bluuebunny/crossref_metadata_2025 huggingface dataset:
Dephosphorylation and activation of chicken liver acetyl‐coenzyme‐A carboxylase
Authors: WADA Kenji , TANABE Tadashi
Published: March 2005

Studies were made on the dephosphorylation and activation of chicken liver acetyl‐coenzyme‐A carboxylase. The enzyme isolated by avidin‐Sepharose affinity chromatography in the presence of protein phosphatase inhibitors contained 4.9 ± 0.2 mol of alkali‐labile phosphate/mol subunit and had a specific activity of 3.5 ± 0.4 units/mg protein. The purified enzyme was dephosphorylated and activated concomitantly when incubated in the presence of protein phosphatase with a release of approximately 2 mol of phosphate/mol subunit. Limited tryptic digestion of the native and dephosphorylated forms of the enzyme (Mr 220000) containing 4.9 and 2.9 mol of phosphate/mol of subunit, respectively, gave almost quantitatively similar polypeptides of Mr 215000 containing 4.0 mol and 3.0 mol of phosphate per mol, respectively, which were indistinguishable by dodecylsulfate‐polyacrylamide gel electrophoresis. A peptide of Mr approximately 5000 was lost from both enzymes. This result suggests that at least one of the two protein‐phosphatase‐labile phosphorylation sites is sensitive to trypsin. The native enzyme and those modified by protein phosphatase or by limited tryptic digestion exhibited a bi‐phasic dependence on citrate. Activation of the native enzyme by protein phosphatase occurred at all the concentrations of citrate used. However, activation of the enzyme by limited tryptic digestion was found at concentrations greater than 5 mM citrate. The dephosphorylation by protein phosphatase caused an approximately fivefold activation in the enzyme activity when assayed at physiological concentrations of citrate (0.5–2.0 mM).
Increased methyl esterification of membrane proteins in aged red‐blood cells
Authors: GALLETTI Patrizia , INGROSSO Diego , NAPPI Ausilia , GRAGNANIELLO Vincenzo , IOLASCON Achille , PINTO Luciano
Published: March 2005

The enzymatic carboxyl methyl esterification of erythrocyte membrane proteins has been investigated in three different age‐related fractions of human erythrocytes. When erythrocytes of different mean age, separated by density gradient centrifugation, were incubated under physiological conditions (pH 7.4, 37°C) in the presence of l‐[methyl‐3H]methionine, the precursor in vivo of the methyl donor S‐adenosylmethionine, a fourfold increase in membrane‐protein carboxyl methylation was observed in the oldest cells compared with the youngest ones.

The identification of methylated species, based on comigration of radioactivity with proteins stained with Coomassie blue, analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, shows, in all cell fractions, a pattern similar to that reported for unfractionated erythrocytes. However in the membrane of the oldest erythrocytes the increase in methylation of the cytoskeletal proteins, bands 2.1 and 4.1, appears to be significantly more marked compared with that observed in the other methylated polypeptides. Furthermore the turnover rate of incorporated [3H]methyl groups in the membrane proteins of the oldest cells markedly increases during cell ageing. Particularly in band 4.1 the age‐related increase in methyl esterification is accompanied by a significant reduction of the half‐life of methyl esters.

The activity of cytoplasmic protein methylase II does not change during cell ageing, while the isolated ghosts from erythrocytes of different age show an age‐related increased ability to act as methyl‐accepting substrates, when incubated in presence of purified protein methylase II and methyl‐labelled S‐adenosylmethionine, therefore the relevance of membrane structure in determining membrane protein methylation levels can be postulated.

Finally the possible correlation of this posttranslational protein modification with erythrocyte ageing is discussed.
Developmental changes in poly(A) polymerase activity in Artemia
Authors: SASTRE Leandro , SEBASTIÀN Jesús
Published: March 2005

The levels of poly(A) polymerase activity have been determined during Artemia early development. Poly(A) polymerase activity increases steadily during postgastrular embryonic development reaching a maximum shortly after hatching. The rise of poly(A) polymerase is concomitant with an increase in poly(A) content and with a change in the subcellular distribution of the enzyme activity, the major increase corresponding to the nuclear fraction. Only one isoenzyme of poly(A) polymerase has been identified in Artemia embryos and nauplii despite changes in enzyme levels and subcellular changes during early development. Poly(A) polymerase is not associated with the cytoplasmic poly(A)‐containing ribonucleoprotein particles stored in Artemia dormant embryos.
Short‐term regulation of glycolysis by insulin and dexamethasone in cultured rat hepatocytes
Authors: PROBST Irmelin , JUNGERMANN Kurt , Unthan Kirsten
Published: March 2005

Evidence for a direct metabolic effect of insulin in isolated liver preparations is scarce. The stimulation of glycolysis by insulin previously demonstrated in monolayer cultures of adult rat hepatocytes [(1982) Eur. J. Biochem. 126, 271–278] was further investigated.

The degree of stimulation varied with the age of the culture and amounted to 250%, 200%, 500% and 200% of the control value using cells at the culture age of 2 h, 24 h, 48 h, and 72 h, respectively.

Half‐maximal dose of insulin was 0.1 nM. Maximal stimulation was reached within 5 min and lasted for at least 4 h.

Dexamethasone acted both as a long‐term and short‐term modulator. Long‐term pretreatment of the cells with dexamethasone proved necessary to permit insulin action. In addition to this permissive action, pretreatment with dexamethasone reduced the insulin‐independent basal glycolytic rate.

In short‐term experiments dexamethasone decreased the basal glycolytic flux, however, it did not affect the absolute increase in glycolysis brought about by insulin. The half‐maximal dose of dexamethasone was 10 nM.

The stimulatory effects of insulin may in part be attributed to the activation of pyruvate kinase. Insulin produced a left‐shift of the substrate saturation curve, decreasing the K0.5 value for phosphoenolpyruvate.
The initation of glycogen biosynthesis in rat heart
Authors: BLUMENFELD Marta L. , WHELAN William J. , KRISMAN Clara R.
Published: March 2005

A rat heart extract synthesizes glycogen‐like material from UDP‐glucose in the absence of added primer. Most of this material is precipitable by dilute trichloroacetic acid, in contrast to glycogen synthesized in presence of added glycogen primer. It is postulated that the endogenous initiation of glycogen synthesis occurs by apposition of glucose residues to a protein and that the material so synthesized in vitro corresponds to the protein‐bound glycogen reported to exist in rat heart. The ability to label the putative protein primer in vitro with [14C]glucose should assist the isolation of the primer and the elucidation of the nature of the association between the carbohydrate and protein moeities.
Peroxidase catalysed aerobic degradation of 5,6,7,8‐tetrahydrobiopterin at physiological pH
Authors: ARMAREGO Wilfred L. F. , RANDLES David , TAGUCHI Hiroyasu
Published: March 2005

The aerobic degradation of 5,6,7,8‐tetrahydrobiopterin at neutral pH is catalysed by peroxidase (EC 1.11.1.7) and provides quinonoid 7,8‐dihydro(6H)biopterin which readily loses the side chain to yield 7,8‐dihydro(3H)pterin. The latter is in equilibrium with trace amounts of 6‐hydroxy‐5,6,7,8‐tetrahydropterin (covalent hydrate) Which is irreversibly oxidised to quinonoid 6‐hydroxy‐7, 8‐dihydro(6H)pterin, and this finally rearranges to 7,8‐dihydroxanthopterin.

Spectroscopic evidence (ultraviolet, 1H NMR and 13C NMR) is presented for the reversible addition of water across the 5,6‐double bond of 7,8‐dihydro(3H)pterin.

The intermediate quinonoid 6‐hydroxy‐7,8‐dihydro(6H)pterin is a good substrate for dihydropteridine reductase (EC 1.6.99.7) with a Km of 16.3 μM and kcat of 22.5s−1.

The rate of aerobic degradation (oxidation and loss of the side chain) of natural (6R)‐5,6,7,8‐tetrahydrobiopterin is several times slower than the rate for the unnatural (6S) isomer.

By using a modified assay procedure the kinetic parameters for dihydropteridine reductase are as follows: with (6R)‐7,8‐dihydro(6H)biopterin Km= 1.3μM and kcat= 22.8s−1; with (6S)‐7,8‐dihydro(6H)biopterin Km= 13.5μM and kcat= 51.6s−1; and with (6RS)‐7,8‐dihydro(6H)neopterin km= 19.2μM and kcat= 116s−1.
Short‐term modulation of glycogen metabolism, glycolysis and gluconeogenesis by physiological oxygen concentrations in hepatocyte cultures
Authors: WÖLFLE Detlef , SCHMIDT Helga , JUNGERMANN Kurt
Published: March 2005

The influence of different oxygen concentrations (0% to 20%, v/v) on the main pathways of carbohydrate metabolism was studied in rat hepatocyte cultures. Cells resembling the periportal or the perivenous cell type were obtained after 48 h culture under different hormonal conditions; they are referred to as ‘periportal’ or ‘perivenous’, respectively. Using radiochemical techniques the metabolic rates of the two cell types were measured between 48 h and 50 h under different oxygen tensions. Standard physiological substrates of 5mM glucose, 2mM lactate and endogenous glycogen were used.

In cells incubated under 4% O2, mimicking hepatovenous oxygen levels, compared to cells assayed under 13% O2, mimicking arterial levels, glycogen degradation to free glucose and to lactate was faster while glycogen synthesis was slower; glycolysis, from glucose to lactate, was faster and gluconeogenesis, from lactate to glucose, was only slightly slower.

Under anoxic conditions glycogen breakdown was maximal and glycogen synthesis minimal; gluconeogenesis was also minimal, but glycolysis was not maximal, it reached its peak rate at 4% O2.

CO2‐formation increased up to 6% and then stayed essentially constant at higher O2tensions

Net glycogen metabolism: in ‘perivenous’ cells net glycogen synthesis was observed above, net glycogen degradation below 4% O2. In ‘periportal’ cells, which had a very low glycogen content, net glycogen metabolism was very small.

Net glucose metabolism: in ‘perivenous’ cells net glucose formation was only seen under anoxic conditions. Net glucose utilization was observed at about the same rate under all physiological O2tensions. In ‘periportal’ cells net glucose formation increased clearly up to 6% O2and then remained almost constant.

Net lactate metabolism: in ‘perivenous’ cells net lactate formation occurred below, and net lactate utilization above, 6% O2. In ‘periportal’ cells a pronounced net lactate utilization was observed under all physiological O2tensions.

Net flow between glucose‐6‐phosphate and pyruvate was observed in the glycolytic direction in ‘perivenous’ and in the gluconeogenic direction in ‘periportal’ cells except under anoxic conditions. When O2tensions were lowered, the percentage of ‘futile cycling’ was decreased in the ‘perivenous’, glycolytic hepatocytes; conversely, it was increased in the ‘periportal’, gluconeogenic hepatocytes.

It is concluded that physiological oxygen concentrations modulate hepatic carbohydrate metabolism and that they contribute further to the proposed metabolic differences between periportal and perivenous cellsin vivo.
Cooperative effects in the peptidyltransferase center of Escherichia coli ribosomes
Authors: BOURD Saul B. , KUKHANOVA Marina K. , GOTTIKH Boris P. , KRAYEVSKY Alexander A.
Published: March 2005

We have measured the binding isotherms of C‐A‐C‐C‐A(3′NH)‐[14C]Phe to the 70S ribosomes and 50S subunits of Escherichia coli and proposed a theoretical model for adsorption when cooperative interaction occurs between ligands that are adsorbed on ribosomes. Analysis of the experimental binding isotherms leads to the following conclusions. A ribosome (or subunit) binds two C‐A‐C‐C‐A(3′NH)‐Phe molecules. The binding of C‐A‐C‐C‐A(3′NH)‐Phe to a ribosome (or subunit) is a cooperative process, characterized by a cooperativity coefficient τ=40±5 or more.

The binding of C‐A‐C‐C‐A(3′NH)‐AcPhe at the donor site of the peptidyltransferase center (association binding constant 1.5×106 M−1) and the binding of puromycin at the acceptor site also occur cooperatively with a coefficient of 10–25, the association binding constant of puromycin at the acceptor site being (1–2)×104 M−1. The puromycin association binding constant at the donor site multiplied by the cooperativity coefficient of two interacting puromycin molecules adsorbed on a ribosome equals 100–200 M−1.
Isolation and comparison of arylsulfatase A from rat liver and Morris hepatoma 7777
Authors: HESS Grzegorz
Published: March 2005

Rat liver and Morris hepatoma 7777 arylsulfatase A were isolated from the soluble lysosomal extract by a procedure involving blue‐Sepharose affinity chromatography, DEAE‐cellulose chromatography, hydrophobic chromatography on phenyl‐Sepharose and preparative polyacrylamide gel electrophoresis. The preparation obtained by this method was apparently homogenous in disc electrophoresis and in immunoelectrophoresis.

The comparative studies revealed that the properties of arylsulfatase A from rat liver and Morris hepatoma 7777 are very similar, considering molecular weight of the native monomer and its subunits, the ability to form tetramers, isoelectric point, Michaelis constant and the anomalous kinetics of the reaction.

The twofold elevation of arylsulfatase B activity found in Morris hepatoma 7777 suggests that the enzyme may have certain functions in tumor growth.
Reaction mechanism of flavin‐dependent hydroxylation
Authors: VISSER Cornelis M.
Published: March 2005

Analysis of the partially refined X‐ray crystallographic structure of the active site of p‐hydroxybenzoate hydroxylase shows clearly that the enzyme is complementary to a flavin C4, C4a‐dioxetane derivative. A mechanism is proposed based on such an intermediate as the oxenoid transferring agent. A non‐enzymatic analogue of this reaction is not known in organic chemistry. A possible evolutionary pathway for such a non‐imitable enzyme is discussed.
A pulse‐radiolysis study of the reduction of flavodoxin from Megasphaera elsdenii by viologen radicals. A conformational change as a possible regulating mechanism
Authors: van LEEUWEN Johan W. , van DIJK Cees , VEEGER Cees
Published: March 2005

Pulse‐radiolysis experiments were performed on solutions containing methyl or benzyl viologen and flavodoxin. Viologen radicals are formed after the pulse. The kinetics of the reaction of these radicals with flavodoxin were studied. The kinetics observed depend strongly on the concentration of oxidized viologen. Therefore one must conclude that a relatively stable intermediate is formed after the reduction of flavodoxin. The midpoint potential of the intermediate state is –(480+30)mV, and is hardly dependent on the pH between 7 and 9.2. Due to a conformational change (k2∼ 105 s−1) the intermediate state decays to the stable semiquinone form of flavodoxin. The ΔG of the conformational change at pH 8 is about 29 kJ mol−1 (0.3 eV). This means that the upper limit for the pK of N‐5 in the semiquinone form will be 13. The activation energy of the conformational changes is 43 kJ mol−1 (0.45 eV).

The reaction between methyl viologen radicals and the semiquinone of flavodoxin can be described by a normal bimolecular reaction. The reaction is diffusion‐controlled with a forward rate constant of (7 ± 1) × 108 M−1 s−1 (pH 8, I= 55 mM). The midpoint potential of the semiquinone/hydroquinone was found to be –(408 ± 5) mV.

A consequence of the intermediate state is that flavodoxin (Fld) could be reduced by a two‐electron process, the midpoint potential of which should be located between –440 mV < Em (Fld/FldH−) < ‐290 mV. The exact value will depend on the ΔG of the conformational change between the fully reduced flavodoxin with its structure in the oxidized form and the fully reduced flavodoxin with its structure in the hydroquinone form. The conditions are discussed under which flavodoxin could behave as a two‐electron donor.
Phenolic and tyrosyl ring deiodination in thyroxine from rat retina during postnatal development
Authors: IENTUILE Riccardo , MACAIONE Salvatore , RUSSO Pietro , PUGLIESE Gaetano , DI GIORGIO Rosa Maria
Published: March 2005

To elucidate tetraiodothyronine (T4) metabolism in developing rat retina 5‐monodeiodinating and 5′‐monodeiodinating activities were studied. T4 was incubated with aliquots of homogenate or crude primary subcellular fraction, and the 3,3′,5′‐triiodothyronine (rT3) or 3,5,3′‐triiodothyronine (T3) produced were measured by radioimmunoassay. Reaction rates were dependent on incubation time, tissue amount, temperature and pH. The optimum pH values were 7.8 and 7.2 respectively for rT3 forming and T3‐forming systems. Conversion of T4 to either T3 or rT3 was dependent on dithiothreitol concentration, and the T4‐5′‐deiodioatrag activity was inhibited by propylthiouracil. Deiodinase activities were mainly found in the crude microsomes. The retinal 5′‐monodeiodination rate of T4 was immeasurably low by the 2nd day and the highest values were reached on 15th day of postnatal development. On the other hand deiodination of the T4 tyrosyl ring shows a progressive decline from birth, and adult values were reached on the 15th day.

Data support the hypothesis that, in developing rat thyroxine, phenolic and tyrosyl‐ring deiodinase activities are present in the retina and their reciprocal changes may regulate morphological and biochemical cell maturation.
Electron allocation to H+ and N2 by nitrogenase in Rhizobium leguminosarum bacteroids
Authors: HAAKER Huub , WASSINK Hans
Published: March 2005

Electron allocation to H+ and N2 by nitrogenase in intact Rhizobium leguminosarum bacteroids has been studied. Nitrogenase activity was measured in intact cells with succinate and oxygen substrates. When whole cell nitrogenase activity was inhibited by oxygen‐limitation or by the addition of the H+‐conducting ionophore carbonylcyanide m‐chlorophenylhydrazone, both inducing a low intracellular ATP/ADP ratio, the electron allocation to H+ was favoured over that to N2. When whole cell nitrogenase activity was inhibited by excess oxygen or by the addition of the K+‐conducting ionophore valinomycin, both inhibiting electron transport to nitrogenase without affecting the intracellular ATP/ADP ratio, no effect upon the electron allocation to H+ and N2 was observed.

The whole cell experiments could be confirmed by experiments with bacteroids treated with hexadecyltri‐methylammonium bromide. Nitrogenase is highly active in these preparations with Na2S2O4 and MgATP as substrates. No effect was observed upon electron allocation to H− and N2 when nitrogenase was inhibited by limitation of reductant (Na2S2O4) or MgATP. Only when nitrogenase was inhibited by MgADP, electron allocation to H+ was favoured.

The amount of nitrogenase component 1 and 2 in bacteroids was estimated with protein blotting, followed by an immunological detection. It was found that 17%± 3% of total bacteroid protein is component 1 and 12%±2% is component 2. The specific nitrogenase activity of bacteroids treated with hexadecyltrimethylammonium bromide is 178±62 nmol C2H4 formed · min−1· mg total protein−1. Despite the high protein concentrations, nitrogenase is not inhibited.

With cell‐free extracts or with purified nitrogenase components isolated from R. leguminosarum bacteroids, electron allocation to H+ was favoured over that to N2, independently of the mechanism of inhibition. The discrepancies between the whole cell studies and those with isolated enzyme will be discussed with respect to the present mechanism of action of nitrogenase.
On auxotrophy for pyrimidines of respiration‐deficient chick embryo cells
Authors: GRÉGOIRE Michel , MORAIS Réjean , QUILLIAM Michael A. , GRAVEL Denis
Published: March 2005

Chick embryo cells treated with chloramphenicol are inherently resistant to the growth‐inhibitory effect of the drug when cultured in the presence of tryptose phosphate broth. The cells were found to be auxotrophic for pyrimidines and the presence in the broth of compounds of pyrimidine origin is demonstrated by chromatographic procedures and mass spectral analyses. They are in the form of ribonucleotides, ribonucleotides and pyrimidine‐containing oligoribonucleotides. To understand the mechanism responsible for pyrimidine auxotrophy, the activity of enzymes involved in the pyrimidine biosynthetic pathway was determined. Measurement of the conversion of dihydroorotic acid to orotic acid in cell‐free extracts revealed that chloramphenicol‐treated chick embryo cells are deficient in dihydroorotate dehydrogenase activity. The data in vitro are supported by studies on the nutritional requirements of the respiration‐deficient cells and by the incorporation in vivo of labelled dihydroorotic acid into the acid‐insoluble fraction of the cells. Although the activity of the dehydrogenase in vitro is decreased by 95%, the enzyme is present in chloramphenicol‐treated cells and its activity is unmasked by the artificial electron acceptor menadione. A study of the activity of other enzymes of the pyrimidine biosynthetic pathway demonstrated that their activity is comparable to that in control cells. The present results indicate that auxotrophy for pyrimidines results from the inhibition of the flow of electrons along the mitochondrial electron transport chain.
Studies on the transmembrane disposition of the neural cell adhesion molecule N‐CAM
Authors: GENNARINI Gianfranco , HIRN Michel , DEAGOSTINI‐BAZIN Hermine , GORIDIS Christo
Published: March 2005

The transmembrane orientation of the polypeptide chains present in preparations of adult and neonatal mouse N‐CAM was studied using, as a model system, liposome‐inserted purified N‐CAM preparations. N‐CAM purified from adult or neonatal mouse brain was 125I‐labeled and reconstituted into artificial lipid vesicles. After trypsin digestion, the peptides that remained associated with the liposomes were isolated by floatation of the vesicles on sucrose gradients. In control experiments the liposomes were lysed before trypsin treatment. Large, overlapping peptides were obtained after this treatment, several of which were protected by the liposome membrane. Sialic‐acid‐bearing peptides were revealed by their sensitivity to neuraminidase. To distinguish between peptides corresponding to intracellular or extracellular domains use was made of the P61 and H28.123 monoclonal antibodies, which recognize determinants located on the cytoplasmic and the extracellular part of the molecules respectively.

There was no indication that the N‐CAM chains were inserted in an inside‐out configuration. Peptides protected from trypsin attack by the liposomes and recognized only by P61 had Mr values of 92000, 42000 and 35000. The H28.123 determinant could be mapped to a 32000‐Mr peptide located close to the membrane at the vesicle's exterior. The bulk of the sialic acid seemed to be carried by a rather short sequence distal to the H28.123‐reactive peptide but at some distance from the N terminus. Fragments of very similar Mr were generated from young and adult material. However, a 45000‐Mr peptide from neonatal N‐CAM appeared to migrate in the high‐Mr region of sodium dodecyl sulfate/polyacrylamide gels in its fully sialylated form.

It is concluded that (a) identical polypeptide chains are present in young and adult preparation, (b) the 180000‐Mr, 140000‐Mr and 120000‐Mr chains differ by the length of their cytoplasmic extensions and (c) the largest cytoplasmic sequences have a Mr close to 90000. A tentative linear model of the transmembrane topography of the N‐CAM polypeptides is presented.
Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine‐linked oligosaccharides
Authors: HETTKAMP Harald , LEGLER Günter , BAUSE Ernst
Published: March 2005

Trimming glucosidase I and II have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p‐nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and II and the nonspecific α‐glucosidase(s) were inhibited by 1‐deoxynojirimycin with median inhibitory concentration of 3 μM, 20 μM, 12 μM, respectively. Discrimination between these enzymes was strongly enhanced by N‐alkylation of 1‐deoxynojirimycin and formed the basis for the design of the affinity ligand.

Glucosidase I has been purified to homogeneity by affinity chromatography on AH‐Sepharose 4B with N‐carboxypentyl‐ 1–deoxynojirimycin as ligand. Sodium dodecyl sulfate gel eletrophoresis of the purified enzyme revealed a subunit molecular mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was ∼ 320–350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4°C in the presence of detergent (tl/2∼ 20 days) and showed high specificity for the hydrolysis of the terminal (α 1,2)‐linked glucose residue in the natural substrate Glc3‐Man9‐(GlcNAc)2.
Corticosteroids as effectors of lipid polymorphism of dielaidoylglycerophosphoethanolamine
Authors: GALLAY Jacques , DE KRUIJFF Ben
Published: March 2005

The influence of corticosteroids on the lipid polymorphism of dielaidoylglycerophosphoethanolamine was studied by31P NMR spectroscopy and differential scanning calorimetry. Both techniques evidenced two transitions in the pure lipid samples. The first one corresponded to the gel→liquid crystalline phase transition. It occurred at a temperature of 38.9°C, as measured by differential scanning calorimetry and at 35–40°C as detected by31P NMR. The second transition corresponded to the bilayer→hexagonal H11phase transition. It occurred at 64.2°C as measured by differential scanning calorimetry and at 60°C as detected by NMR. Addition of corticosteroids led to different specific effects on the bilayer→hexagonal H11phase transition, according to their chemical structure. These effects appear to be the result of low amounts of incorporated steroids, according to binding studies (partition coefficient values range between 5 and 54). The presence of a conjugated 3‐keto group in the steroid molecule (progesterone) promoted a downward shift in the bilayer→hexagonal H11phase transition temperature by about 6–7°C as compared to the 3β‐OH‐bearing compound (pregnenolone), which did not exhibit any appreciable effect. No change in the ΔHof transition could be measured. The presence of the 21‐OH group (like in deoxycorticosterone) induced the formation of a structure, characterized by an isotropic lineshape of the31P NMR spectrum at temperatures where the ‘exagonal’ type of lineshape is present, without steroid. The transition from the bilayer to this other structure occurred at a slightly higher temperature than the bilayer→hexagonal H11phase transition. It corresponded to a peak in differential scanning calorimetry scans with a ΔHof 2.1 kJ · mol−1. The presence of the 17β‐OH group as present in 17β‐OH‐progesterone and 11‐deoxycortisol suppressed the two former effects. These compounds had no influence on the bilayer→hexagonal H11phase transition. The additional presence of the 11β‐OH group like in corticosterone and cortisol, evoked a stabilization of the bilayer organization as the bilayer→hexagonal H11phase transition temperature is shifted upward by about 10°C. This was accompanied by a decrease of the ΔHto 0.8 kJ · mol−1. Besides this, the corticosteroids did not affect to a large extent the gel→liquid crystalline phase transition: a general slight downward shift of the transition temperature and a small broadening of the transition were observed without significant change in the ΔH.The effects of corticosteroids on the bilayer→hexagonal H11phase transition are qualitatively correlated with their influence on biological membranes.
Fatty acid synthesis in mitochondria of Euglena gracilis
Authors: INUI Hiroshi , MIYATAKE Kazutaka , NAKANO Yoshihisa , KITAOKA Shozaburo
Published: March 2005

A malonyl‐CoA‐independent fatty acid synthetic system, different from the systems in other subcellular fractions, occurred in mitochondria of Euglena gracilis. The system had ability to synthesize fatty acids directly from acetyl‐CoA as both primer and C2 donor using NADH as an electron donor. Fatty acids were synthesized by reversal of β‐oxidation with the exception that enoyl‐CoA reductase functioned instead of acyl‐CoA dehydrogenase in degradation system. A fairly high activity of enoyl‐CoA reductase was found on various enoyl‐CoA substrates (C4–C12) with NADH or NADPH. Three species of enoyl‐CoA reductase, distinct from each other by their chain‐length specificity, were found in Euglena mitochondria, and one of them was highly specific for crotonyl‐CoA. It is also discussed that the mitochondrial fatty‐acid synthetic system contributes to wax ester fermentation, the anaerobic energy‐generating system found in the organism.
Induction of phytoalexin synthesis in soybean. Stereospecific 3,9‐dihydroxypterocarpan 6a‐hydroxylase from elicitor‐induced soybean cell cultures
Authors: HAGMANN Marie‐Luise , HELLER Werner , GRISEBACH Hans
Published: March 2005

A microsomal preparation from elicitor‐challenged soybean cell suspension cultures catalyzes an NADPH‐dependent and dioxygen‐dependent 6a‐hydroxylation of 3,9‐dihydroxypterocarpan to 3,6a,9‐trihydroxypterocarpan. The latter is a precursor for the soybean phytoalexin glyceollin. No reaction is observed with NADH. The 6a‐hydroxylase is inhibited by cytochrome c.

Optical rotatory dispersion spectra of the enzymatic product formed from racemic dihydroxypterocarpan and of the remaining unreacted substrate proved that the product has the natural (6aS, 11aS)‐configuration and that hydroxylation proceeds with retention of configuration.

The 6a‐hydroxylase was also found in elicitor‐challenged soybean seedlings. The results indicate that the 6a‐hydroxylase is specifically involved in the biosynthesis of glyceollin.
A 45‐kDa human T‐cell membrane glycoprotein functions in the regulation of cell proliferative responses
Authors: GMÜNDER Hans , LESSLAUER Werner
Published: March 2005

A 45‐kDa human T cell surface glycoprotein which is tightly bound in the membrane or the resting T cell is released into the cell medium in soluble form after cell growth activation by phytohemagglutinin or neuraminidase/galactose oxidase treatments. In limited proteolysis by Staphylococcus aureus V8 protease, two major 35‐kDa and 27‐kDa peptide fragments of the surface‐iodinated 45‐kDa protein are common to the membrane‐bound and the released forms, but a third 18‐kDa fragment is observed exclusively with the released protein. The apparent molecular masses of the deglycosylated peptide backbones of the membrane‐bound and the released molecule are 30 ± 1 kDa, although a small size difference cannot be excluded. A polyclonal rabbit anti‐(T cell membrane protein) antiserum precipitates the 45‐kDa protein. A monoclonal anti‐(45‐kDa protein) antibody precipitates the membrane‐bound 45‐kDa protein solubilized with octyl glucoside, but does not precipitate the released protein. In cell culture assays, the monoclonal anti‐(45‐kDa protein} antibody specifically enhances the cell proliferative responses in phytohemagglutinin‐treated and mixed lymphocyte cultures. These observations suggest that the 45‐kDa protein has a specific receptor function in the regulation of cell proliferative responses.
Sex and age alter plasma membranes of cultured fibroblasts
Authors: SCHROEDER Friedhelm , GOETZ Ingeburg , ROBERTS Eugene
Published: March 2005

Human skin fibroblasts were taken from age‐matched male and female subjects. The cells were then cultured under identical conditions and passage‐number matched. Plasma membranes were isolated and membrane enzyme activities, lipid composition, and structure of isolated plasma membranes were measured in order to determine the presence of significant sex differences in human fibroblast membrane properties. The results indicated that plasma membranes from normal female subjects had a 1.6‐fold and 3.6‐fold higher cholesterol/phospholipid ratio and oleic acid (18:2) content than normal male subjects. The limiting anisotropy and the rotational relaxation time of fluorescence probe molecules such as trans‐parinaric acid and 1,6‐diphenyl‐1,3,5‐hexatriene in the plasma membranes was not significantly different from fibroblasts of male versus female normal subjects. The total activity of plasma membrane (Na+, K+)‐ATPase was significantly higher in female than male normal subjects. A potential ‘membrane structural disorder’, Huntington's disease, was confirmed in fibroblast membranes from male but not from female Huntington's disease subjects. The possibility that Huntington's disease was a ‘premature membrane aging’ phenomenon was considered. A comparison of plasma membrane enzymes, lipids, and structure from old and young Huntington's disease subjects did not show differences consistent with accelerated membrane aging as explaining the molecular basis for the disease. The age‐dependent differences noted in aged Huntington's disease subjects: increased phosphatidylcholine/phosphatidylethanolamine ratio and sphingomyelin + lysophosphatidylcholine content of fibroblast plasma membranes were not significantly altered when compared to normal age‐matched controls. However, (Na+, K+)‐ATPase activity was significantly enhanced in fibroblast plasma membranes of older Huntington's disease subjects unlike those of control subjects. In conclusion, sex and age differences in membrane properties of cultured cells represent important potential variables in the elucidation of human genetic disorders that may be membrane‐related.
Comments on ‘Reaction mechanism of flavin‐dependent hydroxylation: evolution of a non‐imitable enzyme’ by C. M. Visser
Authors: Humphrey I. X. MAGER
Published: March 2005

Dear Sir,

Studies on bioorganic model systems have provided the evidence that dihydroflavin and tetrahydropteridine hydroperoxides can rearrange to other reactive peroxide forms including 4,4a‐dioxetanes. Such rearranged peroxides were already proposed as alternative hydroxylating agents in 1976. This original proposal is ignored in recent biochemical papers in which some incorrect claims and statements are made.
Lactoperoxidase, a dithionite ion dismutase
Authors: OHLSSON Per‐Ingvar
Published: March 2005

The dithionite ion is catalytically disproportionated by lactoperoxidase with Km= 0.36 mM in 100 mM glycine HCl pH 3.0. The products formed are thiosulfate and hydrogensulfite ions. The rate of reaction is considerably increased at low pH with a pKa at 3–3.5 possibly indicating the involvement of a carboxyl group. The reaction is competitively inhibited by hydrogensulfite, Ki= 5.5 mM in 100 mM glycine HCl pH 3.50.

Four different spectral forms of reduced lactoperoxidase appear during the reaction. The first two forms are found during the lag phase of the reaction. The third form, which is interpreted as a ternary complex, exists under the dismutation phase. After exhaustion of the substrate a visible spectrum similar to that of lactoperoxidase H2O2 compound III appears.

A mechanistic model for the lactoperoxidase dismutation of the dithionite ion is proposed and discussed.
A synthetic hapten for induction of thymine‐dimer‐specific antibodies
Authors: KLOCKER Helmut , AUER Bernhard , BURTSCHER Helmut J. , HIRSCH‐KAUFFMANN Monica , SCHWEIGER Manfred
Published: March 2005

High specificity and sensitivity of thymine cyclobutane dimer (thy[]thy) detection were obtained by a radioimmunoassay. Attempts to raise thy[]thy‐monospecific antibodies with antigens produced according to conventional methods were unsuccessful. Thy[]thy‐specific antibodies could only be raised by using a new strategy to bind thy[]thy to protein: thymine was activated by trimethylsilylation and alkylated at N1 yielding N1‐thyminebutanoic acid which was dimerised by ultraviolet treatment. The resulting derivative of thymine cyclobutane dimer was coupled to bovine serum albumin by the active‐ester method. The new strategy appears to be generally applicable for binding haptens, such as DNA bases, photoproducts etc. to proteins via a derivative containing a carboxyl group. Immunisation of rabbits with the thy[]thy‐bovine‐serum‐albumin conjugate prepared by the new method resulted in a highly specific antiserum which allows detection of thy[]thy down to 0.06 p mol (15pg).

The thy[]thy‐specific radioimmunoassay was applied to measure thy[]thy formed in human fibroblasts which were exposed to sunlight at altitudes of 600 m or 2300 m. The amounts of thy[]thy formed in an hour corresponded to doses of 14 J m−2 and 24 J m−2, respectively, of an ultraviolet light lamp emitting predominantly 245‐nm light.
Differences in the binding of sulfate, selenate and thiosulfate ions to bovine liver rhodanese, and a description of a binding site for ammonium and sodium ions
Authors: LIJK Leo J. , TORFS Chris A. , KALK Kor H. , DE MAEYER Marc C. H. , HOL Wim G. J.
Published: March 2005

The binding of sulfate, selenate and thiosulfate by the sulfur‐transferase rhodanese (EC 2.8.1.1) in the crystalline state has been studied by X‐ray analysis at resolutions between 0.23 nm and 0.4 nm. The three ions appear to occupy a common site between the N" atoms of Arg‐29 and the main‐chain NH group of Glu‐148 at the surface of the enzyme molecule.

A second binding site for the three ions is situated at the entrance to the active centre, between the side chains of Arg‐186 and Lys‐249. Selenate and thiosulfate are bound equally well at both anion‐binding sites. Sulfate, however, binds better at the first position, near Arg‐29, than at the second site near Arg‐186.

In the complex of sulfur‐rhodanese with thiosulfate, the outer sulfur atom of the anion near the active centre points towards the extra sulfur atom which is bound as a persulfide to the Sγ of the essential Cys‐247. The distance between the outer sulfur atom of the thiosulfate ion and the persulfide sulfur atom appears to be about 0.3 nm.

The thiosulfate difference Fourier also shows a distinct, localized conformational change involving residues 71, 72 and 249. This is the result of the replacement of an ammonium ion in the sulfate and selenate media by a sodium ion in the sodium thiosulfate solution. Rhodanese is apparently able to accomodate ions with different radii at this cation‐binding site by minor structural alterations.
Factors determining the relative contribution of the adenine‐nucleotide translocator and the ADP‐regenerating system to the control of oxidative phosphorylation in isolated rat‐liver mitochondria
Authors: WANDERS Ronald J. A. , GROEN Albert K. , VAN ROERMUND Carlo W. T. , TAGER Joseph M.
Published: March 2005

The control exerted by the adenine nucleotide translocator and the ADP‐regenerating system on oxidative phosphorylation was studied in isolated rat‐liver mitochondria respiring with succinate.

At intermediate rates of respiration the flux control coefficient (control strength) of the adenine nucleotide translocator on respiration was much higher with creatine/creatine kinase than with glucose/hexokinase as the ADP‐regenerating system. In contrast, at the same rate of respiration the flux control coefficient of creatine kinase was much lower than that of hexokinase.

On addition of a small amount of carboxyatractyloside to mitochondria respiring in the presence of glucose/hexokinase or creatine/creatine kinase, the rate of respiration decreased abruptly and then increased again to a new steady state which was lower with creatine/creatine kinase than with glucose/hexokinase. In the new steady state, the extramitochondrial ATP/ADP ratio was lower with glucose/hexokinase than with creatine/creatine kinase.

At the same rate of respiration, the elasticity coefficient of creatine kinase towards the extramitochondrial ATP/ADP ratio was much higher than that of hexokinase.

The connectivity theorem [Kacser, H. and Burns, J. A. (1973) in Rate Control of Biological Processes (Davies, D. D., ed.) pp. 65–104, Cambridge University Press, London] which relates the flux control coefficients of two adjacent enzymes to their elasticity coefficients towards the common intermediate, provides an explanation for the difference in flux control coefficient of the adenine nucleotide translocator with the two ADP‐regenerating systems.

Using principles developed by R. Heinrich and T. A. Rapoport [BioSystems 7, 130–136 (1975)], the flux control coefficients of the adenine nucleotide translocator and hexokinase on phosphorylation were also calculated from the elasticity coefficients of these enzymes towards the extramitochondrial ATP/ADP ratio and the controls exerted by these enzymes on the extramitochondrial ATP/ADP ratio. The calculated values were approximately 30% lower than the values measured directly.

The elasticity coefficient of the adenine nucleotide translocator towards the extramitochondrial ATP/ADP ratio was determined, using either the connectivity theorem of Kacser and Burns (loe. cit.) or a new connectivity theorem developed by Westerhoff (following paper in this journal). Good agreement was obtained using the different methods of calculation.

It is concluded that flux through the adenine nucleotide translocator, although in principle dependent on ([ATP]/[ADP])out, ([ATP]/[ADP])in and ΔΨ, is regulated primarily by ([ATP]/[ADP])out under the conditions used in this study.
Solution conformation of the C‐terminal domain of skeletal troponin C
Authors: DRABIKOWSKI Witold , DALGARNO David C. , LEVINE Barry A. , GERGELY John , GRABAREK Zenon , LEAVIS Paul C.
Published: March 2005

Proton magnetic resonance spectroscopy has been used to study the cation(Mg2+, Ca2+)‐dependent conformational states of the C‐terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the cation binding reaction. Complex formation with troponin I and the drug trifuloperazine is observed to adjust the solution conformation of the C‐terminal domain of troponin C. The interactive conformational response to cation coordination and of the binding of the drug and troponin I are discussed.
Induction of cytochrome P‐450 b, e‐type isozymes by polychlorionated biphenyls in rat liver
Authors: SCHOLTE Bob J. , te VELDE Bert , GROOT Gert S. P. , de VRIES John
Published: March 2005

We have studied the induction of cytochrome P‐450 b, e‐type antigen and mRNA by phenobarbital, 4,4′‐dichlorobiphenyl and 2,4,5,2′,4′,5′‐hexachlorobiphenyl in male Wistar rat liver. Chronic treatment of rats with 4,4′‐dichlorobiphenyl leads to a relatively slow, 20‐fold increase in the cytochrome P‐450 b,e‐type antigen level and to an equivalent increase in the concentration of the corresponding mRNA. Treatment of rats with phenobarbital or 2,4,5,2′,4′,5′‐hexachlorobiphenyl results in a faster and more pronounced increase of cytochrome P‐450 b,e‐type antigen and mRNA levels. Analysis of clones from cDNA banks showed that two types of sequences are induced by phenobarbital corresponding to cytochrome P‐450 b and cytochrome P‐450 e respectively. 2,4,5,2′,4′,5′‐Hexachlorobiphenyl appears to induce primarily a cytochrome P‐450b‐type sequence. The implications of these results for the study of the mechanism of induction are discussed.
Synthesis and stability of arylsulfatase A and B in fibroblasts from multiple sulfatase deficiency
Authors: STECKEL Friedhelm , HASILIK Andrej , VON FIGURA Kurt
Published: March 2005

Fibroblasts from patients with multiple sulfatase deficiency were analyzed for activities of arylsulfatase A and B, iduronate 2‐sulfatase and sulfamatase. A. group of patients (group I) severely deficient in all sulfatases (residual activites ≤ 10% of control) were differentiated from patients (group II) with residual sulfatase activities of up to 90% of control. The synthesis and stability of arylsulfatase A and B were determined in pulse‐chase labelling experiments. The apparent rate of synthesis of arylsulfatase A and B varied from 30% to normal in both fibroblasts from group I and II multiple sulfatase deficiency. In group I the molecular activity of the arylsulfatase A and B was more than 10‐fold lower than in control fibroblasts. In group II the molecular activity of the arylsulfatase A was twofold to threefold lower and that of arylsulfatase B half of normal. In fibroblasts of both groups the stability of arylsulfatase A polypeptides was significantly diminished. For arylsulfatase B the instability was restricted to the mature 47 000‐Mr polypeptide and was variable withiin both groups. These results demonstrate that multiple sulfatase deficiency is a heterogeneous disorder, in which the primary defects can impair both the catalytic properties and the stability of sulfatases.
The structure of the double‐stranded RNA pentamer 5′(CACAG) · 5′(CUGUG) determined by nuclear Overhauser enhancement measurements: Interproton distance determination and structure refinement on the basis of X‐ray coordinates
Authors: CLORE G. Marius , GRONENBORN Angela M. , McLAUGHLIN Larry W.
Published: March 2005

The structure in solution of the duplex RNA pentamer 5′(CACAG) · 5′(CUGUG), comprising the stem of the TΨC loop of yeast tRNAPhe, has been investigated by means of one‐ and two‐dimensional nuclear Overhauser enhancement measurements. All non‐exchangeable base and sugar proton resonances with the exception of the H5′/H5” sugar resonances are assigned in a sequential manner. From the relative intensities of the cross‐peaks obtained in the pure‐phase absorption two‐dimensional nuclear Overhauser enhancement spectra at several mixing times, it is deduced that the RNA pentamer adopts an A‐type conformation in solution. Cross‐relaxation rates and interproton distances are determined from the time dependence of the nuclear Overhauser effets, principally by one‐dimensional measurements. The structure of the RNA pentamer is then refined by restrained least‐squares minimization on the basis of both distance and planarity restraints using fibre diffraction data as an initial model. The refined structure of the RNA pentamer is of the A type but exhibits local structural variations in glycosidic bond and backbone torsion angles as well as in propellor twist, base roll and base tilt angles.