bluuebunny/crossref_metadata_2025 huggingface dataset:
An Insoluble Receptor for Oestrogens in the “Residual” Nuclear Proteins of Chick‐Liver
Authors: Lebeau Marie‐Claire , Massol Nelly , Baulieu Etienne‐Emile
Published: March 2005

When chick liver nuclei are incubated at 37°C with [3H]oestradiol in buffer or in liver cytosol, the majority of the incorporated radioactivity is not extractable by high or low salt solutions or by the usual detergents. Total extraction is obtained only by using polar organic solvents, such as methanol. This was also observed when liver minces were incubated with the radioactive hormone and the nuclei were prepared subsequently.

The hormone recovered in this insoluble fraction of the nuclei is bound to a protein component, as determined by treatment with various hydrolytic enzymes. This protein binds oestrogens specifically (oestradiol with a Kd∼ 1 nM at 37°C) and is found in the liver but not in the lung nor the kidney. There is a larger number of oestradiol binding sites in the “residual” nuclear fraction of oestradiol‐treated chicks or laying hens than in immature or male chickens.
Adenylate‐Cyclase Receptors for Adrenergic Neurotransmitters in Rat Cerebral Cortex
Authors: VON Hungen Kern , Roberts Sidney
Published: March 2005

The response of adenylate cyclase systems to putative neurotransmitter agents has been studied in cell‐free preparations from various brain regions of young adult rats. Homogenates of cerebral cortex contained adenylate cyclase systems which were responsive to norepinephrine and dopamine. A large proportion of this catecholamine‐sensitive adenylate cyclase activity was concentrated in the crude mitochondrial fraction. Adenylate cyclase activity in this fraction was also responsive to epinephrine and isoproterenol, showed very small stimulation with histamine and serotonin, and was unresponsive to acetylcholine and 4‐aminobutyric acid. The natural l‐forms of the catecholamines were much more active than the d‐forms. Adrenocorticotrophic hormone, glucagon, thyroid stimulating hormone and luteinizing hormone were without significant effect in the cerebral preparation. Basal values for adenylate cyclase activity and the degree of stimulation by the catecholamines were critically dependent upon appropriate concentrations of ATP, an ATP‐regenerating system, H+, Mg2+ and Ca2+ in the assay medium. Addition of the membrane phospholipids, phosphatidylserine and phosphatidylinositol, enhanced stimulation by the catecholamines. Concentrations of norepinephrine or dopamine as low as 0.005 mM produced significant increases in adenylate cyclase activity. The maximum response to norepinephrine was two to three times that with dopamine. The response of cerebral adenylate cyclase to 0.05 mM norepinephrine was strongly blocked by concentrations (0.1 mM) of the β‐adrenergic blocking agents, propranolol and dichloroisoproterenol, which had no effect on the response to 0.05 mM dopamine. Haloperidol eliminated the dopamine response at very low concentration (0.01 mM). The data suggest that norepinephrine and dopamine activate adenylate cyclase systems in rat cerebral cortex by reacting with specific receptors which have the properties of β‐adrenergic and dopaminergic receptors, respectively.
Relationships between Intracellular Contents of Nucleotides and 5‐Phosphoribosyl 1‐Pyrophosphate in Escherichia coli
Authors: Bagnara Aldo S. , Finch Lloyd R.
Published: March 2005

The intracellular contents of nucleotides and 5‐phosphoribosyl 1‐pyrophosphate (P‐Rib‐PP) in cells of Escherichia coli were quantitatively determined under various conditions of growth. When the growth rate was limited by the carbon source available in the growth medium the intracellular content of each of ATP, CTP, GTP, UTP, ADP and GDP was proportional to the growth rate. The intracellular content of P‐Rib‐PP showed a similar, though stronger dependence, suggesting that at the slower growth rates, the availability of P‐Rib‐PP could become limiting for nucleotide biosynthesis.

Several treatments which are known to specifically stimulate the rate of net RNA synthesis in E. coli had a common effect in causing a transient depletion of intracellular ribonucleoside triphosphates. For each treatment, the depletion of triphosphates coincided with transiently elevated levels of P‐Rib‐PP, despite increases in the ADP:ATP ratio, an effect which might be expected to inhibit P‐Rib‐PP synthetase activity in E. coli (Atkinson and Fall [15], Klungsøyr et al. [16]). Explanations of this apparent anomaly might be made on the basis of the properties of P‐Rib‐PP synthetase from Salmonella typhimurium, (Switzer, [22], Switzer and Sogin, [21]). One explanation is that the enzyme is regulated by total concentrations of nucleotides so that decreases in nucleoside triphosphate concentrations result in stimulation of the reaction. Another is that the decrease in nucleoside triphosphate concentrations releases from chelation a pulse of free Mg2+ which stimulates the synthetase. And thirdly, the concentration of the substrate, ribose 5‐phosphate, might be initially high enough to exert its inhibitory effect on the enzyme, so that stimulation of the reaction can occur if the substrate concentration falls.
Les stérols et leurs précurseurs chez Astasia longa Pringsheim
Authors: Rohmer Michel , Brandt Roger D.
Published: March 2005

Sterols and sterol precursors of Astasia longa were characterised by thin‐layer chromatography, gas chromatography and mass spectrometry. Sterols were found in liposoluble fractions in the form of free alcohols, esters or glycosides, in hydrosoluble fractions and in the culture medium in the form of free alcohols and esters. Triterpenes and 4α‐methyl sterols were encountered only in the form of free alcohols in both liposoluble and hydrosoluble fractions of the cell.

The following compounds have been found in cell cultures: cycloartenol, 24‐methylene‐cycloartanol, cycloeucalenol, obtusifoliol, 4α‐methyl‐Δ8‐ergostenol, Δ7‐cholestenol, cholesterol, Δ7‐ergostenol, Δ5‐ergostenol, isofucosterol, Δ7‐chondrillastenol, clionasterol and poriferasterol.

In one culture, some new compounds were accumulated for unknown reasons: 4α,14α‐di‐methyl‐(24S)‐Δ8,25(26)‐stigmastadienol, 14α‐methyl‐(24S)‐Δ8,25(26)‐stigmastadienol, 24‐methylene pollinastanol and 24‐methylpollinastanol.

The sterol composition of Astasia longa is consistent with the hypothesis that this alga is a natural mutant of Euglena which has lost all potential for photosynthesis. The presence of cycloartenol and its metabolites shows that in this nonphotosynthesizing organism sterol biosynthesis follows a plant‐type pathway. It is also suggested that several biosynthetic pathways coexist and are expressed differently depending upon the physiological condition of the alga.
The Neutral Proteases of Human Granulocytes
Authors: Ohlsson Kjell , Olsson Inge
Published: March 2005

Two electrophoretic variants of a collagenolytic enzyme from the cytoplasmic lysosome‐like granules of leukaemic myeloid cells were purified by chromatography of the granule extract on Sephadex G‐75, Bio Rex 70 and collagen–Sepharose, and electrophoresis on polyacrylamide gel. The granulocyte collagenases degraded native collagen in solution at about neutral pH with the production of the two specific fragments. They also acted on other substrates, such as fibrinogen and proteoglycans. The granulocyte collagenases were inhibited by α1‐antitrypsin and by α2‐macroglobulin. The two collagenases gave reactions of identity on Ouchterlony immunodifusion and crossed immunoelectrophoresis. Amino acid analyses suggested that the collagenases differed only in respect of a few residues. Ultracentrifugation indicated that both proteins were homogeneous and had an s020,w of 4.5 S. Thin‐layer chromatography on Sephadex G‐150 suggested that both collagenases had a molecular weight of 76000. Electrophoresis after treatment of the collagenases with sodium dodecylsulfate revealed two components with molecular weights of 42000 and 33000, respectively, suggesting that the native enzymes are built up of to two subunits.
The Actinidin‐Catalysed Hydrolysis of N‐α‐Benzyloxycarbonyl‐l‐lysine p‐Nitrophenyl Ester
Authors: Boland Michael J. , Hardman Michael J.
Published: March 2005

The hydrolysis of N‐α‐benzyloxycarbonyl‐lysine p‐nitrophenyl ester catalysed by the thiol protease, actinidin, from Actinidia chinensis has been studied under steady state and non‐steady state ([E]0 > [S]0) conditions.

The Michaelis constant Km is dependent on ionising groups of pKa 3.75 and 8.1 and lowest at neutral pH. The catalytic centre activity, kcat, is almost pH independent below pH 7.0 but increases greatly at high pH with a pKa of 8.1.

A biphasic reaction was observed under conditions of [E]0 > [S]0 indicating that the simple acylation‐deacylation mechanism does not apply and at least one additional step is necessary. The rapid phase of reaction involves production of p‐nitrophenol bound to an acylated enzyme; p‐nitrophenol is released in the slow phase.

From the amplitude and rate constant of the rapid phase, and the steady state parameters, the rate and equilibrium constants of some of the steps of the reaction were determined.

The biphasic behaviour under conditions of excess enzyme was found from pH 5.0 to 7.9 but the reaction was monophasic at pH 4.0.
Effect of pH on Electron‐Paramagnetic‐Resonance Spectra of Ceruloplasmin
Authors: Gunnarsson Per‐Olov , Nylén Ulf , Pettersson Gösta
Published: March 2005

Analysis of electron paramagnetic resonance spectra of human and porcine ceruloplasmin indicated the presence of one type 2 and two type 1 paramagnetic copper ions per enzyme molecule. The two type 1 copper ions are spectrometrically distinguishable, exhibiting different hyper‐fine splittings, and in the case of human ceruloplasmin respond differently to variation of pH over the range 4.5–7.0. Comparative experiments carried out with porcine enzyme provided evidence that there is no direct relationship between observed pH‐dependent changes of the type 1 copper signal in spectra of human ceruloplasmin and the variation with pH of the rate of reduction of the enzyme.
Cyclopeptin und Dehydrocyclopeptin
Authors: Framm Joachim , Nover Lutz , Azzouny Aida El , Richter Heinrich , Winter Klaus , Werner Sabine , Luckner Martin
Published: March 2005

Feeding tritium labeled N‐methyl phenylalanine and a number of N‐methylated and unmethylated cyclic and acyclic anthranilic‐acid‐phenylalanine‐peptides, the biosynthesis of the benzodiazepine alkaloids cyclopenin and cyclopenol by Penicillium cyclopium was investigated. The results show that only the two N‐methylated cyclic peptides, cyclo‐(An‐MePhe) and 3,10‐dehydro‐cyclo‐(An‐MePhe) were incorporated without degradation and marked depression of the incorporation rate by non‐radioactive phenylalanine.

The negative results with the acyclic dipeptides, N‐methylphenylalanine and cyclo‐(An‐Phe), some of which should be intermediates in the biosynthesis of cyclo‐(An‐MePhe), indicate that the formation of the latter proceeds via enzyme bound intermediates as it was found also for more complex cyclic peptide derivatives like gramicidine S. Aside the formation of the peptide bonds, the N‐methylation seems to be part of the reaction sequence on this enzyme complex, too.

By looking for cyclo‐(An‐MePhe) and its dehydroderivative in cultures of P. cyclopium, two hitherto unknown benzodiazepine derivatives were isolated. Their structures were elucidated by physico‐chemical methods and comparison with synthetic material. The compounds were called cyclopeptine and dehydrocyclopeptine and identified as cyclo‐[anthranoyl‐(S)‐N‐methyl phenylalanyl] and 3,10‐trans‐dehydro‐cyclo‐[anthranoyl‐N‐methyl phenylalanyl] respectively. m‐Hydroxylated derivatives of both substances, the structure of which would correspond to that of cyclopend were not detectable.

The biosyntheses of cyclopeptine and dehydrocyclopeptine from phenylalanine and the coordinated formation of both compounds together with cyclopenin and cyclopenol during the mould's idiophase further prove that they are true intermediates in the synthesis of benzodiazepine alkaloids in P. cyclopium. Cyclopeptine can be reversibly transformed into dehydrocyclopeptine.

According to these and former experimental results the biosynthesis of the benzodiazepine and quinoline alkaloids of P. cyclopium seems to proceed in the following manner: The precursor amino acids (S)‐phenylalanine and anthranilic acid together with the methylgroup of methionine form an enzyme bound acyclic dipeptide intermediate (presumably an anthranoyl‐(S)‐N‐methyl phenylalanyl thioester) which undergoes cyclization to the benzodiazepin derivatives to release cyclopeptin from the enzyme. Cyclopeptin with NAD as hydrogen acceptor forms dehydrocyclopeptin in the presence of cyclopeptine dehydrogenase. Dehydrocyclopeptine by an epoxydation requiring molecular oxygen is transformed to cyclopenin, which upon m‐hydroxylation yields cyclopenol. Both, cyclopenin and cyclopenol, in the conidiospores are converted by cyclopenase to the quinoline derivatives, viridicatin and viridicatol.
Changes in the Activity of Inorganic Pyrophosphatase in a Temperature‐Sensitive DNA‐Synthesis Mutant of Escherichia coli
Authors: Heinonen Jukka , Kärenlampi Sirpa
Published: March 2005

CRT 46, a derivative of Escherichia coli K12, has temperature‐sensitive initiation of DNA synthesis. The specific activity of inorganic pyrophosphatase in the cells of CRT46 decreased during 30 min to a new constant level (approximately 2/3 of the initial value), when DNA synthesis was arrested after 1 h at restrictive temperature 42 °C. When the culture was shifted back to permissive temperature 30 °C, the normal specific activity of the enzyme was restored in 2.5 h, while DNA was synthesized with abnormally high rate. Both the decrease and the increase in the activity of the enzyme were prevented, if protein synthesis was arrested by chloramphenical or by amino acid starvation. The activity of inorganic pyrophosphatase was stable at all temperatures up to 80 °C in the extracts of the mutant strain. Therefore the observed changes in the activity of the enzyme were evidently due to the corresponding changes in the differential rate of synthesis of the enzyme.

In the cultures of the wild‐type parent strain Escherichia coli CR34 the differential rate of synthesis of inorganic pyrophosphatase was similar both at 30 °C and at 42 °C. Valyl‐tRNA synthetase was produced normally by both strains at both temperatures.

The synthesis of inorganic pyrophosphatase continued normally, if DNA synthesis was prevented by nalidixic acid or thymine starvation at 30 °C in the cultures of CRT46 or both at 30 °C and at 42 °C in the cultures of CR34.

By inhibiting the initiation of new rounds of DNA replication by amino acid starvation or the proceeding of DNA replication by nalidixic acid at 42 °C it was observed that the defect in the synthesis of inorganic pyrophosphatase appeared only when the rounds of DNA replication had reached the end and the temperature was restrictive.

In the cultures of spontaneous revertants the differential rate of synthesis of the enzyme slowed down immediately after the shift to 42 °C, although DNA synthesis continued normally. Another derivative of CR34, BT313, which is temperature‐sensitive in the DNA synthesis, produced the enzyme with the lower differential rate both at 30 °C and at 42 °C.

The results show that, although the defect in the synthesis of inorganic pyrophosphatase is somehow dependent on the defect in DNA synthesis those two phenomena are separable. Possible divA mutation, which causes the formation of DNA‐less minicells in the cultures of CRT46 at 42 °C is also responsible for the abnormal synthesis of the enzyme.
Primary Structure of Bovine Growth Hormone
Authors: Santomé José A. , Dellacha Juan M. , Paladini Alejandro C. , Peña Clara , Biscoglio Mirtha J. , Daurat Silvia T. , Poskus Edgardo , Wolfenstein Carlota E. M.
Published: March 2005

The study of the structure of the peptides arising from native, oxidized or reduced and maleinized bovine growth hormone on incubation with trypsin, chymotrypsin and pepsin is reported.

The data obtained permitted the assembly of a unique sequence of amino acids for the poly‐peptide chain of the protein.

Various corrections and one addition to the sequence previously communicated are made.
The Subunit Structure of Yeast Pyruvate Kinase
Authors: BORNMANN Lothar , HESS Benno
Published: March 2005

Yeast pyruvate kinase (Saccharomyces carlsbergensis) contains four identical subunits based on the following observations.

1. Cyanogen bromide cleavage yields eight peptides as expected for identical subunits from the amino acid composition.

2. No free N‐terminal residue could be detected. Upon acid hydrolysis 1 mole of acetate is liberated per mole of subunit. Differential hydrazinolysis indicates that anN‐acetylated terminus of the subunit is the source of the acetate molecule.

3. Cleavage with carboxypeptidase A in the presence of sodium dodecylsulphate revealed that valine is the C‐terminal amino acid.
Immunologic Properties of Aα Chain of Human Fibrinogen
Authors: LEFVERT Ann‐Karin , BLOMBÄCK Birger , KUDRYK Bohdan , MURANO Genesio
Published: March 2005

An antisera to carboxymethylated Aα chain of human fibrinogen was prepared in rabbits. After appropriate absorption the antiserum reacted in immunodiffusion specifically with Aα chain. The antiserum was used in a radioimmunoassay with carboxymethylated and iodinated Aα chain. The labeled chain was bound (60–100%) by appropriate dilutions of the antiserum. It was found that 0.5 ng unlabeled Aα chain could be detected in this system whereas even large excess of Bβ and γ chain did not compete. Fibrinogen, reduced and alkylated fibrinogen, as well as the NH2‐terminal CNBr fragment of fibrinogen (N‐DSK) were all found to compete with labeled Aα chain.
Initiation Does Not Limit the Rate of Globin Synthesis in Message‐Injected Xenopus Oocytes
Authors: LINGREL Jerry B. , WOODLAND Hugh R.
Published: March 2005

Frog oocytes synthesize haemoglobin when injected with globin mRNA. Studies using this mRNA have shown that Xenopus oocytes have a finite unused translational capacity. When injected with roughly equal amounts of α‐and β‐globin mRNA, five times as many β chains as α chains are made. To help understand the underlying basis for these observations we have investigated the size of the polyribosomes which synthesize α‐and β‐globin after injecting various amounts of rabbit globin mRNA. Both α and β chains appear to be made on the same size of polyribosomes, whether or not the translational machinery of the cell is saturated.

We interpret these results to mean that neither the defiency in α chain synthesis, nor the rate‐limiting component at saturation operates by limiting initiation. We suggest that the agent that limits total globin synthesis is a pre‐initiation “recruitment” or a “stabilisation” factor. The deficiency of α‐globin synthesis can best be explained by a partial block in elongation on the α globin mRNA.
Conformational Changes in Myosin under ATP Hydrolysis
Authors: BLUMENFELD Lev A. , IGNATÉVA Lubov G.
Published: March 2005

During ATP hydrolysis by spin‐labelled myosin the time‐dependent local conformational changes near S1 thiol groups have been observed. In the steady‐state stage of ATP hydrolysis practically all myosin molecules are in conformationally changed state M̃. After reaction, part of the myosin molecules returns to the initial conformational state M, and another part remains in conformation M̃ forming a complex with ADP.
Active Transport of Maltose in Escherichia coli K12
Authors: KELLERMANN Odile , SZMELCMAN Sevec
Published: March 2005

A “periplasmic” maltose binding protein was purified from Escherichia coli. This protein is shown to be under the same positive control as the whole maltose system, and its synthesis is inducible by maltose. This binding protein is coded by malE, one of the three cistrons of the malB region involved in the transport of maltose.

The binding characteristics of this protein, as well as the kinetics of release of bound maltose, suggest that the binding protein can exist in two states differing by their affinity towards maltose. The significance of this result is discussed in view of the role that the protein is believed to play in maltose transport.
Phenylalanyl‐tRNA Synthetase from Yeast
Authors: BERTHER Jean‐Martin , MAYER Peter , DUTLER Hans
Published: March 2005

It has been shown that the initial rates, v, of the formation of aminoacyl‐tRNA, catalyzed by phenylalanine‐tRNA synthetase, do not depend on the concentration of the substrates ATP and phenylalanine in a simple way. In contrast a normal Michaelis‐Menten behaviour has been observed for the substrate tRNA. The reciprocal plots of v versus v/[S] for ATP and phenylalanine are curved and only approach linearity at low and high concentrations of the varied substrate. From these limiting slopes two Km and two V values corresponding to low and high concentrations of ATP and phenylalanine have been determined. Since the reciprocal plots for tRNA are linear, only one Km and V value has been obtained for this substrate. In order to show that this kinetic behaviour is an intrinsic property of the enzyme and not an artifact, a new procedure for the purification of the enzyme has been devised and the purity and stability of the preparation carefully checked. In addition the influence of ammonium ions and of spermine on the initial rates has been measured to verify that the non‐linearity of the reciprocal plots is not the result of the substrate acting as a base. Inhibition experiments with tRNAox (a modified tRNA obtained from native tRNA by periodate oxidation of the 2′:3′‐diol in the 3′‐terminal ribose to the corresponding dialdehyde) at low and high phenylalanine concentrations have shown that it is also not a consequence of two fundamentally different reaction mechanisms being followed at the two concentration ranges. On the basis of this and other evidence it is proposed that two mutually interacting sites are engaged, of which one or both are operative, depending on the concentration of the substrates. Studies of pyrophosphate inhibition at low tRNA concentration have provided evidence that the aminoacylation of tRNA proceeds via two catalytic steps in which aminoacyl‐AMP occurs as an intermediate. At high tRNA concentrations it has been found that this substrate is adding prior to the dissociation of pyrophosphate.
Preparation of Highly Purified Human Chorionic Gonadotrophin by Isoelectric Focusing
Authors: QAZI Mahmood H. , MUKHERJEE Greeta , JAVIDI Kouchek , PALA Alessandro , DICZFALUSY Egon
Published: March 2005

Highly purified human chorionic gonadotrophin, assaying 15000–17000 IU/mg in three different bioassay systems, has been obtained from a crude commercial preparation.

The purification method is a simple and rapid two‐step procedure, based on preparative iso‐electric focusing on sucrose density gradient (Ampholine pH range 3–10), followed by chromatography on Sephadex G‐100. The purified gonadotrophin (mol. wt 65000), obtained from an iso‐electric pH of 4.3, moved as a single band in polyacrylamide gel electrophoresis at a pH 8.3 and showed a single precipitation line in immunoelectrophoresis.

The immunological and biological activities of protein components obtained from various iso‐electric zones during fractionation are reported.
The Mechanism of Water‐Proton Relaxation in Enzyme · Paramagnetic‐Ion Complexes
Authors: JONES Robert , DWEK Raymond A. , FORSÉN Sture
Published: March 2005

The water proton relaxation times, T1 and T2, have been measured in solutions of free Gd(III) ions and in Gd(III) solutions containing lysozyme over a wide range of temperatures (0–40°C) and nuclear frequencies (4–60 MHz) using pulsed nuclear magnetic resonance techniques. The equilibrium between lysozyme and Gd(III) has also been studied as a function of temperature. Water relaxation rates in both the free and enzyme‐bound metal sites have been determined and closely fitted to existing theories over the complete range using a combination of graphical and computing techniques. Values for the water co‐ordination number (q) and the correlation time (τc) for the dipolar relaxation processes have been obtained and the rate processes contributing to τc have been analysed as a function of temperature and frequency. The values obtained for τc in the enzyme complex differ considerably from some earlier estimates. In the aquo‐ion relaxation is dominated by the rotational correlation time (τR) but in the enzyme‐metal complex the electron spin relaxation time (τs) becomes more important than other processes at low frequencies. Possible sources of error in the data and assumptions made in the theoretical treatment are discussed. The implications of the results for mapping studies of lysozyme and other macromolecules, using magnetic resonance techniques, are stressed.
Abnormal Mitochondrial DNA Molecules in Megamitochondria from Cuprizone‐Treated Rats
Authors: GUERINEAU Michel , GUERINEAU Suzanne , GOSSE Charles
Published: March 2005

The formation of giant mitochondria was induced in the livers of rats maintained on a diet containing 1 or 2% cuprizone for two weeks. Mitochondrial DNA was extracted, purified from the livers and observed under the electron microscope. In the control rats, mitochondrial DNA was made of monomers of 5 μm and of catenated oligomers. On the contrary, in the treated rats mitochondrial DNA was made of molecules of 4.7 μm to 7 μm, which could be classified into subpopulations of 4.84 μm, 5.00 μm, 5.17 μm, 5.37 μm. These molecules seem thus differ from each other by deletion of one 0.17‐μm sequence or by addition of one or several copies of this sequence. Size analogy with the D loop is discussed. Catenated oligomers and about 5% unicircular dimers were observed in the treated rats.
Determination and Analysis of the Buffer Capacity of Isolated Chloroplasts in the Light and in the Dark
Authors: WALZ Dieter , GOLDSTEIN Leon , AVRON Mordhay
Published: March 2005

The ability of isolated chloroplasts to bind protons has been measured by means of titration with acid or alkali over the range of pH 3.7 to 10.8. It was found that the total amount of protons bound over the whole range titrated was equal for chloroplasts in the light and in the dark (around 6 μmol H±/mg chlorophyll), whereas the buffer capacity, i.e. the pH‐dependence of the binding, drastically changed. Values for the buffer capacity at different pH‐values in the dark and in the light were calculated from titration curves and estimated directly with similar results. An analysis of these changes shows that most, if not all of the light‐minus‐dark differences can be accounted for by the previously demonstrated large change in the pH gradient across the thylakoids in the light versus that in the dark.
An Immunological Approach to Ferredoxin Specificity and Activity
Authors: TEL‐OR Elisha , AVRON Mordhay
Published: March 2005

1. Antibodies to Swiss‐Chard ferredoxin were prepared and were shown to interact with plant ferredoxin and blue green algal ferredoxin, but not with clostridial ferredoxin.

2. Ferredoxin‐dependent electron transport and photophosphorylation reactions were markedly inhibited by the antibodies, but N‐methylphenazonium‐methosulfate‐mediated phosphorylation was not.

3. The antibody preparation inhibited endogenous cyclic and pseudocyclic photophosphorylations, in the absence of added ferredoxin.
Studies with Ubiquinone‐Depleted Submitochondrial Particles
Authors: NORLING Birgitta , GLAZEK Elzbieta , NELSON B. Dean , ERNSTER Lars
Published: March 2005

1. The incorporation of small amounts of ubiquinone (Q) into pentane‐extracted submitochondrial particles has been studied. A procedure is described which allows nearly quantitative incorporation of as little as 0.2 to 0.3 nmol Q/mg protein, i.e. less than 10% of the Q present in the lyophilized particles prior to extraction.

2. It is shown that both NADH oxidase and succinate oxidase activities can be restored to 100% of that in lyophilized particles by incorporation of 6–8 nmol Q/mg protein. The relative activities of both oxidases show a parallel response to an increase of the Q content of the particles between 0.2 and 22 nmol Q/mg protein, 50% reactivation being obtained at about 2 nmol Q/mg protein.

3. 50 to 60% of the incorporated Q can be reduced by NADH or succinate, independent of the total concentration of Q in the membrane. Similar reduction values were obtained for lyophilized particles prior to extraction, suggesting that incorporated Q functions similarly to that originally present in the particles.

4. Succinate dehydrogenase activity, which is decreased 50% by extraction of Q, can be completely restored upon incorporation of only 1.5 nmol Q/mg protein.

5. Extraction of lyophilized particles with pentane +10% acetone results in a more effective removal of Q, but also in a differential Q requirement for NADH oxidase and succinate oxidase. Thin‐layer chromatography shows that extraction with pentane + acetone removes an additional, unidentified, nonpolar lipid.

6. The present data do not support the belief that succinate oxidase and NADH oxidase communicate with separate pools of Q.
Disulfide Bonds of Toxin II of the Scorpion Androctonus australis Hector
Authors: KOPEYAN Charles , MARTINEZ Gérard , MIRANDA François , ROCHAT Hervé , LISSITZKY Serge
Published: March 2005

The positions of the disulfide bridges in toxin II of Androctonus australis Hector were investigated using proteolytic enzymes. The resulting peptides were separated by column and paper chromatography and high‐voltage electrophoresis. Determination of the amino acid compositions of the cystine‐containing peptides or their oxidized derivatives and, when necessary, dansyl Edman degradation allowed to locate the disulfide bonds in the toxin. The four disulfide bridges were found to link the half‐cystine residues number 12 and 63, 16 and 36, 22 and 46, 26 and 48. These positions are probably the same in all scorpion neurotoxins.
Protein A from Staphylococcus aureus The Effect of Iodination on the Reactivity with Immunoglobulin G
Authors: SJÖHOLM Ingvar , SJÖDIN Torgny
Published: March 2005

Protein A, a cell‐wall protein from Staphylococcus aureus containing 4 residues each of histidine and tyrosine, was iodinated at 0°C and pH 8.6 with about a 35‐fold molar excess of KI3. The incorporation of iodine as followed with 125I was comparatively slow, i.e. 8 atoms were incorporated in about 14 h and 12 atoms in about 12 days. An electrophoretically homogeneous fraction of iodo‐protein A containing 3.5 iodine atoms was isolated and shown to contain only 0.68 residues of mono‐iodotyrosine and 0.12 residues of di‐iodotyrosine. However, iodohistidine was identified in enzymatic digests, and thus most probably the histidine residues are iodinated faster than the tyrosine residues. Catalytic dehalogenation of iodoprotein A in tritium was unsuccessful. The circular dichroism spectrum of iodoprotein A was compared with that of native protein A at different pH values and the changes seen could be fully related to the increased acidic properties of iodotyrosine. The reactivity of protein A towards the Fc part of immunoglobulin G was decreased as a consequence of the iodination. However, the reactivity towards specific anti‐protein A serum was unchanged.
DNA‐DNA Hybridization on Nitrocellulose Filters
Authors: FLAVELL Richard A. , BIRFELDER E. Joyce , SANDERS Johan P. M. , BORST Piet
Published: March 2005

1. The kinetics of DNA‐DNA filter hybridization have been studied using model systems. The results suggest that two limiting reactions occur.

a) When the hybridization reaction is fast (i.e. with a DNA of low complexity such as phage T7 at ⋝ 5 μg DNA per filter) the reaction becomes controlled by the diffusion of DNA to the filter. This is shown by the increase in the reaction rate by shaking the hybridization vials, the inverse dependence of the rate on the molecular weight of the DNA in solution and its independence on the kinetic complexity of the DNA.

b) When the hybridization reaction is slow (i.e. with a DNA of high complexity or, for T7 DNA ⋜ 2.5 μg DNA per filter disc) the reaction is limited by the hybridization reaction itself. This is shown by the inverse relationship of the hybridization rate to the complexity of the DNA and the direct proportionality of initial rate to concentration. The reaction rate is independent of shaking.

2. The hybridization‐limited reaction shows two anomalies. The reaction is independent of the molecular weight of the DNA instead of the expected dependence on the square root of the molecular weight, and the calculated rate constant is nearly 10 times too low. We suggest that these anomalies result from inadequate accessibility of the DNA fixed on the filter.

3. Our results provide practical guide lines to minimize competing renaturation reactions in filter hybridization experiments.
DNA‐DNA Hybridization on Nitrocellulose Filters
Authors: FLAVELL Richard A. , BORST Piet , BIRFELDER E. Joyce
Published: March 2005

1. Comparison of DNA‐DNA filter hybridization using either denaturated duplex DNA or artificial mixtures of the two complementary strands of T7 DNA shows that the competing renaturation reaction has two effects: (a) sequestering of 20–30% of the input DNA as duplex, unavailable for hybridization, and (b) hybridization of partially renatured DNA as a concatenane to the filter‐bound DNA.

2. Experiments using artificial mixtures of 32P‐labelled and 3H‐labelled L and H strands of T7 show that 10–20% of the total hybrid bound results from concatenation. With a 1:1 input ratio of complementary strands in solution, 10% of the input bound to filter DNA of a single strand (e.g. L) is of the homologous strand (i.e. L). This effect is relatively unaffected by the concentration of DNA in solution.

3. Variation of the L:H ratio in solution shows that the strand in excess hybridizes and renatures more efficiently. This effect gives an underestimation of the amount of the minor strand present in solution. The numerical values obtained here are likely to be strongly dependent on the complexity of the DNA and the precise hybridization conditions.

4. The implication of the results for homology experiments and determination of strand specificity in transcription is discussed.
Cell Envelope Composition of Escherichia coli K12: A Comparison of the Cell Poles and the Lateral Wall
Authors: GOODELL Ernest W. , SCHWARZ Uli , TEATHER Ron M.
Published: March 2005

Minicells and filaments of Escherichia coli strain P678–54 provide a convenient means of preparing cell‐envelope fractions that are, respectively, greatly enriched or reduced in material derived from the cell poles. The inner (cytoplasmic) membrane, outer membrane, and sacculus were prepared both from minicells and from filaments purified from a single culture. One protein component and phosphatidylglycerol were found to be enriched in minicell inner membrane.
Sandhoff Disease: Defective Glycosaminoglycan Catabolism in Cultured Fibroblasts and Its Correction by β‐N‐Acetylhexosaminidase
Authors: CANTZ Michael , KRESSE Hans
Published: March 2005

1. Fibroblasts cultured from the skin of patients with Sandhoff disease accumulate excessive amounts of sulfated glycosaminoglycans, probably chondroitin sulfate and dermatan sulfate. In addition, Sandhoff fibroblasts show a lengthened turnover time for these compounds. Only little such abnormality in sulfated glycosaminoglycan metabolism has been found in Tay‐Sachs fibroblasts.

2. The impaired catabolism of glycosaminoglycans in Sandhoff fibroblasts can be normalized, i.e. corrected, by addition of β‐N‐acetylhexosaminidase isozyme A from normal human urine to the culture medium. β‐N‐Acetylhexosaminidase isozyme B from urine is ineffective because it is not taken up by the cells. However, if homogenates of normal or Tay‐Sachs fibroblasts are used as a source of the enzymes, metabolic correction is obtained in both instances. When β‐N‐acetylhexosaminidase isozymes A and B of normal fibroblasts are separated by isoelectric focusing and tested individually, isozyme A exhibits about five times the corrective activity of isozyme B, when compared on the basis of their activity toward the synthetic substrate.

3. Although β‐N‐acetylhexosaminidase isozyme B participates in the degradation of sulfated glycosaminoglycans in cultured cells, only fibroblast homogenates containing isozyme A release N‐acetylglucosamine from [14C]glucosamine‐labelled hyaluronic acid oligosaccharides in vitro.
Effects of Octanoate and Oleate on Energy Metabolism in the Perfused Rat Liver
Authors: DEBEER Luc J. , MANNAERTS Guy , DE SCHEPPER Paul J.
Published: March 2005

The effects of octanoate and oleate were studied in the isolated fasted rat liver perfused without substrate and were compared with the effects of lactate. Measurements were made of hepatic adenine nucleotide content, formation of ketone bodies, urea and glucose and uptake of fatty acids, lactate and oxygen. Calculations of the Krebs cycle activity and the flux through the oxidation at C‐3 were carried out. Comparing the extra oxygen consumption with extra ATP needs after addition of the fatty acids or lactate, ADP: O ratios were estimated for fatty acid oxidation and lactate gluconeogenesis.

1. Octanoate as well as oleate induced a net decrease in ATP and an increase in AMP content of the liver. Total adenine nucleotides were unaltered. ATP:ADP ratios were lowered while the adenylate kinase mass‐action ratio increased.

2. The Krebs cycle activity was suppressed by both fatty acids and enhanced by lactate. Oxidation at C‐3 was strongly stimulated by the fatty acids and was unaffected by lactate.

3. An apparent ADP:O ratio of 2.7 was obtained after lactate addition, indicating a tightly coupled oxidative phosphorylation for the liver preparations used. Octanoate and oleate gave extremely low ratios of near one and near zero respectively.

4. Perfusion with oligomycin caused a severe drop in oxygen uptake by the liver, which was unaltered after addition of fatty acids. Oligomycin added after the fatty acids caused an immediate fall in oxygen uptake to the level observed with oligomycin alone. 2,4‐Dinitrophenol was able to stimulate the oligomycin‐depressed respiration, both in the absence and in the presence of fatty acids. These results indicate that under our experimental conditions the fatty acids had no uncoupling effect and that microsomal fatty acid oxidation had to be minimal.

5. Incorporation of [3H]leucine into proteins in liver and medium remained unchanged after addition of fatty acids, indicating that the low apparent ADP:O ratios are not the result of an enhanced synthesis of proteins.

6. The possible relationship between fatty acid transport, energy‐wasting futile cycles and the low apparent ADP:O ratios observed during fatty acid oxidation is discussed.
On the Use of Affinity Chromatography in Demonstrating the Transfer of Thioester‐Bound d‐Phenylalanine from the Light Enzyme of Gramicidin S Synthetase to an Acceptor Site for this Amino Acid on the Heavy Enzyme
Authors: PASS Lidia , ZIMMER Trine‐Use , LALAND Søren G.
Published: March 2005

When a mixture of the light enzyme of gramicidin S synthetase containing thioester‐bound d‐[14C]phenylalanine and the heavy enzyme is passed through a Sepharose column containing carboxyl‐bound proline (substrate amino acid) as ligand, the two enzymes are retained as the result of the affinity between the heavy enzyme and the ligand and the affinity between the two enzymes. Upon elution and separation of the two enzymes it was demonstrated that the d‐[14C]phenylalanyl groups had been transferred to the heavy enzyme. This transfer did not take place when similar experiments were carried out using as ligands valine, ornithine or leucine which are also substrates for the heavy enzyme. Hence binding of proline to the heavy enzyme is required for transfer to take place. Upon incubation of the heavy enzyme containing the transferred d‐[14C]phenylalanyl group with the light enzyme, ATP and the five unlabelled substrate amino acids, radioactivity is incorporated into gramicidin S. This suggests that in the biosynthesis of gramicidin S, the d‐phenylalanyl group is transferred to an acceptor site on the heavy enzyme and subsequently to the imino group of thioester‐bound proline. In view of what is known about gramicidin S synthesis it seems likely that the acceptor site is a thiol site.
O‐Acetylation and Glucosylation of Lipopolysaccaride in Hybrids between Salmonella Groups B and C2
Authors: RUDÉN Ulla , MÄKELÄ P. Helena
Published: March 2005

The structure of the lipopolysaccharide in two hybrid strains formed between a Salmonella donor of O group B and a Salmonella recipient of O group C2 was analyzed. Both the hybrids had the coredetermining rfa genes from the C2 parent, and the rfb genes, which determine the biosynthesis of the O‐units, from group B. The core was found to be modified by alkali‐labile substituents in a way not found in group B and probably typical of group C2. As expected, the O side chain had O‐acetylabequose, (O factor 5) when the group B oafA genes were present, and d‐glucose linked to C‐4 of d‐galactose (O factor 122) when the group B (or D) oafR,E,F genes were present. Furthermore the terminal d‐glucose was O‐acetylated at C‐2. The l‐rhamnose of the side chain as well as its d‐galactose were found in both strains to carry an alkali‐labile group apparently corresponding to group C2‐type modifications. It was inferred that the genes determining these modifications were not alleles of the known oaf genes of group B. The previous conclusion, that the lipopolysaccharide‐modifying genes function independently to modify, e.g. by O‐acetylation or glucosylation, any sort of lipopolysaccharide offered to them, provided this has suitable attachment sites for the substituents, gets further support from these results.
The Flux of Intermediates and Products in Aromatization of C19 Steroids by Human Placental Microsomes
Authors: BRASELTON W. Emmett , ENGEL Lewis L. , ORR James C.
Published: March 2005

The appearance of intermediates and products in the transformation of 4‐androstene‐3,17‐dione and testosterone to estrone and 17β‐estradiol by human placental microsomes was studied. The variables investigated were time, temperature and the concentrations of reduced pyridine nucleotides and of testosterone. The relative amounts of metabolites were markedly influenced by all of the factors studied except temperature, although the reaction velocity at 37 °C was about twice that at 27 °C. Oxidoreduction at C‐17 in starting material, intermediates and products was examined using a mixture of [4‐14C]testosterone and [7β, 17α‐2H2]testosterone as substrate. Oxidation and reduction at C‐17 was most prominent at the beginning and the end of the reaction sequence. 17α‐Hydroxy‐5α‐androst‐1‐en‐3‐one was found to be an inhibitor of the C19 17β‐hydroxysteroid dehydrogenase activity of human placental microsomes.
Depression of Reducing‐Equivalent Translocation from Substrate to Oxygen in Hepatic Metabolism of Sorbitol or Glycerol in Hypothyroidism
Authors: WERNER Harold V.
Published: March 2005

The metabolism of sorbitol or glycerol in isolated liver cells from hypothyroid rats was studied in order to determine whether the rate of reducing‐equivalent translocation limits hepatic metabolism in a hypometabolic state. Rates of utilization of these substrates were compared with analogous data in euthyroid animals. When a saturating concentration of sorbitol or glycerol was added to the cells, gluconeogenic rates were only 54% and 29%, respectively, of the values in cells from euthyroid rats. Reducing‐equivalent translocation to O2, as well as oxygen consumption by the cells, was depressed in a similar fashion. However, addition of pyruvate, acting as a cytoplasmic hydrogen acceptor, increased the utilization of sorbitol by 1.75 μmol. g−1. min−1 and that of glycerol by 0.50 μmol. g−1. min−1. This maneuver restored sorbitol utilization equal to rates in controls, whereas it partially corrected the depression in glycerol metabolism resulting from hypothyroidism. It was inferred from the data that the disposal of hydrogen generated in the cytoplasm is a limiting factor in the metabolism of sorbitol or glycerol in hypothyroid animals. At the same time rates of gluconeogenesis from fructose or dihydroxyacetone, which utilize similar pathways, were not depressed. It was concluded that the enzymes of the gluconeogenic route are not responsible for the diminished metabolism of sorbitol or glycerol in hypothyroidism; rather a diminished capacity for hydrogen traslocation between cell compartments results in lowered rates of substrate oxidation, and subsequently, decreased rates of gluconeogenesis.

Pathways of hydrogen translocation that are depressed in hypothyroidism and responsible for the decreased utilization of sorbitol or glycerol were investigated by the use of specific inhibitors. In the hypothyroid state hydrogen translocation from each substrate was depressed by 0.91–0.96 μmol. g−1. min−1. It was calculated that 82% and 88% of the depressed metabolism of sorbitol or glycerol, respectively, was due to decreased activity of a pathway insensitive to rotenone and sensitive to antimycin, most likely involving mitochondrial glycerol‐phosphate dehydrogenase. Most of the remaining loss was attributed to a diminished capacity of shuttles which were sensitive to fluoromalate and had malate dehydrogenase as a component.

These studies support the concept that thyroid hormone may play an important role in the regulation of hepatic metabolism by modulating the capacities of the shuttles which translocate reducingequivalents between cell compartments.
Interaction of Ganglioside GGtet1 and Its Derivatives with Choleragen
Authors: STAERK Joseph , RONNEBERGER Hans Joerg , WIEGANDT Herbert , ZIEGLER Wolfgang
Published: March 2005

It was confirmed that ganglioside GGtet1, i.e. Galβ1 → 3GalNAcβ1 → 4[NeuNAcα2 → 3]Galβ1 → 4Glc‐ceramide, specifically interacts with choleragen (cholera‐cxoenterotoxin) as shown by precipitate formation and inhibition of the toxicity. The isolated carbohydrate moiety of ganglioside GGtet1. i.e. GGtet1 minus ceramide, neither precipitated choleragen nor interferred with the reaction between the toxin and its antibody. However, polyacrylamide gel electrophoresis revealed a specific interaction between the sialo‐oligosaccharide and choleragen. Identical results were obtained with a sialo‐sugar derivative prepared from the carbohydrate moiety of ganglioside GGtet1 by reductive aminatio followed by N‐acetylation of its glucose unit at the reducing end.

Variations in the lipohilic moiety of ganglioside GGtet1 yielded compounds (gangliosidoides) which completely retained the specific ability to precipitate choleragen, but did not interfere with its toxicity. The choleragenicity of gangliosidoide · choleragen complexes could still be abolished by ganglioside GGtet1. Polyacrylamide gel electrophoresis of galgioside · choleragen as well as gangliosidoide · choleragen complexes, in the presence of sodium dodecylsulphate, showed only the protein subunits (approx. 8000) molecular weight of the toxin to be irreversibly aggregated. When incubating canine ileal loops with ganglioside‐GGtet1 · choleragen complex or with ganglioside GGtet1 in presence of sodium deoxycholate, followed by washing of the loops with buffer after a certain period of time, an inhibition of the secretory response to a subsequent challenge with choleragen was observed. Similar treatment of ileal loops with Vibrio cholerae neuraminidase dissolved in buffer without sodium deoxycholate followed by washing of the loops, caused an enhancement as well as an inhibition of the secretory response to the subsequent challenge with choleragen depending upon the time of incubation with neuraminidase.