bluuebunny/crossref_metadata_2025 huggingface dataset:
The antiglycogenolytic action of 1‐deoxynojirimycin results from a specific inhibition of the α‐1,6‐glucosidase activity of the debranching enzyme
Authors: BOLLEN Mathieu , STALMANS Willy
Published: March 2005

The glucose analogue 1‐deoxynojirimycin (dNOJ) and some of its N‐substituted derivatives have recently been described as potent inhibitors of the hepatic glycogenolysis induced by glucagon, Ca2+ ionophores or anoxia. The inhibition increased with time, in spite of a persistently high level of phosphorylase a [Bollen, M., Vandebroeck, A. & Stalmans, W. (1988) Biochem. Pharmacol. 37, 905–909].

dNOJ equilibrates within 1 min across the plasma membrane of hepatocytes. It is not phosphorylated or oxidized in the cell. The observation that dNOJ did not affect gluconeogenesis excludes the possibility that glucose‐6‐phosphatase is the target for the inhibition of glucose production from glycogen. Neither were the catalytic activities of phosphoglucomutase and phosphorylase a affected by the compound.

dNOJ and two N‐substituted derivatives inhibited instantaneously and completely the α‐1,6‐glucosidase activity of the debranching enzyme, with I50 values in the μmolar range. In contrast, the glucanotransferase activity of the latter enzyme was not inhibited by the compounds at 0.2 mM.

The effect of dNOJ was further studied in an in vitro model system of glycogenolysis. The results were compatible with a block of glycogenolysis at the time when phosphorylase has removed the available glucosyl residues from the outer chains of the glycogen particles. This mechanism appears to account for the lag in the response of glycogenolysis to dNOJ.
The effects of multiple amino acid substitutions on the polypeptide backbone of tuna and horse cytochromes c
Authors: GAO Yuan , LEE A. Daniel J. , WILLIAMS Robert J. P. , WILLIAMS Glyn
Published: March 2005

The cytochromes c provide a wide range of natural and mutant homologous proteins which may be used to study structure/function relationships in biological electron‐transfer reactions. A description of the cytochrome c structure has been provided by high‐resolution X‐ray crystallography for the cytochromes from tuna, bonito, rice and yeast (Saccharomyces iso‐1). Correlation of these structures with NMR parameters is necessary to confirm the structure of the protein in solution and to permit the routine characterisation of cytochromes c with novel sequences. We have previously reported a method based on the analysis of pseudocontact shifts which allowed us to compare the conformations of some amino acid side chains of tuna cytochrome c in solution and in the crystalline state. Here we report a comparison of the conformations of the polypeptide backbone of cytochromes c in proteins from tuna and horse, using the chemical shifts of the amide NH and CαH protons. It is found that the backbone conformation and hydrogen‐bond network is closely conserved between these proteins, despite 19 amino acid substitutions. Appreciable differences occur in two regions, around Asn 31 and at the beginning of the 60s helix. Evidence for some rotational or translational motion of the C‐terminal helix is also presented.
Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase
Authors: CELERIER Jérôme , RISLER Yanick , SCHWENCKE Jaime , JANOT Jean‐Marc , GERVAIS Michel
Published: March 2005

The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an l‐Lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the purification of the resulting l‐lactate: dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr= 4 × 39000) containing FMN as expected and not heme. It has high l‐lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2, but it has no l‐lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b2. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated.
The primary structure of rat ribosomal protein S10
Authors: GLÜCK Anton , CHAN Yuen‐Ling , LIN Alan , WOOL Ira G.
Published: March 2005

The amino acid sequence of rat ribosomal protein S10 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2‐terminal amino acid sequence of the protein. Ribosomal protein S10 contains 165 amino acids and has a molecular mass of 18917 Da. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 17–20 copies of the S10 gene. The mRNA for the protein is about 750 nucleotides in length. Ribosomal protein S10 has several possible internal duplications; one is a tandem repeat of ten residues that is basic and contains two or three prolines.
Mechanism of interaction of citrate synthase with citryl‐CoA
Authors: PETTERSON Gösta , LILL Ute , EGGERER Hermann
Published: March 2005

Arguments are presented which indicate that the low steady‐state rates of citrate production governing the catalytic interaction of citrate synthase from pig heart with citryl‐CoA reflect the formation of a non‐productive enzyme · citryl‐CoA complex. The kinetic predictions of such an extended reaction mechanism are examined and are shown to account in satisfactory detail for the complex multiphasic rate behaviour exhibited by the enzyme under a variety of conditions in reactions involving citryl‐CoA as a substrate.
Effect of hydrophobic sulphydryl reagents on the uncoupling protein and inner‐membrane anion channel of brown‐adipose‐tissue mitochondria
Authors: RIAL Eduardo , ARÉCHAGA Ignacio , SAINZ‐DE‐LA‐MAZA Eduardo , NICHOLLS David G.
Published: March 2005

The effects of three sulphydryl reagents of differing hydrophobicity (N‐ethylmaleimide, N‐benzylmaleimide and N,N′‐o‐phenylenedimaleimide) on ion permeation through the inner membrane of brown‐adipose‐tissue mitochondria are investigated. GDP‐sensitive permeation of chloride and protons (hydroxyl ions) through the uncoupling protein is increased exponentially with time by all three reagents. With increasing hydrophobicity of the reagents, modification is enhanced and an initial inhibited state becomes apparent. Results are interpreted in terms of a two‐stage modification via a non‐transporting intermediate, which does not bind GDP, to a final highly conducting product. The reagents also react with a hydrophilic sulphydryl group on an independent protein to induce a GDP‐insensitive pathway which allows chloride, phosphate and sulphate to cross the membrane. The use of different sulphydryl reagents allows the two pathways to be clearly distinguished.
Different membrane‐bound forms of acetylcholinesterase are present at the cell surface of hepatocytes
Authors: PERELMAN Alejandra , BRANDAN Enrique
Published: March 2005

In the present study we have determinated the acetylcholinesterase molecular forms present in rat liver hepatocytes; we have also studied the association of acetylcholinesterase with the cell surface of the hepatocytes. Subcellular fractionation indicated that rough endoplasmic reticulum and plasma‐membrane‐enriched fractions contains G4 and G2 acetylcholinesterase forms bound to membranes. Hepatocytes incubated with phosphatidylinositol‐specific phospholipase C released about 70% of the surface acetylcholinesterase. Sedimentation analysis showed that all the solubilized acetylcholinesterase activity comes exclusively from a G2 dimer. The G4 hydrophobic form of acetylcholinesterase accounts for the additional cell‐surface activity. The existence of these two forms of acetylcholinesterase on the surface of hepatocytes was further established by analyzing the phosphatidylinositol‐specific phospholipase C sensitivity‐of the acetylcholinesterase molecular forms present in isolated rat liver plasma membranes.
Calcium chelator Quin 2 prevents hydrogen‐peroxide‐induced DNA breakage and cytotoxicity
Authors: CANTONI Orazio , SESTILI Piero , CATTABENI Flaminio , BELLOMO Giorgio , POU Susan , COHEN Myron , CERUTTI Peter
Published: March 2005

Exposure of cultured Chinese hamster ovary (CHO) cells to hydrogen peroxide results in the production of extensive DNA breakage which can be prevented by the intracellular calcium chelator Quin 2. This effect occurs at Quin 2 AM concentrations as low as 0.1 μM and is maximal at 1 μM. Addition of the extracellular calcium chelator, EGTA, does not affect the level of DNA breakage generated by H2O2. Quin 2 also significantly reduces cellular toxicity caused by the oxidant. Experiments with spin‐trapping techniques demonstrate that Quin 2 does not affect the formation of hydroxyl radicals generated by the action of Fe2+ on H2O2. Quin 2 at high concentrations, similar to those reached within the cell, actually enhanced generation of hydroxyl radical in the absence of other iron chelators under our experimental conditions. These results suggest that H2O2 or H2O2‐derived radicals do not directly induce DNA strand breakage in intact mammalian cells; rather, these radicals may disturb intracellular Ca2+ homeostasis which results in secondary reactions ultimately leading to DNA strand breakage. In addition to strand breakage, membrane and protein oxidation probably contribute to the cytotoxic effect of H2O2.
Isolation and characterization of a rat liver alkaline phosphatase gene
Authors: TOH Yasushi , YAMAMOTO Mikio , ENDO Hideya , MISUMI Yoshio , IKEHARA Yukio
Published: March 2005

Structural analysis of 55 nearly full‐length cDNA clones revealed heterogeneity in the 5′‐untranslated regions of rat liver alkaline phosphatase mRNAs. The 5′ extremities diverged into two totally unrelated sequence stretches at the position 88 nucleotides upstream of the initiation codon ATG. These two sequences, referred to as E1 and E2, were assigned on the genome about 36000 base pairs (36 kbp) and 10 kbp upstream, respectively, of the exon coding for the 5′‐most part of the common region. The gene consisted of 13 exons, including E1 and E2, and spanned about 56 kbp. The 11 exons (E3 to E13) following E1 and E2 were shared in common by the E1‐type and the E2‐type mRNAs. Analyses by S1 nuclease mapping and primer extension revealed the presence of two independent transcription‐initiation sites specific to each of the E1 and E2 sequences. These results are interpreted as indicating a possible alternative usage of two leader exons, hence the presence of two independent promoters. Structural features of these putative promoters are described in the context of transcriptional fundamental and regulatory cis‐elements.
The influence of substoichiometric concentrations of myosin subfragment 1 on the state of aggregation of actin under depolymerizing conditions
Authors: GRAZI Enrico , MAGRI Ermes , RIZZIERI Luciana
Published: March 2005

In 3 mM KCl, 2 mM Tris/HCl pH 7.5, 22°C, 0.38 μ myosin subfragment 1 delays the depolymerization of F‐actin (7.2 μ measured as monomer). The depolymerization proceeds rapidly for a few minutes and then slows down suddenly when the ratio between the monomers in the actin filaments and myosin subfragment 1 reaches the value of 11. At this time myosin subfragment 1 is substantially all bound to the actin polymers which form an irregular and discontinuous network of filaments running in doublets and in triplets, perhaps cross‐linked by myosin subfragment 1. Depolymerization proceeds then for several hours, apparently ending up with the formation of the 1:1 actin‐S1 heteropolymer. The ratio between the monomers in the actin filaments and myosin subfragment 1 at the end of the rapid depolymerization proceeds is different for different protein preparations and may be as low as 5.5.

In 2 mM Tris/HCl pH 7.5, 25°C, 1 μ myosin subfragment 1 is able to induce the formation of undecorated actin filaments from 12 μ ATP–G‐actin. These filaments probably originate by redistribution of myosin subfragment 1 between the newly formed 1/1 actin‐S1 heteropolymer and G‐actin in the medium, a process which allows the transient formation of undecorated actin filaments.
Chemical characterization of p17gag from human immunodeficiency virus as an N‐terminally myristoylated protein
Authors: GODDARD Colin , AQUINO Angelo , GLAZER Robert I. , FELSTED Ronald L.
Published: March 2005

The N‐terminal p17gag protein of the human immunodeficiency virus has been shown to incorporate radioactivity following labelling of infected cell lines with [3H]myristic acid. We investigated p17gag to determine whether the incorporated radioactivity was the consequence of N‐terminal myristoylation. The virus was purified by density gradient centrifugation after labelling chronically infected H9 cells with [3H]myristic acid. The p17gag was isolated by immunoprecipitation and subjected to partial acid hydrolysis. [3H]Myristoylglycine generated by the hydrolysis was derivatized to 4‐(p‐nitrobenzylidene)‐2‐tridecanoyloxazol‐5‐one and identified against a co‐eluting, derivatized, unlabelled N‐myristoylglycine standard by reverse‐phase high‐performance liquid chromatography. This study unequivocally demonstrates that p17gag is an N‐myristoylated protein.
A novel l‐glutamate oxidase from Streptomyces endus
Authors: BÖHMER Annette , MÜLLER Anita , PASSARGE Margit , LIEBS Peter , HONECK Horst , MÜLLER Hans‐Georg
Published: March 2005

A new flavoenzyme using molecular oxygen to oxidize l‐glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2‐oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including d‐glutamic acid, l‐glutamine and l‐aspartic acid, only l‐glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273, 355 and 457 nm and the isoelectric point is at pH 6.2.

The Km value for l‐glutamic acid in air‐saturated phosphate pH 7.0 was estimated to be 1.1 mM, the Km for oxygen was calculated to be 1.86 mM at saturating concentration of l‐glutamic acid. The enzymic reaction is inhibited by Ag+ and Hg2+ ions. The enzyme described here distinctly differs from two microbial l‐glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure.
Insulin‐induced dephosphorylation of hormone‐sensitive lipase
Authors: STRÅLFORS Peter , HONNOR Rupert C.
Published: March 2005

The effect of insulin on the state of phosphorylation of hormone‐sensitive lipase, cellular cAMP‐dependent protein kinase activity and lipolysis was investigated in isolated adipocytes. Increased phosphorylation of hormone‐sensitive lipase in response to isoproterenol stimulation was closely paralleled by increased lipolysis. Maximal phosphorylation and lipolysis was obtained when the cAMP‐dependent protein kinase activity ration was ≥ 0.1, and this corresponded to a 50% increase in the state of phosphorylation of hormone‐sensitive lipase.

Insulin (1 nM) reduced cAMP‐dependent protein kinase activity and also reduced lipolysis with both cAMP‐dependent and cAMP‐independent antilipolytic effects up to an activity ratio of ∼ 0.4, above which the antilipolytic effect was lost. Insulin caused a decrease in the state of phosphorylation of hormone‐sensitive lipase at all levels of cAMP‐dependent protein kinase activity. Under basal conditions, with cAMP‐dependent protein kinase activity at a minimum, this reflected a dephosphorylation of the basal phosphorylation site of hormone‐sensitive lipase in a manner not mediated by cAMP. When the cAMP‐dependent protein kinase was stimulated to phosphorylate the regulatory phosphorylation site of hormone‐sensitive lipase, the insulin‐induced dephosphorylation occurred both at the basal and regulatory sites. At low levels of cAMP‐dependent protein kinase activity ratios (0.05–0.1), dephosphorylation of the regulatory site correlated with reduced cAMP‐dependent protein kinase activity, but not at higher activity ratios (> 0.1).

Stimulation of cells with isoproterenol produced a transient (1–5 min) peak of cAMP‐dependent protein kinase activity and of phosphorylation of hormone‐sensitive lipase. The state of phosphorylation also showed a transient peak when the protein kinase was maximally and constantly activated. In the presence of raised levels of cellular cAMP, insulin (1 nM) caused a rapid (t1/2∼ 1 min) dephosphorylation of hormone‐sensitive lipase. In unstimulated cells the reduction in phosphorylation caused by insulin was distinctly slower (t1/2∼ 5 min).

These findings are interpreted to suggest that insulin affects the state of phosphorylation of hormone‐sensitive lipase and lipolysis through a cAMP‐dependent pathway, involving reduction of cAMP, and through a cAMP‐independent pathway, involving activation of a protein phosphatase activity that dephosphorylates both the regulatory and basal phosphorylation sites of hormone‐sensitive lipase.
Stimulation of insulin of glycolysis in cultured hepatocytes is attenuated by extracellular ATP and puromycin through purine‐dependent inhibition of phosphofructokinase 2 activation
Authors: PROBST Irmelin , QUENTMEIER Armin , SCHWEICKHARDT Christa , UNTHAN‐FECHNER Kirsten
Published: March 2005

Activation of glycolysis by insulin in cultured rat hepatocytes is preceeded by an activation of phosphofructokinase 2 (PFK 2) and subsequent rise of the fructase 2,6‐bisphosphate [Fru(2,6)P2] level. Extracellular addition of ATP or puromycin prevented the hormonal effect on glycolysis. The mechanism through which the purines abolished glycolytic stimulation was investigated.

50 μM ATP completely prevented the 3–5‐fold insulin‐dependent increase of glycolysis, irrespective of whether the cells initially possessed a low or a high Fru(2,6)P2 content 50 μM puromycin prevented the stimulation of glycolysis by insulin only in cells whose initial Fru(2,6)P2 levels were low and had to be increased by insulin prior to the increase in glycolysis. It did not antagonize the action of insulin in cells with initial high Fru(2,6)P2 content.

ATP exerted effects on its own; it decreased initially high Fru(2,6)P2 levels by 95% within 10 min and decreased the basal glycolytic rate by 60%. Half‐maximal effects on the Fru(2,6)P2 level were obtained with about 25 μM ATP or 15 μM adenosine 5′[β,γ‐methylene]triphosphate. ADP and adenosine‐5‐(γ‐thio]triphosphate were as effective as ATP, whereas 100 μM adenosine 5′[α,β‐methylene]triphosphate elicited no effect. Puromycin neither decreased high Fru(2,6)P2 levels nor inhibited basal glycolysis.

Extracellular ATP (100 μM) led to inhibition of the active form of PFK 2. Intracellular levels of Glc6P, citrate. ATP, ADP and AMP were increased by extracellular ATP, the phosphoenolpyruvate content was decreased, Fru6P and glycerol 3‐phosphate levels stayed constant. Puromycin did not inhibit PFK 2.

Both puromycin and ATP prevented the insulin‐dependent rise of the Fru(2,6)P2 level, they abolished the activation of PFK 2 by the hormone. Puromycin did not block the accumulation of Fru(2,6)P2 provoked by glucose addition; ATP also antagonized the glucose‐dependent increase.

100 μM ATP elevated the cAMP‐dependent protein kinase activity ratio from 0.1 to 0.38 and increased the level of inositol trisphosphate by 16‐fold within 5 min, whereas puromycin was without effect on either level.

It is concluded that the two purines block the insulin effect on glycolysis by preventing the hormone increasing the Fru(2,6)P2 level. The mode of action, however, seems to be different: ATP antagonizes insulin action in that it leads to increased inhibition of PFK 2 whereas puromycin prevents the activation of PFK 2 by insulin.
The effects of acetylated low‐density lipoproteins on fluid‐phase pinocytosis by macrophages
Authors: KNOWLES Marion E. , LEAKE David S.
Published: March 2005

A simple method has been set up to measure the rate of fluid‐phase pinocytosis in resident mouse peritoneal macrophages in culture. The method uses 125I‐labelled polyvinylpyrrolidone as a nondegradable marker of fluidphase pinocytosis. The accumulation of 125I‐labelled polyvinylpyrrolidone by the cells was directly proportional to its concentration in the culture medium up to at least 200 μg/ml. The estimates of the rate of fluid‐phase pinocytosis were reproducible within each experiment (coefficient of variation 8.5%) but varied between individual experiments. Fluid‐phase pinocytosis was undetectable at 4°C and reduced greatly at 37°C by metabolic inhibitors and 1 mM ZnSO4. High concentrations of human acetylated low‐density lipoproteins, which are taken up rapidly by macrophages, decreased the rate of fluid‐phase pinocytosis by up to about 70%. The inhibition was seen after only 2 h of incubation. Unmodified low‐density lipoproteins, which are taken up only slowly by macrophages, did not usually inhibit fluid‐phase pinocytosis (in fact, they sometimes increased it). Modified low‐density lipoprotein uptake, leading to massive lipid accumulation in macrophages in the arterial wall, has been postulated to be involved in the pathogenesis of atherosclerosis. This study raises the possibility that the rate of fluid‐phase pinocytosis in these lipid‐laden arterial macrophages may be reduced.
Probing the environment of lanthanide binding sites in yeast tRNAPhe by specific metal‐ion‐promoted cleavages
Authors: CIESIOŁKA Jerzy , MARCINIEC Tadeusz , KRZYŻOSIAK Wlodzimierz J.
Published: March 2005

Specific yeast tRNAPhe hydrolysis brought about by europium ions has been studied in detail using the 32P‐end‐labeled tRNA and polyacrylamide gel electrophoresis. The dependence of the induced cleavages on pH, temperature and concentration of the europium ions has been determined. Europium hydrolyzes yeast tRNAPhe in the D‐loop at phosphates 16 and 18, and the anticodon loop of phosphates 34 and 36. The two D‐loop cuts are thought to take place from two distinct europium binding sites, while the two anticodon loop cleavages from a single site. Eight other members of the lanthanide series and ytrium give basically the same pattern of cleavages as europium. The specific cleavages taking place in the anticodon loop occur in an intramolecular mode from the lanthanide binding site that has not been found in yeast tRNAPhe crystal structure. It appears from the comparison of the europium‐promoted cuts with those generated by magnesium and lead that the former two ions give more similar but not identical cleavage patterns. The usefulness of the specific cleavages induced by lanthanides for probing their own and magnesium binding sites in tRNA is discussed.
The human lysozyme gene
Authors: PETERS Christoph W. B. , KRUSE Ulrich , POLLWEIN Renate , GRZESCHIK Karl‐Heinz , SIPPEL Albrecht E.
Published: March 2005

We have isolated two overlapping recombinant λ‐phage clones from a genomic λ‐EMBL3 library containing 25 kb of the human lysozyme gene region. Furthermore a full‐lenght human lysozyme cDNA clone of 1.5 kb was isolated from a human placenta cDNA library. Nucleotide sequences of the entire structural gene and the cDNA clone were determined. The human lysozyme gene spans 5856 bp and its sequence organization with four exons and three introns is homologous to the chicken lysozyme gene and the human α‐lactalbumin gene. Human and chicken lysozyme genes differ mainly in the size of their introns and 3′ non‐coding region. Four Alu repetitive elements were found in the human lysozyme gene, one in each intron and one on the fourth exon. Lysozyme transcripts of 1.6 kb and 0.6 kb in size were detected in human myeloid cell lines U‐937, HL‐60 and THP‐1 and surprisingly in human hepatoma cell lines HepG2 and Hep3B. The lysozyme gene locus was assigned to human chromosome 12 by hybridization to a panel of DNAs from human‐rodent somatic cell hybrids.
Correlation between internal motion and emission kinetics of tryptophan residues in proteins
Authors: KOUYAMA Tsutomu , KINOSITA Kazuhiko , IKEGAMI Akira
Published: March 2005

Time‐resolved fluorescence anisotropy measurements of tryptophan residues were carried out for 44 proteins. Internal rotational motion with a sub‐nanosecond correlation time (0.9 ± 0.6 ns at 10°C) was seen in a large number of proteins, though its amplitude varied from protein to protein. It was found that tryptophan residues which were almost fixed within a protein had either a long (> 4 ns) or short (< 2 ns) fluorescence lifetime, whereas a residue undergoing a large internal motion had an intermediate lifetime (1.5–3 ns). It is suggested that the emission kinetics of a tryptophan residue is coupled with its internal motion. In particular, an immobile tryptophan residue emitting at long wavelength was characterized by a long lifetime (>4 ns). It appears that a tryptophan residue fixed in a polar region has little chance of being quenched by neighboring groups.
The nuclear‐coded chloroplast 22‐kDa heat‐shock protein of Chlamydomonas
Authors: GRIMM Bernhard , ISH‐SHALOM Dvorah , EVEN Dena , GLACZINSKI Heike , OTTERSBACH Peter , OHAD Itzhak , KLOPPSTECH Klaus
Published: March 2005

A cDNA clone, pCHS62, was isolated using poly(A)‐rich RNA from heat‐shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat‐shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat‐shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat‐shock protein of 22 kDa synthesized in vivo during heat‐shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)‐rich RNA from heat‐treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5′ region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non‐coding 3′ region of 600 nucleotides. Northern hybridization indicates a lenght of 1.7 kb for the messenger RNA of heat‐shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat‐shock proteins demonstrates that this protein belongs to the small‐molecular‐mass plant heat‐shock protein family and also shows similarities with animal heat‐shock proteins including the presence of a short region possesing similarity with bovine α‐crystalline as reported for other heat‐shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.
The effect of oligonucleotides complementary to the 3′ end of the 16S rRNA on the formation of the bacterial 30S initiation complex
Authors: ECKERT Volker , KYRIATSOULIS Apostolos , GASSEN H. Günter
Published: March 2005

Although much attention was focused on the role of the 16S RNA in mRNA selection by th3 30S ribosomal subunit no true consensus site has emerged as yet. Oligonucleotides such as GAGG, UGAU and CCAA which are complementary to the 3′ end of the 16S RNA stimulate the AUG‐dependent binding of fMet‐tRNA to 30S subunits. If those tetranucleotides are used in combination or if the octanucleotides GAGGUGAU and UGAUCCAA are applied, the degree of stimulation remians unchanged. Effects are strictly dependent on the presence of iniiaton factor 2 (IF‐2) and cannot be produced by using A4 or U4. With sequences covalently linked to the AUG as in CCAAAUG and UGAUCCAAAUG, the efficiency of the initiation complex formation decreases significantly as compared to AUG with UGAUCCAAAUG being the least efficient mRNA analogue. The pentadecanucleotide GAGGUGAUCCAAAUG, however, shows the highest efficiency in directing the binding of the fMet‐tRNA to 30S subunits and is clearly superior to AUG. Initiation factor 2 (IF‐2), which stimulates tRNA binding significantly with AUG and CCAAAUG, both in terms of slope and plateau values of the binding curves, does not effect the initial rate of tRNA binding to GAGGUGAUCCAAAUG. In another set of experiments, where GAGG and AUG are separated by oligo(U) or oligo(A) sequences, the effect of chain length was investigated. mRNA analogues with a spacer of 6–9 nucleotides show the highest binding efficiencies, with a U spacer being superior to an A spacer, indicating that a more flexible spacer favours tRNA binding.
Sequence and characterization of the sperm‐specific protein φ3 from Mytilus californianus
Authors: AUSIO Juan , McPARLAND Reginald
Published: March 2005

We have shown that the sperm‐specific protein Ø3 from Mytilus californianus (Conrad) exhibits compositional microheterogeneity. For the first time, we have isolated and characterized the three major components of this protein. These fractions display different electrophoretic mobilities on Triton/urea/acetic acid polycrylamide gels. However, they have a very similar molecular mass of 5±0.1 kDa as measured by sedimentation equilibrium in the analytical ultracentrifuge. All of them show a marked trend toward aggregation. We have also established the sequence for each of these three fractions. The sequencing data suggest an even greater extent of microheterogeneity for this protein. The predicted secondary structure from the sequences, as well as infrared analyzes carried out on the native protein, suggest an structure organization into an α helix.
The ATP/ADP‐antiporter is involved in the uncoupling effect of fatty acids on mitochondria
Authors: ANDREYEV Alexander Yu. , BONDAREVA Tatjana O. , DEDUKHOVA Vera I. , MOKHOVA Elena N. , SKULACHEV Vladimir P. , TSOFINA Lilya M. , VOLKOV Nickolaj I. , VYGODINA Tatjana V.
Published: March 2005

The ATP/ADP‐antiporter inhibitors and the substrate ADP suppress the uncoupling effect induced by low (10–20 μM) concentrations of palmitate in mitochondria from skeletal muscle and liver. The inhibitors and ADP are found to (a) inhibit the palmitate‐stimulated respiration in the controlled state and (b) increase the membrane potential lowered by palmitate. The degree of efficiency decreases in the order: carboxyatractylate (CAtr) > ADP > bongkrekic acid, atractylate. GDP is ineffective, Mg > ADP is of much smaller effect, whereas ATP is effective at much higher concentration than is ADP. Inhibitor concentrations, which maximally suppress the palmitate‐stimulated respiration, correspond to those needed for arresting the state 3 respiration. The extent of the CAtr‐sensitive stimulation of respiration by palmitate has been found to decrease with an increase in palmitate concentration. Stimulation of the controlled respiration by p‐trifluoromethoxycarbonylcyanide phenylhydrozone (FCCP) and gramicidin D at any concentrations of these uncouplers is CAtr‐insensitive, whereas that caused by a low concentrations of 2,4‐dinitrophenol and dodecyl sulfate is inhibited by CAtr.

The above effect of palmitate develops immediately after addition of the fatty acid. It is resistant to EGTA as well as to inhibitors of phospholipase (nupercain) and of lipid peroxidation (ionol). Moreover, palmitate accelerates spontaneous release of the respiratory control, developing in rat liver mitochondria under certain conditions. This effect takes several minutes, being sensitive to EGTA, nupercain and ionol. Like the fast uncoupling, this slow effect is inhibited by ADP but CAtr and atractylate are stimulatory rather than inhibitory.

In artificial planar phospholipid membrane, palmitate does not increase the membrane conductance, FCCP increases it strongly and dinitrophenol only slightly.

In cytochrome oxidase proteoliposomes, FCCP, gramicidin and dinitrophenol (less effectively) lower, whereas palmitate enhances the cytochrome‐oxidase‐generated membrane potential. In this system, monensin substitutes for palmitate.

It is concluded that the ATP/ADP antiporter is somehow involved in the uncoupling effect caused by low concentrations of palmitate and, partially, of dinitrophenol, whereas uncoupling produced by FCCP and gramicidin is due to their action on the phospholipid part of the mitochondrial membrane. A possible mechanism of this effect is discussed.
Purification and partial characterization of 1‐aminocyclopropane‐1‐carboxylate synthase from tomato pericarp
Authors: van der STRAETEN Dominique , van WIEMEERSCH Luc , GOODMAN Howard M. , van MONTAGU Marc
Published: March 2005

1‐Aminocyclopropane‐1‐carboxylate synthase was purified 5000‐fold from LiCl‐induced tomato fruit slices by conventional and high‐performance liquid chromatography. The final preparation was estimated to be between 25% and 50% pure. Two‐dimensional gel electrophoresis indicates that 1‐aminocyclopropane‐1‐carboxylate synthase activity is associated with a 45‐kDa polypeptide, with a pI of 5.8 ± 0.2. The enzyme is inactivated both by its substrate, S‐adenosyl‐l‐methionine (AdoMet) and by one of its products, 1‐aminocyclopropane‐1‐carboxylate.

Due to the extremely low abundance of the protein it was necessary to scale up the extraction in order to obtain reasonable amounts for sequence analysis. Therefore, 200 kg tomatoes were extracted on semi‐industrial scale and 1‐aminocyclopropane‐1‐carboxylate synthase purified. This yielded approximately 150 μg enzyme.
Glutamate:4,5‐dioxovaleric acid transaminase from Euglena gracilis
Authors: LOMBARDO María Elisa , ARAUJO Lidia Susana , JUKNAT Adela Ana , BATLLE Alcira M. del C.
Published: March 2005

The kinetic properties of the enzyme l‐glutamate:4,5‐dioxovaleric acid aminotransferase (Glu: DOVA transaminase) from Euglena gracilis have been studied. 5‐Aminolevulinic acid formation was linear with time for at least 45 min at 37°C and l‐glutamate was the most effective amino‐group donor. Lineweaver‐Burk double‐reciprocal plots suggested a ping‐pong reaction mechanism, with Km values for l‐glutamate and DOVA of 1.92 mM and 0.48 mM respectively. Competitive parabolic substrate inhibition by DOVA at concentrations greater than 3.5–4.5 mM was observed. Glyoxylate (4–10 mM) was found to be a competitive inhibitor with respect to DOVA, whereas at low concentrations (0–4 mM) noncompetitive plots were obtained.

An analysis of the possible enzyme forms involved, was carried out. In more crude preparations most of the enzyme is found to be in the form of an enzyme‐glutamate complex.
Tumor necrosis factor stimulates human neutrophils to release leukotriene B4 and platelet‐activating factor
Authors: CAMUSSI Giovanni , TETTA Ciro , BUSSOLINO Federico , BAGLIONI Corrado
Published: March 2005

Tumor necrosis factor stimulates polymorphonuclearneutrophils to synthesize leukotriene B4 and plateletactivating factor (PAF), but α1‐proteinase inhibitor and α1‐antichymotrypsin block this response. However, proteinases such as elastase and cathepsin G induce preferentially synthesis of PAF. An acetyltransferase required, together with phospholipase A2, in the remodeling pathway of PAF synthesis is activated in polymorphonuclearneutrophils stimulated by tumor necrosis factor and elastase. In contrast, 1‐oleyl‐2‐acetylglycerol, a protein kinase C activator, promotes PAF formation by the de novo biosynthetic pathway without activating the acetyltransferase. Staurosporine, an inhibitor of protein kinase C, blocks PAF production apparently by inhibiting phospholipase A2. This suggests that diacylglycerols are involved in activating both pathway of PAF synthesis.
Solubilisation of methane monooxygenase from Methylococcus capsulatus (Bath)
Authors: SMITH D. Drummond S. , DALTON Howard
Published: March 2005

The membrane‐bound (particulate) form of methane monooxygenase from Methylococcus capsulatus (Bath) has been solubilised using the non‐ionic detergent dodecyl‐β‐d‐maltoside. A wide variety of detergents were tested and found to solubilise membrane proteins but did not yield methane monooxygenase in a form that could be subsequently activated. After solubilisation with dodecyl‐β‐d‐maltoside, enzyme activity was recovered using either egg or soya‐bean lipids. Attempts to further purify the solubilized methane monooxygenaser protein into its component polypeptides were unsuccessful and resulted in complete loss of enzyme activity. The major polypeptides present in the solubilised enzyme had molecular masses of 49 kDa, 23 kDa and 22 kDa which were similar to those seen in crude extracts [Prior, S. D. & Dalton H. (1985) J. Gen. Microbiol. 131, 155–163]. Studies on substrate and inhibitor specificities indicated that the membrane‐associated and solubilised forms of methane monooxygenase were quite similar to each other but differed substantially from the well‐characterised soluble methane monooxygenase found in cells grown in a low copper regime and synthesised independently of the particulate methane monooxygenase.
A B‐type PDGF receptor lacking most of the intracellular domain escapes degradation after ligand binding
Authors: SEVERINSSON Liv , CLAESSON‐WELSH Lena , HELDIN Carl‐Henrik
Published: March 2005

The characteristics of the human B‐type platelet‐derived‐growth‐factor (PDGF) receptor expressed in Chinese hamster ovary (CHO) cells, were compared with those of a mutant receptor lacking all but 19 amino acids of the intracellular domain. The transfected wild‐type receptor was synthesized as a 160‐kDa precursor that was processed to 190 kDa. Each CHO cell expressed 30000–100000 receptors which bound PDGF‐BB with a Kd of about 0.5 nM. Analysis of PDGF‐AB binding yielded non‐linear Scatchard plots; the major part of the binding sites had a Kd of 6 nM. PDGF‐AA was not bound. The receptors expressed in CHO cells were down‐regulated after binding of PDGF‐BB, and mediated degradation of 125I‐PDGF‐BB with similar efficiency as PDGF‐B‐type receptors in human fibroblasts. The transfected receptor also transduced a mitogenic signal.

The mutant receptor was synthesized as a 90‐kDa precursor and was processed to 120 kDa with a slightly faster rate than the wild‐type receptor. Cells expressing the mutant receptor generally had around 106 ligandbinding sites/cell, with a Kd for binding of PDGF‐BB of 3 nM. The mutant receptor, which did not transduce a mitogenic response, mediated degradation of 125I‐PDGF‐BB, albeit less efficiently compared to the wild‐type receptor. In contrast to the wild‐type receptor, it was down‐regulated only to a limited extent and not degraded in response to ligand binding. These findings indicate a role for the intracellular part of the receptor, not only in mitogenic signaling, but also in receptor internalization and intracellular routing.
Down‐regulation of α3(VI) chain expression by γ‐interferon decreases synthesis and deposition of collagen type VI
Authors: HECKMANN Marc , AUMAILLEY Monique , HATAMOCHI Atsushi , CHU Mon‐LI , TIMPL Rupert , KRIEG Thomas
Published: March 2005

Treatment of cultured human skin fibroblasts with increasing doses of γ‐interferon produces a distinct reduction of steady‐state levels of the α3 chain of collagen VI mRNA by about 60% but not of the α1 and α2 chain mRNAs. A similar decrease was also observed for collagen I and III mRNA while fibronectin mRNA remained at the same level. The decrease in α3(VI) mRNA is accompanied by a reduced synthesis of collagen VI and by a reduced deposition of both collagen VI and fibronectin in urea‐insoluble form in the cell matrix. No other γ‐interferon effects were observed for fibronectin biosynthesis. Immunoprecipitation of metabolically labeled collagen VI demonstrated a strongly reduced synthesis (by 65–80%) of intracellular α3(VI) chains with no decrease found for α1(VI) and α2(VI) chains. All three chains were, however, found to be reduced in the culture medium. Pepsin treatment of immunoprecipitated collagen VI showed similar chain ratios for material in the culture medium obtained in the absence or presence of γ‐interferon. It indicates that correctly assembled heterotrimers of the composition [α1(VI) α2(VI) α3(VI)] are formed and secreted also in the absence of an equivalent α3(VI) chain synthesis but at a reduced rate. The data support previous predictions from sequence analyses [Chu et al. (1988) J. Biol. Chem. 263, 18601–18606] that collagen VI molecules composed of all three constituent chains are more stable than other assembly alternatives.
Rapid partial degradation of DDT by a cytochrome P‐450 model system
Authors: LANGEN Hanno , EPPRECHT Thomas , LINDEN Michael , HEHLGANS Thomas , GUTTE Bernd , BUSER Hans‐Rudolf
Published: March 2005

Heme compounds, in combination with a reducing agent and oxygen, can express various activities of cytochrome P‐450 enzymes. In the present study it was found that a mixture of hemin and excess cysteine was able to degrade the insecticide DDT partially. The major products were three water‐soluble, non‐toxic conjugates of DDT metabolites with cysteine which had lost two or three of the five chlorine atoms of DDT per molecule and whose structures were elucidated by gas chromatography/mass spectrometry. In 0.05 M NH4HCO3, pH 7.7/ethanol (5:6, by vol.), the degradation reaction catalyzed by the hemin‐cysteine model system was at least 8 × 104 times faster than the uncatalyzed reaction. In the presence of a designed 24‐residues polypeptide or β‐casein, two DDT‐binding proteins, an additional fourfold increase in the rate of DDT degradation was observed. Although the concentrations of DDT and cysteine occurring in an organism would be expected to be lower than those in the expriments described, the formation of water‐soluble conjugates of DDT metabolites with cysteine (and other amino acids) could also play a role in metabolism and excretion of DDT in vivo.
DNA polymerase α from normal rat liver is different than DNA polymerases α from Morris hepatoma strains
Authors: POPANDA Odilia , THIELMANN Heinz Walter
Published: March 2005

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase α complex, was partially purified from a slow‐growing (TC5123) and a fast‐growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase α complexes contained RNA primase. DNA polymerase α activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values.

The sedimentation coefficients of native DNA polymerases α from Morris hepatoma cells were found to be lower than that of polymerase α from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl‐dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino‐dATP were lower for DNA polymerase α from the fast‐growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base‐pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis‐Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single‐stranded DNA as a template was found to be lower for DNA polymerase α from the fast‐growing hepatomal MH3924 than for that from normal rat liver. Thus, the DNA‐polymerizing complex from MH3924 combines both higher binding strength to single‐stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.
Subunit association defects in Escherichia coli ribosome mutants lacking proteins S20 and L11
Authors: GÖTZ Frank , FLEISCHER Carola , PON Cynthia L. , GUALERZI Claudio O.
Published: March 2005

The subunit association capacity of 30S and 50S ribosomal subunits from Escherichia coli mutants lacking protein S20 or L11 as well as of 50S subnits depleted of L7/L12 was tested by sucrose gradient centrifugation and by a nitrocellulose filtration method based on the protection from hydrolysis with peptidyl‐tRNA hydrolase of ribosome‐bound AcPhe‐tRNA. It was found that the subunits lacking either S20 or L11 display an altered association capacity, while the 50S subunits lacking L7/L12 have normal association behavior. The association of S20‐lacking 30S subunits is quantitatively reduced, especially at low Mg2+ concentrations (5 – 12 mM), and produces loosely interacting particles which dissociate during sucrose gradient centrifugation. The association of L11‐lacking 50S subunits is quantitatively near‐normal at all Mg2+ concentrations and produces loosely associating particles only at low Mg2+ concentrations (5 – 8 mM); the mechanism of their association with 30S subunits, however, or the structure of the resulting 30S – 50S couples is altered in such a way as to cause the ejection of an AcPhe‐tRNA molecule pre‐bound to the 30S subunits in response to poly (U).
Factors relevant in the reaction of pyroloquinoline quinone with amino acids
Authors: van KLEEF Mario A. G. , JONGEJAN Jaap A. , DUINE Johannis A.
Published: March 2005

In order to reveal the stability of pyrroloquinoline quinone (PQQ) in complex samples, its reaction on incubation with amino acids was followed spectrophotometrically by monitoring oxygen consumption, and with a biological assay. For several α‐amino acids, the formation of a yellow coloured compound (λmax= 420 nm) was accompanied by oxygen uptake and disappearnace of biological activity from the reaction mixture. The yellow product appeared to be an oxazole of PQQ, the exact structure depending on the amino acid used. Oxazole formation also occurred under anaerobic conditions with concomitant formation of PQQH2, suggesting that PQQ is able to oxidize the presumed oxazoline to the oxazole. Besides the condensation reaction, there is also a catalytic cycle in which an aldimine adduct of PQQ and the amino acid is converted into the aminophenol form of the cofactor and an aldehyde resulting from oxidative decarboxylation of the amino acid. Addition of NH4+ salts, as well as that of certain divalent cations, greatly stimulated both the cyclic and the linear reaction. With basic amino acids, oxazole formation scarcely occurred. However, as oxygen consumption was observed (provided that certain divalent cations were present), conversion of these compounds took place. A reaction scheme is proposed accounting for the products formed and the effects observed. Since NH4+ ions activate several quinoproteins (PQQ‐containing enzymes) and divalent cations (Ca2+, Fe2+, and Cu2+) are additional (co)factors in certain metallo quinoproteins, the effects of metal ions observed here could be related to the mechanistic features of these enzymes. Although all oxazoles were converted to PQQ by acid hydrolysis, PQQ was not detected when hydrolysis was carried out in the presence of tryptophan, a compound which appeared to have a deleterious effect on the cofactor under this condition. The results here described explain why analysis methods for free PQQ in complex samples fail in certain cases, or are not quantitative.