bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Requirements for Maximal Inhibitory Allosteric Effect of Estrogens and Estrogen Analogues on Glutamate Dehydrogenase
Authors: PONS Michel , MICHEL Françoise , DESCOMPS Bernard , DE PAULET André CRASTES
Published: March 2005

The inhibition of glutamate dehydrogenase by estrogens, estrogen analogues or polyphenylethylene derivatives (about one hundred molecules, most of them having estrogenic or antiestrogenic activities) was measured. The efficiency of these compounds in inducing allosteric inhibition of the enzyme was compared and correlated to their chemical structure: an aromatic ring A, a free phenolic group in the region of carbon 3 of the steroid nucleus and a lipophilic substitution in the region of C‐12, C‐13 or C‐17 were found to be the main structural features required for maximal efficiency on glutamate dehydrogenase.

A tentative model for the relative orientation of the main inhibitor families is proposed. It accounts for most of the kinetic results and can be used as a tool for the selection of affinity labels directed towards the estrogen binding site of glutamate dehydrogenase.
Affinity Labelling of the Estrogen Binding Site of Glutamate Dehydrogenase with Iodoacetyldiethylstilbestrol
Authors: MICHEL Françoise , PONS Michel , DESCOMPS Bernard , DE PAULET André CRASTES
Published: March 2005

Iodoacetyldiethylstilbestrol was used as an affinity label to alkylate the estrogen binding site of bovine liver glutamate dehydrogenase.

This reagent induced inactivation and alkylation of the enzyme. The non‐alkylating analogues diethylstilbestrol and estradiol protected the enzyme towards alkylation. The apparent constant of alkylation was of the order of magnitude of I50 for the allosteric inhibition by diethylstilbestrol. These two results suggest that alkylation occurred at the estrogen binding site.

The stoichiometry of alkylation was between one and two, depending on the experimental conditions. When the stoichiometry was found to be ≤1, 90% of the label was bound on cystein residues, 70% of which was carried by cysteine ‐89, a cysteine residue which is known to be inacessible to iodoacetamide in phosphate buffer in the same conditions of temperature and pH.
Enzymic Hydrolysis of 3‐Acetyl‐estrogens or Analogues by Glutamate Dehydrogenase with Specific Acylation of the Estrogen Binding Site
Authors: MICHEL Françoise , PONS Michel , JULLIARD Jacques , DESCOMPS Bernard , DE PAULET André CRASTES
Published: March 2005

The estrogen analogues or derivatives 4‐acetyldiethylstilbestrol, 3‐acetyl‐estradiol and 3‐acetyl‐2‐nitroestrone were hydrolysed by glutamate dehydrogenase with covalent incorporation of an acetyl group into the enzyme molecule, probably through a transacylation process.

When 3‐acetyl‐2‐nitroestrone was used as a ‘substrate’, spectrophotometric titration of the free product (a nitrophenol) allowed kinetic studies: there was a linear relationship between initial hydrolysis rates and enzyme concentration and Michaelis kinetics were observed suggesting an enzyme catalyzed process.

The covalent incorporation of the acetyl group on the enzyme was studied using 3‐[2′‐14C]acetyl‐2‐nitroestrone: in the absence of NADH, lysine residues 419 and 422 were specifically acetylated without enzyme inactivation. In the presence of NADH, the labelling was distributed over two more lysine residues, 143 and 155, with progressive inactivation.

Estradiol lowered the hydrolysis rate of 3‐acetyl‐2‐nitroestrone by glutamate dehydrogenase, protected the enzyme against acylation and against the inactivation which is observed in the presence of NADH. Moreover glutamate dehydrogenase acylated by 3‐acetyl‐2‐nitroestrone became less sensitive to its allosteric effector: estradiol. Thus, it is suggested that hydrolysis of acetyl‐estrogen or analogues by glutamate dehydrogenase and acylation of the enzyme occur in or near the estrogen binding site.
Response of Fluorescamine during the Interaction of Cloacin DF 13 with the Isolated Cytoplasmic Membrane of Susceptible Cells
Authors: OUDEGA Bauke , SMIT Hendrik , STRAATHOF Jos W. M. , DE GRAAF Frits K.
Published: March 2005

The fluorescence spectra of fluorescamine‐labeled cloacin complex and fluorescamine‐labeled cloacin in 0.1 M potassium phosphate buffer pH 7.0 at 25 °C show an emission maximum at 478 nm and 476 nm, respectively, and an excitation maximum at 395 nm.

The binding of an increasing number of fluorescamine molecules to the cloacin complex with a maximum of about 20 molecules, results in a decreasing receptor binding activity and specific killing activity to about 5%, but has no effect on the endoribonucleolytic activity in vitro. Increasing binding of fluorescamine to cloacin with a maximum of about 30 molecules results in a strong reduction of the activities in vivo and in vitro.

The addition of cytoplasmic membrane vesicles isolated from susceptible cells of Enterobacter cloacae to fluorescamine‐labeled cloacin DF 13 preparations (about half of the maximum number of dye molecules bound per molecule of bacteriocin) causes an enhancement of the fluorescence intensity of 40% for cloacin complex or 75% for cloacin, and a blue shift in the maximum emission wavelength from 478 nm to 475 nm or from 476 nm to 471 nm, respectively. The observed fluorescence changes have, most probably, to be attributed to changes in local solvent polarity of the labeled bacteriocin molecules.

The specificity of the interaction between the labeled bacteriocin and the cytoplasmic membrane was shown using various labeled proteins and vesicles isolated from various insensitive bacteria.

Trypsin‐digested or denatured labeled bacteriocin molecules do not cause any significant changes in the fluorescence characteristics, while proteolytic cloacin fragments possessing receptor‐binding and/or endoribonucleolytic activity are still capable of interacting significantly with the cytoplasmic membrane vesicles.

The interaction of the bacteriocin with the vesicles as determined by the fluorescence response shows a pH optimum between 6.5 and 7.0 at 25 °C but is only slightly affected by variation of the temperature above 20 °C at pH 7.0 or the ionic strength of the incubation buffer. Furthermore, the ‘specific’ interaction is dependent on the presence of molecular oxygen.

Fluorimetric titration experiments under ‘optimal’ conditions demonstrate ‘specific’ binding of labeled cloacin complex and labeled cloacin to vesicles isolated from susceptible cells with dissociation constants of 1.46 nM and 1.20 nM, respectively. The concentration of ‘specific’ sites for labeled cloacin complex as well as for labeled cloacin is about 5 × 105 per vesicle. In addition ‘aspecific’ sites are demonstrated in a concentration of 7–8 × 105 per vesicle. The dissociation constant for these lowaffinity ‘aspecific’ binding sites is about 23.5 nM. Vesicles isolated from insensitive bacterial strains possess only about 9 × 105‘aspecific’ binding sites with a dissociation constant of 9.6 nM.
The Dependence of Acetylcholinesterase Aggregation at Low Ionic Strength upon a Polyanionic Component
Authors: BON Suzanne , CARTAUD Jean , MASSOULIÉ Jean
Published: March 2005

The low ionic strength aggregation of Electrophorus asymmetric forms of acetylcholinesterase has been found to depend upon a distinct aggregating agent, present in large excess in the electric organ extracts. The aggregates appeared, in electron micrographs, as bundles of a few molecules, the tails of which were linked in a side‐to‐side association, leaving the head groups at both ends. The proportion of aggregated enzyme, but not the sedimentation properties of the aggregates, depended in a complex manner upon ionic conditions and aggregating agent levels. The aggregates were not dissociated by dilution even at very low concentrations. Properties of the aggregating agent suggested that it might be a non‐proteic polymer, interacting with positively charged groups of the enzyme tail.

Various polyanions were found to promote aggregation of acetylcholinesterase, but induced aggregates of different sedimentation coefficients. Polycarboxylates were effective only at high concentrations while sulfate and phosphate polyanions were very efficient. Aggregates induced by these latter polyanions possessed the same morphological features as those produced by the endogenous preparation.

Analysis of the Electrophorus aggregating preparation demonstrated that its chondroitin sulfate content fully accounts for its aggregating capacity, and this was in agreement with its sensitivity to specific mucopolysaccharidases.

Aggregates were also obtained in Electrophorus/Torpedo mixed systems but their characteristics were found to depend both on the nature of the enzyme and of the aggregating agent. Aggregation had no effect on catalytic efficiency, and we discuss its relevance to the synaptic localization of the enyme, through interactions between the tails of acetylcholinesterase and chondroitin‐sulfatetype glycosaminoglycan components.
Valine Uptake and Incorporation into Protein in Isolated Rat Hepatocytes
Authors: SEGLEN Per O. , SOLHEIM Anne E.
Published: March 2005

In isolated rat hepatocytes, valine is transported by a high‐Kmsystem (Km= 43 mM, V= 22 nmol · mg protein−1· min−1), which is non‐concentrative and energy‐independent. Valine transport proceeds equally well in both directions (influx and efflux) and is subject to accelerative exchange. By these properties, the transport system can be defined as a facilitated diffusion (passive transport). There is no evidence for valine uptake by simple (physical) diffusion (i.e. no uptake at O°C), and it is suggested that the latter takes place only in damaged or dysfunctional cells.

Valine transport is temperature‐dependent, with an activation energy of 60 kJ/mol in the temperature range 30–40 °C. Below 25 °C the activation energy increases to 103 kJ/mol, possibly reflecting a phase transition in the cell membrane. Protein synthesis and protein degradation both have an activation energy of about 100 kJ/mol throughout the temperature range 10–40 °C.

At 37 °C, a tracer dose of [14C]valine equilibrates so rapidly across the cell membrane that it is impossible to tell whether the radioactive valine incorporated into protein is taken from an intracellular or extracellular amino acid pool. In addition, interpretation of the protein labelling pattern is complicated by endogenous protein degradation and subsequent amino acid release, which results in both isotope dilution and a gradual stimulation of protein synthesis.

Since valine transport is temperature‐dependent, a slow influx or efflux of valine can be produced by lowering the incubation temperature to the range 15–25 °C. Low temperatures also reduce the disturbing influence of protein degradation, while protein synthesis still proceeds at a sufficient rate to obtain analyzable incorporation kinetics.

With only a tracer dose of [14C]valine in the extracellular medium, the rate of protein labelling under these conditions can be seen to follow closely the changes in the intracellular rather than in the extracellular radioactivity, both during influx and efflux of [14C]valine. However, when the extracellular valine concentration is maintained several‐fold higher than the intracellular concentration, protein labelling correlates better with the extracellular than with the intracellular radioactivity. These results suggest that both the intracellular and the extracellular valine pools can directly provide precursors for protein synthesis, the relative contribution from each pool being proportional to the relative valine concentration in that pool, as suggested by Khairallah and co‐workers [Biochem. J. (1974) 140, 539–548; J. Biol. Chem. (1976) 251, 1375–1384; Biochem. J. (1977) 162, 257–266].
Requirement for ‘Factor(s)’ for Tubulin Assembly during Brain Development
Authors: FRANCON Jacques , FELLOUS Arlette , LENNON Ana Maria , NUNEZ Jacques
Published: March 2005

The polymerisation of neurotubulin to neurotubules in vitro was studied using brain supernatants from rats of different ages (from the 15th day of gestation till 30 days post‐partum).

When the concentration of tubulin was kept low the initial rates of polymerisation of both fetal and new born material were markedly lower than that of the adult. Both the critical concentration and the lag period were, however, increased. When higher tubulin concentrations were used the initial rates of the young stages ressembled those of the adult (3 days after birth) or were even higher (9 days after birth).

These differences in the relationship between the initial assembly velocities and tubulin concentration were interpreted as resulting either from the presence of an inhibitor of microtubule assembly in the young brain or the fact that a factor is required for the formation of nucleating centers and for tubulin polymerisation, which may be limiting in early developmental stages. The first possibility has been disproved by mixing small amounts of fetal supernatants with adult preparations; no inhibition was found under these conditions. Support for the second possibility has been gained by adding small quantities of adult supernatant to young brain preparations. Clear stimulation of the initial rates of polymerisation and overall turbidity was found. These results suggest that a factor (and therefore the number of nucleating centers) is in excess in the adult and limiting in the young brain.

Further experiments were performed to better characterize the limiting entity(ies). Additions of sonicated microtubule fragments, of isolated microtubule associated proteins and of purified τ protein, all produced a clear increase in assembly rate and a decreased lag period. These results suggest that the τ protein plays an important role in promoting the initiation and the elongation of the microtubule. The overall turbidity, however, was never completely restored. Moreover, the more the ‘factor’ were purified the more the overall polymerisation was diminshed, suggesting that another factor involved in the elongation process is probably present in the brain but is eliminated during the purification of the τ protein.

In conclusion these results demonstrate that tubulin alone is unable to assemble into microtubules in the absence of one (or several) factors which are limiting in the early stages of brain development.
Membrane Lipid Metabolism in Acholeplasma laidlawii A EF 22
Authors: CHRISTIANSSON Anders , WIESLANDER Åke
Published: March 2005

Membrane lipid metabolism in Acholeplasma laidlawii A EF 22 has been studied under different conditions by applying three different techniques for changing membrane viscosity: fatty acid and cholesterol supplementation and temperature changes.

The molar relationship between the two dominating membrane lipids, monoglucosyldiglyceride and diglucosyldiglyceride, is to a large extent determined by membrane viscosity properties. This is shown by the varying metabolic responses occurring during incorporation of different fatty acids with and without cholesterol and by temperature shift‐down experiments. Higher viscosity in membranes stimulates synthesis of monoglucosyldiglyceride at the expense of diglucosyldiglyceride. Synthesis of phospho and phosphoglucolipids is affected as well.

Temperature shift‐down from 37°C to 17°C results in an immediate synthesis of monoglucosyldiglyceride accompanied by an increased incorporation of unsaturated fatty acids into this lipid. Synthesis of the other membrane lipid species (containing more unsaturated fatty acids) lags behind temporarily.

Incorporation from an equimolar mixture of palmitic and oleic acids together with cholesterol yields greater amounts of oleic acid in membrane lipids than incorporation in the absence of cholesterol, indicating that incorporation is viscosity dependent.

Studies of precursor relationships reveal that all main lipids have an active turnover which differs depending on membrane composition and conditions. Furthermore, this turnover proceeds with different intra‐lipid pools.

Isolated membranes contain no detectable lipolytic enzymes capable of hydrolyzing membrane phospho or glycolipids. It is suggested that lipid turnover is partly mediated by enzymatic interlipid conversions, thus not allowing intermediates to accumulate.
Lanthanum as a Tool to Study the Role of Phosphatidylinositol in the Calcium Transport in Rat Parotid Glands upon Cholinergic Stimulation
Authors: KERYER Guy , ROSSIGNOL Bernard
Published: March 2005

We describe the effects of lanthanum on protein secretion, potassium efflux, calcium uptake and phosphatidylinositol turnover stimulated by cholinergic agonists in rat parotid glands. Carbachol increases in vitro calcium uptake, protein secretion and K+ efflux through muscarinic receptor; however it fails to stimulate protein discharge or K+ release in a incubation medium free of calcium. Lanthanum inhibits calcium uptake, protein secretion and K+ efflux induced by carbachol without impairing protein discharge stimulated by norepinephrine through the β‐adrenergic receptor. Norepinephrine, in the presence of calcium in the incubation medium, stimulates the K+ efflux through the α‐adrenergic receptor: this effect is suppressed by lanthanum. These results emphasize the role of increased influx of calcium in the cellular phenomena controlled by muscarinic or α‐adrenergic receptors. Carbachol increases phosphatidylinositol turnover in the absence of calcium in extracellular medium; indeed it is shown that carbachol increases the rate of phosphatidylinositol breakdown and that lanthanum impairs this cholinergic effect. From these data it is suggested that the interaction between cholinergic agonist and muscarinic receptor could induce a stimulation of ‘phosphatidylinositol turnover' which could control the calcium influx according to the gradient through the plasmalemma membrane.
Characterization of Proteins from Nucleolar Preribosomes of Mouse Leukemia Cells by Two‐Dimensional Polyacrylamide Gel Electrophoresis
Authors: AUGER‐BUENDIA Marie‐Annick , LONGUET Michèle
Published: March 2005

Proteins of isolated 80‐S and 60‐S nucleolar preribosomal particles were characterized by means of two‐dimensional polyacrylamide gel electrophoresis, in the lymphocytic mouse leukemia cells L5178Y. Their identification and metabolic relationships with ribosomal subunit proteins were investigated using co‐electrophoresis of unlabeled polysomal proteins with labeled proteins of either nucleolar preribosomes or ribosomal subunits. The large and small ribosomal subunits contain 40 and 31 proteins, respectively. The nucleolar 80‐S preribosomes were analysed after 2 and 5 h of incubation with tritiated valine and leucine and were shown to contain about 55 proteins. Most of them were identical to cytoplasmic ribosomal subunit proteins. The nucleolar 60‐S preribosomes contain all the proteins which are common to 80‐S preribosomes and large ribosomal subunits, and one additional protein (L10). The ribosomal proteins which were absent from nucleolar particles were found to be labeled in the cytoplasmic ribosomes after the same incubation period. Thus, in addition to the association of the bulk of ribosomal proteins with 45‐S RNA within the 80‐S preribosomes, results indicate that a group of ribosomal proteins and particularly from the small subunits, become associated at later stages of the maturation process of mammalian ribosomes. It was further shown that a set of 10 proteins, different from ribosomal polypeptides, were present in nucleolar preribosomal particles. Several of them were associated with polyribosomes in the cytoplasm, whereas the others were unique to the nucleolus.
Changes in Chromatin Properties after Partial Extraction of Non‐histone Proteins
Authors: BOKHON'KO Andrei I. , RAZUMOVA Valentina V.
Published: March 2005

By treatment with tRNA in the presence of 1 mM MgCl2, a chromatin preparation was obtained containing all five major histone fractions but lacking a considerable portion of non‐histone proteins. This chromatin preparation as well as chromatin extracted with 0.6 M NaCl (depleted of H1 histone and some non‐histone proteins) were characterized in respect of solubility and chromatin DNA accessibility.

Both samples possessed practically the same solubility in the presence of 0.15 M NaCl and 1 mM MgCl2. The solubility of tRNA‐treated chromatin in 5 and 10 mM MgCl2 was higher than that of salt‐extracted chromation.

The accessibility of the DNA of these chromatin preparations was tested with DNA‐dependent RNA polymerase of Escherichia coli as a probe, using procedure that permits measurement of binding site frequency. Both tRNA‐treated and salt‐extracted chromatin contained as many as 33% and untreated chromatin as few as 4% of the number of binding sites found on protein‐free DNA.

These results demonstrate that at least in part the non‐histone proteins are reponsible for salt‐induced insolubility and low DNA accessibility of chromatin, thus revealing the importance of nonhistone proteins in the maintainance of an overall chromatin structure.
Purification and Properties of Reduced Ferredoxin: CO2 Oxidoreductase from Clostridium pasteurianum, a Molybdenum Iron‐Sulfur‐Protein
Authors: SCHERER Paul A. , THAUER Rudolf K.
Published: March 2005

Reduced ferredoxin: CO2 oxidoreductase from Clostridium pasteurianum, a ferredoxin‐dependent soluble formate dehydrogenase, was purified 3000‐fold with an overall yield ranging between 10 and 20%. The enzyme was at least 85% pure as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by the molybdenum content. The enrichment was performed in the presence of 10 mM azide, a competitive inhibitor of the enzyme, and under strictly anaerobic conditions (E∼–400 mV) in order to protect the CO2 reductase from rapid inactivation. The purification procedure included a heat step, a precipitation by polyethyleneimine and by ammonium sulfate, a sedimentation by ultracentrifugation, and a chromatography on DEAE‐cellulose in 39% ammonium sulfate.

The highly purified CO2 reductase contained 7.2 nmol of molybdenum, 170 nmol of non‐heme iron and 150 nmol of acid‐labile sulfur per mg of protein. The spectrum of the enzyme was typical of an iron‐sulfur protein. Selenium, tungsten, flavins or heme could not be detected in significant amounts. The enzyme consisted of a number of aggregated forms with molecular weights near 700000, 460000, and 240000 which could be partially dissociated in the presence of 1% Triton X‐100 into an enzymatically active species of molecular weight 118000. Treatment with sodium dodecl sulfate led to a dissociation of the enzyme into two enzymatically inactive polypeptide chains of molecular weights near 34000 and 86000. The purified enzyme will catalyze the following reactions at the same relative rates as the cell‐free extracts: (a) the reduction of CO2 to formate with reduced ferredoxin, (b) the oxidation of formate to CO2 with oxidized ferredoxin, (c) an isotopic exchange between 14CO2 and formate in the absence of ferredoxin, and (d) the reduction of methyl viologen with formate.

It is concluded that CO2 reductase from C. pasteurianum is a molybdenum iron‐sulfur protein containing 1 mol of molybdenum, and 24 mol of non‐heme iron and acid‐labile sulfur per mol enzyme of molecular weight 118000.
Kinetic Evidence for Interaction between Aldolase and d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: OVÁDI Judit , KELETI Tamás
Published: March 2005

The Possibility of interaction between purified rabbit muscle aldolase and d‐glyceraldehyde‐3‐phosphate dehydrogenase was studied by rapid kinetic methods, by analyzing the kinetics of the consecutive reaction catalyzed by the coupled enzyme system.

The Km of the intermediary product, glyceraldehyde 3‐phosphate, produced by aldolase was determined in the coupled reaction for glyceraldehyde‐3‐phosphate dehydrogenase. Its value corresponds to that of the aldehyde (active) from of glyceraldehyde 3‐phosphate, although in the given conditions the aldehyde → diol interconversion is faster than the enzymic reaction catalyzed by glyceraldehyde‐3‐phosphate dehydrogenase.

We suggest that above a certain concentration of the enzymes the glyceraldehyde 3‐phosphate produced by aldolase gets direct access to glyceraldehyde‐3‐phosphate dehydrogenase without participating in the aldehyde → diol interconversion which otherwise would occur if the substrate were to mix with the bulk medium.
Immunological Studies on CTP:Phosphocholine Cytidylyltransferase from the Livers of Normal and Choline‐Deficient Rats
Authors: CHOY Patrick C. , SCHNEIDER Wolfgang Johann , VANCE Dennis E.
Published: March 2005

Chickens were immunized with the purified low‐molecular‐weight form of CTP:phosphocholine cytidylyltransferase from rat liver cytosol. The antiserum was obtained and fractionated to yield immunoglobulin. The antibodies specifically inhibited the enzymatic activity of the partially purified low‐molecular‐weight form of the enzyme from pH 6.0 to 8.5. Antibodies against the low‐molecular‐weight form of the enzyme cross‐related with the high‐molecular‐weight form of the enzyme from cytosol as well as with the cytidylyltransferase associated with the microsomal fraction. The antibodies were used for the immunochemical determination of the amount of cytosolic phosphocholine cytidylyltransferase in the livers of normal and choline‐deficient rats. The amount of enzyme in rat liver cytosol was not changed for at least 18 days of choline deficiency. The decrease in specific activity of the enzyme in choline‐deficiency may be caused by factors other than adaptive changes in the level of enzymes.
Nucleotide Sequence of Part of the Simian Virus 40 Hind‐D Restriction Fragment
Authors: YSEBAERT Marc , VAN HEUVERSWYN Hugo , VAN DE VOORDE André , FIERS Walter
Published: March 2005

The nucleotide sequence at the beginning of the restriction fragment Hind‐D from Simian virus 40 DNA has been derived by partial chemical degradation of (5′‐32P)‐labeled restriction fragments followed by analysis on polyacrylamide gel according to Maxam and Gilbert [Proc. Natl Acad. Sci. U.S.A. 74, 560–564 (1977)]. The sequence reported here is 140 nucleotides long. It contains an ATG codon which presumably corresponds to the initiator codon of the VP2 protein. This codon is preceded by an untranslated region which shows several interesting features, such as an alternation of (dA+dT)‐rich and (dG+dC)‐rich blocks.
Nucleotide Sequence of the Simian Virus 40 Hind‐K Restriction Fragment
Authors: ROGIERS Raphaël , VAN DE VOORDE André , SOEDA Eiichi , FIERS Walter
Published: March 2005

The restriction fragment Hind‐K represents 4.2% of the genome of Simian virus 40 (SV40) and is located near the middle of the late region. Its nucleotide sequence is reported here. It was mainly established by analysis of transcription products, synthesized by means of Escherichia coli RNA polymerase and nucleoside triphosphates, one of which was (α‐32P)‐labeled. Strand assignment was possible by hybridization of asymmetric, labeled transcripts of total SV40 DNA to filter‐bound Hind‐K fragment. Further information and unambiguous confirmation of the sequence was obtained by the use of direct DNA‐sequencing methods. For this purpose the fragment was labeled at the 5′ ends by means of polynucleotide kinase and [γ‐32P]ATP and redigested with a suitable restriction enzyme. The separated products were then either partially degraded with snake venom diesterase for analysis by the ‘wandering spot' method or partially degraded with the base‐specific reagents dimethylsulphate or hydrazine for direct sequence analysis on gel. The Hind‐K sequence is 219 base pairs long. The message strand is particularly rich in adenosine (39%) and purines. The nucleotide sequence can unambiguously be translated into an amino acid sequence and the N‐terminal codon of the viral protein VP1 gene could be identified. The amino‐terminal part of VP1 is rich in proline and lysine. The nucleotide sequence of Hind‐K codes also for the carboxyl‐terminal part of the viral protein VP2 and VP3 genes, which partly overlap the VP1 gene.
Properties of Microsomal and Soluble Diacylglycerol Kinase in Rat Liver
Authors: KANOH Hideo , ÅKESSON Björn
Published: March 2005

The properties of ATP: diacylglycerol phosphotransferase (ATP + 1,2‐diacyl‐sn‐glycerol → phosphatidate + ADP, EC 2.7.1.‐) activity found in rat liver microsomal and 105000 ×g supernatant fractions were investigated in detail using emulsified and membrane‐bound diacylglycerol. The microsomal (specific activity about 0.4 nmol · min−1· mg of protein−1) and soluble (0.3 nmol · min−1· mg−1) fractions contained about 30 and 50% of the recovered enzyme activity, respectively. The microsomal activity was more unstable on storage at 0°C and was more sensitive to fenfluramine [1‐(m‐trifluoromethylphenyl)‐2‐ethylaminopropane hydrochloride] than the soluble enzyme. Except for these differences, both enzyme activities had similar properties. They required Mg2+ and deoxycholate for activity and had pH optimum around 7.0. Both enzymes stereospecifically utilized 1,2‐diacyl‐sn‐glycerol, and did not possess marked selectivity in utilizing emulsified or membrane‐bound diacylglycerols of different unsaturation. Other lipid substrates, including monoacylglycerol and ceramide were hardly utilized. The apparent Km values for ATP and diacylglycerol emulsion were 0.16 mM and 0.018 mM, respectively, for microsomal and 0.17 mM and 0.022 mM for soluble enzymes. Both kinase activities were similar when ATP was replaced by GTP (Km for GTP, 0.63 mM for microsomal and 0.42 mM for soluble enzyme).

Membrane‐bound diacylglycerol was prepared by treatment of microsomes with CMP or phospholipase C. Although microsomal diacylglycerol kinase was inactivated considerably by these treatments, soluble enzyme could utilize membrane‐bound diacylglycerol 3–5‐fold more actively than emulsified substrate. Deoxycholate was required also for maximal utilization of membrane‐bound substrates. The metabolic role of diacylglycerol kinase(s) is discussed.
Interaction between Membrane Functions and Protein Synthesis in Reticulocytes: Specific Cleavage of 28‐S Ribosomal RNA by a Membrane Constituent
Authors: WRESCHNER Daniel , MELLOUL Daniele , HERZBERG Max
Published: March 2005

A factor isolated from rabbit reticulocyte white ghosts by Triton X‐100 treatment blocks protein synthesis at the elongation‐termination stage. Factor‐treated ribosomes were found to have an identical buoyant density to that of control ribosomes. When incubated with either reticulocyte ribosomes or ribosomal RNA, the factor products specific cuts in the 28‐S ribosomal RNA component without damaging the 18‐S RNA. Incubations of pancreatic or T1 RNase, with ribosomal RNA, at similar protein‐synthesis inhibitory concentrations effected a complete breakdown to oligo and mononucleotides.

When challenged with isolated 28‐S or 18‐S reticulocyte ribosomal RNA, the highly purified factor only attacked the 28‐S RNA species. There was no accumulation of nucleotides or oligonucleotides and we concluded that the membrane factor causes inhibition of protein synthesis by having a specific endonucleolytic cleavage activity.
Studies on the Conformation of the 3′Terminus of 18‐S rRNA
Authors: VASS J. Keith , MADEN B. Edward H.
Published: March 2005

We have studied the conformation of the 3′ end of 18‐S RNA from human, hamster and Xenopus laevis cells. The 3′‐terminal oligonucleotide in a T1 ribonuclease digest of 18‐S RNA from HeLa cells was identified, using a standard fingerprinting method. The sequence (G)‐A‐U‐C‐A‐U‐U‐A, established by Eladari and Galibert for HeLa 18‐S rRNA, was confirmed. An identical 3′ terminus is present in hamster fibroblasts and Xenopus laevis cells. The ease of identification of this oligonucleotide has enabled us to quantify its molar yield relative to several other oligonucleotides, and hence to analyse the 3′ terminus by several conformation probes. Its sensitivity to S1 nuclease, limited T1 ribonuclease digestion, bisulphite modification and carbodiimide modification was consistent with the terminal oligonucleotide being in a highly exposed conformation. The m62‐m62A‐C‐containing sequence of 18‐S rRNA also appears to be in an exposed location on the basis of three of these probes.
ABH‐Blood‐Group Antigens and Glycolipids of Human Saliva
Authors: SLOMIANY Bronislaw L. , SLOMIANY Amalia
Published: March 2005

The nature of ABH‐blood‐group antigens in saliva was investigated. Human saliva was examined serologically for ABH‐blood‐group activity in its native form and after various treatments. The activity of the native form persisted in the delipidated samples, but was entirely lost after alkaline degradation. The lipid portion of saliva was completely inactive in the ABH hemagglutination inhibition system. The same results were obtained when purified glycolipid fraction of saliva was used instead of whole lipid extract. Neither alkaline treatment nor excessive amounts of salivary lipids effected antigenic activity of A‐active glycosphingolipids of hog gastric mucosa admixed to saliva samples before alkaline degradation and/or in presence of large amounts of salivary lipids.

The isolated glycolipid fractions contained at least eight glycolipids, each of which was composed of glucose, glyceryl ethers and fatty acids and differed from others with respect to number of glucose residues.

Sphingosine and sugar residues involved in formation of ABH antigenic determinants were not detected.

These findings together with data on stomach secretion [1,2] led us to the conclusion that ABH‐blood‐group antigens of saliva are exclusively of glycoprotein nature.
Isolation and Characterization of Free Cytoplasmic Messenger Ribonucleoproteins from Rabbit Reticulocyte
Authors: HUYNH‐VAN‐TAN  , SCHAPIRA Georges
Published: March 2005

Free cytoplasmic messenger ribonucleoproteins have been prepared with a high yield in a zonal rotor. They are rapidly labelled by [3H]uridine. Their sedimentation coefficients are 14.6 S and 19.4 S. Their protein contents are higher than in ribosomes, as indicated by a shift towards greater wavelength in their ultraviolet spectra and a buoyant density of 1.39 g cm−2 in CsCl. They do not cross‐react with antiribosome antibodies and their protein patterns in sodium dodecylsulfate and two‐dimensional polyacrylamide gel electrophoresis are different from ribosomes'. They may contain up to 30 proteins. The proteins are acidic and their molecular weights range from 22000 to 100000. When compared to chromatin they behave more like non‐histone proteins than histones.
l‐Glutamate Dehydrogenase. The Catalytic Properties of the Mercurial‐Activated Enzyme
Authors: COSSON Marie Paule , PANTALONI Dominique , IWATSUBO Motohiro
Published: March 2005

Glutamate dehydrogenase can be modified by reaction of cysteine residue 319 with p‐chloromercuribenzoate or methyl mercuric chloride. The catalytic properties of these modified enzymes have been investigated.

Both mercurial enzymes are able to catalyse glutamate oxidation, but their apparent affinities for substrate are decreased, particularly at those sites involved in the inhibition process at high glutamate concentration. Like the native enzyme, both mercurial enzymes exhibit downward curvature in Lineweaver‐Burk plots of reaction velocity against either substrate or coenzyme concentration.

Study of the pH‐dependence of the first prestationary step of glutamate oxidation demonstrates that the maximum rate constant (120 s−1 at 15°C) occurs with the ionization of a charged group of pKa 8.2, possibly involved in the hydride transfer. The pKa of this charged group is shifted 0.7 in the case of the methyl mercurial enzyme (pKa 8.9 and kapp= 100 s−1 at 15 °C), and 0.2 in the case of p‐mercuribenzoate enzyme (pKa 8.4 and kmaxapp= 30 s−1).

The modifications with mercurial reagents also induce a change in the pH‐dependence of the steady‐state velocity. The maximum velocity is 2 s−1 between pH 7.5 and 8.0 for the native enzyme, 4 s−1 above pH 8.8 for the methyl mercurial enzyme and 2 s−1 above pH 8.5 for the p‐mercuribenzoate enzyme. This capacity of methyl mercuric chloride to increase the rate of enzyme turnover has been correlated with an increase in the rate of dissociation of products.

The mercurial‐induced perturbations of the rate constants of the prestationary steps and stationary state, and of their pH‐dependence, modify the time resolution of the absorbance and fluorescence during the glutamate oxidation in a manner similar to those induced in the native enzyme by the binding of ADP.
On the Mechanism of the Cytochrome c Oxidase Reaction
Authors: PETERSEN Lars Chr.
Published: March 2005

The anaerobic‐aerobic transition of the CO‐inhibited ascorbate → cytochrome c→ cytochrome aa3 system is investigated using purified oxidase.

In this system the presence of CO in the medium facilitates the measurements of the oxygen kinetics and at the same time allows a spectral resolution of cytochromes a and a3. Measurements of the oxygen consumption, and the steady‐state reduction of the cytochromes c, a, and a3 as a function of the oxygen concentration are reported.

A double reciprocal plot of oxygen consumption rate versus oxygen concentration is nonlinear at low concentration of cytochrome c but becomes linear as the cytochrome concentration is increased.

The plot of oxygen consumption rate against [O2] × [a2+3· CO], where a2+3· CO is the CO complex of the oxidase measured at 590 nm, is a straight line through the origin. This suggests that the binding of CO to the oxidase does not depend on the redox state of cytochrome a but is described by a single dissociation constant and also that the reaction between reduced cytochrome a3 and O2 is a second‐order reaction, k+1= 1.4 · 108 M−1 s−1.

The results indicate that transfer of electrons from cytochrome a to cytochrome a3 during turnover is an irreversible second‐order reaction. But a similar simple relation seems not to be valid for the transfer of electrons from cytochrome c to a or from cytochrome c to a3.

The assumption of a quasi‐equilibrium between cytochrome c and cytochrome a leads to the conclusion that the midpoint potential of cytochrome a changes with the redox state of cytochrome a3, probably as a consequence of allosteric interaction between the hemes.
4‐Aminobutyrate Aminotransferase Fluorescence Studies
Authors: BEELER Troy , CHURCHICH Jorge E.
Published: March 2005

The kinetics of resolution of the pyridoxamine phosphate form of the enzyme 4‐aminobutyrate aminotransferase were monitored by fluorescence spectroscopy. 2 mol pyridoxamine phosphate are released/mol enzyme, indicating that two molecules of cofactor are involved in catalysis. The apoprotein is reconstituted by addition of pyridoxal phosphate; the apparent rate constant corresponding to the formation of active species is not a linear function of the concentration of cofactor. A multistep mechanism is proposed for the reconstitution of 4‐aminobutyrate aminotransferase. A slow phase of reactivation of the aminotransferase is observed when the apoprotein is allowed to reconstitute in the presence of pyridoxal kinase, ATP and pyridoxal.

The enzyme 4‐aminobutyrate aminotransferase is a dimeric protein made up of subunits of identical molecular weight. It is characterized by a rotational relaxation time of 110 ns. The dimeric structure does not dissociate into subunits over a wide range of protein concentration (4–0.2 μM) at neutral pH.
Role of Acetylglutamate in Ureotelism
Authors: SHIGESADA Katsuya , AOYAGI Kazumasa , TATIBANA Masamiti
Published: March 2005

The level of N‐acetyl‐l‐glutamate showed a diurnal rhythm, which was associated primarily with dietary ingestion. Since the hepatic level of arginine, activator for acetylglutamate, is one of the plausible principle factors responsible for the regulation of acetylglutamate level, the effect of arginine was further studied. The intraperitoneal administration of a large amount of arginine increased the level of acetylglutamate and the effect was augmented by the combined administration of glutamine. The arginine administration stimulated the hepatic synthesis of acetylglutamate as measured with [14C]glutamate as tracer, but did not affect the half‐life (about 20 min) of the synthesized 14C‐labeled acetylglutamate.

The effect of arginine in raising the acetyglutamate level was also demonstrated in vitro in isolated rat liver cells. On the other hand, pent‐4‐enoic acid, known to inhibit fatty acid oxidation and to decrease acetyl‐CoA production in the liver, lowered the level of acetylglutamate in the rat liver cells. It was shown that the cellular ureogenic capacity, as measured with NH4Cl as nitrogen source, showed a close correlation with the varying levels of acetylglutamate, brought about by arginine or pent‐4‐enoic acid.

The carbamoyl phosphate level, which may reflect the balance between its synthesis and utilization for citrulline synthesis, was low (about 1 nmol/g) and remained relatively constant in the livers of intact mice even when the rate of urea synthesis was expected to vary with changes in the dietary protein content. A transient increase in the level was brought about by agents expected to increase the synthesis or to inhibit the utilization, such as ammonia, N‐acetylglutamate diethyl ester, or norvaline. These results, together with others, suggest that in intact animals the production of carbamoyl phosphate is coupled to its utilization for citrulline synthesis.
The Carboxyl‐Terminal Sequence of the Heavy Chain of a Hong Kong Influenza Haemagglutinin
Authors: DOPHEIDE Theo A. , WARD Colin W.
Published: March 2005

The reduced carboxymethylated heavy chain (HA1) of a Hong Kong influenza haemagglutinin (H3) was digested with cyanogen bromide to give five peptides. The sequences of three of these peptides, together with the sequences of two methionine overlap peptides were combined to give the C‐terminal 68‐residue sequence of the heavy chain.

Threonine is the C‐Terminal residue of the heavy chain, an unexpected result in veiw of the generation of the heavy and light chains by the proteolytic cleavage of a pro‐haemagglutinin during virus assembly. Three half‐cystine residue occur close together at positions 24, 48 and 52 residues in from the C‐terminus. A carbohydrate group is attached to an asparagine residue 44 residues from the C‐terminus; it contains 5 residues mannose and 2 residues glucosamine.
The Androgenic Regulation of Abundant mRNA in Rat Ventral Prostate
Authors: PARKER Malcolm G. , SCRACE Geoffrey T.
Published: March 2005

The most abundant poly(A)‐containing RNA accounting for approximately half the total is regulated by testosterone in rat ventral prostate. A complementary DNA probe to these sequences was isolated by hybridizing the total cellular poly(A)‐containing RNA with its complementary DNA to an rot1/2 value of 2 × 10−2 mol l−1 s and separating the abundant cDNA, from the non‐abundant cDNA by hydroxyapatite chromatography. The abundant cDNA, complementary to about three different poly(A)‐containing RNA sequences comprising 45% of the total poly(A)‐containing RNA, has been used to investigate the effect of androgens on their metabolism.

Following castration there is a progressive decrease in the abundant sequences from 45% of the total in normal animals to less than 0.03% after 14 days; testosterone administration in vivo results in their regeneration. The sequences were not detected in seminal vesicle, liver, heart or spleen but represented about 0.01% of the poly(A)‐containing RNA of kidney.

Translation of the poly(A)‐containing RNA in vitro in a cell‐free system derived from wheat germ resulted in the synthesis of four major proteins which could be separated by polyacrylamide gel electrophoresis. Their synthesis, as measured by [35S]methionine incorporation, accounted for 40 – 50% of the total incorporation and declined after castration but was restored by testosterone treatment. We conclude that a class of àbundant poly(A)‐containing RNA which codes for four major proteins is regulated by testosterone in rat ventral prostate.
Methionyl‐tRNA Synthetase from Escherichia coli
Authors: HYAFIL Fran¸ois , BERNASSAU Jean‐Marie
Published: March 2005

Enhancement of the nuclear relaxation rates by manganese has been used to derive manganese‐purine‐ring distances in the activation site of methionyl‐tRNA synthetase. This is possible with the help of an abortiv complex between the enzyme, methionine, adenosine, pyrophosphate and manganese which simulates an intermediate species of the activation reaction. It is found that the distances between the manganese ion and the purine ring are too high (>0.8 nm) to allow interaction between them. Thus, metal‐purine interaction is involved neither in the catalytic mechanism nor in the stabilization of abortive synergistic complexes [S. Blanquet, G. Fayat and J. P. Waller (1975) J. Mol. Biol. 94, 1–15].
Further Evidence for the Structure of the Teichoic Acids from Bacillus stearothermophilus B65 and Bacillus subtilis var. niger WM
Authors: DE BOER Willem R. , WOUTERS Jan T. M. , ANDERSON Alistair J. , ARCHIBALD A. Ronald
Published: March 2005

Bacillus stearothermopnilus B65 and Bacillus subtilis var, niger WM both contain teichoic acids in their walls composed of glycerol, phophate and glucose. The 13C nuclear magnetic resonance spectrum of B. stearothermophilus teichoic acid showed 13C‐31P coupling on the signals from the C‐5 and C‐6 carbon atoms of the glucose molecule and an α‐glucosidic linkage between glucose and the C‐1 atom of the glycerol moiety. These data are consistent with a poly[glucosylglycerol phospate] as the cell‐wall teichoic acid in the organism.

B. subtilis var. niger WM teichoic acid was oxidized by periodate and incubated in glycine buffer at pH 10.5. This treatment did not significantly increase the phosphomonoester content (by β‐elimination of the phosphate groups) of the teichoic acid molecule (7.1 to 9.5%). which is in accordance with earlier data derived from 13C nuclear magnetic resonance spectrocopy [De Boer et al. (1976) Eur. J. Biochem. 62, 1–6], that in this organism the glucose is not an integral part of the polymer chain. Similar treatment of B. stearothermophilus B65 teichoic acid increased the phosphomonoester content of the preparation from 0.15 to 68.1%.
Electron Transport in the cni‐1 Mutant of Neurospora crassa
Authors: EDWARDS David L. , KLEIN Jerry L. , WARDEN Joseph T.
Published: March 2005

Mitochondria from the nuclear mutant cni‐1 have no optically detectable cytochrome aa3 in early log phase growth. These mitochondria have a high level of respiration that is not inhibited by cyanide but is inhibited by salicylhydroxamic acid. They also show a substantial amount of cyanide‐sensitive respiration.

As cultures of mutant cni‐1 age, flux through the hydroxamate‐sensitive pathway decreases markedly while flux through the cytochrome chain remains constant.

Growth studies with mutant cni‐1 indicate that the cytochrome chain in this mutant is more important in supporting growth than the hydroxamate‐sensitive pathway.

Measurements of the steady‐state level of reduction of cytochrome c in mutant cni‐1 indicate that the rate‐limiting step in the cytochrome chain is at the position occupied by cytochrome oxidase.

Electron spin resonance studies with cni‐1 mitochondria show normal cytochrome oxidase signals in the g ≅ 6 region although there is little or no optically detectable cytochrome aa3.