bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparisons of the Tryptophan Synthase Inactivating Enzymes with Proteinases from Yeast
Authors: Saheki Takeyori , Holzer Helmut
Published: March 2005

The partially purified tryptophan synthase inactivating enzymes I and II were compared with the earlier described proteinases A, B and C by studying their catalytic properties, their sensitivity against a serine proteinase inhibitor and their temperature stability.

Tryptophan synthase inactivase activities of inactivase I and II coincided with hemoglobin hydrolyzing and azocoll hydrolyzing activities, respectively, on hydroxyapatite column chromatography.

Inactivase and hemoglobin hydrolyzing activities of the inactivase I fraction were insensitive to the treatment with phenylmethylsulfonyl fluoride, whereas inactivase and azocoll hydrolyzing activities of the inactivase II fraction were almost completely inhibited by phenylmethylsulfonyl fluoride.

The N‐acetyltyrosine ethyl ester (Ac‐Tyr‐OEt) hydrolyzing activity of an inactivase I fraction from an hydroxyapatite column could be separated from the inactivase activity by DEAE‐Sephadex column chromatography. Because of its esterolytic properties, the separated fraction corresponds to proteinase C.

Inactivase and hemoglobin hydrolyzing activities of the inactivase I fraction were stable at pH 6.0 and 37°C, but unstable at pH 8.0 and 37°C, while inactivase and azocoll hydrolyzing activities of the inactivase II fraction were unstable even at pH 6.0 and 37°C.

From these studies it is concluded that inactivase I is identical to proteinase A, inactivase II is identical or very similar to proteinase B and that proteinase C had no tryptophan synthase inactivase activity.
Purification and Properties of N‐Acetylglucosamine Kinase from Human Gastric Mucosa
Authors: GindzieŃSKI Andrzej , Glowacka Danuta , Zwiez Krysztof
Published: March 2005

N‐Acetylglucosamine kinase from the human gastric mucous membrane was purified about 170‐fold. The purified enzyme is specific for N‐acetylglucosamine with slight phosphorylation of N‐acetylmannosamine. The aminosugar product of the enzymatic reaction was identified as N‐acetylglucosamine 6‐phosphate. The Km for N‐acetylglucosamine is 0.11 Km and for ATP 3.03 mM. The pded enzyme is stable at +4 °C for three days, labile to dialysis and irreversibly inhibited by p‐chloromercuribenzoate. The molecular weight of the enzyme was 77000.
Immunochemical and Enzymic Properties of the Electric Eel Acetylcholinesterase‐Antiacetylcholinesterase System
Authors: Gurari Dalia , Silman Israel , Fuchs Sara
Published: March 2005

Antibodies to acetylcholinesterase from the electric organ of the electric eel were elicited in rabbits by immunization with the different molecular forms of the enzyme, namely, the globular 11‐S and the “native” 14‐S + 18‐S acetylcholinesterase.

The antigenic specificity of the two acetylcholinesterase preparations was analysed by the reaction of antisera to each of them with both the homologous and heterologous enzyme antigens. The immunological techniques employed included precipitation, immunoelectrophoresis and complement fixation. No differences in immunodominant antigenic determinants were observed between the two forms of the enzyme. The difference in the pattern of reaction between the 11‐S and 14‐S + 18‐S preparations with either antibody, in both the precipitation reaction and complement fixation, is ascribed to the higher aggregative state of the “native” 14‐S + 18‐S acetylcholinesterase preparation.

The effect of antibodies to acetylcholinesterase on the enzymatic activity of the 11‐S and 14‐S + 18‐S preparations towards the substrates acetylcholine, acetylthiocholine and indophenyl acetate was studied. The enzymic activity towards acetylcholine and acetylthiocholine is markedly inhibited by specific antibodies whereas the activity towards indophenyl acetate is hardly affected under similar conditions. The monovalent fragments, Fab, of the antibodies have no inhibitory effect on acetylcholinesterase activity. From kinetic studies it appears that the antigenic determinants involved in enzyme‐antibody interactions are not located in the active site, and that the inhibition caused by antibodies under certain experimental conditions results from microenvironmental effects on enzyme activity.
Transport of 2‐Keto‐3‐deoxy‐D‐gluconate in Isolated Membrane Vesicles of Escherichia coli K12
Authors: Lagarde Alain E. , Stoeber François R.
Published: March 2005

Escherichia coli membrane vesicles isolated from strains derepressed for 2‐keto‐3‐deoxy‐d‐gluconate transport system were found to be able to take up 2‐keto‐3‐deoxy‐d‐gluconate and d‐glucuronate when an energy source was added. The artificial pair ascorbate plus phenazine methosulfate and the physiological electron donors d‐(—)‐lactate and dl‐glycerol 3‐phosphate specifically stimulated transport activity.

Vesicular uptake was lacking in kdgT– mutants and was restored in membranes from a revertant strain. Km values for 2‐keto‐3‐deoxy‐d‐gluconate and for d‐glucuronate closely agree with those previously determined in whole cells. From the additive fact that no binding activity was detected in the periplasm, it was concluded that all 2‐keto‐3‐deoxy‐d‐gluconate transport activity coded by the kdgT gene was restricted to membrane.

By recording difference spectra between oxidized and reduced membrane vesicles, l‐glycerol 3–phosphate oxidation was shown to follow the terminal cytochrome chain common to d(–)‐lactate and other substrates. d‐(–)‐Lactate and d‐glycerol 3‐phosphate oxidation primarily occurs via two membrane‐bound, flavin‐linked specific dehydrogenases and this step was found to be rate‐limiting for 2‐keto‐3‐deoxy‐d‐gluconate uptake in vesicles.

2‐Keto‐3‐deoxy‐d‐gluconate uptake is inhibited by classical electrontransfer inhibitors, by proton‐conducting compounds and thiol reagents. Except for oxamate and rotenone, all inhibitors induced rapid and complete efflux of previously accumulated 2‐keto‐3–deoxy‐d‐gluconate. Considering the site of action of these inhibitors along the respiratory chain it was concluded that (a) the site coupling electron flux to substrate uptake was lying between the primary dehydrogenase and cytochrome b1; and that (b) the carrier coupling site or a coupling component, yet speculative, rather than the recognition site for 2‐keto‐deoxygluconate had active sulfhydryl groups.
The Biochemical Requirements for Bacterial Division
Authors: Pardee Arthur B.
Published: March 2005

We are searching for biochemical events related to the process of bacterial division. Data obtained with intact bacteria, using inhibitors primarily, suggest that protein synthesis must occur through most of the division cycle in order that a bacterium can divide at the end of the cycle. Hence, one approach is to look for a protein that is made throughout the division cycle and whose production is related to ability of the bacteria to divide. Dr Masayori Inouye modifled the ability of the bacterium Escherichia coli to divide, using mutants, inhibitors or nutritional deficiencies, and then looked for alterations in the pattern formed by electrophoresis of membrane proteins on polyacrylamide gels. One protein peak on the gels was strongly increased whenever the cells were prevented from dividing. We named this protein “X”; it has a molecular weight of about 50000.

A role was sought for the protein made throughout the cycle but showing its effect only at the end. Earlier observations by Dr Helene Smith showed that brief heating of the bacteria (16 min at 45 °C) produced synchronous cell division; heating delayed division in a way suggesting that a protein needed for division had been damaged and must be resynthesized before division occurs. Damage to this protein causes the cells to swell; this swelling appears to be the prime basis for the delay of cell division, as shown recently by Dr Po Chi Wu. The swelling caused by an osmotic shift similarly delayed cell division; a result that is support for this hypothesis. A model is proposed for the role of the X protein as a sort of “girdle” that must be built up during the cycle, and that constricts the cells at the time of division.
Distribution of Newly Synthesized Histones during DNA Replication
Authors: Tsanev Roumen , Russev Georgi
Published: March 2005

A procedure has been developed which allows the separation of chromatin into two fractions highly enriched in preexisting and in newly synthesized DNA, respectively. The procedure includes the following steps. (a) Partial substitution of thymine with 5–bromouracil in the newly synthesized DNA strand. (b) Labelling of proteins with 14C‐labelled amino acids and DNA with [3H]thymidine during the period of DNA replication. (c) Introduction of breaks in the new DNA strand at the points containing 5‐bromouracil by irradiation at 313 nm. (d) Denaturation of chromatin at pH 12.2 and separation of the intact from the fragmented material by centrifugation in alkaline sucrose density gradient. (e) Purification of the two fractions by a recentrifugation under the same conditions.

The results from such experiments have shown that during DNA replication the preexisting histones remain bound to the parental DNA strand while the newly synthesized histones join the new DNA strand.
Studies on Seed‐Oil Triglycerides
Authors: Gurr Michael I. , Blades Jon , Appleby Robert S. , Smith Colin G. , Robinson Malcolm P. , Nichols Bryan W.
Published: March 2005

Crambe seeds harvested 6—30 days after flowering actively incorporated radioactive precursors into lipids. The distribution of label among the different lipid classes depended on the precursor, the age of the seed, and the duration of the incubation.

Labelled acetate was incorporated preferentially into phospholipids of whole seeds but the proportion of the radioactivity located in triglycerides increased as the age of the seed increased.

Uniformly labelled glucose was incorporated readily into the glycerol moiety of glycerolipids but not at all into the fatty acid moiety. The largest proportion was found in the phospholipid fraction and this proportion increased as the age of the seed increased, in contrast to labelled acetate.

Uniformly labelled glycerol was actively incorporated into glycerolipids of whole seeds. Phospholipids were the first fraction to be labelled, and in this fraction labelled phosphatidic acid could be detected at early time periods. Subsequently, diglycerides became labelled and finally triglycerides. Labelled glycerol was quickly converted into a number of water‐soluble metabolites, one of the most rapidly labelled of which was glycerol phosphate. Taken together these data are consistent with the presence of the glycerol phosphate pathway for triglyceride synthesis in maturing Crambe seeds.

A fat fraction has been isolated from mature and maturing Crambe seeds by homogenisation of the seeds followed by flotation in the centrifuge. Electron microscopic examination indicated that the fraction contains the oil bodies that are the storage form of the oil in the seed in vivo.

The isolated oil bodies contain 75% by weight of lipid and 19% of protein. About 90% of the lipid is triglyceride, the remainder is mostly phospholipid. No glycolipid is present.

The isolated fat fraction catalysed the conversion of different substrates, malonyl‐CoA, long chain acyl‐CoA and glycerol 3‐phosphate into lipids. Evidence is presented that this is not due to contamination from other cell fractions.
Mößbauer Effect and Electron Spin Resonance of the (Iron)4‐Sulfur Clusters of Ferredoxin from Clostriclium pasteuriunum
Authors: Gersonde Klaus , Schlaak Heinz‐Eberhard , Breitenbach Michael , Park Fritz , Eicher Hermann , Zgorzalla Werner , Kalvius Michael G. , Mayer Aldabert
Published: March 2005

Ferredoxin from Clostridiurn pasteurianum which is characterized by two clusters containing four sulfur‐linked iron atoms each, was studied by MöBbaner and elect)ron spin resonance spectroscopy.

The iron of oxidized ferredoxin is high‐spin Fe(III) (S=5/2) coupled antiparallel magnetically as conaludcd from the magnitude of the quadrupole splitting and its slight temperature dependence. Thus the net spin of the cluster is 8=0. This explains why no magnetic hyperfine splitting was observed in the Mößbauer absorption spectrum of oxidized ferredoxin at 4.2 K. The observation of at least two quadrupole doublets with a ratio of intensities deviating from unity points to a slight inequivalence of the ligand field symmetry of the individual iron atoms.

Reduction of oxidized ferredoxin leads to an uptake of one electron per cluster. The net electron spin of each cluster of reduced ferredoxin is thus S=1/2. Spin‐spin coupling of both the clusters prevents the appearance of a magnetic hyperfine splitting at low temperature. In an external magnetic field of 20 kG the coupling is removed and a hyperfine structure is observed.

Under the conditions of X‐band electron spin resonance spectroscopy, i.e. in a magnetic field of 3.5 kG, the spin‐spin coupling is not totally removed. Thedore, an anisotropic 7‐line spectrum originating from a system with S = 1, is observed. Under chemical conditions producing partly reduced ferrcdoxin a 2‐line spectrum from the uncoupled S=1/2 systems is also found. It indicates axial symmetry of the innermolecular electric field. Möβhauer spectroscopy enabled the detection of various amounts of non‐cluster iron in samples of oxidized and reduced ferredoxin.
The Reactivation of C55‐Isoprenoid‐Alcohol‐Phosphokinase Apoprotein by Lipids
Authors: Sandermann Heinrich
Published: March 2005

The reactivation of C55‐isoprenoid‐alcohol phosphokinase apoprotein by a variety of lipids has been investigated.

A considerable lack of specificity for the chemical structure of the activating lipid is demonstrated.

Reactivation appears to occur by some physical mechanism. An electrostatic interaction of protein and lipid does not appear to be important.

All active lipids found are able to swell and become hydrated in aqueous buffer. Some of these lipids were active only after sonication or in the presence of detergent.

The hydrated lipid phase is concluded to activate by solubilization, as a liquid‐crystalline or micellar solvent for the extremely hydrophobic enzyme apoprotein and for the non‐hydrating C55‐isoprenoid alcohol.
Influence of Temperature on the Action of Rifampicin on RNA Polymerase in Presence of DNA
Authors: Kerrich‐Santo Regina E. , Hartmann Guido R.
Published: March 2005

When rifampicin, together with ribonucleoside triphosphates, is added at 25 °C to a mixture of RNA polymerase and DNA which had been preincubated for a few minutes, RNA is synthesized despite the presence of the inhibitor. With 0.8 mM Mg2+ present during the preincubation an optimum of RNA synthesis is observed which equals the synthesis in the absence of rifampicin. The extent of RNA synthesis at 25 °C is dependent on the temperature at which the enzyme has been preincubated with DNA. RNA synthesis in the presence of the inhibitor increases almost linearly with preincubation temperatures from 5 to 25 °C. The same results are obtained by applying either the chain initiation assay or the chain elongation assay for measuring RNA synthesis. The effect of temperature during preincubation of enzyme and template on subsequent RNA synthesis at 25 °C proved to be reversible. Experiments with poly[d(A‐T)] as competing template are consistent with the notion that during preincubation RNA polymerase is bound to the template at low as well as at higher temperatures under the conditions used. Essentially the same results concerning the initiation of RNA synthesis in the presence of rifampicin are obtained with T4 DNA, T2 DNA and poly[d(A‐T)] as templates. These results support the hypothesis that RNA polymerase bound to DNA exists in at least two different states which differ in their susceptibility to rifampicin. These different states are in a rather rapid and temperature‐dependent equilibrium. According to this hypothesis rifampicin prevents the conversion of the sensitive state of the complex of RNA polymerase and DNA to the resistant form. Subsequent initiation of RNA synthesis by addition of substrate is consequently blocked.
Lateral Mobility and Surface Density of Lipopolysaccharide in the Outer Membrane of Salmonella typhimurium
Authors: Mühlradt Peter F. , Menzel Johannes , Golecki Jochen R. , Speth Volker
Published: March 2005

Salmonella typhimurium 1195 mutant bacteria were allowed to produce clusters of lipopolysaccharide with new, wild‐type specificity over the export sites. Ferritin‐labeling experiments showed that the lipopolysaccharide diffuses away from the export sites at 37 °C and is found dispersed on the cell surface, whereas at 0 °C it remains immobile.

The density of lipopolysaccharide molecules on the bacterial surface, measured as number of lipopolysaccharide molecules per μm2, or as percentage of the bacterial surface covered by lipopolysaccharide was determined in two wild‐type S. typhimurium strains and one mutant strain, which was grown under conditions either permissive or nonpermissive for the formation of wild‐type lipopolysaccharide. There are 0.7—1.0 × 105 lipopolysaccharide molecules per μm2 surface area. About 15—25% of the bacterial surface was found to be covered by lipopolysaccharide. The mean intermolecular distance at hypothetical uniform distribution of lipopolysaccharide was calculated to be in the range of 3—4 nm. It was of no consequence to the lipopolysaccharide surface density whether the mutant strain produced wildtype or mutant lipopolysaccharide.

An estimate of the lateral diffusion constant D (D∼ 3 × 10−13 cm2/s) of lipopolysaccharide in the outer membrane was made from earlier published data [2]. Possible reasons for the low order of magnitude of D, based on the observed surface density, are discussed.
Purification and Properties of Ovalbumin Messenger RNA
Authors: Haines Michael E. , Carey Norman H. , Palmiter Richard D.
Published: March 2005

Ovalbumin messenger RNA was purified 67‐fold from hen oviduct polysomal RNA by sucrose gradient centrifugation and oligo(dT)‐cellulose hybridization. Ovalbumin messenger RNA activity was coincident with the main peak of absorbance after acrylamide gel electrophoresis of the purified RNA. Each purified messenger was translated over 20 times in a rabbit reticulocyte lysate, protein‐synthesizing system. An apparent molecular‐weight of approximately 875 000 was obtained by electrophoresis under both native and denaturing (formamide) conditions. In contrast, on sucrose gradients this messenger sedimented at approximately 15 S, corresponding to a molecular weight of about 550 000. Under certain conditions, ovalbumin messenger RNA activity was observed on both gels and gradients at a position approximately double these molecular weights. The larger messenger RNA species could be dissociated to the smaller one by either heating at 65 °C or formamide treatment, thus demonstrating messenger RNA aggregation.

We conclude that ovalbumin messenger RNA contains between 1670 and 2640 nucleotides; a value significantly greater than the number (1161) that are translated into ovalbumin.
l‐Phenylalanyl‐tRNA Synthetase of Escherichia coli K‐10
Authors: Hanke Till , Bartmann Peter , Hennecke Hauke , Kosakowski Heinz M. , Jaenicke Rainer , Holler Eggehard , Böck August
Published: March 2005

The molecular weight of l‐phenylalanyl‐tRNA synthetase was redetermined. A new value of 267 000 was obtained by high‐speed analytical ultracentrifugation and Sephadex G‐200 gel chromatography. The value is in contrast to the molecular weight of 180000 determined previously

From the molecular weight 267000 and the sedimentation coefficient s20,w= 8.6 S the ratio f/f0= 1.67 was calculated. The magnitude of f/f0 is in accord with the molecular weight 183000 obtained from sucrose gradient centrifugation. The results indicate that the enzyme is highly hydrated and/or assumes the shape of a rod or a disc.

The subunit composition was reinvestigated by sodium dodecylsulfate gel electrophoresis, guanidinium chloride gel filtration and by reactivation experiments of pH‐2 inactivated enzyme. The results are consistent with a subunit structure of the α2β2 type. Molecular weights of the subunits are 39000 and 94000, respectively.
Glycoproteins of Chick Embryo Fibroblasts in Cultures with a Finite Life Span
Authors: Courtois Yves , Hughes R. Colin
Published: March 2005

Chick embryo fibroblasts, that cease to grow after a finite number of passages in culture, have been cultured in the presence of radioactive d‐glucosamine. Incorporation of radioactivity into cellular macromolecules is very similar for cells regardless of their position in the finite life span.

The labelled substances removed by trypsin from the surface of non‐confluent cells growing in different parts of the life span were analysed. Smooth membrane fractions isolated from trypsinised cells were also compared.

The labelled substances were very similar, in particular the extent of sialylation of the total glycoprotein fraction of early phase or late phase cells. Minor differences were found, however.

It was found that mucopolysaccharides constitute a larger proportion of the labelled material from the surface of late phase cells than from the surface of early phase cells. Conversely early phase cells were relatively rich in several glycopeptides compared to late phase cells.
The Amino‐Acid Sequences of Three Tryptic Glycopeptides from Human Ceruloplasmin
Authors: Rydén Lars , Eaker David
Published: March 2005

The isolation and amino‐acid and carbohydrate composition of three tryptic glycopeptides, containing 16,17 and 20 amino‐acid residues, respectively, from the neuraminidase‐treated major form of human ceruloplasmin have been described earlier. It has also been suggested that the peptides possibly represent all of the carbohydrate‐binding sites in the protein. The present paper describes the amino‐acid sequences of these peptides as obtained by stepwise Edman degradation and carboxypeptidase digestions of the whole peptides and their fragments after cleavage by chymotrypsin, pepsin and thermolysin. The results confirm that the carbohydrate in all cases is attached via an asparagine (or aspartic acid) residue, that is followed by an X‐Thr/Ser sequence. No sequence homologies are found when the peptides are compared to each other or to other glycopeptides of known sequence.
The Rate‐Limiting Step of Adrenal Steroidogenesis and Adenosine 3′:5′‐Monophosphate
Authors: Kahnt Friedrich W. , Milani Alfred , Steffen Hans , Neher Robert
Published: March 2005

The rate‐limiting step of adrenal steroidogenesis is first defined in detail. The kinetics of the conversion in vitro of double‐labeled exogenous cholesterol as potential precursor by intact and ruptured bovine adrenocortical mitochondria are compared under identical conditions. The results strongly suggest that the transport of cholesterol from the outside to the inside of the mitochondrial membrane is the proper rate‐limiting process and not the subsequent enzymatic conversion of cholesterol to steroids.

The involvement of a transport process is supported by findings such as increase of conversion rates in intact mitochondria by (a) preincubation of exogenous precursor with mitochondria; (b) addition of carrier proteins such as non‐defatted bovine serum albumin as against proteins strongly binding cholesterol; (c) addition of Ca2+ concentrations sufficient to increase membrane permeability.

The immediate stimulation by adrenocorticotrophic hormone of adrenocortical steroidogenesis seems to be mediated by cyclic AMP formed intracellularly. Such a messenger may be considered to act directly or indirectly upon the rate‐limiting step. It was found that the presence of cyclic AMP (1–5 mM) or cyclic monobutyryl AMP (0.04–1 mM) under optimal and various suboptimal conditions of cholesterol utilization did in no instance increase the conversion rate nor did it influence the utilization of endogenous precursor in intact mitochondria.

It is concluded that cyclic AMP has no direct effect on (a) the transport process for exogenous cholesterol; (b) the intramitochondrial enzymatic conversion; (c) the utilization of endogenous steroid precursor. If cyclic AMP is involved, its action seems to have a premitochondrial localization and to be related indirectly only to a transport mechanism or a precursor supply process.
Antamanide. Dynamics of Metal‐Complex Formation
Authors: Burgermeister Wolfgang , Wieland Theodor , Winkler Ruthild
Published: March 2005

Overall rate constants for complex formation of Na+ and Ca2+ with antamanide and of Na+ with perhydro‐antamanide were determined by using the temperature‐jump relaxation technique. Measurements were carried out in methanol or acetonitrile as solvents. Murexide was used as Na+‐indicator. The reaction enthalpy of the Na+‐antamanide system was determined from relaxation amplitudes. Values of the rate constants are considerably lower than those for similar macrocyclic complex‐forming compounds.
Hexokinase of Human Erythrocytes
Authors: Gerber Gerhard , Preissler Heidemarie , Heinrich Reinhart , Rapoport Samuel M.
Published: March 2005

Hexokinase was purified 680‐fold from erythrocytes of man. The preparation was not contaminated by other glycolytic enzymes.

Initial reaction rate measurements were performed at pH 7.2, ionic strength 0.15 and 37°C by use of coupled optical assays. The experimental data were used to construct a kinetic model of the enzyme, the kinetic parameters of which were estimated by means of a computer‐fit program.

The hexokinase of erythrocytes operates by a rapid‐equilibrium random mechanism. The dissociation constant of the E · glucose complex is 0.038 to 0.054 mM, of the E · MgATP complex 1.0 to 2.1 mM. Uncomplexed Mg2+ up to 4 mM is capable of activating the enzyme velocity twofold (KMg2+= 1.0 mM). An inhibition was observed at higher Mg2+ concentrations. α‐d‐Glucose 6‐phosphate and α‐d‐glucose 1,6‐bisphosphate inhibit the hexokinase competitive to MgATP with dissociation constants of 0.069 mM. The enzyme is also inhibited by 2,3‐bisphosphoglycerate competitive with respect to MgATP with a KP2G= 2.7 mM. An inhibition by uncomplexed ATP was not detected.

The parameters of the kinetic model of the enzyme in conjunction with the free intracellular Concentrations of substrates and effectors were used to calculate the actual intracellular activity of the hexokinase in dependence on variations of pH, temperature and the state of haemoglobin oxygenation. The calculated hexokinase activities correspond to the glycolytic flux in all conditions considered, except at pH 8.
Interaction of Biguanides with Mitochondrial and Synthetic Membranes
Authors: Schäfer Günter
Published: March 2005

Biguanides inhibit a large scale of metabolic processes which in most cases are membrane‐linked. Oxidative phosphorylation is strongly inhibited in that part of the respiratory chain located on the substrate site of cytochrome c.

It is assumed that inhibition is due to physical changes within the membrane phospholipids, rather than a result of direct interaction with functional intermediates of energy conservation. The following observations have been made.

Binding affinity to mitochondrial, submitochondrial and other membranes is identical with that to pure phospholipids.

Synthetic phospholipid vesicles exactly resemble the binding properties and kinetics of mitochondrial membranes.

Gradual lipid extraction of mitochondrial membranes causes a proportional decrease of binding capacity but no change of affinity constants.

The stoichiometry shows that the capacity for inhibitor binding is indeed in the range of mitochondrial phospholipid content.

Binding of the inhibitors occurs to the aqueous lipid interface of the membrane. Possibilities are discussed for the mode of action of the inhibitors representing firmly fixed positive charges.
Physical and Functional Homology between Ribosomal Protein S1 and Interference Factor i
Authors: Inouye Hiroshi , Pollack Yaakov , Petre Jean
Published: March 2005

The homology between the 30‐S ribosomal protein S1 and interference factor i has been investigated.

The two proteins are indistinguishable by gel electrophoresis under various conditions. Similar peptides are obtained by cleavage with cyanogen bromide. After labelling with N‐ethylmaleimide, enzymatic digestion yields homologous labelled peptides. This homology is confirmed by immunological data.

Characteristic activities of the two proteins have been compared. Both bind polyuridylic acid and stimulate polypeptide synthesis on this template by S1‐depleted ribosomes. Furthermore, S1 inhibits the translation of the coat protein cistron on MS2 RNA, while it has little effect on the overall translation of T4 mRNA, two characteristic properties of interference factor i.

S1 and factor i are thus physically and functionally indistinguishable, which suggests that they have identical polypeptide chains. Implications are discussed in terms of function.
Effect of Calcium Ions on Steroid‐Binding Spectra and Pregnenolone Formation in Rat–Adrenal Mitochondria
Authors: Simpson Evan R. , Jefcoate Colin R. , Mccarthy John L. , Boyd George S.
Published: March 2005

The effect of calcium ions on steroid‐induced difference spectra of cytochrome P‐450, and on pregnenolone formation, has been studied in rat adrenal mitochondria. In the presence of phosphate, addition of calcium chloride to suspensions of these mitochondria resulted in an increase in the inverted type‐I difference spectrum induced by pregnenolone or methylandrostenediol, and a disappearance of the type I spectrum induced by 25‐hydroxycholesterol. The difference in the inverted type I spectrum between mitochondria from ether‐stressed animals and those given cycloheximide was abolished. Cholesterol side‐chain cleavage was increased, providing an NADPH generator was used as source of reducing equivalents. The difference in side‐chain cleavage activity between mitochondria from ether‐stressed animals and those given cycloheximide, was maintained.

In the absence of phosphate, addition of calcium chloride again resulted in a loss of the type I difference spectrum induced by 25‐hydroxycholesterol, but this time there was almost no change in the inverted type I spectrum induced by pregnenolone in mitochondria from rats subjected to ether anaesthesia, and the change in cycloheximide‐treated rats was only about 50%, of the change in the 25‐hydroxycholesterol‐induced difference spectrum. These observations are interpreted in terms of interconversions of the various forms of cytochrome P‐450 associated with side‐chain cleavage proposed to exist in intact adrenal mitochondria.

Factors such as bovine serum albumin and EDTA, which increase respiratory control in these mitochondria, also stimulate cholesterol side‐chain cleavage activity, as do magnesium ions, but without altering the magnitude of the steroid‐binding spectra.
On the Mechanism of Glucose‐6‐Phosphate Dehydrogenase Regulation in Mouse Liver
Authors: Hizi Amnon , Yagil Gad
Published: March 2005

Glucose‐6‐phosphate dehydrogenase is purified from C57BL mouse liver to a specific activity of 116 units/mg protein. The molecular weight of the main active unit is 121000 and it is composed of three interconvertible subforms, as revealed by acrylamide electrophoresis. The main active unit is composed of two subunits of molecular weight 62000, and may aggregate to form higher oligomers. Amino acid composition and dependence of rate on pH and substrate concentration are reported. The kinetic data suggest the existence of two types of active sites with separate sets of Km and V values. The electrophoretic and kinetic properties of the enzyme from induced and non‐induced liver homogenates are compared.
Separation of the Two Non‐Identical Subunits of Lombricine Kinase from Lumbricus terrestris Muscle by Chromatography on Sepharose‐Mercurial
Authors: Der terrossian Elisabeth , Pradel Louise‐Anne , Kassab Ridha , Desvages Gisèble
Published: March 2005

Of the dimeric phosphagen kinases (Mr= 80000) studied, lombricine kinase is the only one which contains only one essential thiol group and one binding site for ADP‐Mg. These facts suggest a non‐identity between the two subunits of this enzyme.

The reversible labeling of the essential thiol group of this protein with 2,4‐dinitrofluorobenzene, followed by alkylation of the remaining thiol groups, allowed the separation of the two subunits by means of a Sepharose‐mercurial column. The Sepharose‐mercurial was prepared by treatment of Sepharose 4B with p‐aminophenyl mercuric acetate.

Amino‐acid analyses, N‐ and C‐terminal determinations and fingerprintings were performed to detect the differences in the primary structure between the two subunits.

Furthermore, the Sepharose‐mercurial column was used to purify the tryptic peptide containing the essential thiol group of lombricine kinase. By this process, it was possible to obtain, in one step, the peptide in a pure state.

It is interesting to point out the applicability of this method for the purification of peptides or proteins containing essential thiol groups which can be specifically labelled by reversible inhibitors.
Conformational Properties of Some Viral DNAs
Authors: Champell P. , Brahms J.
Published: March 2005

Oriented films of bacteriophage DNAs of low and intermediate (G + C) content and of different chemical composition were investigated by infrared linear dichroism: PBS1, DNA (28% G + C, uracil replaces thymine), T4, DNA (34%, G + C, glucosylated), T4* DNA (34% G + C, unglucosylated), T5, DNA (39% G + C). None of these phage DNAs exhibit a transition to a predominantly pure A form when relative humidity is lowered. At lower relative humidity, the infrared dichroism of the 1230‐cm‐1 band changes to mainly parallel orientation with respect to the DNA axis. The pattern of the tansition as a function of relative humidity indicates the presence at low relative humidity of a well‐defined form for which the O2–O3 line of the phosphate group was found to be directed approximately at 52°C with respect to the helix axis. This allows one to assign the lower‐humidity form of these phage DNAs as a predominant C‐like form.

The comparison of T4 with T4* DNA indicates that glucosylation has no effect on conformational properties of phage DNAs, and particularly on the absence of the B to A structural change. The replacement of the thymine by uracil in DNA of PBS1, has not affected the structural properties of the B form at high humidity nor the absence of the main A form at lower humidity; the transition from the B form to a predominant C‐like form was observed.
The Role of Dolicholmonophosphate in Glycoprotein Biosynthesis in Saccharomyces cerevisiae
Authors: SHARMA Chandra Bhan , BABCZINSKI Peter , LEHLE Ludwig , TANNER Widmar
Published: March 2005

1. In a crude particulate fraction from yeast mannose is transferred from GDP‐mannose to an endogenous “lipid” fraction, the monophosphates of dolichol‐14 to dolichol‐18. The same membrane fraction also catalyzes the mannosylation of glycoproteins. More than 80% of the total radioactivity in the glycoprotein fraction obtained from GDP‐[14C]mannose is released by β‐elimination. The small‐sized radioactive products of β‐elimination are mannose, mannobiose and mannotriose.

2. Ageing of the particulate fraction leads to a drastic loss of mannosyl transfer activity from GDP‐[14C]mannose to dolichol monophosphate. To the same extent the amount of [14C]mannose obtained after β‐elimination of the glycoproteins decreases. The amount of radioactive mannobiose and mannotriose is, however, much less affected.

3. With decreasing GDP‐mannose concentrations the amount of [14C]mannose obtained after β‐elimination increases as compared to the amount of radioactive mannobiose and mannotriose. The Km‐value for the incorporation of mannosyl groups directly linked to serine and/or threonine, therefore, is lower than the Km‐value for the transfer of subsequent mannosyl residues.

4. The mannosylation of dolichol monophosphate from GDP‐[14C]mannose proceeds in the presence of Mg2+ and Mn2+ ions as well. When solely Mg2+ is present in the incubation mixture, β‐elimination of the glycoprotein produced yields almost exclusively mannose, and it is only in the presence of Mn2+ that the normal β‐elimination pattern is observed.

5. Incubation of the particulate fraction with dolichol‐monophosphate‐[14C]mannose results in the formation of radioactive glycoprotein; again most of the radioactivity is released by β‐elimination but only [14C]mannose is obtained as product. When dolichol‐monophosphate [14C]mannose is incubated together with non‐radioactive GDP‐mannose, β‐elimination yields radioactive mannose, mannobiose and mannotriose. Mannobiose is found to be labelled in the reducing end only.

6. The results are consistent with the assumption that dolichol monophosphate is involved in the mannosylation of a specific position in yeast glycoproteins, i.e. in the formation of mannosyl linkages to serine and/or threonine. The subsequent mannosylation proceeds directly from GDP‐mannose in a reaction obligatorily requiring Mn2+.

7. After β‐elimination of the methanol‐insoluble material mannosylated in the presence of dolichol‐monophosphate [14C] mannose the residual insoluble radioactivity is to a large extent transformed into dialyzable material by pronase. Mannose, therefore, is transferred from dolichol‐monophosphate mannose also to glycoprotein positions not involving OH‐groups of serine or threonine.
Rapid and Selective Inhibition of RNA Synthesis in Yeast by 8‐Hydroxyquinoline
Authors: FRASER Ronald S. S. , CREANOR James
Published: March 2005

A selective inhibitor of RNA synthesis is an essential tool for studies of the mechanisms of control of enzyme synthesis. Yeast is a particularly suitable organism for such studies, but until recently no suitable inhibitor of RNA synthesis has been available. 8‐HydroxyquinolineY at concentrations of 10–50 μ;g/ml, rapidly and selectively inhibits RNA synthesis in fission yeast. Protein synthesis, cell growth and uridine uptake are not immediately affected. The mechanism of inhibition appears to be by chelation of divalent cations required for RNA synthesis. An explanation of the selectivity of inhibition is proposed. An estimate of 20 min for the upper limit of messenger RNA half‐life was obtained by following the rate of protein synthesis after inhibition of RNA synthesis by 8‐hydroxyquinoline. At low concentrations, 8‐hydroxyquinoline inhibits the synthesis of ribosomal RNA and messenger RNA, but has no effect on the synthesis of transfer RNA or 5‐S RNA. This differential effect could arise from differing divalent cation requirements of different RNA polymerases.
On the 3α‐Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni
Authors: SKÅLHEGG Bjømn A.
Published: March 2005

The steroid‐induced 3α‐hydroxysteroid:NAD+‐oxidoreductase from Pseudomonas testosteroni (ATCC 11996) exhibits reversible, concentration‐dependent monomer‐dimer transitions. The dimerization is demonstrable by gel chromatography and by precipitations with ammonium sulphate in concentrated solutions of the enzyme. The dimer formation leads to a considerable decrease in the specific activity, especially when androsterone is used as substrate. In this case the formation of dimers results in a 90 % reduction of the specific activity found with the monomers. The described effect is demonstrable, but less pronounced, when tetrahydrocortisone or deoxycholate are the substrates.

Higly purified preparations of the 3α‐hydroxysteroid dehydrogenase prepared by repeated isoelectric focusings at different pH gradients have shown the enzyme to be composed of at least two isoenzymes. One major part focusing at pH 6.2 and a minor part focusing at pH 5.8.
Properties of the Assimilatory Nitrate Reductase from Aspergillus nidulans
Authors: McDONALD Donald W. , CODDINGTON Alan
Published: March 2005

1. Nitrate reductase has been partially purified from Aspergillus nidulans. It has a sedimentation coefficient of 7.6 (± 0.1) S and a molecular weight of 190000 (± 10000) from sucrose density gradient centrifugation and gel‐filtration studies. There is spectroscopic evidence for the presence of a haemoprotein component.

2. It possesses two associated activities, cytochrome c reductase and reduced viologen dye: nitrate reductase. A study of the effects of heat and inhibitors on the three activities suggests that the molecule consists of two functional parts. One of these is heat labile, contains FAD and a haemoprotein and catalyses the transfer of electrons from NADPH to cytochrome c. The other part is heat stable, contains molybdenum and catalyses the transfer of electrons from reduced viologen dyes to nitrate.

3. The Km values for nitrate, NADPH and cytochrome c are reported. In each case the Km for one substrate is not affected by varying the concentration of the other, suggesting a lack of interaction between their respective binding sites. Product inhibition by NADP+ and nitrate under saturating and non‐saturating concentrations of NADPH and nitrate suggests a random order rapid‐equilibrium mechanism for the enzyme.
The Lipid Composition and Na+‐K+‐Dependent Adenosine‐Triphosphatase Activity of the Salt (Nasal) Gland of Eider Duck and Herring Gull
Authors: Karlsson Karl‐Anders , Samuelsson Bo E. , Steen Göran O.
Published: March 2005

1. The lipid composition of the salt (nasal) gland of eider duck and herring gull, an organ with a high transport capacity for sodium ions, was characterized in detail.

2. The three major lipids of both animals were cholesterol, ethanolamine phosphoglycerides and choline phosphoglycerides. The major fatty acids of ethanolamine phosphoglycerides were stearic and arachidonic acid, and the major fatty acids of choline phosphoglycerides were palmitic, oleic and arachidonic acid. Small amounts of plasmalogens were found.

3. Other phospholipids identified were sphingomyelins, diphosphatidylglycerols, inositol phosphoglycerides and serine phosphoglycerides.

4. The major glycosphingolipids were sulphatides, with similar structure for the two animals. A sulphate group was identified in position 3 of galactose, bound to ceramide mainly containing the C20 homologue of sphingosine and saturated 2‐d‐hydroxy C22, C23 and C24 fatty acids. No branched‐chain or trihydroxy bases were present.

5. An earlier unknown glycolipid, xylopyranosylceramide, was found in small amounts in herring gull but not in eider duck. Glucosyl‐and galactosylceramides were present in both animals.

6. The ratio of Na+‐K+‐dependent ATPase and sulphatides was similar to ratios found for other tissues with increased capacity for the transport of sodium ions. No other lipid resembled sulphatides in this respect.
Comparison of Template‐Property Changes after Salt Extraction of Avian Erythrocyte and Liver Chromatin
Authors: SELIGY Verner L. , MIYAGI Masakazu
Published: March 2005

An analysis was made of the induced changes in template properties of mature erythrocyte and liver chromatin which occur after extraction with 0.1 to 2.0 M NaCl pH 8.0. DNA‐bound proteins soluble in acid at pH 1.0 and in phenol at pH 9.0 were identified by disc gel electrophoresis. Purified histones were added during the extraction regime to assess endogenous proteolytic activity and to serve as internal markers. Removal of proteins by 0.4 M NaCl from either chromatin resulted in little or no change in original template activity, solubility and thermal denaturation properties. Significant changes in these parameters were observed after extraction with NaCl 0.6 M. For liver chromatin, template activity and thermal melting properties gradually approached those of deproteinized DNA. The largest change occurred after the extraction of non‐histone proteins which are not removed during 2.0‐M NaCl treatment. In contrast, the largest single change in erythrocyte template properties occurred with 0.6‐M NaCl treatment. At this salt concentration, erythrocyte template availability was the same as for liver chromatin. The only major component extracted was the erythrocyte specific histone, V or f2c. Annealing studies using saturating amounts of labelled RNA synthesized from the various types of chromatin confirmed the template activity studies. More of the DNA in chromatin appears to be transcribed as chromatin is extracted with increasingly higher concentrations of salt, with little indication that reassociation of protein and loss of transcripts occurs. Pre‐saturation competition studies using [3H]RNA synthesized from 0.6‐M and 2.0‐M salt‐extracted chromatin indicates that histones are probably more important for maintaining the highly restricted template of the mature erythrocyte chromatin than for the more active template of liver chromatin.
A Study of Drug Metabolism Linked to CytochromeP‐450 in Isolated Rat‐Liver Cells
Authors: MOLDÉUS Peter , GRUNDIN Robert , VADI Helena , ORRENIUS Sten
Published: March 2005

Isolated rat liver cells were found to catalyze the cytochrome P‐450 dependent, oxidative metabolism of alprenolol [1‐(2‐allylphenoxy)‐3‐isopropylaminopropanol] at a rate similar to that obtained with the isolated microsomal fraction in the presence of a NADPH‐generating system. Alprenolol was rapidly taken up into the liver cells and the apparent Michaelis constant was similar to that obtained with liver microsomes. Further, inhibitors of drug metabolism such as SKF 525‐A and metyrapone showed similar inhibitory patterns in the cellular and microsomal systems. With liver cells isolated from control rats, NADPH generation from endogenous substrates was sufficient to support optimal alprenolol metabolism, whereas the addition of glucose or lactate stimulated this rate in liver cells isolated from starved, phenobarbital‐treated rats. Inhibitors of mitochondrial respiration such as rotenone, antimycin A and KCN and the uncoupler carbonyl cyanide‐p‐trifluoromethoxy‐phenyl hydrazone inhibited cellular alprenolol metabolism, presumably by lowering the ATP/ADP ratio thereby interfering with NADPH generationviathe malic enzyme system; this effect was more pronounced in liver cells from starved rats. Furthermore, in analogy with previous findings, the rate of gluconeogenesis from lactate was inhibited during alprenolol metabolism; this effect in turn was most pronounced in liver cells from phenobarbital‐treated rats. The findings indicate that under certain conditions, the level of cytoplasmic NADPH may become rate‐limiting for drug metabolism linked to cytochromeP‐450 and support the previous assumption that in fed rats, generation of cytoplasmic NADPH is mainlyviathe pentose phosphate shunt whereas the malic enzyme system becomes more important for this purpose during starvation.
Effect of Allopurinol (4‐Hydroxypyrazolo[3,4‐d]pyrimidine) on Xanthine Accumulation by Milk Xanthine Oxidase in vitro
Authors: JEżEWSKA Maria M.
Published: March 2005

An accumulation of xanthine and a decrease of uric acid formation as the effect of 4‐hydroxy‐pyrazolo[3,4‐d]pyrimidine (allopurinol) and other inhibitors (8‐azaadenine, 8‐azaguanine, 6‐thio‐purine) on hypoxanthine hydroxylation by milk xanthine oxidase in vitro was found. A remaining activity of xanthine oxidoreductases seems to be the main base of enhanced formation of xanthine due to allopurinol treatment in vivo. Xanthine oxidoreductases, increasing and decreasing the pool of oxypurines (hypoxanthine and xanthine) must play an important role in purine biosynthesis regulation.