bluuebunny/crossref_metadata_2025 huggingface dataset:
UDP‐Apiose/UDP‐Xylose Synthase
Authors: MATERN Ulrich , GRISEBACH Hans
Published: March 2005

The UDP‐apiose/UDP‐xylose synthase from cell suspension cultures of parsley has been purified 1400‐fold by an improved method. The ratio of apiose to xylose formed from UDP‐D‐glucuronic acid (UDP‐GlcUA) remained constant throughout the purification procedure. Dodecylsulfate‐gel electrophoresis and sedimentation equilibrium measurements showed that this enzyme preparation is composed of two proteins with molecular weights of 65000 and 86000. The two proteins which are present in a molar ratio of about 1:0.7 to 1:0.9 could not be separated by ammonium sulfate fractionation, chromatography on DEAE‐cellulose at different pH‐values, and on ω‐aminoalkyl‐Sepharose, and by gel filtration on Acrylex P‐100. Each protein is composed of two apparently identical subunits. The presence of only two different subunits was confirmed by end group analysis in which glycine was found as N‐terminal amino acid for the larger and lysine for the smaller protein. Crosslinking with dimethylsuberimidate gave dimers of the identical subunits but no hybrids.

Separation of the two proteins was achieved on DEAE‐cellulose in the presence of urea. After dialysis only the 86000‐Mr protein showed enzyme activity with no significant change in the apiose/xylose ratio. However, in the absence of the 65000‐Mr protein enzyme stability was decreased drastically.

By equilibrium dialysis it was found that 0.5 mol UDP‐GlcUA are bound per mole of 86000‐Mr protein. NAD+ alone was not bound, but in the presence of UDP it was also bound in a ratio of 0.5 mol/mol catalytic protein. Experiments in which sodium borohydride was added to the enzyme incubation gave no indication that the 4‐keto intermediate is bound as a Schiff base to the enzyme. Also no evidence for epimerization at C‐3 of the 4‐ulose intermediate prior to ring contraction to apiose was found.
Transport of Hexoses across the Liver‐Cell Membrane
Authors: BAUR Hartmut , HELDT Hans W.
Published: March 2005

The uptake of radioactively labelled hexoses into the cellular space of isolated liver cells has been studied using silicone layer filtering centrifugation. From the hexoses tested, D‐glucose is transported most rapidly followed by D‐galactose and D‐fructose. The rate of L‐glucose transport is only 5% of that of D‐glucose. This transport enables the concentration of free aldohexoses in the cellular space to reach the external concentration.

For D‐glucose the rate of transport into the cell largely exceeds the rate of metabolic conversion. This is different with D‐fructose where the rate of transport is considerably lower but the rate of metabolism higher. Here the transport may even be a limiting step.

The transport of D‐glucose and D‐galactose shows a saturation characteristic, whereas D‐fructose appears not to be saturatable. The Km for D‐glucose is found to be 30 mM. All these hexoses compete with each other for transportation.

The temperature dependency of D‐glucose reveals an activation energy of 22 kcal (92 kJ)/mol (4–18 °C) and 7 kcal (29 kJ)/mol (18–37 °C). The transport of D‐glucose, D‐galactose and D‐fructose in inhibited by cytochalasin B, phloretin and phlorizin. It is not dependent on Na+ ions. Preliminary results showed no stimulation of the transport by insulin.

It is concluded that D‐glucose and other hexoses are transported by carrier‐mediated diffusion across the plasma membrane of liver cells. This transport shows a large resemblance to the transport of D‐glucose into human erythrocytes.
Least‐Squares Analysis of Circular Dichroic Spectra of Proteins
Authors: HAMMONDS R. Glenn
Published: March 2005

It is shown that the method proposed by Baker and Isenberg [Biochemistry, 15, 629 (1976)] for estimating secondary structure composition of proteins from circular dichroic spectra is a leastsquares fitting technique. Estimates obtained by this method for myoglobin, lysozyme, lactate dehydrogenase, papain, and ribonuclease are not substantively different from those obtained using unconstrained linear least squares.
4‐Amino‐hex‐5‐enoic Acid, a Selective Catalytic Inhibitor of 4‐Aminobutyric‐Acid Aminotransferase in Mammalian Brain
Authors: LIPPERT Bruce , METCALF Brian W. , JUNG Michel J. , CASARA Patrick
Published: March 2005

Incubation of rat brain 4‐aminobutyrate aminotransferase with 4‐amino‐hex‐5‐enoic acid, a substrate analog of 4‐aminobutyric acid, results in a time‐dependent irreversible loss of enzymatic activity. In the presence of 0.1 mM inhibitor the half‐life of the inactivation process is approximately 6 min. Low concentrations of L‐glutamic acid or 4‐aminobutyric acid protect against this inactivation. while 2‐oxoglutarate prevents this protection, suggesting that only the pyridoxal form of the enzyme is susceptible to inhibition by 4‐amino‐hex‐5‐enoic acid.

The irreversible inhibition of mammalian 4‐aminobutyrate aminotransferase by 4‐amino‐hex‐5‐enoic acid is selective. There is no inhibition of this enzyme from Pseudomonas fluorescens with the inhibitor at mM concentrations. Even at 10 mM there is no irreversible inhibition of mammalian glutamate decarboxylase or of aspartate aminotransferase, while alanine aminotransferase is inhibited over 500 times more slowly than rat brain 4‐aminobutyrate transaminase.
Poly(4‐thiouridylic Acid) as Messenger RNA and Its Application for Photoaffinity Labelling of the Ribosomal mRNA Binding Site
Authors: FISER Ilja , SCHEIT Karl H. , KUECHLER Ernst
Published: March 2005

Poly(4‐thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4‐thiouridine 5′‐diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein‐synthesizing system from E. coli. It stimulates binding of Phe‐tRNA to ribosomes both in the presence of EF‐Tu · Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70‐S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16‐S RNA. Ribosomes preincubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16‐S RNA can be reduced by treatment of ribosomes with colicin E3.
Deoxyribonuclease Activity Associated with Adenovirus 5 and 7
Authors: CAJEAN‐FEROLDI Chantal , CHARDONNET Yvette , CHANTEPIE‐AURAY Jacqueline
Published: March 2005

A DNase activity was found associated with isolated components of adenovirus types 5 and 7. The enzymatic activity was associated with the purified virus and viral components extracted from the soluble material of infected cells. The DNase activity of adenovirus type 5 was maximum at pH 5.5 and 7.0 whereas adenovirus 7 was active only at pH 7.0. Both DNases were shown to be located in the penton fraction.
Properties of ATPase Activity in Coupling Factor from Chromatium Strain D Chromatophores
Authors: GEPSHTEIN Amira , CARMELI Chanoch
Published: March 2005

Coupling factor extracted from chromatophores of the photosynthetic bacteria Chromatium strain D was partially purified. The enzyme catalyzed ATPase activity in the presence of Ca2+ and Mg2+ ions. Higher Vapp values were obtained when the activity was measured as a function of the divalent cation‐ATP complex rather than as a function of either the divalent cation or ATP because the free components competitively inhibited the activity in the presence of the cation‐ATP complex. The Km values were lower than or equal to the Ki values for free ATP indicating that the cation‐ATP complex is bound tighter than the free ATP to the enzyme. Based on these results a possible mode of binding of substrate to the active site of the enzyme was suggested.

A comparative study indicated no changes in the temperature dependance of ATPase activity when the enzyme was solubilized. However, possible conformation changes could have caused a decrease in the Km values for the (Ca‐ATP)2− and (Mg‐ATP)2− and in the Ki for free Mg2+ ions and ATP. The Ki for free Ca2+ ions increased on solubilization of the coupling factor. ATPase activity was inhibited by dicyclohexylcarbodiimide both in the soluble and in the membrane‐bound coupling factor.
Biosynthesis of Cyclopentenyl Fatty Acids
Authors: CRAMER Udo , SPENER Friedrich
Published: March 2005

In seeds of Hydnocarpus anthelminthica of Flacourtiaceae, cyclopentenylglycine and cyclopentenyl fatty acids are found naturally. The non‐proteinogenic amino acid may serve as precursor of cyclo‐pentenyl fatty acids via aleprolic acid, the starter molecule for these long‐chain compounds.

After administration of cyclopentenyl[2‐14C]glycine to maturing seeds of H. anthelminthica, labelled cyclopentenyl fatty acids were synthesized. Comparative activities were observed, when [1‐14C]aleprolic acid was supplied to the seeds. Incorporation studies with [1‐14C]acetate revealed that the chain‐lengthening systems for straight‐chain and cyclic fatty acids were still functioning in mature seeds.

Endosperm and embryo of H. anthelminthica seeds synthesized cyclopentenyl fatty acids from cyclopentenyl[2‐14C]glycine, [1‐14C]aleprolic acid and [1‐14C]acetate. In embryonic tissue, a dilution experiment proved the following path for cyclopentenyl fatty acid biosynthesis: cyclopentenylglycine → aleprolic acid → cyclopentenyl fatty acids. The conversion of cyclopentenylglycine to aleprolic acid may occur via transamination and oxidative decarboxylation; activated aleprolic acid is then lengthened by C2‐units to cyclopentenyl fatty acids.
The Superhelical Density of Nuclear DNA from Human Cells
Authors: COOK Peter R. , BRAZELL A.
Published: March 2005

Structures resembling nuclei may be released by gently lysing human cells in solutions containing non‐ionic detergents and high concentrations of salt. These structures, which we call nucleoids, sediment in sucrose gradients containing the intercalating agent, actinomycin D, in the manner characteristic of superhelical DNA. We have determined the concentration of actinomycin that minimises the rate of sedimentation of nucleoids. At this concentration, we have determined the amount of drug bound per base pair of DNA by means of a double‐labelling procedure. Assuming that each molecule of actinomycin bound to nucleoid DNA unwinds the double helix by 26°, we calculate that there is one supercoil every 90–180 base pairs in nucleoid DNA. These values lie within the range found for the circular DNA molecules of plasmids and viruses.
Are the Aerobic and Anaerobic Phosphofructokinases of Escherichia coli Different?
Authors: BABUL Jorge , ROBINSON John P. , FRAENKEL Dan G.
Published: March 2005

Phosphofructokinase has been purified from Escherichia coli strain K‐12 grown in a glucose‐limited chemostat, both aerobically and anaerobically. The enzymes migrated together in polyacrylamide gel electrophoresis, had the same subunit size in denaturing (dodecylsulfate) gels (Mr approx. 34000) and the same kinetic characteristics as described earlier for E. coli phosphofructokinase [e.g. Blangy et al. (1968) J. Mol. Biol. 31, 13–35]: a sigmoid curve of velocity vs. fructose 6‐phosphate concentration, activation by ADP, and inhibition by phosphoenolpyruvate. Findings [e.g. Doelle (1975) Eur. J. Biochem. 50, 335–342] of quite different enzymes in aerobic and anaerobic cells were not confirmed.
Measurement of Progesterone and Pregnenolone 16α‐Hydroxylase Activities by a Tritium‐Exchange Method
Authors: DE GRAEVE Jean , KREMERS Pierre , GIELEN Jaques E.
Published: March 2005

A new isotopic method, based upon the stereospecific replacement of a proton (3H) by a hydroxyl group, has been developed for the measurement of progesterone and pregnenolone‐16α‐hydroxylase activity. The incubation medium consists of a phosphate buffer (pH 7; 150 mM), NADPH (1 mM), nicotinamide (10 mM) and magnesium chloride (4 mM). Tween‐80 (1 mg/ml) is used to solubilize saturating concentrations of [16‐3H]progesterone (200 μM) or [16‐3H]‐pregnenolone (50 μM). The microsomal fraction isolated from a male rat liver is used as the enzymic source.

The enzymically released tritium is recovered in the incubation medium as molecules of tritiated water which are distilled under reduced pressure. The amount of radioactivity present in the water exactly reflects the 16α‐hydroxylase activity.

The method is easy to perform and is completely independent of any further metabolism of the 16α‐hydroxylated products.
Role of Divalent Ions in Folding of tRNA
Authors: LEROY Jean‐Louis , GUÉRON Maurice , THOMAS Gilles , FAVRE Alain
Published: March 2005

The native structure of tRNA is not achieved in low salt (4.5 mM Na+, 25 C), but can be restored by addition of divalent ions. We have explored the structure of the central region in Escherichia coli tRNAfMet by absorption and emission spectroscopy of 4‐thiouracil, and the structure of the anticodon loop in yeast tRNAPhe by fluorescence of the ‘Y’ base, versus the number of manganese ions bound to tRNA, which was derived from electron spin resonance. The fluorescence of the reduced 8–13 photoproduct (in which 4‐thiouracil at position 8 is crosslinked to cytosine at position 13) was also analysed.

In low salt (e.g. 4.5 mM Na+), the region of 4‐thiouracil is affected strongly as the first eight Mn2− bind to tRNA, whereas the fluorescence of the ‘Y’ base is affected only after four Mn2+ are bound. Considering the structural similarities of the two tRNAs, this suggests that the reorganisation brought about by divalent ions starts in the central region, the anticodon loop being affected later. The binding of divalent ions to each region starts together with its restructuration.

Monovalent ions can substitute for divalent ions in this process, a 15 mM sodium concentration being equivalent to the binding of the first five Mn2+. If divalent ions are then added, even the first ones distribute themselves between both the central and the anticodon region. Alternatively, the renaturation may be achieved by monovalent ions only, implying that no sites exist whose occupancy by divalent ions is crucial for the native structure. These observations suggest that the role and means of divalent ion binding to tRNA are largely explainable in terms of a simple maganese‐phosphate binding supplemented by electrostatic interaction with distant phosphates.
The Purification and Properties of Human α1‐Antitrypsin (α1‐Antiprotease), Variant Z
Authors: HERCZ Albert , BARTON Marcela
Published: March 2005

After three stages of preliminary purification, variant Z was chromatographed on a DEAE‐cellulose column. Upon elution with a linearly increasing concentration of NaCl, variant Z was recovered in two separate peaks, the first of which contained 81% and the second 19% of the total. The preparation corresponding to the first peak was homogeneous by various criteria. The trypsin and chymotrypsin inhibiting capacities and the specific antigenic activity of the preparation were nearly the same as those of an authentic sample of variant M. Variant Z contained 8 or 9 more glycine residues than variant M, but no appreciable difference was found between their carbohydrate contents. By analytical isoelectrofocusing the isoinhibitors of purified variant Z overlapped with those in the plasma of the donor and were cathodal to, but partially overlapped with purified variant M. After desialysation, the overlap between the different variants became complete, but variant Z contained a larger proportion of cathodal and smaller proportion of anodal components than variant M. Both variants formed five distinct isoinhibitor‐protease complexes after incubation with trypsin and chymotrypsin and the corresponding complexes in the different variants completely coincided.
Extracellular Protein A from a Methicillin‐Resistant Strain of Staphylococcus aureus
Authors: LINDMARK Roger , MOVITZ Jan , SJÖQUIST John
Published: March 2005

Protein A has been isolated from a methicillin‐resistant strain of Staphylococcus aureus, A676, which produces only extracellular protein A. The material is homogeneous after a one‐step separation and has a molecular weight of 41000. Its physicochemical and immunochemical properties have been studied.
Regulation of ATP Pools, rRNA and DNA Synthesis in 3T3 Cells in Response to Serum or Hypoxanthine
Authors: GRUMMT Friedrich , PAUL Dieter , GRUMMT Ingrid
Published: March 2005

The serum‐induced transition of 3T3 fibroblasts from resting to growing state is characterized by a marked increase in cellular ATP content, the maximal level of which is reached at the onset of DNA replication. This increase in cellular ATP during the G1 period of the cell cycle is correlated with about 3‐fold stimulation of transcription of rRNA measured in permeabilized cells in vitro. Addition of hypoxanthine to serum‐depleted quiescent 3T3 cells gives rise to an increase in both the ATP pool and the rate of rRNA synthesis. The expansion of cellular ATP pools after growth induction by serum seems to be a prerequisite for initiation of DNA synthesis since inhibition of purine de novo biosynthesis by azaserine inhibits both ATP pool expansion and DNA replication. This effect of azaserine can be abolished by addition of hypoxanthine to the culture medium.

It is concluded that (a) the increase of the rate of rRNA synthesis in 3T3 cells in response to growth factors or serum is controlled by the cellular purine nucleoside triphosphate concentration and (b) an increased ATP level is necessary for initiation of DNA synthesis but is not sufficient to trigger the events that lead to DNA replication.
Particle‐Bound Enzymes in the Bloodstream Form of Trypanosoma brucei
Authors: OPPERDOES Fred R. , BORST Piet , SPITS Hergen
Published: March 2005

We have screened the bloodstream form of Trypanosoma brucei for the presence of enzymes that could serve as markers for the microbodies and the highly repressed mitochondrion of this organism. None of seven known microbody enzymes were detected at all, but glycerol‐3‐phosphate oxidase, ATPase, isocitrate dehydrogenase, acid phosphatase and part of the hyperoxide dismutase and malate dehydrogenase activities were found to be particle‐bound after fractionation of homogenates by differential centrifugation. Part of the ATPase activity was sensitive to oligomycin, an inhibitor of oxidative phosphorylation. This oligomycin‐sensitive activity can serve as a specific marker for the mitochondria.

More than 80% of the NAD+ ‐linked glycerol‐3‐phosphate dehydrogenase in T. brucei was found to be particulate and latent. The enzyme could be activated by Triton X‐100, by the combined action of sonication and salt, but not by salt alone, and partially by freezing and thawing. We conclude that the NAD+ ‐linked glycerol‐3‐phosphate dehydrogenase is located inside an organelle.
Analysis of the relA Gene Product of Escherichia coli
Authors: PEDERSEN Finn Skou , KJELDGAARD Niels Ole
Published: March 2005

The relA gene product, ATP:GTP 3′‐pyrophosphotransferase (stringent factor) has been isolated in homogeneous form from an Escherichia coli strain polyploid for this gene at a yield of 1 mg/100 g cells and at a specific activity in a ribosome‐activated assay at 37 °C of 120 μmol guanosine pentaphosphate formed min−1 mg protein−1. The specific activity in a methanol‐activated assay at 25°C was found to be 4 μmol guanosine pentaphosphate formed min−1 mg protein−1. These values are about 100 times higher than reported by others.

Our further studies of this enzyme led to the following results.

Antibodies raised against this enzyme inhibit the ribosome‐activated synthesis of guanosine tetraphosphate and pentaphosphate but have no effect on the much slower synthesis, detected in the absence of ribosomes.

The amount of stringent factor in the relA+ strain CP78 is estimated to about 1 copy per 200 ribosomes.

The amount of antibody‐binding material in CP79 (relA) is at least 5 times lower.
Specific Casein Phosphorylation by a Casein Kinase from Lactating Bovine Mammary Gland
Authors: MACKINLAY Antony G. , WEST David W. , MANSON William
Published: March 2005

A preparation which contains protein kinase activity capable of rephosphorylating dephosphorylated αs1 and β‐caseins has been prepared from lactating bovine mammary gland. This activity is localised in the Golgi fraction and in this respect is similar to that obtained previously from rat tissue. It differs from the rat preparation in being unable to rephosphorylate dephosphorylated ϰ‐casein.

Progressive dephosphorylation of αs1 and β‐caseins increases the rate of their rephosphorylation in the presence both of Ca2+ and of Mg2+. For β‐casein significant non‐specific phosphorylation occurs in the presence of Mg2+ while in the presence of Ca2+ the level of phosphorylation is very low.

Comparison of the products of the action of this kinase on partially dephosphorylated αs1 and β‐caseins indicates that seryl residues grouped with pre‐existing phosphoseryl residues are phosphorylated preferentially in the presence of Mg2+, while the phosphorylation of isolated single seryl residues is promoted by Ca2+.

Digests of αs1‐casein, prepared by treatment with cyanogen bromide, have been fractionated and the three major peptides, separated. After dephosphorylation two of these, which between them contain all of the phosphate of αs1‐casein, are rephosphorylated at sites occupied in the native protein by phosphoseryl residues, i.e., at positions 46, 48, 75 and 115 of the αs1‐casein molecule. Evidence is presented which shows that this specificity of action also operates when intact αs1‐casein is used as phosphate acceptor. It is concluded that the enzyme responsible for specific phosphorylation acts through recognition of an amino acid sequence and does not require the provision of a specific structural conformation in the protein substrate.
Immunochemical Evidence for the Difference between Coenzyme‐B12‐Dependent Diol Dehydratase and Glycerol Dehydratase
Authors: TORAYA Tetsuo , FUKUI Saburo
Published: March 2005

Klebsiella pneumoniae ATCC 25955 (formerly named Aerobacter aerogenes PZH 572, Warsaw), which is known to produce coenzyme‐B12‐dependent glycerol dehydratase when grown anaerobically in a glycerol medium, formed coenzyme‐B12‐dependent diol dehydratase in a 1.2‐propanediol‐containing medium. Both the diol dehydratase and the glycerol dehydratase produced by the organism catalyzed the conversion of glycerol, 1,2‐propanediol and 1,2‐ethanediol to the corresponding aldehydes and underwent concomitant inactivation during the catalysis of glycerol dehydration, as does the diol dehydratase of K. pneumoniae (A. aerogenes) ATCC 8724. However, the two enzymes were distinguishable from each other by the monovalent‐cation‐selectivity pattern and by substrate specificity; that is, glycerol dehydratase preferred glycerol to 1,2‐propanediol as a substrate, whereas diol dehydratase preferred 1,2‐propanediol to glycerol, as judged from initial velocity studies. Ouchterlony double‐diffusion analysis and immunochemical titration with rabbit antiserum against diol dehydratase of K. pneumoniae ATCC 8724 established clearly that the diol dehydratase of K. pneumoniae ATCC 25955 is immunologically similar to that of K. pneumoniae ATCC 8724, while the glycerol dehydratase of the former is different from the diol dehydratase of both strains. Both the enzymes were found to be distributed in several bacteria of the family Enterobacteriaceae.
The Action of Lysozyme on Peptidoglycan with N‐Unsubstituted Glucosamine Residues
Authors: AMANO Ken‐ichi , HAYASHI Hideyuki , ARAKI Yoshio , ITO Eiji
Published: March 2005

A peptidoglycan preparation N‐acetylated at about 30% of glucosamine residues was obtained by the treatment of the lysozyme‐resistant cell wall peptidoglycan of Bacillus cereus with acetic anhydride at pH 7. Fractionation of dialyzable material resulting from lysozyme digestion of the glycan component of this peptidoglycan preparation yielded five oligosaccharides designated as S1 to S5 besides the disaccharide GlcNAc‐MurAc.

Oligosaccharide S3, which accounted for about 30% of the disaccharide units recovered as disaccharides and oligosaccharides, was identified as GlcN‐MurAc‐GlcNAc‐MurAc. Oligosaccharide S1, accounting for about 20% of the disaccharide units recovered, was characterized as GlcN‐MurAc‐GlcN‐MurAc‐GlcNAc‐MurAc, while oligosaccharide S2, present in a smaller amount, as GlcNAc‐MurAc‐GlcN‐MurAc‐GlcNAc‐MurAc. Oligosaccharides S4, and S5, present in small amounts, were identified as GlcNAc‐MurAc‐GlcNAc‐MurAc and MurAc‐GlcNAc‐MurAc, respectively.

Oliogsaccharides S1, S3 and S5 proved to be completely insusceptible to lysozyme, whereas S2 was digested by lysozyme to produce GlcNAc‐MurAc and S3. S1 was found to act as a more potent inhibitor than S3 in lysozyme‐catalyzed digestion of polysaccharides.

The results obtained show that the lysozyme‐catalyzed hydrolysis of peptidoglycan oligosaccharides has an obligatory requirement for the N‐acetyl group on the glucosamine residue located in subsite C in the enzyme‐substrate complex.
Effects of Fatty Acid Unsaturation on Chylomicron Metabolism in Normal and Hepatectomized Rats
Authors: FLORÉN Claes‐Henrik , NILSSON Ake
Published: March 2005

1. Hepatectomized rats were injected intravenously with doubly labelled ([14C]linoleic acid and [3H]palmitic acid) thoracic duct lymphs from rats fed cream, triolein or corn oil. The disappearance of the radioactive fatty acids of different molecular triacylglycerol species and of phospholipids from plasma was studied.

2. 73–93% of the injected triacylglycerols had been cleared from plasma within 15 min. At all stages of lipolysis the 3H/14C ratio of the plasma triacylglycerol was the same as in the injected material. If the cream chyle had been cooled to 4 °C before use there was, however, an enrichment of [3H]palmitic acid and of fully saturated triacylglycerols in the remnant particles formed.

3. Only 38–50% of the radioactive chyle phosphatidylcholine was eliminated from plasma in 30 min. At this time most of the remaining phosphatidylcholine was, however, in other lipo‐protein classes than the chylomicron remnants.

4. Also in intact rats data were obtained, indicating that the major portion of chylomicron phospholipids is transferred to other serum lipoproteins by exchange or net movement rather than being hydrolysed in the 1‐position by lipoprotein lipase or taken up intact by the liver.

5. More of both the labelled fatty acids appeared in liver triacylglycerols in experiments with cream chyle than in experiments with corn oil chyle. Data were obtained suggesting that this may be due to a higher uptake of intact triacylglycerol as remnant particles.

6. When linoleic acid is fed as a tracer dose in cream, a high proportion (16–36%) is incorporated into chyle phospholipids.
Studies on the Role and Mode of Operation of the Very‐Lysine‐Rich Histone H1 in Eukaryote Chromatin
Authors: HARTMAN Peter G. , CHAPMAN George E. , MOSS Thomas , BRADBURY E. Morton
Published: March 2005

Limited digestion with trypsin of both calf thymus H1 histone and the fragment 1‐120 of the H1 molecule has resulted in the isolation of the fragment 35‐120. This fragment assumes a globular structure under physiological conditions of pH and ionic strength. The variable N‐terminal portion of the molecule, up to residue 34, is not required for the formation of the H1 globular structure.

Proton nuclear magnetic resonance (NMR) and ultracentrifugation studies show that the H1 histone molecule consists of three distinct structural domains under structuring conditions: a random coil ‘nose’ consisting of 35 to 40 residues from the N‐terminal end; a globular ‘head’ involving the next approximately 80 residues; and a random‐coil ‘tail’ of the remainder of the molecule.
Structure de la bacillomycine L, antibiotique de Bacillus subtils
Authors: BESSON Françoise , PEYPOUX Françoise , MICHEL Georges , DELCAMBE Lucien
Published: March 2005

The Structure of Bacillomycin L, an Antibiotic from Bacillus subtilis

The structure of bacillomycin L, an antifungal agent isolated from the culture medium of a strain of Bacillus subtilis, has been investigated. The peptide moiety contains one mole each of D‐aspartic acid, L‐aspartic acid, L‐glutamine, L‐serine, D‐serine, L‐threonine, and D‐tyrosine. The lipid moiety is a mixture of 3‐amino‐12‐methyltridecanoic acid (46%), 3‐amino‐12‐methyltetrade‐canoic acid (38%), 3‐amino‐14‐methylpentadecanoic acid (11%), and two minor homologues.

The peptide sequence and the cyclic structure were determined by structural analysis of the peptides obtained by mild acid hydrolysis.

The molecular weight was determined by the thermoosmotic method; this showed that bacillomycin L has a monomeric structure which is given in Formula 1.
Purification and Comparative Study of Adenine and Guanine Phosphoribosyltransferases from Schizosaccharomyces pombe
Authors: NAGY Maria , RIBET Anne‐Marie
Published: March 2005

1. Adenine and guanine phosphoribosyltransferases from the yeast Schizosaccharomyces pombe were purified respectively 1200‐fold and 1000‐fold from the crude extracts giving specific activities respectively of 18 and 5.7 μmol purine nucleotide monophosphates mg protein−1 min−1. The purified enzymes remain stable for at least a month when conserved at −20°C in the presence of 6 mM 5‐phosphoribosyl‐1‐pyrophosphate (P‐Rib‐P2) and 2 mM MgCl2.

2. Both phosphoribosyltransferases have multiple, apparently buffer‐independent, pH optima; both have an absolute requirement for Mg2+, but, contrary to other organisms studied, the effects of inhibitory concentrations of Mg2+ are non‐competitive towards P‐Rib‐P2 binding.

3. The hyperbolic P‐Rib‐P2versus rate curve of guanine phosphoribosyltransferase (but not that of adenine phosphoribosyltransferase) becomes sigmoidal in the presence of fixed concentrations of the reaction product (GMP).

4. Guanine phosphoribosyltransferase is ten times more sensitive to inhibition by reaction products in comparison to adenine phosphoribosyltransferase. The inhibition of the first enzyme by other purine and pyrimidine nucleotides appears much more specific relative to the last enzyme.

5. Both activities are separated in two active forms by filtration through a controlled‐pore glass column CPG‐10. The estimation of molecular weights of these forms gives 48000 and 42000 for guanine phosphoribosyltransferase and 50000 and 44000 for adenine phosphoribosyltransferase. In the presence of 5 mM MgCl2 the last enzyme, but not the first, dissociates into a single form of apparent molecular weight 30000.

6. Electrophoresis on polyacrylamide gel of crude as well as of purified extracts reveals two active bands of adenine phosphoribosyltransferase but only a single active band of guanine phosphoribosyltransferase.
Kinetics of Sheep‐Liver Cytoplasmic Aldehyde Dehydrogenase
Authors: MacGIBBON Alastair K. H. , BLACKWELL Leonard F. , BUCKLEY Paul D.
Published: March 2005

1. Sheep liver cytoplasmic aldehyde dehydrogenase showed little pH dependence of V or kcat. Some buffer anion effects were noted.

2. The oxidation of aldehydes at pH 7.6 was quantitative but irreversible. The initial velocity data indicated a sequential mechanism for the addition of substrates. Inhibition by NADH and the product analogue 2‐bromo‐2‐phenylacetic acid, together with the known tight binding of NADH to the free enzyme, indicated an ordered mechanism with NAD+ as leading substrate.

3. Values for the rate of binding and dissociation of NAD+ were obtained from the steady‐state data. The values obtained were virtually identical with those which could be calculated from the data for the horse liver cytoplasmic enzyme. Close similarities are in general apparent for the horse and sheep liver cytoplasmic enzymes and with other tissue aldehyde dehydrogenases.

4. Apparent substrate activation was observed with high concentrations of both acetaldehyde and propionaldehyde, a limiting value of 0.25 s−1 being obtained for kcat. No isotope effect was observed on V using [1‐2H]propionaldehyde as substrate suggesting that NADH release might be rate‐limiting in the steady‐state.

5. The implications of the non‐linear steady‐state behaviour are discussed.
Frequency Distribution of Messenger Sequences within Polysomal mRNA and Nuclear RNA from Rat Liver
Authors: SIPPEL Albrecht E. , HYNES Nancy , GRONER Bernd , SCHÜTZ Günther
Published: March 2005

DNA complementary to polysomal poly(A)‐containing mRNA (cDNA) of male rat liver was used to study the diversity of messenger sequences in the nucleus and in polysomes.

1. Hybridization of cDNA against an excess of its own polysomal mRNA template revealed that about 10000 different mRNA species are expressed in the liver tissue. They are distributed in a wide frequency range derived from approximately 0.5% of the total genome.

2. Hybridization of the cDNA against total nuclear RNA shows that messenger sequences comprise less than 1% of the mass of total nuclear RNA. Messenger sequences have a different frequency distribution in nucleus and cytoplasm.

3. In hybridizations using cDNA, which had been fractionated into sequences representing abundant and scarce polysomal mRNA molecules, it was found that although abundant cytoplasmic messenger sequences are also abundant in the nucleus, they exist in a significantly lower frequency range in the nuclear compartment.
The Lipid Bilayer Structure of the Abnormal Human Plasma Lipoprotein X
Authors: LAGGNER Peter , GLATTER Otto , MÜLLER Karl , KRATKY Otto , KOSTNER Gerhard , HOLASEK Anton
Published: March 2005

The structure of the abnormal lipoprotein X occurring in the plasma of patients with obstructive jaundice was investigated by X‐ray small‐angle scattering. The data were analyzed by discussing the distance distribution functions obtained directly from the experimental data by Fourier transformation, involving no a priori assumptions. The results provide evidence for lipoprotein X being essentially a random distribution of lamellae with a thickness of 5.1 nm and are consistent with hollow spherical (vesicular) structures of outer diameters greater than 30 nm with some overall size heterogeneity. Under the experimental conditions chosen, lipoprotein concentrations between 0.01 and 0.18 g/ml in buffers of low ionic strength, lateral stacking as observed in negative‐stain electron microscopy does not occur. The electron density profile perpendicular to the lamellar plane indicates that a lipid bilayer is the underlying structural principle, with the protein moieties partly bound within the polar head‐group regions and partly occluded in soluble form in the vesicle interior.
Detergent Influence on Rat‐Liver Galactosyltransferase Activities towards Different Acceptors
Authors: BRETZ Rolf , STÄUBLI Willy
Published: March 2005

1. Galactosyltransferase activities in postnuclear supernatants and Golgi fractions from rat liver were assayed with two improved and simplified methods, using high‐ and low‐molecular‐weight acceptors. Transfer to N‐acetylglucosamine was measured after the separation of the reaction product N‐acetyllactosamine from all other radioactive molecules (including galactose) on an ion‐exchange column partially converted to the borate form. To determine the transfer of galactose to a glycoprotein acceptor we used ovomucoid, which accepts galactose without any previous chemical or enzymic modification.

2. Both enzymic activities were enriched 60–80‐fold (compared with the post‐nuclear supernatant) in Golgi fractions, which were isolated on two subsequent sucrose gradients and identified morphologically by their high contents of stacked Golgi elements. The two activities could not be resolved by isolation of the Golgi fractions or by detergent solubilization. Each acceptor inhibited the galactose transfer to the other one (up to 95%), presumably because both compete for the same enzyme.

3. The transferase activities were enhanced by the nonionic detergent Triton X‐100. The degree of activation depended directly on the amount of Triton bound to the membrane, i.e. the Triton/phospholipid ratio and not the w/v concentration of the detergent in the assay medium. This relationship persisted, regardless of the purity of the Golgi preparation: Half‐maximal activation occurred at the same Triton/phospholipid ratio in postnuclear supernatants as well as in isolated Golgi fractions. The activation could not be explained by complete solubilization, because 50% of the fully activated enzyme could still be sedimented (1 h, 100000 ×g).

4. Galactose transfer to the high‐molecular‐weight acceptor required a higher Triton/phospholipid ratio for half‐maximal activation than did the transfer to the monosaccharide N‐acetylglucosamine (1 mg/mg compared with 0.5 mg/mg). The degree of activation maximally achieved was much higher with the protein acceptor (400%) than with the sugar (150%). Because both activities are probably due to the same enzyme, it is suggested that these differences in activation reflect properties of the membrane rather than the enzyme, e.g. the presence of a tight diffusion barrier for ovomucoid and the breakdown of this barrier by the detergent.
A Kinetic Study of the pH‐Dependent Properties of the Ferric Undecapeptide of Cytochrome c (Microperoxidase)
Authors: WILSON Michael T. , RANSON Richard J. , MASIAKOWSKI Piotr , CZARNECKA Eva , BRUNORI Maurizio
Published: March 2005

The ferric form of the haem undecapeptide, derived from horse cytochrome c by peptic digestion, undergoes at least three pH‐induced transitions with pK values of 3.4, 5.8 and 7.6. Temperature‐jump experiments suggest that the first of these is due to the binding of a deprotonated imidazole group to the ferric iron while the second and third arise from the binding of the two available amino groups present (the α‐NH2 of valine and the ɛ‐NH2 of lysine).

Molecular models indicate that steric restraints on the peptide dictate that these amino groups may only coordinate to iron atoms via intermolecular bonds, thus leading to the polymerization of the peptide.

Cyanide binding studies are in agreement with these conclusions and also yield a value of 3.6 × 106 M−1 s−1 for the intrinsic combination constant of CN− anion with the haem.

A model is proposed which describes the pH‐dependent properties of the ferric undecapeptide.
The Purification and Properties of Two Low‐Molecular‐Weight Proteins Required for the Initiation of Translation in Ascites Tumour Cells
Authors: DAHL Hans‐Henrik M. , TRUELSEN Ebbe , BLAIR G. Eric
Published: March 2005

A fractionated cell‐free protein‐synthesising system from Krebs II ascites tumour cells is described. It is dependent on the addition of exogenous mRNA. In this system the translation of natural mRNAs requires the presence of four initiation factors: the high‐molecular‐weight complex IF‐M3, IFEMC and two low‐molecular‐weight proteins IF‐Mα and IF‐Mβ. None of these factors are required for the translation of the synthetic messenger poly(A,U,G). The purification of IF‐Mα and IF‐Mβ was achieved by the use of gel and ion‐exchange chromatography. Analysis of the purified proteins by sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed that the molecular weight of IF‐Mα is 19000 and that of IF‐Mβ is 15000. The initiation factor IF‐E1 isolated from rabbit reticulocytes can substitute for IF‐Mβ in the translation of encephalomyocarditis virus RNA. IF‐Mα does not correspond to any previously recognised eukaryotic initiation factor.
Mapping of 23‐S rRNA at the Ribosomal Peptidyl‐Transferase Center by Photo‐Affinity Labeling
Authors: SONENBERG Nahum , WILCHEK Meir , ZAMIR Ada
Published: March 2005

Photo‐sensitive peptidyl‐tRNA's were used to scan the environment of the peptidyl‐transferase center of the ribosome. The specificity of the previously described labeling in the 18‐S fragment of 23‐S rRNA by Boc‐Phe(N3)‐Phe‐tRNA (4‐azido‐N‐t‐butoxycarbonyl‐phenylalanyl‐phenylalanyl‐tRNA) was demonstrated by the ability of the covalently anchored molecule to serve as donor substrate in peptide bond formation. Labeling patterns were also obtained with Boc‐Phe(N3)‐Phe‐Phe‐tRNA bound at the acceptor site and with Boc‐Phe(N3)‐(Gly)n‐Phe‐tRNA (n= 2,4). The results indicate that sequences within the 18‐S fragment of 23‐S rRNA are located close to the acceptor site as well as along the path where the peptide moiety adheres to the ribosome. Identification of the labeled sequences is expected to shed light on the spatial arrangement as well as functional role of rRNA in the peptidyl transferase center.
A Steady‐State‐Kinetic Random Mechanism for Glutathione S‐Transferase A from Rat Liver
Authors: JAKOBSON I. , WARHOLM M. , ASKELÖF P. , MANNERVIK B.
Published: March 2005

The steady‐state kinetics of glutathione S‐transferase A from rat liver were studied with the substrates glutathione and 3,4‐dichloro‐1‐nitrobenzene varying from 1 μM to 5 mM and from 1 μM to 0.5 mM, respectively. The mathematical description of the experimental data required a rate law which was at least third degree with respect to concentration of each of the substrates. The rate equation of a generalized random sequential mechanism was fitted by a nonlinear regression program to the kinetic‐data set and was found to be superior to other two‐substrate models tested. Product inhibition studies with S‐(2‐chloro‐4‐nitrophenyl)‐glutathione were also in accordance with a random sequential mechanism for the forward reaction. The second product of the reaction, chloride, was a very poor inhibitor. In the regression analysis of the data, the correlation of experimental error with velocity was taken into account by weighting. For this purpose an empirical error function was used.

Binding of the product S‐(2‐chloro‐4‐nitrophenyl)‐glutathione was studied by the method of equilibrium partition in an aqueous two‐phase system. The binding could be described by a rectangular hyperbola which showed binding at a ratio of 1.95 molecules of product per molecule of enzyme at saturating product concentration. No cooperativity could be established by the binding studies and it is assumed that each of the two subunits of the enzyme can bind one molecule of S‐(2‐chloro‐4‐nitrophenyl)‐glutathione.

The results of the different experiments were all in accordance with the proposition of a steady‐state random sequential model for the mechanism of action of GSH S‐transferase A, provided that kinetically significant enzyme‐product complexes were introduced. In structural and functional terms the model has the following simple features: the two subunits of the enzyme are not assumed to display any cooperativity, and a subunit contains only one catalytically relevant binding site for GSH and GSH derivatives and one for the electrophilic substrate; these sites constitute subsites of the catalytically active center of the enzyme.
Biosynthesis of N‐Glycolylneuraminic Acid in Porcine Submandibular Glands
Authors: BUSCHER Hans‐Peter , CASALS‐STENZEL Jorge , SCHAUER Roland , MESTRES‐VENTURA Pedro
Published: March 2005

1. The morphology of porcine submandibular gland tissue is demonstrated by light and electron microscopy.

2. Subcellular particles from porcine submandibular glands were prepared by a procedure adapted to the presence of viscous mucus, and were fractionated on continuous and discontinuous sucrose gradients.

3. Six particulate fractions were obtained by discontinuous gradient centrifugation. These were identified by electron microscopy, marker enzymes and nucleic acid content and were designated as follows: (1) plasma membranes, (2) smooth membranes and light Golgi membranes, (3) heavy Golgi membranes, (4) membrane mixture and mitochondria, (5) mitochondria and (6) membrane mixture and mucin residues. The bulk of rough membranes and cell nuclei was found in the 3000 ×g sediment before fractionation.

4. Sialyltransferase and N‐acetylneuraminate monooxygenase showed highest activities in the Golgi membrane fractions representing 45% and 36%, respectively, of total tissue activity. The specific activities of these membrane‐bound enzymes were respectively 13 and 10 times higher when compared with the homogenate.

5. The acylneuraminic acid/protein ratio was lowest in the Golgi membranes when compared with the other particulate fractions and the cytosol glycoprotein. The molar N‐acetylneuraminic acid/N‐glycolylneuraminic acid ratio was highest in the two Golgi membrane fractions (average 45:55) and in the pool of free acylneuraminic acids (41:59), and lowest in the cytosol glycoprotein (10:90).

6. Approximately 2.6% of the total acylneuraminic acids occurring in porcine submandibular glands are freely diffusible, 80% of them representing free N‐acetylneuraminic acid and N‐glycolyl‐neuraminic acid in a molar ratio of about 4:6 and 20% representing cytidylyl‐glycosides of N‐acetylneuraminic acid and N‐glycolylneuraminic acid in a ratio of 6:4.

7. In radioactive labelling experiments with surviving slices of porcine submandibular glands the highest specific radioactivities were found in the pool of free acylneuraminic acids, followed by N‐acetylneuraminic acid and N‐glycolylneuraminic acid bound to the Golgi membranes. N‐Acetylneuraminic acid and N‐glycolylneuraminic acid of the cytosol glycoproteins have lowest turnover rates.

8. Based on these experiments a model is presented describing two pathways of N‐acetylneuraminic acid modification in the course of glycoprotein biosynthesis. At least 40% of N‐acetylneuraminic acid is hydroxylated before its transfer onto Golgi membranes and linkage to growing glycoprotein molecules and about 50% thereafter. This results in a molar N‐acetylneuraminic acid/N‐glycolyl‐neuraminic acid relationship of 1:9 in the secreted glycoprotein. It is assumed that free N‐acetylneuraminic acid is modified by the soluble cytosol monooxygenase whereas the glycoprotein‐bound N‐acetylneuraminic acid is hydroxylated by the corresponding membrane‐bound enzyme.
Incorporation of the 1‐pro‐R and the 1‐pro‐S Hydrogen Atoms of Ethanol into Steroids and Phosphatidylcholines in vivo
Authors: CRONHOLM Tomas , CURSTEDT Tore
Published: March 2005

The transfer of deuterium from chiral 1‐monodeuteroethanols to various metabolites formed in the liver was studied in order to investigate the coupling of metabolic reductions to the alcohol dehydrogenase and the aldehyde dehydrogenase reactions. The ethanols were administered to female bile fistula rats for 10 h. The hydrogen at C‐2 in the glycerol moiety of newly formed phosphatidylcholine molecules in bile, liver and plasma was derived to 22‐25% from the 1‐pro‐R position and to 5‐6% from the 1‐pro‐S position in the ethanol. sn‐Glycerol 3‐phosphate isolated from liver had a lower deuterium content at C‐2. The ratio between the contributions from the two positions in ethanol to C‐2 of free sn‐glycerol 3‐phosphate was the same as in the phosphatidylcholines. This indicates that the higher degree of labelling of this position in phosphatidylcholines is not due to a specific coupling between alcohol dehydrogenase and the formation of a phosphatidylcholine precursor. Cholesterol and chenodeoxycholic acid in bile became increasingly labelled, and the ratio between the incorporations from the 1‐pro‐S and the 1‐pro‐R positions of ethanol was about 0.37 in cholesterol and 0.46 in chenodeoxycholic acid. Thus, these NADPH‐dependent reactions utilized hydrogen from the 1‐pro‐S position to a larger extent than NADH‐dependent reactions.
Transcription Fidelity and Structural Integrity of Isolated Nucleoli
Authors: BEEBEE Trevor J. C. , BUTTERWORTH Peter H. W.
Published: March 2005

1. RNA was transcribed in vitro using isolated nucleoli, the endogenous form A RNA polymerase and mercurated UTP as one of the nucleoside triphosphate substrates. The products were isolated from endogenous nucleolar RNA sequences by chromatography on sulphydryl‐Sepharose and analysed by hybridisation in vast DNA excess. The results of competition‐hybridisation experiments suggested that a large proportion of the transcript was ribosomal RNA although some sequences had been transcribed from DNA of lower reiteration frequency.

2. Analyses of the constituents of nucleoli following isolation by the sonication procedure suggested that the nucleolar DNA, particularly the ribosomal cistrons, are severely degraded. Furthermore, indications were obtained that the transcription complexes were damaged and many of the nascent RNA chains appeared to have been sheared from near the growing points.