bluuebunny/crossref_metadata_2025 huggingface dataset:
Reconstitution of the partially purified membrane component of the superoxide‐generating NADPH oxidase of pig neutrophils with phospholipid
Authors: NOZAKI Masahiko , TAKESHIGE Koichiro , SUMIMOTO Hideki , MINAKAMI Shigeki
Published: March 2005

The NADPH‐dependent superoxide‐generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell‐free system. The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat‐germagglutinin – agarose column chromatography. The chromatography resulted in loss of the O2−‐generating activity in the cell‐free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N‐acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre‐mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent‐free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 ± 0.53 nmol/mg protein (mean ± SD, n= 5). The potency of fraction B in the reconstitution of the O−2‐generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O−2‐generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O−2‐generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p‐chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0–7.5. The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 ± 3.5 μmol O−2· min−1· mg−1 membrane‐derived protein (mean ± SD, n= 5) which shows that the membrane component was purified about 100‐fold. These findings indicate that cytochrome b558 is probably a membrane component of the O−2‐generating NADPH oxidase and its activation in the cell‐free system requires the reconstitution with phospholipids.
Selection of the 5′‐proximal translation initiation site is influenced by mRNA and eIF‐2 concentrations
Authors: DASSO Mary C. , MILBURN Susan C. , HERSHEY John W. B. , JACKSON Richard J.
Published: March 2005

A cDNA clone of the influenza virus NS (non‐structural protein) gene in a vector carrying a bacteriophage T7 RNA polymerase promoter was manipulated so as to reiterate the initiation site to give two in‐frame AUG codons 57 nucleotide residues apart. Each initiation site was in either a preferred context (…AUAAUGG…) or a less favourable context (…UUUAUGG…) and the four possible permutations were constructed. When capped mRNA transcripts of these clones were translated in the rabbit reticulocyte lysate system, products from initiation at both AUG codons were observed. At low RNA concentrations the frequency of initiation at the 5′‐proximal AUG codon rather than the second was higher when the first AUG codon was in the preferred context, in qualitative agreement with the scanning ribosome model. However, a completely unexpected finding was that the ratio of initiation at the first AUG codon to initiation at the second decreased with increasing mRNA concentration, irrespective of the particular context involved. Several lines of evidence indicated that the increased frequency of initiation at the second AUG codon was not due solely to the lower density of ribosome loading per mRNA at high RNA concentrations, and may therefore be the result of high RNA concentrations out‐titring the capacity of endogenous reticulocyte factors responsible for preferential initiation at the 5′‐proximal AUG codon. The effect of supplementing the system with purified initiation factors was examined. Only eIF‐2 was capable of decreasing the frequency of initiation at the second AUG codon and promoting use of the first AUG at high mRNA concentrations; eIF‐3, 4A, 4B, 4C + 4D, 4F and 5 were inactive.
Metabolism of adenosine through adenosine kinase inhibits gluconeogenesis in isolated rat hepatocytes
Authors: LAVOINNE Alain , CLAEYSSENS Sophie , CHEDEVILLE Arlette
Published: March 2005

In isolated hepatocytes from fasted rats, 0.5 mM adenosine inhibited gluconeogenesis from glutamine, lactate and pyruvate. This inhibition was due to adenosine conversion through adenosine kinase. An increase in ketone‐body release was only observed in the presence of lactate or pyruvate, and the two phenomena (i.e. inhibition of gluconeogenesis and increased ketone‐body release) were linked.

With alanine, dihydroxyacetone or serine as substrates, adenosine did not change gluconeogenesis; however, its conversion through adenosine kinase also inhibited gluconeogenesis. With asparagine as substrate, 0.5 mM adenosine increased gluconeogenesis; this increase was due to adenosine conversion through adenosine deaminase. However, adenosine conversion through adenosine kinase inhibited gluconeogenesis from asparagine. Thus, whatever the substrate used, adenosine conversion through adenosine kinase inhibited gluconeogenesis.

The inhibitory effect of adenosine on gluconeogenesis cannot be related to the decrease in Pi concentration and to the increase in ATP pool.

Beside its effect on gluconeogenesis, adenosine inhibited ketogenesis measured without added substrate; adenosine conversion through adenosine kinase was also involved in the inhibition of ketogenesis.
Characterisation of a monoclonal antibody against native human type I collagen
Authors: WERKMEISTER Jerome A. , RAMSHAW John A. M. , ELLENDER Graham
Published: March 2005

A monoclonal antibody against a pepsin‐soluble mammalian type I collagen has been produced. This antibody, subclass IgG1,K, was specific for type I collagen and did not cross‐react with a range of other collagen types or connective tissue proteins. The epitope recognized by the antibody was dependent upon an intact triple‐helical structure for the collagen, and was shown by rotary shadowing and by immunoblotting of collagenase‐derived fragments to be near the C‐terminal of the pepsin‐soluble collagen. Although the antibody had a low affinity, with Kd= 4×10−7 M, it could be used for immunohistology of tissue sections and for studies of collagen produced by cells in culture. The antibody, which was raised against human collagen, also recognized type I collagens from certain other species, including calf, pig, sheep, goat and dog.
Multiple coordinate controls contribute‐to a balanced expression of ribulose‐1,5‐bisphosphate carboxylase/oxygenase subunits in rye leaves
Authors: WINTER Ute , FEIERABEND Jürgen
Published: March 2005

In the leaves of rye (Secale cereale L.), control mechanisms acting at multiple molecular levels contribute to a coordinate expression of the subunit polypeptides of ribulose‐1,5‐bisphosphate carboxylase. The relevance and hierarchy of the different control steps were evaluated by comparing the time courses of changes in levels of translatable mRNA, rates of in vivo amino acid incorporation, and the turnover of subunit polypeptides after selective interference with translation at either cytoplasmic 80S ribosomes, or at the 70S ribosomes of the chloroplast, by compartment‐specific inhibitors, or by the use of 70S‐ribosome‐deficient leaves. The latter were generated by growing the plants at a non‐permissive elevated temperature of 32°C. The rates of synthesis of the two ribulose‐1,5‐bisphosphate carboxylase subunits were most rapidly adapted to each other by translational controls. Within 0.5–2.5 h after selective inhibition of the synthesis of either subunit, that of the other subunit made in the unaffected compartment also declined by more than 90% without any marked change in its mRNA. After prolonged inhibition (24 h) of either cytoplasmic or chloroplast protein synthesis, the levels of mRNAs for both subunits were greatly diminished. In rye, the mRNA levels for both subunits changed under all experimental conditions tested in a closely parallel manner and appeared to be always maintained in a balanced, fairly constant ratio by strong coordinate controls. Even 70S‐ribosome‐deficient leaves contained mRNAs for both the small and the large subunits, although only in small amounts. The mRNAs for both subunits were also markedly further decreased in 70S‐ribosome‐deficient leaves after application of an inhibitor of cytoplasmic translation, MDMP [2‐(4‐methyl‐2,6‐dinitroanilino)‐N‐methylpropionamide], suggesting that the suppression of the large subunit mRNA in the plastids was not mediated through feedback effects of accumulating unassembled large subunits. Coordinate controls at both the mRNA and the translational level require a bidirectional exchange of regulatory signals between chloroplast and cytoplasm. However, these controls were not absolutely restrictive and allowed low rates of uncoupled synthesis of either large or small subunits. Large subunits made in the presence of MDMP were stable over 24 h. However, unassembled small subunits synthesized in 70S‐ribosome‐deficient leaves were degraded with a half‐time of 10.5 h, in contrast to their behavior after integration into the holoprotein in normal leaves, where no turnover was detected. The proteolytic removal of surplus free small subunits is regarded as a final post‐translational fine‐tuning step to establish a balanced subunit stoichiometry in leaves.
Enzyme‐enzyme interactions and control analysis
Authors: KACSER Henrik , SAURO Herbert M , ACERENZA Luis
Published: March 2005

Two usual assumptions of the treatment of metabolism are: (a) the rates of isolated enzyme reactions are additive, i.e. that rate is proportional to enzyme concentration; (b) in a system, the rates of individual enzyme reactions are not influenced by interactions with other enzymes, i.e. that they are acting independently, except by being coupled through shared metabolites. On this basis, control analysis has established theorems and experimental methods for studying the distribution of control. These assumptions are not universally true and it is shown that the theorems can be modified to take account of such deviations. This is achieved by defining additional elasticity‐coefficients, designated by the symbol π, which quantify the effects of homologous and heterologous enzyme interactions. Here we show that for the case of non‐proportionality of rate with enzyme concentration, (πii≠ 1), the summation theorems are given by

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The example of monomer‐oligomer equilibria is used to illustrate non‐additive behaviour and experimental methods for their study are suggested.
Enzyme‐enzyme interactions and control analysis
Authors: SAURO Herbert M. , KACSER Henrik
Published: March 2005

The association of different enzymes into a complex may induce changes in the kinetic parameters of its component enzymes. This implies that they cannot be treated as independent catalysts. It will affect the formulations and theorems of control analysis and necessitates the introduction of additional elasticities reflecting the effect of one enzyme on the rate of another. We show how this is achieved as an extension of the classical treatment. We present modified summation and connectivity theorems incorporating both homologous and heterologous interactions. The case of channelling of metabolites in such complexes is considered and an experimental method for its detection is suggested.
Redox‐linked conformational changes in proteins detected by a combination of infrared spectroscopy and protein electrochemistry
Authors: MOSS David , NABEDRYK Eliane , BRETON Jacques , MÄNTELE Werner
Published: March 2005

We have developed a new technique for the study of redox‐linked conformational changes in proteins, by the combination of two established techniques. Fourier‐transform infrared spectroscopy has been used together with direct electrochemistry of the protein at a modified metal electrode surface. The technique has been evaluated with cytochrome c, because of its well‐characterized electrochemistry and because the availability of X‐ray crystallographic and NMR studies of both redox states of the protein provides a reference against which our data can be compared. In electrochemical control experiments, it was confirmed that the spectroelectrochemical cell design allows fast, accurate and reproducible control of the redox poise of the protein. The resulting reducedminus‐oxidized infrared difference spectra show the changes in the frequencies and intensities of molecular vibrations which arise from the redox‐linked conformational change. In contrast to the absolute infrared spectra of proteins, such difference spectra can be sufficiently straightforward to allow interpretation at the level of individual bonds. A complete interpretation of the spectra is beyond the scope of the present paper: however, on the basis of the data presented, we are able to suggest assignments for all except one of the major bands between 1500 cm−1 and 1800 cm−1.
Purification and properties of an endo‐1,4‐xylanase excreted by a hydrolytic thermophilic anaerobe, Clostridium thermolacticum
Authors: DEBEIRE Philippe , PRIEM Bernard , STRECKER Gerard , VIGNON Michel
Published: March 2005

An extracellular xylanase from a thermophilic anaerobe, Clostridium thermolacticum, was purified 400‐fold by ion‐exchange chromatography and gel filtration. The purified enzyme had a specific activity of 31 670 nkat/mg of protein at 60°C, a molecular mass of 39 kDa and a pI of 4.9. The enzyme exhibited maximal activity at 80°C (1 h assay) and at pH 6.0–6.5. There was little loss of activity after 4 days at 60°C and the enzyme was stable in the wide pH range 3–11. Examination of the hydrolysis products of larchwood xylan indicated that it was an endoxylanase; at the early stage of the reaction, xylose (Xyl)‐containing oligosaccharides of 3–12 residues were released and after a prolonged incubation time, the neutral end‐products were Xyl2 and Xyl3. Kinetic studies of the hydrolysis of xylose‐containing oligosaccharides of 4–7 residues showed that the tetrasaccharide was hydrolysed more slowly than the pentasaccharide, while the calculated Km and V values for pentasaccharide and hexasaccharide were similar. The primary structures of the XylnGlcA produced by long‐term hydrolysis of larchwood glucuronoxylan were determined on the basis of their carbohydrate composition, by methylation analysis and by 1H‐NMR and 13C‐NMR spectroscopies. These data allowed us to propose a model for the mode of action of this endoxylanase on larchwood 4‐O‐methylglucuronoxylan.
Conformational changes of tarantula (Eurypelma californicum) haemocyanin detected with a fluorescent probe, 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole
Authors: LEIDESCHER Thomas , DECKER Heinz
Published: March 2005

Different fluorescent labels were tested in order to monitor conformational transitions of the four‐hexamer haemocyanin from the tarantula Eurypelma californicum during the oxygenation process. When the four‐hexamer was labelled with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole, the maximum wavelength λmax of the fluorescence emission spectrum was significantly shifted up to 5 nm, depending on pH and the degree of oxygenation. The values for λmax of the fully oxygenated haemocyanin were 531.5 nm (pH < 7.8) and 530.0 nm (pH > 7.8). For deoxygenated haemocyanin the values were 533.5 nm (pH < 7.2) and 535.2 nm (pH > 7.2). The occurrence of four distinct emission maxima supports the hypothesis of four conformational species for the tarantula haemocyanin, which have been predicted by the nesting model [Robert, C. H., Decker, H., Richey, B., Gill, S. J. & Wyman, J. (1987) Proc. Natl Acad. Sci. USA 84, 1891–1895].

Only four amino acids of the four‐hexamer were labelled with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole. They were identified as lysine 484 on the purified peptide Leu‐Arg‐Lys‐Phe‐His‐Arg. This amino acid is located on the surface of the four copies of subunit d.

The sharp shift of the maxima of the emission wavelengths during oxygenation indicates that the four copies of subunits d synchronously take part in the conformational switch. This points to a concerted mechanism for the conformational transitions of the tarantula haemocyanin.
NMR identification of a partial helical conformation for bombesin in solution
Authors: CARVER John A. , COLLINS J. Grant
Published: March 2005

The conformation of bombesin in trifluoroethanol/water mixtures has been studied using 1H‐NMR spectroscopy. By a combination of two‐dimensional 1H‐NMR techniques and measurement of vicinal NH‐α‐CH spin‐spin coupling constants, the secondary structure of the molecule has been determined. Bombesin adopts a helical structure in the region from Asn‐6 to Met‐14 with the remaining N‐terminal portion existing as a more extended structure. The structure is very similar to that proposed from Fourier‐transform infrared spectroscopic measurements for bombesin inserted into lipid bilayers [D. Erne & R. Schwyzer (1987) Biochemistry 26, 6316–6319]. The absence of a hydrogen bond between the sidechains of Trp‐8 and His‐12 is discussed in terms of the ionization state of His‐12. Stabilisation of the helix results when His‐12 is in the ionized state.
Epidermal growth factor and cyclic AMP stimulate Na+/H+ exchange in isolated rat hepatocytes
Authors: MOULE S. Kelly , McGIVAN John D.
Published: March 2005

Na+/H+ exchange in acid‐loaded isolated hepatocytes was measured using the intracellular pH indicator biscarboxyethyl‐carboxyfluorescein (BCECF) to follow intracellular pH (pHi). The rate of amiloride‐sensitive Na+‐dependent recovery from cytoplasmic‐acid‐loading was found to be increased in cells treated with epidermal growth factor (EGF), 8‐(4‐chlorophenylthio)adenosine 3′,5′‐monophosphate (ClPhScAMP) or phorbol 12‐myristate 13‐acetate (PMA). These three agents increased the rate of Na+/H+ exchange to similar extents and their effects were not additive. The stimulation was shown in all three cases to be due an alkaline shift of 0.1 in the set point pH of the Na+/H+ exchanger. Experiments measuring the uptake of 22Na+ into acid‐loaded primary hepatocyte monolayer cultures confirmed these results. EGF, ClPhScAMP and PMA significantly increased the amiloride‐inhibitable accumulation of 22Na+, thus providing further evidence that Na+/H+ exchange is stimulated by these effectors.
Regulation by butyrate of the cAMP response to cholera toxin and forskolin in pituitary GH1 cells
Authors: ARANDA Ana , TORO Maria Jose , USERA Fernando , PASCUAL Angel
Published: March 2005

In pituitary GH1 cells, a rat growth hormone‐producing cell line, butyrate elicited a dose‐dependent increase in cholera toxin receptors as measured by an increased binding of 125I‐labeled cholera toxin to the intact cells. Butyrate did not alter the affinity of cholera toxin binding, the dissociation constant being 0.4 nM for both control and butyrate‐treated cells. Despite the increased binding, the cAMP response to cholera toxin was strongly reduced after exposure to butyrate. This reduction was dose‐dependent and with butyrate 1–5 mM, intracellular and extracellular (medium) cAMP levels were decreased by more than 70% in cells incubated for 24 h with 1 nM cholera toxin. Forskolin (30 μM) elicited a cAMP response similar to that found with the toxin, and a similar inhibition of cAMP was also found after incubation of GH1 cells with butyrate. Butyrate also affected basal cAMP levels which were reduced by 40–60% in cells cultured for 24–48 h with the fatty acid. In order to study whether butyrate influenced cAMP synthesis and/or cAMP degradation, adenylyl cyclase and phosphodiesterase activities were determined in control cells and in cells incubated for 24 h with cholera toxin or forskolin. Butyrate had a dual effect since, besides activating phosphodiesterase by more than twofold, it also inhibited the cyclase by 40–50% in all groups. The in vitro response of adenylyl cyclase to stimulatory (NaF) and inhibitory (carbachol and adenosine) effectors was also examined. The absolute activity of the cyclase was always 40–50% lower in the cells incubated with butyrate, but the percentage change of activity obtained in butyrate‐treated and untreated cells was unaltered. In addition, ADP‐ribosylation of the guanine nucleotide stimulatory component of the cyclase (Gs) was not affected in the cells incubated with butyrate. These results suggest that the catalytic (C) subunit of adenylyl cyclase and/or its interaction with the regulatory components might be altered in butyrate‐treated GH1 cells.

The inhibition of the cAMP response in GH1 cells was accompanied by an inhibition of a biological action of the nucleotide, namely growth hormone (somatotropin) production which is primarily controlled by thyroid hormones in these cells. Forskolin alone did not affect the somatotropin levels but potentiated the growth hormone response to triiodothyronine. Butyrate produced a dose‐dependent inhibition of this response, which was totally abolished at concentrations of butyrate higher than 1 mM.
Quantitative estimation of minor mRNAs by cDNA‐polymerase chain reaction
Authors: CHELLY Jamel , MONTARRAS Didier , PINSET Christian , BERWALD‐NETTER Yoheved , KAPLAN Jean‐Claude , KAHN Axel
Published: March 2005

Amplification of the mRNA polymerase chain reaction is a very sensitive technique to detect low‐abundance transcripts. We describe in this paper conditions necessary to make this technique quantitative. Quantification is performed in the exponential phase of the amplification process and the results are standardized with respect to those obtained with an exogenous mRNA which is co‐reverse‐transcribed and co‐amplified in the same reaction as the analyzed transcripts. The primers are chosen in different exons to distinguish the amplification of mRNA fragments from the amplification of contaminating DNA. Analysis of the kinetics of amplification and parameters influencing this kinetics shows that: (a) in the exponential phase of amplification, the amount of amplified fragments is proportional to the initial amount of transcripts; (b) in a certain range of length fragment, the yield of amplification is inversely proportional to the length of the amplified fragments.

Using this method we have demonstrated that the dystrophin gene is already activated at the myoblastic stage. A quantitative estimation of the transcript showed that the expression of this gene increases strongly in the course of in vitro myogenesis. In primary culture of mouse brain cells, the dystrophin gene was found to be more expressed in neuronal than in glial cells.
Glutathione‐protein mixed disulfide decreases the affinity of rat liver fatty acid‐binding protein for unsaturated fatty acid
Authors: HITOMI Masahiro , ODANI Shoji , ONO Teruo
Published: March 2005

0.16 ± 0.062% of the fatty acid‐binding protein purified from 50 mM N‐ethylmaleimide‐treated rat liver (L‐FABP) was determined as a form S‐thiolated by glutathione (L‐FABP‐SSG). L‐FABP‐SSG, which was prepared in vitro through thiol – disulfide exchange reaction, showed more acidic pI (∼ 5.0) than the pI (∼ 7.0) of reduced L‐FABP. S‐thiolation of L‐FABP by glutathione decreased the affinity of the protein for unsaturated fatty acids without changing the equimolar maximum binding. The changes in Kd were from 0.63 ± 0.054 μM to 1.03 ± 0.14 μM for oleic acid, from 0.63 ± 0.028 μM to 0.97 ± 0.12 μM for linoleic acid and from 0.85 ± 0.050 μM to 1.45 ± 0.024 μM for arachidonic acid. This modification did not alter the affinity nor the maximum binding for saturated fatty acids, which were determined to be Kd of ∼ 1.0 μM for palmitic acid and ∼ 0.9 μM for stearic acids, and equimolar maximum binding for both fatty acids. The binding affinity of L‐FABP for unsaturated fatty acid may be regulated by redox state of the liver.
Acute stimulation by lutropin of mitochondrial protein synthesis in small luteal cells
Authors: MITTRE Hervé , AUNAI Pascal , BENHAIM Annie , LEYMARIE Pierre
Published: March 2005

A two‐dimensional electrophoretic technique was used to study the effect of acute stimulation of bovine luteal cells with lutropin on protein synthesis. Cells were incubated for 30 min with [35S]methionine in the presence of stimulating levels of luteinizing hormone (lutropin), after which the proteins were analyzed by autoradiography. Lutropin or N6,2′‐O‐dibutyryl‐adenosine 3′,5′‐phosphate (Bt2cAMP) induced the labelling of three proteins, referred to as proteins A, B and C. Protein A, had a molecular mass of 28 kDa and an isoelectric point (pI) of 6.7. Proteins B and C had a molecular mass of 27 kDa and pI of 6.2 and 6.4 respectively. After subcellular fractionation, the three proteins were found to be markedly concentrated in the only fraction enriched in an established mitochondrial marker. Moreover, protein A was one of the major mitochondrial newly synthesized proteins. Its appearance was observed after a 5‐min incubation and was prevented by 100 μM cycloheximide. The acute accumulation of proteins A, B and C in mitochondria, the site of the rate‐limiting step of steroidogenesis, suggest that they could be involved in the mechanism of stimulation by lutropin of progesterone synthesis.
Chemiosmotic energy conversion and the membrane ATPase of Methanolobus tindarius
Authors: SCHEEL Edgar , SCHÄFER Günter
Published: March 2005

Electron transport phosphorylation has been demonstrated to drive ATP synthesis for the methanogenic archaebacterium Methanolobus tindarius: Protonophores evoked uncoupler effects and lowered the membrane potential ΔΨ. Under the influence of N,N′‐dicyclohexylcarbodiimide [(cHxN)2C] the membrane potential increased while methanol turnover was inhibited. 2‐Bromoethanesulfonate, an inhibitor of methanogenesis, had no effect on the membrane potential but, like (cHxN)2C and protonophores, decreased the intracellular ATP concentration.

Labeling experiments with (cHxN)214C showed membranes to contain a proteolipid, with a molecular mass of 5.5 kDa, that resembles known (cHxN)2C‐binding proteins of F0‐F1 ATPases. The (cHxN)2‐sensitive membrane ATPase hydrolysed Mg · ATP at a pH optimum of 5.0 with a Km (ATP) of 2.5 mM (V= 77 mU/mg). It was inhibited competitively by ADP; Ki (ADP) = 0.65 mM. Azide or vanadate caused no significant loss in ATPase activity, but millimolar concentrations of nitrate showed an inhibitory effect, suggesting a relationship to ATPases from vacuolar membranes. In contrast, no inhibition occurred in the presence of bafilomycin A1.

The ATPase was extractable with EDTA at low salt concentrations. The purified enzyme consists of four different subunits, α (67 kDa), β (52 kDa), γ (20 kDa) and δ (< 10 kDa), as determined from SDS gel electrophoresis.
Subunit composition of vacuolar membrane H+‐ATPase from mung bean
Authors: MATSUURA‐ENDO Chie , MAESHIMA Masayoshi , YOSHIDA Shizuo
Published: March 2005

The vacuolar H+‐ATPase from mung bean hypocotyls was solubilized from the membrane with lysophosphatidylcholine and purified by QAE‐Toyopearl column chromatography. The purified ATPase was active only in the presence of exogenous phospholipid and was inhibited by nitrate, dicyclohexyl carbodiimide and Triton X‐100, but not by vanadate or azide. Dodecyl sulfate/polyacrylamide gel electrophoresis of the purifed ATPase yielded ten polypeptides of molecular masses of 68 kDa, 57 kDa, 44 kDa, 43 kDa, 38 kDa, 37 kDa, 32 kDa, 16 kDa, 13 kDa and 12 kDa. All polypeptides remained in the peak activity fraction after glycerol density gradient centrifugation. Nine of them, excluding the 43‐kDa polypeptide, comigrated in a polyacrylamide gradient gel in the presence of 0.1% Triton X‐100. The 16‐kDa polypeptide could be labeled with [14C]dicyclohexylcarbodiimide. The amino‐terminal amino acid sequence of the isolated 68‐kDa polypepide generally agreed with that deduced from the cDNA for the carrot 69‐kDa subunit [Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H. & Taiz, L. (1988) J. Biol. Chem. 263, 9102–9112]. Thus, mung bean vacuolar H+‐ATPase seems to consist of nine distinct subunits.
A structure‐reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I
Authors: BRITTON Hubert G. , GARCÍA‐ESPANÑA Antonio , GOYA Pilar , ROZAS Isabel , RUBIO Vicente
Published: March 2005

The requirements for binding at the N‐acetyl‐L‐glutamate binding site of carbamoyl phosphate synthetase I were studied by the displacement of the activator from the central enzyme complex by analogs. Two carboxyls are essential and the acetamido group, if linked to the α‐carbon, enhances binding 5000‐fold. the subsite for the δ‐carboxyl is mobile with respect to that for the α‐carboxyl. Mixtures of complementary fragment of acetylglutamate do not bind, inidicating a strong ‘chelate’ effect. Substituents revealed the existence of steric constraints around the δ‐carboxyl, the α and γ‐carbons, and the whole of the acetamido group. However, phenyl substituents at the β‐carbon did not hamper binding, indicating that substituents at the β‐carbon face the solution. This is consistent with binding of acetylglutamate as the minimum‐energy conformer. All analogs binding with high affinity are activators. Some analogs that bind poorly are competitive inhibitors. They appear to bind preferentially to a low‐affinity conformation adopted by the site when the products dissociate and the substrates bind. The acetamido group plays no role in the binding of the binding of the inhibitors but it is crucial for the binding of the activators, and the high‐and low‐affinity conformations of the site differ markedly in structural selectivity.
The effect of substituting phosphotyrosine for sulphotyrosine on the activity of hirudin
Authors: HOFSTEENGE Jan , STONE Stuart R. , DONELLA‐DEANA Arianna , PINNA Lorenzo A.
Published: March 2005

Recombinant hirudin (hirudin), which lacks the sulphate group of Typ‐63, has a tenfold‐reduced affinity for α‐thrombin. Incubation of recombinant hirudin with [γ‐32P]ATP and protein tyrosine kinase III from spleen resulted in incorporation of radioactivity into the protein. Phosphatohirudin was purified to homogeneity (overall yield 5%) and shown to contain 1 mol phosphate/mol protein, as a phosphotyrosyl residue at position 63.

The kinetics of the inhibition of human α‐thrombin by phosphatohirudin were determined. It was found that the introduction of the negatively charged phosphate had fully restored the affinity of recombinant hirudin for α‐thrombin to the level of the wild‐type sulphatohirudin. The inhibition constant of phosphatohirudin was 18 fM compared with 20 fM for that of sulphatohirudin. Moreover, the values for the on‐and off‐rate constants of the both forms of hirudin were indistinguishable.
Endocytosis of liposomes containing lysosomal proteins increases intracellular protein degradation in growing L‐132 cells
Authors: VARGAS José Luis , KNECHT Erwin , GRISOLÍA Santiago
Published: March 2005

We have used a new approach to test the possible participation of lysosomes in the degradation of long‐lived proteins. Rat liver lysosomal proteins were introduced, via multilamellar liposomes, into L‐132 cells. Viability and protein synthesis were not impaired by this treatment. The liposomal content was released into the lysosomes of the cultured cells, as revealed b ferritin uptake and electron microscopy. Degradation rated of long‐lived proteins increased with the uptake of lysosomal proteases. However, the increased rates were not affected by chloroquine and leupeptin, in contrast to the inhibition by these reagents of the increased protein degradation of cells ‘starved’ of serum (step‐down conditions.) This approach opens a new way of investigating the degradation on intracellular proteins in cultured cells.
Mechanism of glucocorticoid regulation of alkaline phosphatase gene expression in osteoblast‐like cells
Authors: GREEN Elaine , TODD Bindu , HEALTH David
Published: March 2005

In the rat osteosarcoma cell line ROS 17/2.8, glucocorticoids increase the activity of the plasma membrane enzyme, alkaline phosphatase. To determine the mechanisms responsible for this effect, we have studied the actions of dexamethasone on alkaline phosphatase activity, immunoreactive protein, and steady‐state mRNA levels. Dexamethasone treatment increased both specific activity of alkaline phosphatase and the cell surface expression of immunoreactive protein in a dose‐dependent manner, with a half‐maximal increase at 2 nM. Steady‐state alkaline phosphatase mRNA‐levels were also increased in a dose‐dependent manner. The time course of dexamethasone induction occurred relatively slowly, with a lag period of 12 h before any discernable effect on alkaline phosphatase mRNA levels. The rise in alkaline phosphatase mRNA levels was attributable entirely to changes in gene transcription, with no effect on message stability. Treatment of ROS 17/2.8 cells with actinomycin D completely abolished the dexamethasone‐induced rise in alkaline phosphatase mRNA levels. Measurement of alkaline phosphatase mRNA degradation, by incubation of cells with the transcriptional inhibitor 5,6‐dichlororibofuranosylbenzimidazole, indicated an apparent half‐life of 24 h in both untreated and dexamethasonestimulated cells. The protein synthesis inhibitors cycloheximide and puromycin blocked the dexamethasone induction of alkaline phosphatase mRNA. These data suggest that the dexamethasone‐induced rise in alkaline phosphatase gene transcription requires the synthesis of an unknown mediator protein.
Evidence for a specific mechanism of laminin assembly
Authors: HUNTER Irene , SCHULTHESS Therese , BRUCH Marcel , BECK Konrad , ENGEL Jürgen
Published: March 2005

The specificity of laminin chain assembly was investigated using fragments E8 and C8–9, derived from the long arm of the molecule, whose rod‐like domain consists of the α‐helical regions of the A, B1 and B2 chains. Urea‐induced chain separation and unfolding were monitored by transverse urea/polyacrylamide gel electrophoresis (PAGE) and circular dichroism. Separation of the A and disulphide‐linked B1‐B2 chains occurred at 3.5–4.0 M urea and by 7.0 M urea all residual α‐helicity was lost. Removal of urea by dialysis resulted in high recoveries (87–100%) of renatured protein which in its apparent molecular mass, α‐helix content, chain composition, degree of association and ultrastructural appearance was indistinguishable from native E8. Reduction or reduction and alkylation of the chains did not lead to a decrease in their ability to reassemble specifically. Reformation of the single interchain disulphide, linking the B1 and B2 chains, clearly demonstrates that these chains are correctly aligned in parallel and in register in E8 renatured from its reduced chains. Renaturation of E8 from its reduced and alkylated chains precludes a role for disulphide formation in determining chain alignment but suggests rather that it is involved in the stabilisation of the correctly assembled molecule. These results, together with recent sequence data, provide evidence for the interaction of the α‐helical regions of the A, B1 and B2 chains in the formation of a triple coiled‐coil within the long arm of the molecule. The highly specific nature of this interaction suggests that it is the mechanism by which laminin is assembled in vivo.
Evolution of large‐subunit rRNA structure
Authors: MICHOT Bernard , QU Liang‐Hu , BACHELLERIE Jean‐Pierre
Published: March 2005

During evolution, the potential for sequence (and length) variation of large‐subunit rRNA has been mostly restricted over 12 divergent domains (termed D1–D12) interspersed along the molecule. Here, we have focused our attention onto the D3 divergent domain, through a detailed analysis of its pattern of variation in the phylogeny, both in terms of primary and secondary structures. We have systematically compared all the procaryotic and eucaryotic sequences published so far (i.e. 36 species), together with a series of 10 additional eucaryotic specimens, which were determined by direct RNA sequencing. Secondary structures supported by comparative evidence have been derived for archaebacteria, eubacteria and eucaryotes respectively, which shows that the D3 domain contains a subset of universally conserved structural features interspersed with four variable subdomains. Within the four portions where a structural diversification has taken place, elementary structures specific of large phylogenetic groups can be identified. Remarkably such diversified structures appear to be preserved despite sequence divergence, suggesting they correspond to functionally important structures. Accordingly, the mode of sequence variation of the D3 domain suggests this region of the molecule may encode elementary functions of rRNA which could have significantly diversified during the evolution of the major groups of organisms.
Interaction of upstream stimulatory factor with the human heme oxygenase gene promoter
Authors: SATO Michihiko , ISHIZAWA Shinobu , YOSHIDA Tadashi , SHIBAHARA Shigeki
Published: March 2005

Upstream stimulatory factor (USF), originally identified in HeLa cells, interacts with the upstream promoter sequence of adenovirus 2 major late promoter (Ad2MLP) and activates its transcription. USF is present in uninfected HeLa cells and appears to be involved in the transcription of cellular genes related to stress. Recently, we have proposed that the rat heme oxygenase gene, newly identified heat‐shock protein gene, is regulated at least in partly by a rat homolog of USF [Sato, M., Fukushi, Y., Ishizawa, S., Okinaga, S., Muller, R. M. & Shibahara, S. (1989) J. Biol. Chem. 264, 10251–10260]. We therefore confirm that the heme oxygenase gene is expressed in HeLa cells and its expression is increased by cadmium, suggesting that human heme oxygenase is a stress protein similar to the metallothioneins. Using partially purified USF from HeLa cells, we show that USF binds to the human heme oxygenase gene promoter and stimulates its cell‐free transcription. The cis‐acting element, identified as CACGTGACCCG, is located 34 bp upstream from the transcription initiation site, and contains the core sequence of the upstream promoter sequence of Ad2MLP. We propose that USF contributes to the transcription of the human heme oxygenase gene.
Evidence for an endogenous ATPase inhibitor protein in plant mitochondria
Authors: NORLING Birgitta , TOURIKAS Christos , HAMASUR Beston , GLASER Elzbieta
Published: March 2005

An endogenous ATPase inhibitor protein has been identified and isolated for the first time from plant mitochondria. The inhibitor protein was isolated from potato (Solanum tuberosum) tuber mitochondria and purified to homogeneity. The isolated inhibitor is a heat‐stable, trypsin‐sensitive, basic protein, with a molecular mass ∼ 8.3 kDa. Amino acid analysis reveals a high content of glutamic acid, lysine and arginine and the absence of proline, threonine and leucine. The interaction of the inhibitor with F1‐ATPase requires the presence of Mg2+‐ATP in the incubation medium. The ATPase activity of isolated F1 is inhibited to 50% in the presence of 14 μg inhibitor/mg F1. A stoichiometry of 1.3 mol inhibitor/mol F1 for complete inhibition can be calculated from this value. The potato ATPase inhibitor is also a potent inhibitor of the ATPase activity of the isolated yeast F1. The inhibitor resembles the ATPase inhibitors of yeast and mammalian mitochondria, and does not seem to be related to the inhibitory peptide, ɛ subunit, of chloroplast ATPase.
Regulation of gene expression by transfected subunits of cAMP‐dependent protein kinase
Authors: BÜCHLER Wolfgang , MEINECKE Matthias , CHAKRABORTY Trinad , JAHNSEN Tore , WALTER Ulrich , LOHMANN Suzanne M.
Published: March 2005

cAMP regulates the expression of several genes by activation of a promoter consensus sequence which functions as a cAMP‐response element. Evidence indicated that this is accomplished via cAMP dissociation of cAMP‐dependent protein kinase into its regulatory (R) and catalytic (C) subunits. Our investigations of the role of these two subunits in gene expression provide direct and quantitative evidence that the C subunit is required for cAMP stimulation of the cAMP‐response element in the vasoactive‐intestinal‐peptide gene in rat pheochromocytoma cells. After cotransfection of a metallothionein‐regulated C‐subunit expression vector (pCEV) and a vasoactive‐intestinal‐peptide–chloramphenicol acetyltransferase construct containing a cAMP‐response element, we could demonstrate expression of transfected C‐α‐subunit mRNA (truncated size 1.7 kb) by Northern blot and a concentration‐dependent C subunit stimulation of chloramphenicol acetyltransferase activity. Basal activity was stimulated 12‐ and 50‐fold by pCEV (30 μg), in the absence and presence, respectively, of Zn2+ Metallothionein‐regulated expression of C was demonstrated by results that showed a 2–4‐fold increase in chloramphenicol acetyltransferase activity in the presence versus the absence of 90 μM Zn2+. In contrast, overexpression of the R‐IIβ regulatory subunit did not stimulate chloramphenicol acetyltransferase activity, and R‐IIβ transfected together with C (ratio 2:1 and 4:1) inhibited the stimulation by the C subunit 70% and 90% respectively. Our results indicate that transfection of cAMP‐dependent protein kinase subunits results in functional expression of both C‐α and R‐IIβ subunits. Expression of the C subunit mediated cAMP‐regulated gene expression but this expression could be inhibited by cotransfected R‐IIβ subunit, indicating intracellular reconstitution of the inactive holoenzyme of cAMP‐dependent protein kinase.
Conformational and thermodynamic effects of naturally occurring base methylations in a ribosomal RNA hairpin of Bacillus stearothermophilus
Authors: HEUS Hans A. , FORMENOY Leo J. , VAN KNIPPENBERG Peter H.
Published: March 2005

The 3′‐terminal colicin fragments of 16S ribosomal RNA were isolated from Bacillus stearothermophilus and from its kasugamycin‐resistant (ksgA) derivative lacking N6‐dimethylation of the two adjacent adenosines in a hairpin loop. The fragment from the ksgA strain still contains a naturally occurring N2‐methylguanosine in the loop. An RNA molecule resembling the B. stearothermophilus colicin fragment but without modified nucleosides was synthesized in vitro using a DNA template and bacteriophage T7 RNA polymerase.

Proton‐NMR spectra of the RNAs were recorded at 500 MHz. The imino‐proton resonances of base‐paired G and U residues could be assigned on the basis of previous NMR studies of the colicin fragment of Escherichia coli and by a combination of methylation‐induced shifts and thermal melting of base pairs. The assignments were partly confirmed by NOE measurements. Adenosine dimethylation in the loop has a distinct conformational effect on the base pairs adjoining the loop.

The thermal denaturation melting curve of the enzymatically synthesized RNA fragment was also determined and the transition midpoint (tm) was found to be 73°C at 15 mM Na+. A comparison with previously determined thermodynamic parameters for various colicin fragments demonstrates that base methylations in the loop lead to a relatively strong destabilization of the hairpin helix. In terms of free energy the positive contribution of the methylations are in the order of the deletion of one base pair from the stem. Other data show that recently published free‐energy parameters do not apply for certain RNA hairpins.
Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the ‘top‐down’ approach of metabolic control theory
Authors: HAFNER Roderick P. , BROWN Guy C. , BRAND Martin D.
Published: March 2005

The rate of respiration of isolated mitochondria was set at different values by addition of either oligomycin or an ADP‐regenerating system (glucose and different amounts of hexokinase). We measured the relationship between respiration rate and membrane potential as respiration was titrated by the addition of malonate under each condition. We used the flux control summation and connectivity theorems and the branching theorem of metabolic control theory to calculate the control over respiration rate exerted by the respiratory chain (and associated reactions), phosphorylating system (and associated reactions) and proton leak at each respiration rate. The analysis also yielded the flux control coefficients of these three reactions over phosphorylation rate and proton leak rate and their concentration control coefficients over protonmotive force. We found that respiration rate was controlled largely by the proton leak under non‐phosphorylating conditions, by the phosphorylating system at intermediate rates and by both the phosphorylating system and the respiratory chain in state 3. The rate of phosphorylation was controlled largely by the phosphorylating system itself in state 4 and at intermediate rates, while state 3 control was shared between the phosphorylating system and the respiratory chain; the proton leak had insignificant control. In all states the phosphorylating system had large negative control over the proton leak; the chain and the proton leak both had large positive control coefficients. The protonmotive force was controlled by the chain and by the phosphorylating system; the proton leak had little control.
Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer
Authors: LARK Richard H. , COLMAN Roberta F.
Published: March 2005

Bovine liver glutamate dehydrogenase is known to bind reduced coenzyme at two sites/subunit, one catalytic and one regulatory; ADP competes for the latter site. The enzyme is here shown to be catalytically active with the thionicotinamide analogue of NADPH ([S]NADPH). For native enzyme, ultrafiltration studies revealed that [S]NADPH reversibly occupies about two sites/enzyme subunit in the absence of other ligands; by the addition of ADP, [S]NADPH binding can be limited to one molecule/subunit. The enzyme is irreversibly inactivated by reaction with 4‐(iodoacetamido)salicylic acid (ISA) at lysine126 within the 2‐oxoglutarate binding site [Holbrook, J. J., Roberts, P. A. & Wallis, R. B. (1973) Biochem. J. 133, 165–171]. ISA‐modified enzyme binds 1 molecule [S]NADPH/subunit in the absence of ADP, suggesting that reaction at the substrate site blocks binding at the catalytic, but not at the regulatory site. The fluorescence spectrum of ISA‐modified enzyme overlaps the absorption spectrum of [S]NADPH allowing a distance measurement between these sites by resonance energy transfer. [S]NADPH quenches the emission of ISA‐modified enzyme, yielding 3.2 nm as the average distance between sites. ADP competes for the [S]NADPH site but does not affect the fluorescence of ISA‐modified enzyme, indicating that [S]NADPH quenching is attributable to energy transfer rather than to a conformational change. The 3.2 nm thus represents the distance between the 2‐oxoglutarate and reduced coenzyme regulatory sites of glutamate dehydrogenase.