bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Estimates of Cytoplasmic and Nuclear Oestrogen Receptors in Chick Oviduct
Authors: SUTHERLAND Robert L. , BAULIEU Etienne‐Emile
Published: March 2005

[3H]Oestradiol exchange techniques were developed for the determination of specific cestrogen receptor site concentrations in the cytoplasm and nuclei of chick oviduct cells. Non‐labelled receptor‐bound oestrogens were exchanged with [3H]oestradiol during a 24‐h incubation at 20 °C. 2 h at 30 °C or 45 min at 37 °C. Both ‘soluble’ and ‘insoluble’ nuclear receptors were stable for at least 6 h at 30 °C and 37 °C but a proportion (approx. 30%) of cytoplasmic sites from withdrawn chickens were inactivated after 2 h at 20 °C.

The magnum of 4‐week‐old immature chickens (weight = 15 mg) contained 0.20 pmol of oestrogen receptor which corresponds to 4275 receptor sites/cell, when it is assumed that all magnum cells have equal concentrations of receptor. In primarily stimulated chickens of similar age which had received 10 × 1 mg of oestradiol benzoate/day, the magnum weighed approximately 800 mg and contained 8.65 pmol of oestrogen receptor (4610 sites/cell). Withdrawal from primary oestrogenic stimulation for 3–6 weeks resulted in a 110 mg magnum which contained 1.20 pmol of receptor (2225 sites/cell). Oviducts from immature and withdrawn chickens had the majority (73–77%) of their oestrogen receptor sites in the cytoplasmic fraction, while in primary stimulated chicken oviducts the majority (82%) of receptor sites were located in the nucleus.

A single secondary injection of oestradiol, to oestrogen‐withdrawn chickens, resulted in apparent translocation of cytoplasmic receptors to the nucleus during the first hour after injection. The magnitude of the decline in cytoplasmic receptor, and the concurrent increase in nuclear receptor concentration, was dose‐dependent between 2 and 100 μg oestradiol‐ kg body weight. Larger doses of oestradiol up to 1 mg/kg did not increase the concentration of nuclear receptor above the maximum level seen at 100 μg oestradiol/kg. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. By 15 h after an injection of 100 μg oestradiol kg, the concentration of nuclear sites had reached pre‐injection levels. During the same time period, the depleted cytoplasmic receptor levels were replenished such that they reached control values by 12 h and were about 150% of the pre‐injection level at 24 It.
A Method for Measuring the Internal pH in Illuminated Chloroplasts Based on the Stimulation of Proton Uptake by Amines
Authors: PICK Uri , AVRON Mordhay
Published: March 2005

A simple method for measuring the internal pH of chloroplasts during steady‐state illumination based on the stimulation of proton uptake by monofunctional amines was developed. Predictions of a mathematical derivation concerning the dependence of the stimulation on the amine concentration, the internal volume, the pK of the amine and the external pH have been verified experimentally. To circumvent uncoupling and swelling, due to large internal accumulation of amines extrapolation of the stimulation to low amine concentrations was suggested and shown to lead to valid values. Alternatively, swelling could be largely reduced in a medium containing potassium aspartate and valinomycin.
The Translation in vitro of mRNA from Developing Cysts of Artemia salina
Authors: GROSFELD Haim , LITTAUER Uriel Z.
Published: March 2005

Succcssive stages in the development of the brine shrimp cyst were used as a model for studying differentiation at the level of mRNA transcription and translation. The poly(A)‐containing mRNA from dormant cysts and free‐swimming larvae (nauplii) was found to be efficiently translated in a Wheat‐germ cell‐free system, and electrophoretic patterns of translation products in vitro resembled those of the endogenous proteins extracted from the equivalent developmental stages. Each stage, however, exhibits a characteristic protein pattern. Two low‐molecular‐weight proteins prominent in the cyst disappeared almost completely in the nauplius stage, whereas the proportion of actin increased 3‐fold. Parallel patterns were observed upon translation in vitro of the respective mRNA preparations. The percentage of the acidic protein, tubulin, decreased somewhat during development.
Conformational Studies on Murein‐Lipoprotein from the Outer Membrane of Escherichia coli
Authors: BRAUN Volkmar , ROTERING Heinz , OHMS J.‐Peter , HAGENMAIER Hanspaul
Published: March 2005

Conformational studies on an isolated integral membrane protein are reported. Lipoprotein of the Escherichia coli outer membrane was released from murein by treatment with either lysozyme or trypsin. The isolated lysozyme‐released lipoprotein (lipoprotein I) contained 2 or 3 muropeptides covalently linked at the C‐terminal end, while the trypsin‐released lipoprotein (lipoprotein II) was free of muropeptides and lacked the C‐terminal peptide Tyr‐Arg‐Lys. Circular dichroism spectra of the two preparations were essentially identical, and they show an α‐helix content of about 80% According to calculations based on the Chou‐Fasman rules for proteins of known sequence, lipoprotein is 64%α‐helix and 15%β‐structure. Infrared spectroscopy qualitatively supports these values. The conformation was stable in the pH range of 5‐12. Denaturation of lipoprotein by heat, 8 M urea, or sodium dodecylsulphate was a fully reversible, cooperative process. The thermal denaturation of lipoprotein occurs in two steps with transition points at 79.4°C for lipoprotein I and at 85.1 °C for lipoprotein II. Lipoprotein markedly changes conformation at dodecylsulphate concentrations where micelle formation sets in. The unusual behaviour of the lipoprotein conformation in sodium dodecylsulphate is discussed in relation to the lipoprotein conformation and aggregation within the membrane.
Preparation and Properties of S‐Cyano Derivatives of Creatine Kinase
Authors: DER TERROSSIAN Elisabeth , KASSAB Ridha
Published: March 2005

The two reactive thiol groups of rabbit muscle creatine kinase were stoichiometrically reacted with 5,5′‐dithio‐bis(2‐nitrobenzoic acid). In the resulting inactive mixed disulfide derivative they were subsequently substituted with [14C]cyanide, the smallest uncharged thiol‐blocking group. The modified enzyme contained 1.6 mol label/mol protein and showed by Ellman's titration and amino acid analysis a concomitant loss of about 0.8‐0.9‐Si‐i group per subunit. This mono‐Scyano derivative of creatine kinase was found 73% as active as the native unniodified protein. It was still able to react in the native state with a variety of thiol reagents with the further blocking of another pair of thiol groups; their substitution once more with cyanide resulted ire the his‐Scyano derivative of creatine kinase, which lost 2 thiol groups per subunit and had about 50% of the original catalytic activity. It is concluded that the four cyanylated thiol groups are not required for the catalytic activity of creatine kinase and the cyanoprotein derivatives described are shown to be useful tools for some interesting investigations related to this enzyme.
Ca2+, K+‐Regulated Intramolecular Crosslinking of S‐100 Protein via Disulfide Bond Formation
Authors: CALISSANO Pietro , MERCANTI Delio , LEVI Andrea
Published: March 2005

Reaction of the thiol reagent 5,5′‐dithio‐bis(2‐nitrobenzoic acid) (Nbs2) with the brain‐specific protein S‐100 favours stabilization of the quaternary structure of the protein via disulfide bond formation. This process is modulated by those cations (Ca2+ and K+) which are known to affect the conformation of the protein. Ca2+ markedly favours the reaction of S‐100 with Nbs2 but inhibits subsequent disulfide bond formation; K+, on the contrary, is much less effective in promoting interaction with Nbs2 but strongly stimulates disulfide bond formation. These findings are interpreted assuming that in presence of Ca2+ the three subunits forming the native S‐100 protein have two cysteine residues exposed to the solvent but mismatched to form disulfides while in presence of K+ the sulphydryl groups are in a less accessible position to Nbs2 but suitable for S‐S bond formation.

Crosslinking of S‐100 subunits is characterized by the appearance in dodecylsulphate electrophoresis of two very close protein bands having a molecular weight almost identical to that of the native, undenatured protein but not of higher or lower‐molecular‐weight components. This finding, and the demonstration that both the crosslinked and native S‐100 proteins have identical profiles when analyzed by sucrose density centrifugation or gel chromatography indicate that disulfide bond formation occurs among subunits of the same molecule.
Transcription in Vitro of φ 29 DNA and EcoRI Fragments by Bacillus subtilis RNA Polymerase
Authors: INCIARTE Marta R. , VIÑUELA Eladio , SALAS Margarita
Published: March 2005

EcoRI fragments A, B and C produced from linear φ29 DNA, but not D or E fragments, are transcribed by purified Bacillus subtilis RNA polymerase. The transcription of fragments A and C is initiated preferentially with GTP and to a lesser extent with ATP; the reverse happens in the case of fragment B. The dinucleotides GpU and GpA respectively, compete specifically with the incorporation of [γ‐32P]GTP directed by fragments A and C. The RNA synthesized in vitro by purified B. subtilis RNA polymerase is highly asymmetric. Most of the RNA synthesis directed by fragments A and C is early RNA. However, most of the RNA produced by fragment B is anti‐late‐RNA. Addition of crude extracts inhibit the transcription of fragment B but not that of fragments A and C.
The Effect of Endogenous Phosphate on the H+/Mn2+ Ratio and the State of Mn2+ in the Mitochondrial Matrix
Authors: POZZAN Tullio , BRAGADIN Marcantonio , AZZONE Giovanni Felice
Published: March 2005

Kinetics and stoichiometry of H+ extrusion and reuptake and of Mn2+ uptake and release have been measured in respiring liver mitochondria in the absence of external added Pi. H+ and Mn2+ fluxes are parallel during aerobic cation uptake but not during uncoupler induced cation release. The H+/Mn2+ is 1.24. Addition of SH reagents, in concentrations inhibiting the Pi carrier, modifies the kinetics of H+ extrusion and of Mn2+ uptake and release. The slow phase of uncoupler induced Mn2+ release is diminished. The H+/Mn2+ is increased to 1.72. Addition of SH reagents, after the phase of aerobic uptake is completed, results in a significant reduction of the extent of uncoupler‐induced Mn2+ release. The extent of reuptake of endogenous Pi during aerobic uptake of Mn2+ is about 8 nmol × mg protein−1.

Aerobic uptake of Mn2+ in the absence of external Pi results in an electron spin resonance spectrum which is the sum of two components. One, denoted as S, corresponds to Mn(H2O)62+. Another denoted as E, reflects spin exchange narrowing. In contrast to previous claims the following evidence suggests that the spin exchange component is due to Mn3(PO4)2 precipitate: (a) the dimension of the spin exchange spectrum is markedly reduced by abolition of Pi transport; (b) the spin exchange spectrum is released very slowly by addition of uncouplers under conditions where uncouplers cause a rapid deenergization of mitochondria, reuptake of H+ and release of cations; (c) the free matrix Mn2+ is released slowly after addition of uncoupler if there is a large spin exchange signal; however the free matrix Mn2+ is abolished rapidly by uncoupler when formation of the spin exchange signal is prevented by pretreatment with Ca2+; (d) the band width of the spin exchange fraction is independent of the Mn2+/protein ratio either under kinetic or steady state conditions; (e) the experimental spectrum recalls closely that obtained by computer simulation by assuming it as a combination of Mn(H2O)62+ and Mn3(PO4)2.

It is concluded that endogenous Pi affects the process of aerobic divalent cation uptake. A part of Mn2+ uptake in the absence of externally added anions, consists of a Mn3(PO4)2 precipitate. This accounts for a H+/Mn2+ ratio lower than 2.
Sequence Studies on d‐Serine Dehydratase of Escherichia coli
Authors: SCHILTZ Emil , SCHNACKERZ Klaus D.
Published: March 2005

An improved procedure for large‐scale production of crystalline d‐serine dehydratase (EC 4.2.1.14) from Escherichia coli is described. The N‐terminal sequence of the enzyme (Mr 45 500) was determined in a solid‐phase sequencer as Met‐Glu‐Asn‐Ala‐Lys‐Met‐Asn‐Ser‐Leu‐Ile‐Ala‐Gln‐Tyr‐Pro‐Leu‐Val‐Lys‐Asp‐Leu‐Val‐Ala‐Leu‐Lys. Four of the first five N‐terminal residues are homologeous with tryptophanase, another pyridoxal‐phosphate (P‐Pxy) enzyme that catalyzes α,β‐elimination reactions. After borohydride reduction and tryptic digestion of the enzyme, a peptide was isolated showing the sequence Lys‐Asp‐Ser‐His‐Leu‐Pro‐Ile‐Ser‐Gly‐Ser‐Ile‐Lys(P‐Pxy)‐Ala‐Arg. No clear homology of this portion of the enzyme with tryptophanase or another pyridoxal‐phosphate enzyme was observed.
Effect of Thyroid Hormones on Microsomal Fatty Acid Chain Elongation Synthesis in Rat Liver
Authors: LANDRISCINA Clemente , GNONI Gabriele V. , QUAGLIARIELLO Ernesto
Published: March 2005

Evidence is presented that rat liver microsomal fatty acid chain elongation synthesis and desaturation, as well as acetyl‐CoA carboxylase and fatty acid synthetase, are strongly influenced by thyroid hormone level. Conversely, the fatty acid chain elongation system in mitochondria, unlike the oxidative capacity of palmitate, NADH, succinate and malate, does not seem significantly affected by the thyrotoxic state.

In triiodothyronine‐induced or thyroxine‐induced hyperthyroidism, rat liver acetyl‐CoA carboxylase, fatty acid synthetase and microsomal chain elongation and desaturation reactions are not greatly affected after the first 10 days of treatment, while after longer intervals a respective increase in these activities is shown of up to 87, 116 and 65% after 22 days. In propylthiouracil‐induced hypothyroidism, all the above synthetic activities are strongly reduced immediately after three days of drug administration and diminished no further following longer periods.

Although the pattern of synthesized fatty acids in the thyrotoxic state is similar to that obtained from normal subcellular rat fractions, the esterification process of fatty acids in microsomal lipids appears to be slightly inhibited in hypothyroid rats and increased following triiodothyronine or thyroxine administration. Finally, a reduction in the hepatic cyclic AMP level of about 41% is reported after 19 days of triiodothyronine‐administration to rats.

On the basis of the observed insensitivity of the mitochondrial fatty acid chain elongation system to the thyrotoxic state, a tentative interpretation of its role in the hepatic cell is postulated.
Replenishment and Depletion of Citric Acid Cycle Intermediates in Skeletal Muscle.
Authors: SPYDEVOLD Øystein , DAVIS E. Jack , BREMER Jon
Published: March 2005

The effects of various substrates on the concentrations of free amino acids, citric acid cycle intermediates and acylcarnitines were studied in perfused hindquarter of rat in presence of glucose and insulin in order to assess regulatory mechanisms of the level of citric acid cycle intermediates in skeletal muscle.

Acetate and acetoacetate effected a significant increase in the level of citrate cycle intermediates and accumulation of acetylcarnitine. These changes were accompanied by a reduction in the level of alanine. The concentration of AMP was significantly elevated.

Muscle mitochondria fixed 14CO2 in the presence of pyruvate. The products were identified as malate or citrate when whole and disintegrated mitochondria were used respectively. The fixation was greatly stimulated by acetylcarnitine.

Acetylcarnitine inhibited the production of pyruvate from malate by muscle mitochondria.

Perfusion with 2‐oxoisocaproate and 2‐oxoisovalerate promoted increases in the level of citric cycle intermediates, a drop in both alanine and glutamate, and accumulation of branched‐chain acylcarnitines. 2‐Oxoisocaproate also caused a reduction of alanine released from the muscle.

Perfusion with leucine and valine did not change the concentration of citric acid cycle intermediates, but elevated glutamate and still more the concentration of alanine.

It is concluded that citric cycle intermediate level in the perfused resting muscle is modified by

a) conditions which change the concentration of acetyl‐CoA and thereby modify the rate of pyruvate carboxylation and decarboxylation of malate via malic enzyme

b) conditions which change the concentration of pyruvate cause changes in alanine and cycle intermediates in the same direction via transmination reactions

c) conditions which change the concentrations of 2‐oxoacids which are converted to cycle intermediates via oxidation.
Degradation of Abnormal Proteins in Escherichia coli
Authors: KEMSHEAD John T. , HIPKISS Alan R.
Published: March 2005

Escherichia coli alkaline phosphatase has been purified and modified by either carboxymethylation or treatment with cyanogen bromide (CNBr). Only the CNBr‐treated protein was degradable in an E. coli cell extract. Separation of the CNBr cleavage products by gel filtration in non‐denaturing conditions gave rise to a number of oligomeric complexes, of which only those of molecular weight less than approximately 29000 were degradable in E. coli cell‐free extracts. Carboxymethylation of the non‐degradable complexes (greater than 29000 molecular weight) resulted in the formation of some complexes of less than 29000 molecular weight: such newly formed complexes were degradable by E. coli cell‐free extracts. It is suggested that E. coli cell‐free extracts may contain a protease/peptidase system which is active against peptide complexes below 29000 molecular weight, but inactive against peptide oligomers of greater molecular weight.
Studies on the Synthesis of Casein Messenger RNA during Pregnancy in the Rabbit
Authors: SHUSTER Robert C. , HOUDEBINE Louis‐Marie , GAYE Pierre
Published: March 2005

The quantity of casein mRNA in the rabbit mammary gland was assayed during the course of pregnancy, by translation of casein mRNA in a reticulocyte lysate and by hybridization to DNA complementary to purified casein mRNA. Both methods indicated that the major increases in the concentration of casein mRNA in both polysomal and total cellular RNA occurred between the 18th and 25th day of pregnancy. The change in casein mRNA concentration during this period coincided with a sharp rise in casein synthesis in mammary gland explants suggesting that the levels of casein mRNA determine the rate of casein synthesis in the mammary gland.

The sensitivity of the hybridization assay made it possible to detect the presence of casein mRNA in virgin animals and during the very early stages of pregnancy. At day 5, casein mRNA was found associated with polysomes indicating that there was probably some casein synthesis at this early stage of gestation. These results suggest that the hormones controlling lactogenesis in the rabbit may function by augmenting the rate of casein mRNA synthesis rather than initiating transcription of previously inactive genes.
Isolation and Properties of an Inhibitor of Escherichia coli RNA Polymerasè
Authors: CRIMALDI Albert , IHLER Garret
Published: March 2005

An inhibitor of the Escherichia coli RNA polymerase has been isolated from E. coli and has been partially characterized. The inhibitor, a polypeptide of molecular weight 70000, acts to shut off RNA synthesis at about the time that the first round of RNA synthesis is over, preventing any further RNA synthesis. The inhibitor apparently does not recognize specific termination sequences in the DNA template, since it works equally well with double‐stranded DNA, single‐stranded DNA and poly[d(A‐T)] as templates for RNA synthesis, and because the RNA molecules synthesized from T7 DNA appear to be terminated at the same site either in the presence or absence of the inhibitor. Several experiments indirectly indicate that the inhibitor may reversibly bind to the RNA polymerase at the termination step, in a ratio of approximately one inhibitor molecule per polymerase molecule.
Acetyl‐Coenzyme‐A Carboxylase of Candida lipolytica
Authors: MISHINA Masayoshi , KAMIRYO Tatsuyuki , NUMA Shosaku , TANAKA Atsuo , FUKUI Saburo
Published: March 2005

Acetyl‐coenzyme‐A carboxylase has been isolated in homogeneous form from Candida lipolytica. The homogeneity of the enzyme preparation is evidenced by analytical ultracentrifugation, dodecylsulfate‐polyacrylamide gel electrophoresis and Ouchterlony double‐diffusion analysis. The purified enzyme exhibits a specific activity of 8.0 U/mg protein at 25°C and contains 1 mol biotin/263000 g protein. The sedimentation coefficient (S20, w) of the enzyme is 18 S. It has been shown by dodecyl‐sulfate‐polyacrylamide gel electrophoresis that the enzyme possesses only one kind of subunit with a molecular weight of 230000. This finding, together with the biotin content, indicates that the C. lipolytica enzyme has a highly integrated subunit structure.

The C. lipolytica enzyme is very labile, but is stabilized by glycerol. The enzyme is markedly activated by poly(ethyleneglycol), the activation being due principally to a decrease in the Km values for substrates. Even in the presence of this activator, the Km value for acetyl‐CoA of the C. lipolytica enzyme is much higher than that of the enzyme from Saccharomyces cerevisiae and animal tissues. The C. lipolytica enzyme, unlike the enzyme from animal tissues, is not activated by citrate.
ChorismateMutase/Prephenate Dehydratase from Escherichia coli K12
Authors: GETHING Mary‐Jane H. , DAVIDSON Barrie E.
Published: March 2005

On the basis of amino acid composition, tryptic fingerprints and the determination of amino acid sequences around the four cysteine residues, it can be concluded that chorismate mutase/prephenate dehydratase from Escherichia coli K12 consists of identical, or closely similar subunits. It follows from this that the mutase and dehydratase activities of the enzyme are probably catalysed on the one subunit.
Nucleolar DNA‐Dependent RNA Polymerase from Rat Liver
Authors: MATSUI Takashi , ONISHI Toshio , MURAMATSU Masami
Published: March 2005

DNA‐dependent RNA polymerase I (or A) was purified from rat liver nucleoli. DNA was effectively removed from the solubilized enzyme with a defined concentration of polyethyleneglycol. The enzyme was purified further with successive DEAE‐Sephadex and phosphocellulose column chromatography followed by glycerol gradient centrifugation. The procedure yielded an electro‐phoretically homogeneous enzyme with a specific activity 400 times that of the nucleolar extracts. The recovery of the activity was approximately 20%.

The RNA polymerase I eluted as a single peak from DEAE‐Sephadex was separated into two distinct peaks by a phosphocellulose column. The first peak eluting at about 0.12 M ammonium sulfate was designated as RNA polymerase IA and the second peak eluting at about 0.18 M as RNA polymerase IB. In normal rat liver nucleoli IA enzyme comprised approximately 20% of the total RNA polymerase I activity and the IB enzyme comprised approximately 80%.

On sodium dodecyl sulfate polyacrylamide gel electrophoresis, enzyme IB contained five subunits with molecular weights of 195000 (a), 130 000 (b), 65 000 (c), 40 000 (d), and 19 000 (e) at nearly equimolar amounts. The calculated molecular weight of the enzyme (449000) agreed well with that predicted from the sedimentation coefficient of the enzyme. Enzyme IA contained identical subunits except that subunit c was absent. Preliminary studies could not demonstrate any significant differences in template specificity between IA and IB enzyme.
Nucleolar DNA‐Dependent RNA Polymerase from Rat Liver
Authors: MATSUI Takashi , ONISHI Toshio , MURAMATSU Masami
Published: March 2005

RNA polymerase I (or A) was extracted from nuclear, nucleolar and nucleoplasmic fractions, and resolved into IA and IB forms on a phosphocellulose column. During the course of cyclo‐heximide treatment, the activity of RNA polymerase IB decreased in the nucleoli with concomitant increase in the nucleoplasmic fraction, suggesting strongly that cycloheximide induced specific leakage of IB enzyme from the nucleolus. The activity of IA enzyme did not change in the nucleoli.

When nucleoli were incubated in the presence of actinomycin D, all the IA enzyme activity and approximately 30% of the total IB enzyme activity were released in the incubation medium, whereas 70% of IB activity remained associated with the nucleolar pellet where no IA activity was detected. The enzyme which was released into the incubation medium was tentatively designated as free or unbound RNA polymerase I and that which was associated with the nucleolar pellet as template‐bound enzyme. During the treatment with cycloheximide, the activity of bound enzyme, which contained exclusively IB form, decreased rapidly, with kinetics almost identical to that of nucleolar RNA synthesis in vivo. The activity of free enzyme did not change appreciably.

At 2 h after partial hepatectomy, IB enzyme activity in the free RNA polymerase fraction increased to almost twice the control, while the bound enzyme activity did not increase appreciably until 4 h of regeneration. Enhancement of nucleolar RNA synthesis in vivo was not apparent at 2 h but became significant by 4 h after partial hepatectomy.

These results strongly suggest that (a) the above‐mentioned procedure is actually fractionating RNA polymerase I into free and bound forms, (b) RNA polymerase IB is the transcriptionally active form in vivo, (c) RNA polymerase IB exists in excess in the nucleoli, but the amount of bound IB molecules, which are engaged in transcription in vivo, must be determined by some other factor(s) than the mere concentration of IB enzyme in the nucleolus, and (d) IA form is not an artifact of isolation but is always present in vivo at a certain amount, although the exact nature of this molecule is not known at present.
Excretion of Taurocholate from Isolated Hepatocytes
Authors: SCHWARZ Leslie R. , SCHWENK Michael , PFAFF Erich , GREIM Helmut
Published: March 2005

Efflux of taurocholate from isolated rat hepatocytes was studied to characterize the mechanism of bile acid secretion. Cells were incubated with taurocholate for 15 min. The amount of the intracellularly accumulated bile acid was directly related to the concentration in the medium. Transfer of the loaded cells from the incubation medium to a medium without taurocholate led to taurocholate efflux.

Efflux was saturable, its activation energy amounted to 12 kcal/mol (50 kJ). It was strongly inhibited by the metabolic inhibitor antimycin A and to a lesser extend by the uncoupler carbonyl‐cyanide‐m‐chlorophenylhydrazone. Dinitrofluorobenzene and mersalyl, reagents which react with amino acids, inhibited efflux by about 30% when applied at concentrations of 50 μM. Ouabain increased the rate of efflux.

The observations indicate that secretory functions are maintained in isolated liver cells.
Histone‐Hydrocarbon Interaction. Partition of Histones in Aqueous Two‐Phase Systems Containing Poly(ethylene glycol)‐Bound Hydrocarbons
Authors: AXELSSON Claes‐Göran , SHANBHAG Vithaldas P.
Published: March 2005

The hydrophobic properties of histones have been examined with help of the two‐phase partition technique using dextran‐poly(ethylene glycol)‐water systems. We have found that different fatty acid esters of poly(ethylene glycol) interact with total histones in a manner similar to proteins of the type β‐lactoglobulin and serum albumins. Thus the maximum interaction occurs when the fatty acid contains 16–18 carbon atoms. With less than eight carbon atoms in the polymer‐bound fatty acid, no histone‐hydrocarbon interaction is observed. The interaction of the five individual histone fractions with palmitate depends on the type of salt used and on its concentration. We suggest that the histones can be divided into three groups with decreasing hydrophobic properties: H3, H2a > H4, H2b > H1.
Thermodynamic Studies on the Specificity of l‐Isoleucine · tRNA Ligase of Escherichia coli MRE 600
Authors: HINZ Hans‐Jürgen , WEBER Klaus , FLOSSDORF Josef , KULA Maria‐Regina
Published: March 2005

The association enthalpies, δHa, involved in the reactions between l‐isoleucine:tRNA ligase (AMP‐forming) from Escherichia coli MRE 600 (EC 6.1.1.5) and various amino acids have been determined calorimetrically in 50 mM potassium phosphate buffer, at pH 7.5, in the presence of 1 mM dithioerythritol. The δHa values for binding of l‐isoleucine, l‐leucine, l‐valine, l‐norvaline and l‐2‐amino‐3S,4‐dimethyl pentanoic acid agree within the limits of experimental error in magnitude (−3.7 ± 0.9 kcal mol−1 or −15.5 ± 3.8 kJ mol−1 at 25°C) and variation with temperature (δcp=−430 ± 20 cal mol−1 K−1 or 1799 ± 84 J mol−1 K−1). In view of the large differences in the equilibrium constants for the corresponding binding equilibria, the identical association enthalpies suggest that the enthalpic contribution to the Gibbs free energy of binding, δGa, cannot be responsible for the specificity of the interaction of the enzyme with the amino acids. It has rather to be inferred that the entropic term, δSa, is decisive in discriminating the correct amino acid. Analogous calorimetric binding studies on the reaction between l‐isoleucinol and the enzyme suggest that the absence of the carboxyl group renders the association enthalpy more positive (by 4–5 kcal mol−1 or 16.7–20.9 kJ mol−1) with respect to that of the amino acids. The variation with temperature of the δHa values, however, practically parallels that of the amino acids.
Isolation and Characterization of Hydrophobic Proteins (H Proteins) in the Membrane Fraction of Bacillus subtilis
Authors: KUSAKA Iwao , HAYAKAWA Kou , KANAI Keiko , FUKUI Sakuzo
Published: March 2005

Cytoplasmic membranes of Bacillus subtilis, grown in complex medium containing glucose, were fractionated into three membrane subfractions [light band (1.155–1.158 g/cm3); medium band (1.181–1.183 g/cm3); heavy band (1.21–1.25 g/cm3)] by sucrose density gradient centrifugation. Among these subfractions, the light and medium bands consisted mainly of membranes but the heavy band consisted of an irregular arrangement or aggregate of small globular protein components of 5–8 nm in diameter. We named this H‐protein.

H‐protein formed trilamellar unit membrane structure when combined with lipid. In pulse‐labeling and pulse‐chase experiments with radioactive leucine, it was found that H‐protein consisted of the newest membrane protein synthesized in the cells and the label incorporated into H‐protein was shifted into light and medium band of the membranes during the chase.

Cytochromes were not found in H‐protein. However, when H‐protein was incubated with haem α and protohaem, these compounds were incorporated into the apoproteins of the cytochromes present in H‐protein and form cytochromes a and b. Cytochromes were also formed in H‐protein which were isolated from the cells grown in the presence of haemin (haemin‐grown H protein).

Succinate dehydrogenase activity was increased about 4‐fold by combining H‐protein or haemin‐grown H‐protein with lipid. H‐protein had no cytochrome oxidase activity; however, haemin‐grown H protein was found to have some of the activity and this was increased about 4‐fold by combining the protein with lipid. Haemin‐grown H protein was also found to form succinate: cytochrome c oxidoreductase when combined with lipid and vitamin K2. On the other hand, succinate oxidase was required for the addition of lipid, vitamin K2 and cytochrome c. NADH oxidase was also found in haemin‐grown H protein and was activated about 9‐fold in constituted reaction systems. Vesicles formed by haemin‐grown H protein and lipid, could accumulate alanine and proline by addition of NADH or reduced phenazine methosulfate. Alanine and proline was also accumulated into the vesicles when transport energy was supplied as a membrane potential introduced by K+‐diffusion via valinomycin.

These results would indicate that H‐protein contains the apoprotein of cytochromes, and a carrier involved in the active transport of alanine and proline.
An Assessment of Some of the Methods Available for the Determination of Molecular Weights of Proteins as Applied to Aspartate Aminotransferase from Pig Heart
Authors: BANKS Barbara E. C. , DOONAN Shawn , FLOGEL Mirna , PORTER Patricia B. , VERNON Charles A. , WALKER John M. , CROUCH Thomas H. , HALSEY John F. , CHIANCONE Emilia , FASELLA Paolo
Published: March 2005

The isopotential specific volume of cytoplasmic aspartate aminotransferase from pig heart was found to be 0.763 ml g‐1 whereas the value of the apparent specific volume obtained by summation of contributions from each type of amino acid in the protein is 0.735 ml g‐1. Use of the experimentally determined isopotential specific volume largely abolishes the discrepancy between a previously reported value of the molecular weight of the native (dimeric) enzyme and that of the enzyme subunit obtained from its primary structure (46300).

A new non‐empirical method based on quantitative N‐terminal analysis involving radioisotope dilution is described for the determination of subunit molecular weight of proteins. The method is capable of considerable accuracy and sensitivity.

Some of the methods available for the determination of molecular weights and subunit compositions of proteins are discussed.
Difficulties in Determining Accurate Molecular Motion Parameters from Proton Relaxation Enhancement Measurements as Illustrated by the Immunoglobulin G · Gd(III) System
Authors: BURTON Dennis R. , FORSÉN Sture , KARLSTRÖM Gunnar , DWEK Raymond A. , MCLAUGHLIN Alan C. , WAIN‐HOBSON Simon
Published: March 2005

Longitudinal and traverse proton magnetic relaxation rates for water in the hydration sphere of Gd(III) bound to non‐immune rabbit IgG (immunoglobulin G) have been determined over a wide range of frequencies (4–84 MHz) at constant temperature (19 °C) using pulsed nuclear magnetic resonance spectrometry. The rates have also been determined at temperatures between 0 and 40 °C for two frequencies (61 and 84 MHz). The rates were fitted to existing theory using a computer leastsquares procedure. Further computer analysis was then carried out to determine the sensitivity of the best‐fit error to variation in the variable parameters in the theoretical expressions used. These include the water co‐ordination number (q) for which it was found large variations could occur (between approximately 2 and 8) for only small changes in the error of best‐fit. It was concluded that a slow exchange contribution to the relaxation rates was important in deciding which parameters are poorly determined. A rotational correlation time (τR) was obtained which suggested there might be considerable internal motion of the Fe region (C‐terminal half of heavy‐chain dimer) of the IgG molecule. However the possibility of large errors in this value prevented unequivocal conclusions being drawn.
The Dissociation of Flavin Coenzymes from Trypsin‐Solubilized NADPH/Cytochrome c (P‐450) Reductase of Pig‐Liver Microsomes
Authors: TROUT Gordon E.
Published: March 2005

The change in fluorescence emission at 520 nm after excitation at 365 nm was used to investigate the effect of pH and ionic strength on the dissociation of flavin cofactors from microsomal NADPH/cytochrome c (P‐450) reductase.

In the unmodified enzyme both the FAD and FMN moieties appeared to dissociate at a similar rate and followed first‐order kinetics. The rate constant for the dissociation was increased by low pH and high ionic strength, particularly in the range pH 4.4–3.8 (0.02 M acetate buffer) where the rate constants increased 80‐fold.

Modification of the enzyme by treatment with p‐chloromercuribenzoate enhanced the rate of flavin dissociation and, in the region of pH 4, resulted in a biphasic increase in fluorescence consistent with two simultaneous parallel first‐order dissociations. It was concluded that p‐chloromercuribenzoate treatment modified the protein so that the two flavin cofactors dissociated at different rates.

Using the measured rate constants for the dissociations, and the known variation in fluorescence of flavin nucleotides with pH, an analogue computer simulation of the dissociation as well as a manual curve‐fitting procedure showed that the biphasic response could be explained as a simultaneous rapid dissociation of FAD and a slower loss of FMN from the protein.
Magnetic Studies on the Ferri‐haem Undecapeptide of Cytochrome c
Authors: JEHANLI Ahmed M. T. , STOTTER David A. , WILSON Michael T.
Published: March 2005

The pH‐dependence of the magnetic moment of a ferri‐haem undecapeptide, produced by peptic digestion of cytochrome c, has been measured in aqueous solution using a nuclear magnetic resonance method.

Below pH 3 the magnetic moment is consistent with the presence of hydroxo‐bridged dimers of high‐spin iron(III). Above pH 7 the iron(III) is low‐spin, the spin crossover conforming to a simple titration curve with pK 6.3 (n= 1). This transition is discussed with reference to spectrophotometric studies of ligand binding at the haem.
Rhodanese‐Mediated Sulfur Transfer to Succinate Dehydrogenase
Authors: BONOMI Franco , PAGANI Silvia , CERLETTI Paolo , CANNELLA Carlo
Published: March 2005

The interaction of the sulfurtransferase rhodanese (EC 2.8.1.1) with succinate dehydrogenase (EC 1.3.99.1), yeast alcohol dehydrogenase (EC 1.1.1.1) and bovine serum albumin was studied.

Succinate dehydrogenase incorporates the sulfane sulfur of [35S]rhodanese and, in the presence of unlabelled rhodanese, also incorporates that of [35S]thiosulfate. Rhodanese releases most of its transferable sulfur and is re‐loaded in the presence of thiosulfate. Rhodanese undergoes similar modifications with yeast alcohol dehydrogenase but this latter does not bind 35S in amounts comparable to those incorporated in succinate dehydrogenase: nearly all the 35S released by [35S]rhodanese is with low‐molecular‐weight compounds. Bovine serum albumin also binds very little sulfur and [35S]rhodanese present in the reaction mixture does not discharge its radioactive sulfur nor does it take up sulfur from thiosulfate.

Sulfur release from rhodanese appears to depend on the presence of – SH groups in the acceptor protein.

Sulfur incorporated into succinate dehydrogenase was analytically determined as sulfide. A comparison of the optical spectra of succinate dehydrogenase preparations incubated with or without rhodanese indicates that there is an effect of the sulfurtransferase on the iron‐sulfur absorption of the flavoprotein.

The interaction of rhodanese with succinate dehydrogenase greatly decreases the catalytic activity of rhodanese with respect to thiocyanate formation. This is attributed to modifications in rhodanese associated with the reduction of sulfane sulfur to sulfide. Thiosulfate in part protects from this deactivation.

The reconstitutive capacity of succinate dehydrogenase increased in parallel with sulfur incorporated in that enzyme following its interaction with rhodanese.
Ribitol‐Containing Lipopolysaccharides from Proteus mirabilis and Their Serological Relationship
Authors: GMEINER Jobst , MAYER Hubert , FROMME Inge , KOTELKO Krystyna , ZYCH Krystyna
Published: March 2005

Ribitol phosphate was recently identified as a constituent of lipopolysaccharides obtained from Proteus mirabilis strain D52 giving 1:4‐anhydroribitol during acid hydrolysis (Gmeiner, 1975). Two other Proteus mirabilis strains belonging to serogroups O16 and O33 were shown previously to contain an unknown compound X as lipopolysaccharide constituent (Kotelko et al., 1975).

In this report the identification of compound X as 1:4‐anhydroribotol by gas‐liquid chromatography, mass spectrometry and mass fragmentography is described.

Serological investigations using passive hemagglutination, hemagglutination inhibition and semiquantitative precipitin reactions indicate strongly that ribitol plays a role in the serological specificity of the respective lipopolysaccharides.
Lignoceric Acid Biosynthesis in the Developing Brain. Activities of Mitochondria1 Acetyl‐CoA‐Dependent Synthesis and Microsomal Malonyl‐CoA Chain‐Elongating System in Relation to Myelination
Authors: BOURRE Jean‐Marie , PATURNEAU‐JOUAS Marion Y. , DAUDU Odile L. , BAUMANN Nicole A.
Published: March 2005

Age‐related changes in the activities of microsomal and mitochondrial elongating systems have been determined in mouse brain from birth to maturity.

In microsomes, the components necessary for behenyl‐CoA (docosanoyl‐CoA) elongation have been found to be NADPH and malonyl‐CoA. In mitochondria, both NADH and NADPH are used and acetyl‐CoA is the only donor of two‐carbon‐atoms unit. The synthesised fatty acids were identified by thin‐layer and gas chromatography. The specific activity is higher in microsomes than in mitochondria.

In microsomes, the specific activity for malonyl‐CoA incorporation reached a maximum at 15–20 days of age; this peak was not obtained in the Quaking and Jimpy mutants. The increase in enzyme activity (specific activity and total activity per brain) paralleled the myelin deposition. The activity of the mitochondrial system increases regularly during development: it is not correlated to myelination and it is not affected in the Quaking mutant. The interplay between microsomal and mitochondrial elongation systems is studied.
Assymetry of the myo‐Inositol Transport System in Klebsiella aerogenes
Authors: DESHUSSES Jacques , REBER Guido
Published: March 2005

In Klebsiella aerogenes the influx of myo‐inositol proceeds during the steady state at a rate equal to that of efflux.

The kinetic parameters of influx during the steady state are similar to those observed at the initial time of uptake.

Efflux and influx processes share the same transport system.

The ability of other cyclitols to chase the accumulated scyllo‐inositol is dependent on their affinity for the transport system.

A counter transport can be observed in preloaded cells only in poisoned cells and under anaerobiosis.

The efflux process is not temperature‐dependent.

The KT of influx in poisoned cells is larger than that in normal cells.

Although no saturation kinetics of efflux could be observed, it can be inferred that the KT of efflux is at least 50‐fold larger than the KT of influx;

The results suggest that in the transport of myo‐inositol by K. aerogenes energy coupling enhances the affinity of the carrier for the substrate at the outer side of the cytoplasmic membrane by lowering the KT.
Inequivalent Conformational Response of Chironomus Hemoglobins to Ligation with O2 and CO.
Authors: WOLLMER Axel , STEFFENS Guy , BUSE Gerhard
Published: March 2005

For the monomeric hemoglobins III and IV and the dimeric hemoglobins of Chironomus the tryptophan circular dichroism reports different conformational response to binding of different ligands. The fine structure bands at 292 nm are most strongly developed in the unliganded state and considerably reduced in the oxy form. The spectrum of the CO derivative is intermediate but clearly more deoxy‐like. This finding correlates well with the corresponding Bohr effect magnitudes determined by measuring proton release upon ligation.

Infrared difference spectroscopy on O2versus CO derivatives in aqueous solution shows a normal O2 stretching band position at 1107 cm−1 characteristic of asymmetric oxygen binding. The CO stretching band, however, is consistently blue‐shifted by 11–13 cm−1 from the 1951 cm−1 position observed with mammalian hemoglobins, indicating reduced CO binding strength.

Structural factors relevant to an explanation of the observed phenomena are discussed.
CO2‐Mediated Control of Fatty Acid Metabolism in Isolated Hamster Brown‐Fat Cells during Norepinephrine Stimulation
Authors: PETTERSSON Bertil
Published: March 2005

Addition of norepinephrine to isolated hamster brown‐fat cells suspended in Krebs‐Ringer phosphate buffer induces a pronounced, but temporary, increase in respiratory rate.

If Krebs‐Ringer phosphate buffer is bubbled with CO2 prior to the addition of cells and norepinephrine, the respiratory capacity of the cells is further potentrated and most important, the respiration is maintained at a high rate until the medium becomes depleted of oxygen.

This respiratory pattern cannot be obtained in CO2‐bubbled Krebs‐Ringer bicarbonate buffer.

The results indicate that CO2 has a regulatory effect on fatty acid metabolism in isolated hamster brown‐fat cells.
Role of Thyroid Gland on the Peroxidase and lodinating Enzymes of Submaxillary Gland
Authors: CHANDRA Tarasankar , DAS Rakhahari , DATTA Asoke G.
Published: March 2005

The peroxidase (EC 1.11.1.7) and iodinase (EC 1.11.1.8) activities of rat submaxillary gland were found to be increased after thyroidectomy. The enzyme activities were maximal on the seventh day after operation and then decreased slightly. However, the enzyme activities were still more than 100% even 28 days following operation. Administration of thyroxine (10 μg/100 g body weight) prevented the increase. Puromycin, cycloheximide, and actinomycin D, the inhibitors of protein synthesis, as well as thiouracil partially abolished the increase of activities. These results suggest that thyroxine acts as a regulator of the iodinase and peroxidase enzyme(s) of submaxillary gland.
Organization of Sequences in Avian Globin mRNA
Authors: CRAWFORD Robert John , SCOTT Andrew Charles , WELLS Julian Richard E.
Published: March 2005

Formamide polyacrylamide gel electrophoresis shows that chicken globin mRNA contains about 650 nucleotides, and since only 435 of these code for globin, a further 215 are not translated, and their function and position are not known. This work has produced the following conclusions.

45–50 of these untranslated nucleotides are present as poly(A) at the 3′ terminus.

The 3′ untranslated region of chicken globin mRNA is at least 90 nucleotides in length. This minimal estimate is based on data derived from hybridization of defined lengths of chicken globin cDNA to rabbit globin mRNA. The percentage of avian globin cDNA sequences which hybridize to rabbit globin mRNA is directly proportional to the length of the cDNA in each case. This relationship holds for lengths of cDNA from 115 up to 620 nucleotides. The low percentage homology for short cDNA molecules is not due to their being short per se In homologous mRNA excess hybridizations (chicken cDNA/chicken mRNA), all cDNA preparations were completely protected from S1 nuclease digestion.

It is probable that there is greater evolutionary divergence in the 3′ untranslated region of chicken and rabbit globin mRNA when compared with the coding regions of these molecules.

The combined data is used to formulate a regional map of chicken globin mRNA.