleovorans DSM1045. It conferred on the latter the ability to synthesize and accumulate polyhydroxyalkanoates consisting of medium‐chain‐length 3‐hydroxyalkanoic acids from unrelated substrates in addition to poly(3‐hydroxybutyrate). The sequence of the putative translational product of ORF1 was similar to those of the leukotoxin repressor of Pasteurella haemolytica and to the ORF9 product of Azotobacter vinelandii, and that of ORF4 was similar to the algP product of P. aeruginosa and to eukaryotic histone H1 proteins. The proteins of ORF2 and ORF3 appear to be previously unidentified.
Footprinting studies of DNA‐sequence recognition by nogalamycin
Authors: FOX Keith R. , ALAM Zafir
Published: March 2005

We have studied the DNA sequence binding preference of the antitumour antibiotic nogalamycin by DNase‐I footprinting using a variety of DNA fragments. The DNA fragments were obtained by cloning synthetic oligonucleotides into longer DNA fragments and were designed to contain isolated ligand‐binding sites surrounded by repetitive sequences such as (A)n· (T)n and (AT)n. Within regions of (A)n· (T)n, clear footprints are observed with low concentrations of nogalamycin (< 5 μM), with apparent binding affinities for tetranucleotide sequences which decrease in the order TGCA > AGCT = ACGT > TCGA. In contrast, within regions of (AT)n, the ligand binds best to AGCT; binding to TCGA and TGCA is no stronger than to alternating AT. Within (ATT)n, the preference is for ACGT > TCGA. Although each of these binding sites contains all four base pairs, there is no apparent consensus sequence, suggesting that the selectivity is affected by local DNA dynamic and structural effects. At higher drug concentrations (> 25 μM), nogalamycin prevents DNAse‐I cleavage of (AT)n but shows no interaction with regions of (AC)n· (GT)n. Regions of (A)n· (T)n, which are poorly cut by DNase I, show enhanced rates of cleavage in the presence of low concentrations of nogalamycin, but are protected from cleavage at higher concentrations. We suggest that this arises because drug binding to adjacent regions distorts the DNA to a structure which is more readily cut by the enzyme and which is better able to bind further ligand molecules.
Purification, characterization and structure of protein phosphatase 1 from the cilia of Paramecium tetraurelia
Authors: FRIDERICH Gerald , KLUMPP Susanne , RUSSELL Chris B. , HINRICHSEN Robert D. , KELLNER Roland , SCHULTZ Joachim E.
Published: March 2005

A type 1 serine/threonine protein phosphatase (PP1) which is mostly localized in the excitable ciliary membranes from the protozoan Paramecium, was purified to homogeneity. Approximately 4 μg enzyme of 37 kDa was isolated from 100 1 axenic culture. The enzymic properties were characterized using phosphorylase a from rabbit skeletal muscle as a substrate and several known effectors of mammalian PP1. The protozoan PP1 was enzymically indistinguishable from its mammalian congener.

The amino acid sequence of the Paramecium PP1 was deduced from its cDNA. The full‐length clone was obtained in several steps starting with a pair of degenerate primers made according to the two most conserved peptides of rabbit PP1 and PP2A. The gene encodes a protein of 36 392 Da. The identity of the cloned gene and the isolated ciliary PP1 was unequivocally established by microsequencing of four tryptic and cyanogen‐bromide peptides which were generated from the purified protein. Paramecium PP1 shows 75% amino‐acid‐sequence identity with rabbit PP1α. Areas of major differences are the C‐termini and N‐termini and a sequence between residues 219–242.
Gene cloning and characterization of a novel extracellular ribonuclease of Bacillus subtilis
Authors: NAKAMURA Akira , KOIDE Yoshinao , MIYAZAKI Hiroyuki , KITAMURA Atsunori , MASAKI Haruhiko , BEPPU Teruhiko , UOZUMI Takeshi
Published: March 2005

An extracellular nuclease gene of Bacillus subtilis was cloned in the same organism by detecting the amplified enzyme activity, which was secreted from the transformant cells on an RNA‐containing agar medium. An open reading frame encoding 289 amino acids was identified within the cloned fragment. The transcriptional initiation site was determined by nuclease S1 mapping and the promoter region showed similarity to the conserved recognition sequences for the EσA and/or EσE RNA polymerases. The production of the nuclease by the B. subtilis transformants greatly depends on the liquid medium used. SDS/PAGE analysis of the purified enzyme showed two adjoining bands of molecular mass about 32 kDa, and the NH2‐terminal amino acid sequence analysis suggested that the NH2‐terminal portion of the nuclease was subjected to a limited proteolysis after or during secretion. The nuclease was uniquely characterized as a Mg2+‐activated ribonuclease which hydrolyzes RNA apparently nonspecifically into oligonucleotides with 5′‐terminal phosphate. The deduced amino acid sequence of this enzyme shows no obvious similarity with other nuclease sequences.
Purification and characterization of a rat liver protein‐tyrosine phosphatase with sequence similarity to src‐homology region 2
Authors: HIRAGA Akira , MUNAKATA Hiroshi , HATA Keiko , SUZUKI Yoichi , TSUIKI Shigeru
Published: March 2005

Utilizing three proteins plus tyrosine‐glutamate copolymer as substrates, all of which are subjected to (near) stoichiometrical phosphorylation exclusively on tyrosine residues, we partially purified four different protein‐tyrosine phosphatases (PTPases) from rat liver cytosol which differed in substrate preference. Of the four PTPases, tentatively termed L1, L2, L3, and L4, PTPase L1 was purified to apparent homogeneity by a procedure involving chromatography on DEAE‐cellulose at pH 7.0, Blue Sepharose, DEAE‐cellulose at pH 7.6, hydroxyapatite, Phenyl Sepharose, Mono Q, and TSKgel Heparin. PTPase L1 was purified about 7000‐fold from the extract and 0.27 mg was isolated from 1000 g liver corresponding to a yield of 13% from the Blue Sepharose step where it had become freed from any other PTPases detectable by our assay procedure. The purified PTPase L1 showed a major protein band of 67 kDa on SDS/PAGE. Catalytically, PTPase L1 had a specific activity of about 6500 nmol P1 released min−1mg1 toward tyrosine‐glutamate copolymer phosphorylated on tyrosine residues. PTPase L1 exhibited very low sensitivities to PTPase inhibitors such as zinc acetate, sodium vanadate, and acidic compounds as compared with those of most of the PTPases purified thus far.

Amino acid sequence analysis of the purified PTPase L1 revealed a partial peptide sequence showing similarity to the catalytic domain core sequences conserved in the PTPase family. PTPase L1 was most similar to a PTPase termedPTP1C encoded by a human breast carcinoma cDNA but the identity was 55% over 117 residues spanning nearly half of the catalytic domain of PTP1C. The analysis also revealed another partial peptide sequence (113 residues) 70% identical with the sequence corresponding to 68% of two adjacent copies of the src homology region 2 (SH‐2 domain) identified in PTP1C. Besides those peptide sequences, PTPase L1 had regional sequences which were 70–90% identical with the residues lying between the two SH‐2 domains or between the more C‐terminal SH‐2 domain and the catalytic domain of the carcinoma PTPase.
The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F1F0‐ATPase
Authors: NOLAN Derek P. , VOORHEIS H. Paul
Published: March 2005

Bloodstream forms of Trypanosoma brucei were found to maintain a significant membrane potential across their mitochondrial inner membrane (ΔΨm) in addition to a plasma membrane potential (ΔΨp). Significantly, the ΔΨm was selectively abolished by low concentrations of specific inhibitors of the F1F0‐ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondrial membrane potential is generated and maintained exclusively by the electrogenic translocation of H+, catalysed by the mitochondrial F1F0‐ATPase at the expense of ATP rather than by the mitochondrial electron‐transport chain present in T. brucei. Consequently, bloodstream forms of T. brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F1F0‐ATPase in intact trypanosomes was calculated after solving the two‐compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh3P+) and was shown to have a value of approximately 150mV. When the value for the ΔΨm is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton‐motive force was calculated to be greater than 190 mV. It seems likely that this mitochondrial proton‐motive force serves a role in the directional transport of ions and metabolites across the promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclear‐encoded protein into the promitochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. brucei.
Effects of selective inhibition of cathepsin B and general inhibition of cysteine proteinases on lysosomal proteolysis in rat liver in vivo and in vitro
Authors: OHSHITA Takeyuki , NIKAWA Takeshi , TOWATARI Takae , KATUNUMA Nobuhiko
Published: March 2005

Intraperitoneal administration of N‐(L‐trans‐propylcarbamoyloxirane‐2‐carbonyl)‐l‐isoleucyl‐l‐proline (CA‐074) to rats at a dose of 4 mg/100 g greatly inhibited cathepsin‐B activity in both liver and kidney for at least 4 h. Its inhibitory effect was selective for cathepsin‐B activity in the liver but not in the kidney. The effects of selective inhibition of cathepsin‐B activity by CA‐074 treatment, and general inhibition of cysteine proteinases by N‐(L‐3‐trans‐carboxyoxirane‐2‐carbonyl)‐l‐leucyl‐3‐methylbutylamide (E‐64‐c) on the degradation of fluorescein isothiocyanate (FITC)‐labeled asialofetuin in liver lysosomes, were examined in vivo. Undegraded or partially degraded FITC‐labeled asialofetuin and its FITC‐labeled degradation products were both found in the lysosomes and were easily separated by Sephadex G‐25 column chromatography. The FITC‐labeled degradation products were mainly lysine with an FITC‐labeled ɛ‐amino group. Accumulation of undegraded or partially degraded FITC‐labeled asialofetuin in the lysosomes was marked after E‐64‐c treatment, but slight after CA‐074 treatment. Under the marked inhibition of general lysosomal cysteine‐proteinase activity by E‐64‐c or marked selective inhibition of cathepsin‐B activity by CA‐074 in vitro, degradation of FITC‐labeled asialofetuin by disrupted lysosomes was analyzed on the basis of measurement of FITC‐labeled degradation products by Sephadex G‐25 column chromatography. It was suppressed markedly but incompletely by E‐64‐c as well as by CA‐074, but more weakly than by E‐64‐c. These results shows that E‐64‐sensitive cysteine proteinases are important in lysosomal protein degradation, but cathepsin B has only a role in part and that an E‐64‐resistant proteinase(s) may also be important.
10N‐Nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria
Authors: PETIT Jean‐Michel , MAFTAH Abderrahman , RATINAUD Marie‐Hélène , JULIEN Raymond
Published: March 2005

The acridine orange derivative, 10N‐nonyl acridine orange, is an appropriate marker of the inner mitochondrial membrane in whole cells. We use membrane model systems to demonstrate that 10N‐nonyl acridine orange binds to negatively charged phospholipids (cardiolipin, phosphatidylinositol and phosphatidylserine). The stoichiometry has been found to be 2 mol 10N‐nonyl acridine orange/mol cardiolipin and 1 mol dye/mol phosphatidylserine or phosphatidylinositol, while, with zwitterionic phospholipids, significant binding could not be detected. The affinity constants were 2 × 106 M−1 for cardiolipin‐10N‐nonyl‐acridine‐orange association and only 7 × 104 M−1 for that of phosphatidylserine and phosphatidylinositol association. The high affinity of the dye for cardiolipin may be explained by two essential interactions; firstly an electrostatic interaction between the quaternary ammonium of nonyl acridine orange and the ionized phosphate residues of cardiolipin and secondly, hydrophobic interactions between adjacent chromophores. A linear relationship was demonstrated between the cardiolipin content of model membranes and the incorporated dye. Consequently, a convenient and rapid method for cardiolipin quantification in membranes was established and applied to the cardiolipin‐containing organelle, the mitochondrion.
Leukotriene uptake by hepatocytes and hepatoma cells
Authors: LEIER Inka , MÜLLER Michael , JEDLITSCHKY Gabriele , KEPPLER Dietrich
Published: March 2005

The uptake of tritiated cysteinyl leukotrienes (LTC4, LTD4, LTE4) and LTB4 was investigated in freshly isolated rat hepatocytes and different hepatoma cell lines under initial‐rate conditions. Leukotriene uptake by hepatocytes was independent of an Na+ gradient and a K+ diffusion potential across the hepatocyte membranes as established in experiments with isolated hepatocytes and plasma membrane vesicles. Kinetic experiments with isolated hepatocytes indicated a low‐Km system and a non‐saturable system for the uptake of cysteinyl leukotrienes as well as LTB4 under the conditions used. AS‐30D hepatoma cells and human Hep G2 hepatoma cells were deficient in the uptake of cysteinyl leukotrienes, but showed significant accumulation of LTB4. Moreover, only LTB4 was metabolized in Hep G2 hepatoma cells. Competition studies on the uptake of LTE4 and LTB4 (10 nM each) indicated inhibition by the organic anions bromosulfophthalein, S‐decyl glutathione, 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonate, probenecid, docosanedioate, and hexadecanedioate (100 μM each), but not by taurocholate, the amphiphilic cations verapamil and N‐propyl ajmaline, and the neutral glycoside ouabain. Cholate and the glycoside digitoxin were inhibitors of LTB4 uptake only. Bromosulfophthalein, the strongest inhibitor of leukotriene uptake by hepatocytes, did not inhibit LTB4 uptake by Hep G2 hepatoma cells under the same experimental conditions.

Leukotriene‐binding proteins were analyzed by comparative photoaffinity labeling of human hepatocytes and Hep G2 hepatoma cells using [3H]LTE4 and [3H]LTB4 as the photolabile ligands. Predominant leukotriene‐binding proteins with apparent molecular masses in the ranges of 48–58 kDa and 38–40 kDa were labeled by both leukotrienes in the particulate and in the cytosolic fraction of hepatocytes, respectively. In contrast, no labeling was obtained with [3H]LTE4 in Hep G2 cells. With [3H]LTB4 a protein with a molecular mass of about 48 kDa was predominantly labeled in the particulate fraction of the hepatoma cells, whereas in the cytosolic fraction a labeled protein in the range of 40 kDa was detected.

Our results provide evidence for the existence of distinct uptake systems for cysteinyl leukotrienes and LTB4 at the sinusoidal membrane of hepatocytes; however, some of the inhibitors tested interfere with both transport systems. Only LTB4, but not cysteinyl leukotrienes, is taken up and metabolized by the transformed hepatoma cells.
Evidence for an impaired long‐chain fatty acid oxidation and ketogenesis in Fao hepatoma cells
Authors: PRIP‐BUUS Carina , BOUTHILLIER‐VOISIN Anne‐Claire , KOHL Claude , DEMAUGRE France , GIRARD Jean , PEGORIER Jean‐Paul
Published: March 2005

Fatty acid metabolism has been studied in Fao rat hepatoma cells. In basal conditions of culture, [l‐14C]oleate is mainly esterified (85% of oleate uptake) in Fao cells, phospholipids being the most important esterified products (60% of oleate esterified). Addition of N6,O2′‐dibutyryl‐adenosine 3′,5′‐monophosphate (0.1 mM) in Fao cells does not change the metabolic fate of oleate whereas it induces gluconeogenesis and phosphoenolpyruvate carboxykinase mRNA accumulation. It is shown that the limitation of oleate oxidation is located at the level of the entry into mitochondria since octanoate is actively oxidized in Fao cells. Neither the activities of carnitine palmitoyltransferase (CPT) I and II nor the CPT II protein amount are affected by cAMP addition. The limitation of oleate oxidation in Fao cells results from (a) a high rate of lipogenesis and a high malonyl‐CoA concentration, (b) a CPT I very sensitive to malonyl‐CoA inhibition. The presence of an active oleate oxidation in mitochondria isolated from Fao cells confirms that CPT I is the limiting step of oleate oxidation. Moreover, Fao cells are unable to perform ketogenesis. This particular feature results from a specific deficiency in mitochondrial hydroxymethylglutaryl‐CoA synthase protein, activity and gene expression. The metabolic characteristics observed in Fao cells could be a common feature in hepatoma cell lines with regard to the low capacity for long‐chain fatty acid oxidation and ketone body production observed in the rat H4IIE and the human HepG2 cells.
Further characterization of the [Fe]‐hydrogenase from Desulfovibrio desulfuricans ATCC 7757
Authors: HATCHIKIAN E. Claude , FORGET Nicole , FERNANDEZ Victor M. , WILLIAMS Ruth , CAMMACK Richard
Published: March 2005

The properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans ATCC 7757, previously reported to be a single‐subunit protein [Glick, B. R., Martin, W. G. and Martin, S. M. (1980) Can. J. Microbiol. 26, 1214–1223] were reinvestigated. The pure enzyme exhibited a molecular mass of 53.5 kDa as measured by analytical ultracentrifugation and was found to comprise two different subunits of 42.5 kDa and 11 kDa, with serine and alanine as N‐terminal residues, respectively. The N‐terminal amino acid sequences of its large and small subunits, determined up to 25 residues, were identical to those of the Desulfovibrio vulgaris Hildenborough [Fe]‐hydrogenase. D. desulfuricans ATCC 7757 hydrogenase was free of nickel and contained 14.0 atoms of iron and 14.4 atoms of acidlabile sulfur/molecule and had ɛ400, 52.5 mM−1· cm−1. The purified hydrogenase showed a specific activity of 62 kU/mg of protein in the H2‐uptake assay, and the H2‐uptake activity was higher than H2‐evolution activity. The enzyme isolated under aerobic conditions required incubation under reducing conditions to express its maximum activity both in the H2‐uptake and 2H2/1H2 exchange reaction. The ratio of the activity of activated to as‐isolated hydrogenase was approximately 3. EPR studies allowed the identification of two ferredoxin‐type [4Fe‐4S]1+ clusters in hydrogenase samples reduced by hydrogen. In addition, an atypical cluster exhibiting a rhombic signal (g values 2.10, 2.038, 1.994) assigned to the H2‐activating site in other [Fe]‐hydrogenases was detected in partially reduced samples. Molecular properties, EPR spectroscopy, catalytic activities with different substrates and sensitivity to hydrogenase inhibitors indicated that D. desulfuricans ATCC 7757 periplasmic hydrogenase is a [Fe]‐hydrogenase, similar in most respects to the well characterized [Fe]‐hydrogenase from D. vulgaris Hildenborough.
Purification and characterization of a novel enzyme, arylalkyl acylamidase, from Pseudomonas putida Sc2
Authors: SHIMIZU Sakayu , OGAWA Jun , CHUNG Max Ching‐Ming , YAMADA Hideaki
Published: March 2005

A novel enzyme, arylalkyl acylamidase, which shows a strict specificity for N‐acetyl arylalkyl‐amines, but not acetanilide derivatives, was purified from the culture broth of Pseudomonas putida Sc2. The purified enzyme appeared to be homogeneous, as judged by native and SDS/PAGE. The enzyme has a molecular mass of approximately 150 kDa and consists of four identical subunits. The purified enzyme catalyzed the hydrolysis of N‐acetyl‐2‐phenylethylamine to 2‐phenylethylamine and acetic acid at the rate of 6.25 μmol · min−1· mg−1 at 30°C. It also catalyzed the hydrolysis of various N‐acetyl arylalkylamines containing a benzene or indole ring, and acetic acid arylalkyl esters. The enzyme did not hydrolyze acetanilide, N‐acetyl aliphatic amines, N‐acetyl amino acids, N‐acetyl amino sugars or acylthiocholine. The apparent Km for N‐acetylbenzylamine, N‐acetyl‐2‐phenyl‐ethylamine and N‐acetyl‐3‐phenylpropylamine are 41 mM, 0.31 mM and 1.6 mM, respectively. The purified enzyme was sensitive to thiol reagents such as Ag2SO4, HgCl2 and P‐chloromercuribenzoic acid, and its activity was enhanced by divalent metal ions such as Zn2+, Mg2+ and Mn2+.
Purification and biochemical characterization of the Ecal DNA methyltransferase
Authors: SZILÁK László , VENETIANER Pál , KISS Antal
Published: March 2005

The EcaI GGTNACC‐specific DNA‐adenine modification methyltransferase has been purified to apparent homogeneity. The active form of the DNA methyltransferase is a single polypeptide. The enzyme has a pH optimum at pH 8.0 and a temperature optimum at 25°C. EcaI DNA methyltransferase transfers one methyl group to the adenine of the recognition site in a single binding event. The Km was 170 nM for DNA and 1.8 μM for the methyl donor S‐adenosylmethionine. Methylated DNA is a competitive inhibitor with respect to DNA (Ki= 3.5 nM). The other product of the DNA‐methylation reaction, S‐adenosylhomocysteine was found to be a competitive inhibitor with respect to S‐adenosylmethionine (Ki= 2.7 μM). The S‐adenosylmethionine analog sinefungin was shown to be a very strong inhibitor (Ki= 3.5 nM) of the DNA methyltransferase reaction.
Interaction between bovine trypsin and a synthetic peptide containing 28 residues of the bait region of human α2‐macroglobulin
Authors: THOMSEN Niels Kirk , CHRISTENSEN Ulla , MADSEN Kjeld , SOTTRUP‐JENSEN Lars
Published: March 2005

The time course of the interaction between trypsin and a synthetic peptide corresponding to a segment (residues 676–703) of the bait region (residues 666–706) of human α2‐macroglobulin (α2M) was studied by measuring the generation of cleavage products as a function of time by HPLC. Three primary cleavage sites for trypsin were present in the synthetic peptide. The fastest cleavage occurred at the bond corresponding to Arg696‐Leu in α2M with an estimated kcat/Km= 1–2 × 106 M−1· s−1. This value is of the same magnitude as that characterizing the interaction of α2M and trypsin when taking into account the fact that α2M is a tetramer, kcat/Km= 5 × 106 M−1· s−1 [Christensen, U. & Sottrup‐Jensen, L, (1984) Biochemistry 23, 6619–6626]. The values of kcat/Km for cleavage at bonds corresponding to Arg681‐Val and Arg692‐Gly in α2M were 1.5 × 105 M−1· s−1 and 1.3 × 105 M−1· s−1, respectively. Cleavage of intermediate product peptides was slower, with kcat/Km in the range 13–1.3 × 106 M−1·s−1. The value of Km determined for fast cleavage in the synthetic peptide was 8–10 μM. 1H‐NMR spectroscopy indicated no ordered structure of the peptide. Hence, the very fast cleavage of the peptide is compatible with a loose structure that readily adopts a conformation favorable for recognition and cleavage by trypsin.
Regulation of insulin‐like‐growth‐factor‐II gene expression in rat liver cells
Authors: ZARRILLI Raffaele , COLANTUONI Vittorio , BRUNI Carmelo Bruno
Published: March 2005

The rat insulin‐like‐growth‐factor‐(IGF)‐II gene is expressed at high levels during embryonic and fetal life and at low levels in adult animals. To study the regulation of IGF‐II gene expression, we analyzed the synthesis and localization of the IGF‐II transcripts in cultured rat liver cells either expressing (BRL3A cells) or not expressing (BRL30E and FAO cells) the IGF‐II mRNA. The IGF‐II gene is transcribed at a similar rate in expressing and non‐expressing cells, whereas its nuclear and cytoplasmic RNA levels are diversely distributed in the cells. IGF‐II RNA is more abundant in the cytoplasmic than in the nuclear RNA fraction of BRL3A cells and is present in the nucleus but not in the cytoplasm of the FAO cells. However, both precursor and mature IGF‐II nuclear RNA levels are reduced in FAO cells. Our data indicate that the IGF‐II gene expression is regulated by mechanisms affecting the subcellular distribution and the abundance of the transcripts.
The long‐term combined stimulatory effects of ethanol and phorbol ester on phosphatidylethanolamine hydrolysis are mediated by a phospholipase C and prevented by overexpressed α‐protein kinase C in fibroblasts
Authors: KISS Zoltan
Published: March 2005

The protein kinase C (PKC) activator 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) has been shown to potentiate the stimulatory effect of ethanol on the hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Following an initial 20‐min period, the main product of PtdEtn degradation in cells treated with TPA plus ethanol was ethanolamine phosphate. Here, we have examined the regulatory role of PKC and the possible catalytic role of phospholipase C in the formation of ethanolamine phosphate. TPA, bryostatin, and bombesin, direct or indirect activators of PKC, had similar potentiating effects on ethanol‐induced formation of [14C]ethanolamine phosphate from [14C]PtdEtn in [14C]ethanolamine‐prelabelled NIH 3T3 fibroblasts. At lower concentrations of ethanol (40–80 mM), significant stimulation of ethanolamine phosphate formation required longer treatments (2 h or longer). The combined effects of TPA (100 nM) and ethanol (50–200 mM) on ethanolamine phosphate formation were not inhibited by the PKC inhibitors staurosporine or l‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H7). In contrast, these inhibitors significantly inhibited TPA‐induced formation of ethanolamine, catalyzed by a phospholipase‐d‐type enzyme. In membranes isolated from TPA+ ethanol‐treated cells, enhanced formation of ethanolamine phosphate was maintained for at least 20 min. Down‐regulation of PKC by prolonged (24‐h) treatment of NIH 3T3 fibroblasts by 300 nM TPA enhanced, while overexpression of α‐PKC in Balb/c fibroblasts diminished, the stimulatory effect of ethanol on the formation of ethanolamine phosphate. Finally, addition of the protein phosphatase inhibitor okadaic acid (2 μM) to fibroblasts inhibited TPA + ethanol‐induced formation of ethanolamine phosphate. These results suggest that α‐PKC‐mediated protein phosphorylation may negatively regulate PtdEtn hydrolysis and that the potentiating effect of TPA may result, at least partly, from increased degradation of this PKC isoform.
The elusive nuclear matrix
Authors: JACK Robert S. , EGGERT Harald
Published: March 2005

The structure of the interphase nucleus is a major area of current interest in cell biology. It is thought likely that the nucleus is organised around some form of structural matrix and that this matrix will play a role in processes as diverse as chromosome replication and the integration of gene expression. However, the structure of the matrix within the nucleus has remained elusive, largely because attempts to define it have been dogged by technical problems arising from the great complexity of this organelle. This situation is now being changed by the application of in situ analysis and of molecular genetic methodologies which are opening up this hitherto intractable field.
Localization and identification of actin structures involved in the filamin – actin interaction
Authors: MÉJEAN Catherine , LEBART Marie‐Christine , BOYER Mireille , ROUSTAN Claude , BENYAMIN Yves
Published: March 2005

The interface between gizzard filamin and skeletal muscle actin was located on the actin monomer. Conserved sequences 105–120 and 360–372, in the actin subdomain 1 near the myosin binding sites, were involved in this interaction. The corresponding peptides for these sequences were each found to bind filamin and compete in the actin–filamin interaction. When these two peptides were used together in the presence of filamin and filamentous actin, they dissociated sedimentable complexes formed by these two proteins.