bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.
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.
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.